MXPA06000189A

MXPA06000189A - Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof

Info

Publication number: MXPA06000189A
Application number: MXPA/A/2006/000189A
Authority: MX
Inventors: Bojsen Kirsten; Liu Wei; Tune Berg Casper; M F Derkx Patrick; Fioresi Carol; M Kragh Karsten; Shaw Andrew; Gerritse Gijsbert; R Thoudahl Charlotte
Original assignee: Tune Berg Casper; Danisco A/S; M F Derkx Patrick; Fioresi Carol; Genencor International Inc; Gernot Abel; Gerritse Gijsbert; Kellettsmith Anja H; M Kragh Karsten; Liu Wei; Shaw Andrew; Veltman Oene R
Priority date: 2003-07-07
Filing date: 2006-01-05
Publication date: 2006-12-13

Abstract

This invention relates to amylase polypeptides, and nucleic acids encoding the polpypeptides and uses thereof. The amylases of the present invention have been engineered to have more beneficial qualities. Specifically, the amylases of the current invention show an altered exospecifity.

Description

ZW), Eürasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), For two-letter codes and other abbrevialions. referto the "GuidEurop? an (AT. BE. BG, CH, CY, CZ, DE, DK, EE, ES, FL ance Notes on Codes and Abbreviations" appearing at the begm- FR, GB, GR, HU, EE, IT , LU, MC, NL, PL, PT, RO, SE, YES, ning ofeacli regular issue of the PCT Gazette. "SK, TR), - OAPl (BF, BJ, CF. CG, Cl, CM, GA, GN, GQ, .GW, ML..MR, NE, SN, TD, TG.) Published - without intemational search repon and to be republished upon receipt oftliaí report EXO-SPECIFIC AMYLASE POLYPEPTIDES, NUCLEIC ACIDS THAT CODIFY FOR THOSE POLIPEPTIDES AND USES THEREOF FIELD OF THE INVENTION This invention relates to the amylase polypeptides, and to the nucleic acids encoding the polypeptides and uses thereof. The amylases of the present invention have been engineered to have more beneficial qualities. Specifically, the amylases of the present invention show altered thermostability. BACKGROUND OF THE INVENTION Improved amylases can improve the problems inherent in certain processes, such as baking. The crystallization of amylopectin takes place in the starch granules a few days after baking, which leads to increased bread firmness and causes bread to break up. When the bread was pulled, the bread loses the softness of the crumb and the humidity of the crumb. As a result, the crumbs become less elastic, and the bread develops a chunky crust. Enzymatic hydrolysis (by amylases, for example) of amylopectin side chains can reduce crystallization and increase anti-tear. The crystallization depends on the length of REF..169268 the amylopectin side chains. The longer side chains are, the greater the crystallization. Most starch granules are composed of a mixture of two polymers: amylopectin and amylose, of which about 75% is amylopectin. Amylopectin is a very large branched molecule consisting of chains of a-D-glucopyranosyl units linked by links (1-4), where the chains are linked by a-D- (1-6) bonds to form branches. Amylose is a linear chain of linked α-D-glucopyranosyl units (1-4) that have few a-D- (1-6) branches. The baking of floury bread products, such as white bread, bread made from crushed rye flour and wheat flour and buns, are obtained by baking the bread dough at oven temperatures in the range of 180 to 250 ° C. approximately 15 to 60 minutes. During the baking process, a gradually lower temperature gradient (200 -> 120 ° C) prevails over the outer layers of the dough, where the crust of the baked product is developed. However, due to the steam, the temperature in the crumb is only about 100 ° C at the end of the baking process. Above temperatures of approximately 85 ° C, enzymatic inactivation can take place and the enzyme will not have anti-tear properties. Only the thermostable amylases, in this way, are able to modify the starch efficiently during baking. The present invention is directed to polypeptides having altered thermostability. BRIEF DESCRIPTION OF THE INVENTION In a first aspect, the invention provides a polypeptide that provides a variant of PS4, the variant of PS4 is derivable from a progenitor polypeptide. The progenitor enzyme may preferably be a non-maltogenic exoamylase from Pseudomonas saccharophila, such as the exoamylase having the amino acid sequence described in SEQ ID. ?or. : 1 or SEQ ID: 5. The progenitor enzyme can preferably be a non-maltogenic exoamylase of Pseudomonas stutzeri, such as a polypeptide having the amino acid sequence described in SEQ ID?: 7 or SEQ ID No. : eleven.

Other members of the PS4 family can be used as progenitor enzymes. In preferred modalities, the parent polypeptide is a non-maltogenic exoamylase of Pseudomonas saccharophila having the amino acid sequence described in SEQ ID. : l or described in SEQ ID?: 5. In other preferred embodiments, the parent polypeptide is a non-maltogenic exoamylase of Pseudomonas stutzeri, having the amino acid sequence described in SEQ ID No. : 7 or described in SEQ ID No.: 11. In a preferred embodiment, the PS4 variant differs from the progenitor polypeptide by the inclusion of amino acid substitutions, the substitutions being located in a position comprising at least one position selected from the group which consists of: 4, 9, 13, 33, 34, 42, 70, 71, 87, 99, 100, 108, 113, 121, 131, 134, 135, 141, 153, 157, 158, 160 ,. 161, 166, 170, 171, 178, 179, 184, 188, 198, 199, 221, 223, 238, 270, 277, 290, 307, 315, 334, 335, 342, 343, 372, 392, 398, 399, 405, 415, 425, where the reference to the position of the numbering is with respect to the sequence of Pseudomonas saccharophila shown in SEQ ID No .: 1. In a preferred embodiment, the PS4 variant differs from the parent polypeptide by the inclusion of amino acid substitutions, the substitutions located in a position comprising at least one position selected from the group consisting of: 4, 33, 34, 70, 71, 87, 99, 108, 113, 121, 134, 141 , 157, 158, 171, 178, 179, 188, 198, 199, 223, 290, 307, 315, 334, 343, 399, and 405, where the reference to the numbering of the position is with respect to the sequence of Pseudomonas saccharophila shown in SEQ ID No: 1. Preferably, the position is at least one position selected from the group consisting of: 33, 34, 71, 87, 121, 134, 141, 157, 178, 179, 223, 307, 334, and 343. Prefere The variant of PS4 comprises at least one substitution selected from the group consisting of N33Y, D34N, K71R, G87S, G121D, G134R, A141P, L178F, A179T, G223A, H307L, S334P, and D343E. In yet another embodiment, the exoamylase further comprises an additional substitution at a selected position of 108, 158, 171 and 188. Preferably, the PS4 variant comprises at least one substitution selected from the group consisting of K108R, G158D, Y171S, and G188A . Preferably, the PS4 variant comprises at least one substitution selected from the group consisting of: G4D, N33Y, D34N, G70D, K71R, G87S, A99V, K108R, V113I, G121D, G134R, A141P, I157L, G158D, Y171S, L178F, A179T, G188A, Y198F, Y198L, A199V, G223A, V290I, H307L, I315V, S334P, D343E, S399P, A405F, and A405E. Preferably, the PS4 variant comprises the following substitutions: N33Y, D34N, G134R, A141P, I157L, G223A, H307L and S334P with at least one additional substitution of L178F or A179T. Preferably, the PS4 variant comprises at least one of the following substitutions: N33Y, D34N, I157L, L178F, A179T, G223A or H307L. Preferably, the PS4 variant comprises the following substitutions: G87S, G134R, A141P, or S334P. In other preferred embodiments, the variant PS4 polypeptide comprises a combination selected from: G134R, A141P I157L G223A H307L S334P D343E G121D; G134R A141P I157L G223A H307L S334P D343E N33Y G121D; G134R A141P I157L G223A H307L S334P D343E N33Y; G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G87S G121D S214N T375A; G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D Y171S G188A N138D; G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D; G87S G121D G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T; G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34? K71R L178F A179T G188A; G134R, A141P I157L G223A H307L S334P K71R L178F A179T; G134R, A141P I157L G223A H307L S334P L178F A179T; G134R, A141P I157L G223A H307L S334P? 33Y D34? L178F A179T; G134R, A141P I157L G223A H307L S334P L178F A179T G87S G121D; G134R, A141P I157L G223A H307L S334P L178F A179T G121D; G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T G121D E343D; G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34? K71R L178F A179T; G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T Y33? 34D E343D; G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T G121D; G134R, A141P I157L G223A H307L S334P K71R L178F A179T G121D; G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; 113F, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; A99V, VL131, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, I157L, Y198F, G223A, V290I, H307L, S334P, D343E V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; A199V, D343E, V1131, A141P, I157L, Y198F, G223A, V290I, H307L, S334P; V113I, A141P, I157L, Y198F, G223A, V290I, S334P, D343E; V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, A141P, Y198F, G223A, V290I, H307L; V113I, A141P, Y198F, G223A, V290I, S334P, D343E; V113I, A141P, Y198F, G223A, A268P, V290I, S399P V113I, A141P, Y198F, G223A, V290I, S399P; V113I, A141P, Y198, G223A, V290I; V113I, A141P, Y198F, G223A, V290I; Y198F, G223A, V290I; Y198, G223A, V290I; V113I, A141P, I157L, Y198F, G223A, V290I; V113M; V113A; VL13I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, 1315V, S334P, D343E; D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, G188S, Y198F, G223A, V290I, H307L, S334P, D343E; K71R, V113I, G134R, A141P, I157L, L178L, Y198F, G223A, V290I, H307L, G313G, S334P, D343E; D34N, VL13I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, G313G, S334P, D343E; V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, 11701, Y198F, G223A, V290I, H307L, G313G, S334P, D343E V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L, G313G, S334P, D343E; G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405V; A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198L, G223A, V290I, H307L, S334P, D343E; VL13I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; K71R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; K108R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; D34G, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; G4D, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; and A141P, G134R, G223A, H307L, I157L, V113I, V290I, Y198F, G188A; In other preferred embodiments, the variant polypeptide of PS4 comprises a selected combination of: G134R, A141P, I157L, G223A, H307L and S334P; G121D, G134R ,. A141P, I157L, G223A, H307L and S334; G87S, G121D, G134R, A141P, I157L, G223A, H307L and S334P; G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; and N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P. In other preferred embodiments, the variant PS4 polypeptide comprises a combination selected from the following: N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; and N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P.

In other preferred embodiments, the PS4 variant is an amino acid comprising the sequence described either as SEQ ID No: 2; SEQ ID No: 3; SEQ ID No: 4; SEQ ID No: 4a, SEQ ID No: 4b, SEQ ID No: 4c, SEQ ID No: 8, SEQ ID No: 9 or SEQ ID No: 10. The variant of PS4 can be derived from Pseudomonas sp. In one embodiment, the Pseudomonas species is selected from Pseudomonas saccharophila and Pseudomonas stutzeri. The variant polypeptide of PS4 may comprise one or more mutations in addition to those described above. Other mutations, such as deletions, insertions, substitutions, transversions, transitions and inversions, in one or more other sites, may also be included. Similarly, the polypeptide may be lacking at least one of the substitutions described above. In a preferred embodiment, the polypeptide is truncated. The truncation may be at the N-terminus or at the C-terminus. The progenitor enzyme or PS4 variant may lack one or more portions, such as subsequences, signal sequences, domains or portions, whether active or not. For example, the progenitor enzyme or variant polypeptide of PS4 may lack a signal sequence, as described herein. Alternatively, or in addition, the progenitor enzyme or the PS4 variant may lack one or more catalytic or binding domains. In preferred embodiments, the progenitor enzyme or the PS4 variant may lack one or more of the domains present in non-maltogenic exoamylases, such as the starch binding domain. For example, PS4 polypeptides can have only the sequence up to position 429, relative to the numbering of a non-maltogenic exoamylase of Pseudomonas saccharophila shown in SEQ ID No: 1. In a preferred embodiment, they are provided the variants of PS4, pSac-d34 (SEQ ID No: 4c, Fig.8c), pSac-D20 (SEQ ID No: 4a, Fig.8a) and pSac-D14 (SEQ ID No: 4b, Fig.8b) ), the variants having an amino acid sequence as described in the Figures. The variant of PS4 can also comprise a homologous sequence. A homologous sequence comprises a nucleotide sequence at least 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence encoding a polypeptide enzyme of the PS4 variant. Preferred embodiments also include functional equivalents. The PS4 variant polypeptides described herein are derivatives of, or are variants of, polypeptides that preferably exhibit non-maltogenic exoamylase activity. Preferably, the enzymes . progenitors are non-maltogenic exoamylases by themselves.

Variant polypeptides of PS4 in the preferred embodiments also show non-maltogenic exoamylase activity. The PS4 variants described herein will preferably have exospecificity, for example measured by exo-specificity indices, as described herein, consistent with their exoamylases. In addition, these preferably have higher or increased exospecificity, when compared to the progenitor enzymes or the polypeptides from which they are derived. Thus, for example, the PS4 variant polypeptides may have an exospecificity index of 20 or more, for example, their total amylase activity (including exoamylase activity) is 20 times or more, greater than their endoamylase activity. . In the preferred embodiments, the exo-specificity index of the exoamylases is 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more. In the preferred embodiments, the exospecificity index is 150 or more, 200 or more, 300 or more, or 400 or more. Preferably, the variant of PS4 will be more thermostable than the parent. Preferably, the variant PS4 polypeptide is capable of degrading starch at temperatures from about 55 ° C to about 80 ° C or more. Preferably, the PS4 variant retains its activity after exposure to temperatures of up to about 95 ° C. The PS4 variant polypeptides described herein have prolonged half-lives relative to the progenitor enzyme, preferably by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more, preferably at elevated temperatures of 55 ° C to about 95 ° C or more, preferably at about 80 ° C. Preferably, the sample is heated for 1-10 minutes at 80 ° C or more. Preferably, the variant PS4 polypeptide is more stable at pH. Preferably, it has a higher pH stability than its cognate progenitor polypeptide. Preferably, the variant PS4 polypeptide is capable of degrading the starch at a pH of about 5 to about 10.5. Variant polypeptides of PS4 may have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or longer half-life when compared to their polypeptides progenitors under identical pH conditions. In another embodiment, the degree of stability to the pH can be evaluated by measuring the activity or the specific activity of the enzyme under specific pH conditions. The specific pH conditions can be any pH from pH 5 to pH 10.5. In preferred embodiments, the functional equivalents will have sequential homology to at least one of the members of the PS4 family. Functional equivalents will have sequential homology to either the non-maltogenic exoamylases of Pseudomonas saccharophila and Pseudomonas stutzeri mentioned above, preferably both. The functional equivalent may also have sequential homology with any of the sequences described in _ J3EQ ID _Nos: 1 to 12, preferably SEQ ID No: 1 or SEQ ID No: 7 or both. The sequence homology is preferably at least 60%, preferably 65% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, preferably 90% or more, preferably 95% or more. Sequential homologies can be generated by any or all of the programs described herein. In other embodiments, functional equivalents will be capable of specifically hybridizing to any of the sequences described above. In a second aspect, the invention provides a nucleic acid, the nucleic acid encoding a polypeptide comprising a variant of PS4 that is derivable from a progenitor polypeptide, as described above. The progenitor enzyme may preferably be a non-maltogenic exoamylase from Pseudomonas saccharophila as described in SEQ ID No: as described in SEQ ID No: 5. The progenitor enzyme may preferably be a non-maltogenic exoamylase from Pseudomonas stutzeri, as described in SEQ ID No: 5. described in SEQ ID No: 7 or as described in SEQ ID No. 11. Other members of the PS4 family can be used as progenitor enzymes. In preferred modalities, the parent polypeptide is a non-maltogenic exoamylase derived from the non-maltogenic exoamylase of Pseudomonas saccharophila having a sequence as described in SEQ ID No: 1 or as described in SEQ ID No: 5. In other preferred embodiments, the The parent polypeptide comprises a non-maltogenic exoamylase from Pseudomonas stutzeri having a sequence shown as described in SEQ ID No: 7 or as described in SEQ ID No: 11. In a preferred embodiment, the nucleic acid encoding the variant of PS4 differs from the parent nucleic acid because it encodes for amino acid substitutions, the substitutions are located in a position comprising at least one position selected from the group consisting of: 4, 9, 13, 33, 34, 42, 70, 71, 87, 99, 100, 108, 113, 121, 131, 134, 135, 141, 153, 157, 158, 160, 161, 166, 170, 171, 178, 179, 184, 188, 198, 199, 221, 223, 238, 270, 277, 290, 307, 315, 334, 335, 342, 343, 372, 392, 398, 3 99, 405, 415, 425, where the reference to the numbering of the position is with respect to a sequence of Pseudomonas saccharophila described in the sequence SEQ ID No: 1. Preferably, the positions are. at least one position selected from the group consisting of: 33, 34, 87, 121, 134, 141, 157, 178, 179, 223, 307, and 334. Preferably, the nucleic acid encoding the PS4 variant comprises an acid nucleic that -codes -for at least one substitution in the polypeptide, the substitution is selected from the group consisting of: N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P. Preferably, the nucleic acid encoding the PS4 variant comprises the following substitutions: N33Y, D34N, G134R, A141P, I157L, G223A, H307L and S334P with at least one additional substitution of L178F or A179T. Preferably, the nucleic acid encoding the PS4 variant comprises one of the following substitutions: N33Y, D34N, I157L, L178F, A179T, G223A or H307L.

Preferably, the nucleic acid encoding the PS4 variant comprises at least one of the following substitutions: G87S, G134R, A141P, or S334P. In yet another embodiment, the nucleic acid encoding the PS4 variant comprises one of the following substitutions: K71R, K108R, G158D, Y171S, G188A, and D343E. In another embodiment, the nucleic acid encoding the PS4 variant comprises a nucleic acid sequence encoding a combination selected from the group of: G134R, A141P I157L G223A H307L S334P D343E G121D; G134R A141P I157L G223A H307L S334P D343E N33Y G121D; G134R A141P I157L G223A H307L S334P D343E N33Y; G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T G87S G121D S214? T375A; G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T G121D Y171S G188A? 138D; G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T G121D; G87S G121D G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T; G87S G121D G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T G188A; G134R, A141P I157L G223A H307L S334P K71R L178F A179T; G134R, A141P I157L G223A H307L S334P L178F A179T; G134R, A141P I157L G223A H307L S334P? 33Y D34? L178F A179T; G134R, A141P I157L G223A H307L S334P L178F A179T G87S G121D; G134R, A141P I157L G223A H307L S334P L178F A179T G121D; G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T G121D E343D; G87S G121D G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T; G87S G121D G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T Y33? 34D E343D; G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D; G134R, A141P I157L G223A H307L S334P K71R L178F A179T G121D; G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; 113F, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; A99V, V1131, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, I157L, Y198F, G223A, V290I, H307L, S334P, D343E V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; A199V, D343E, V1131, A141P, I157L, Y198F, G223A, V290I, H307L, S334P; V113I, A141P, I157L, Y198F, G223A, V290I, S334P, D343E; V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, A141P, Y198F, G223A, V290I, H307L; V113I, A141P, Y198F, G223A, V290I, S334P, D343E V113I, A141P, Y198F, G223A, A268P, V290I, S399P V113I, A141P, Y198F, G223A, V290I, S399P; V113I, A141P, Y198, G223A, V290I; V113I ,. A141P, Y198F, G223A, V290I; Y198F, G223A, V290I; Y198W, G223A, V290I; -V113I, A141P, I157L, -Y198F, G223A, V290I; V113M; VI13A; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, 1315V, S334P, D343E; D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, G188S, Y198F, G223A, V290I, H307L, S334P, D343E; K71R, V113I, G134R, A141P, I157L, L178L, Y198F, G223A, V290I, H307L, G313G, S334P, D343E; D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, G313G, S334P, D343E; V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, 11701, Y198F, G223A, V290I, H307L, G313G, S334P, D343E V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L, G313G, S334P, D343E; G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405V; A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198L, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; K71R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; K108R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; D34G, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; G4D, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; and A141P, G134R, G223A, H307L, I157L, V113I, V290I, Y198F, G188A; G134R, A141P, I157L, G223A, H307L and S334P; G121D, G134R, A141P, I157L, G223A, H307L and S334; G87S, G121D, G134R, A141P, I157L, G223A, H307L and S334P; G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; and N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P. N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; and N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P In preferred embodiments, the nucleic acid encoding the PS4 variant codes for an amino acid sequence comprising SEQ ID No : 2; SEQ ID No: 3; SEQ ID No: 4; SEQ ID No: 4a; SEQ ID No: 4b; SEQ ID No: 4c; SEQ ID No: 8, SEQ ID No: 9 or SEQ ID: 10. In a preferred embodiment, the nucleic acid encodes a truncated polypeptide. The truncation may be at the N-terminus or at the C-terminus. The progenitor enzyme or variant of PS4 may lack one or more portions, such as sub-sequences, signal sequences, domains or portions, whether active or not. For example, the progenitor enzyme or the variant polypeptide of PS4 may lack a signal sequence.

Alternatively, or in addition, the progenitor enzyme or the PS4 variant may lack one or more catalytic binding domains. In a preferred mode, the progenitor enzyme or the PS4 variant may lack one or more of the domains present in the non-maltogenic exoamylases, such as the starch binding domain. For example, the PS4 polypeptides may have only the sequences of position 429 with reference to the numbering of the non-maltogenic exoamylase of Pseudomonas saccharophila shown as SEQ ID No: 1. In a preferred embodiment, the nucleic acid encodes the variants of PS4, pSac-d34, pSac-D20 and pSac-D14 as described in the figures. The nucleic acid encoding the variant PS4 polypeptide may comprise one or more mutations in addition to those described above. Other mutations such as deletions, insertions, substitutions, transversions, transitions or inversions in one or more other sites may also be included. Likewise, the polypeptide encoded by the nucleic acid may be lacking at least one of the substitutions described above. The nucleic acid encoding the PS4 variant can also comprise a homologous sequence. A homologous sequence comprises a nucleotide sequence of at least 75, 10, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence encoding a polypeptide enzyme variant of PS4. Preferred embodiments also include a nucleic acid encoding a polypeptide that is a functional equivalent of a variant of PS4. The nucleic acids encoding the PS4 variant polypeptides described herein are derivatives of, or are variants of, the nucleic acids that preferentially encode an enzyme having non-maltogenic exoamylase activity. Preferably, the progenitor enzymes encoded by the nucleic acids are non-maltogenic exoamylases by themselves. PS4 variant polypeptides encoded by nucleic acids in preferred embodiments, also show non-maltogenic exoamylase activity. The PS4 variants encoded by the nucleic acids will preferably have exospecificity, for example measured by exospecificity indices, as described herein. In addition, these preferably have greater or increased exospecificity, when compared to the progenitor enzymes or polypeptides from which they are derived, preferably under identical conditions. Thus, for example, the PS4 variant polypeptides can have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or higher. exospecificity These may have 1.5 x or greater, 2 x or greater, 5 x or greater, 10 x or greater, 50 x or greater, 100 x or greater, when compared to their parent polypeptides, preferably under identical conditions. Preferably, the PS4 variant encoded by the nucleic acid will be more thermostable than the parent counterpart. Preferably, the variant PS4 polypeptide is capable of degrading the starch at temperatures of about 55 ° C to about 80 ° C or more. Preferably, the PS4 variant retains its activity after exposure to temperatures up to about 95 ° C. The PS4 variant polypeptides described herein have long half-lives relative to the progenitor enzyme, preferably by 10%, 20%, 30%, 40%, 50%,. 60%, 70%, 80%, 90%, 100%, 200% or more, preferably at elevated temperatures of 55 ° C to about 95 ° C or more, preferably at about 80 ° C. Preferably, the sample is heated for 1-10 minutes at 80 ° C or more. Preferably, the variant PS4 polypeptide, encoded by the nucleic acid, is stable at pH.

Preferably, it has a higher pH stability than its parent polypeptide. Preferably, the variant PS4 polypeptide is capable of degrading the starch at a pH of about 5 to about 10.5. The specific pH conditions can be any pH from pH 5 to about pH 10.5. The variant PS4 polypeptide encoded by the nucleic acid may have a longer half-life, or. . Increased activity (depending on the assay) when compared to the parent polypeptide under identical conditions. The variant polypeptide of PS4 may have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or longer half-life when compared to its parent polypeptide under identical pH conditions. Alternatively, or in addition, these may have higher activity when compared to the parent polypeptide under identical pH conditions. In a preferred embodiment, the functional equivalents encoded by the nucleic acid will have sequential homology to at least one of the members of the PS4 family. Functional equivalents will have sequential homology to either the non-maltogenic exoamylases of Pseudomonas saccharophila and Pseudomonas stutzeri mentioned above, preferably to arabas, in a preferred embodiment. The functional equivalent may also have sequential homology in any of the sequences described as SEQ ID Nos: 1 to 12, preferably SEQ ID No: l or SEQ ID No: 7 or both. The sequence homology is preferably at least 60%, preferably 65% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, preferably 90% or more, preferably 95% or more. In other embodiments, a nucleic acid complementary to a nucleic acid encoding any of the PS4 variants described herein is provided.

In addition, a nucleic acid capable of hybridizing to complement.

In a preferred embodiment, a nucleic acid encoding functional equivalents is provided herein, which will be capable of specifically hybridizing to any of the sequences described above, as well as to its complement. In a preferred embodiment, the sequence for use in the methods and compositions described herein, is a synthetic sequence, this includes, but is not limited to, the sequence prepared with the optimal use of codon for host organisms - such as methylotrophic yeasts Pichia and Hansenula. A third aspect of the invention provides the compositions comprising at least one polypeptide variant of PS4 and yet another ingredient. The other ingredient may be an enzyme selected from the group consisting of oxidoreductases, hydrolases, lipases, esterases, glucosidases, amylases, pullulanases, xylanases, cellulases, hemicellulases, enzymes that degrade starch, proteases, and lipoxygenases. In a preferred embodiment, the composition comprises at least one variant of PS4 and a maltogenic Bacillus amylase, as described in 091/04669. A preferred embodiment comprises a variant of PS4 and flour. The additional enzyme can not be added together with any dough ingredient including flour, water or other ingredients or optional additives or the dough-improving composition. The additional enzyme can be added before or after the flour, water and optionally other ingredients or additives or the dough-improving composition. The additional enzyme may be a liquid preparation or be in the form of a dry composition. A fourth aspect provides the vectors comprising a polypeptide variant of PS4, the cells comprising a polypeptide variant of PS4 and the methods for expressing a polypeptide variant of PS4. In a preferred embodiment, the invention is directed to a recombinant replicable vector with a nucleic acid encoding a variant PS4 polypeptide. The vector may further comprise any of the elements described herein. Another preferred embodiment provides a host cell comprising a nucleic acid encoding a variant of PS4. The host cell can be any of the bacterial cells, fungal or yeast described herein. In a preferred embodiment, the invention is written for a method of expressing a PS4 polypeptide as provided herein. Other aspects of the invention may be found in the related applications (attorney's case No. 674510-2007 and GC807 incorporated by reference herein, including any drawings, references and figures.) BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graph that shows the improvement of thermostability of the PS4 variants.

PS4ccl is an expressed control enzyme, derived from Pseudomonas saccharophila, without signal sequence and lacking the binding domain to starch. The half-life in minutes is plotted against temperature in degrees C for PS4ccl, pSac-D3, pSac-D20 and pSac-D14. Figure 2 is a graph showing the dose effect of PSac-D34 in a model test of the mass system. The solid content of the migajon was measured in Nuclear Magnetic Resonance (NMR). The firmness measured by the solids content is plotted against the days after baking for the control, 0.5, 1, 2 ppm of D34. Figure 3 is a graph showing the results of a baking test showing the reduced firmness and the rate or rate of affirmation after adding PSac-D3 and Psac-D14 in a dose of 1 mg per kg of flour. The firmness measured by hPa is plotted against the days after baking for control. Figure 4 shows a baking test, showing ^ the effect of increased softening of PSac-D3 (G134R, A141P, I157L, G223A, A307L, S334P, K71R, D343E, N33Y, D34N, L178F, A179T) compared to Psac-D3 without N33Y, D34N, K71R, L178F, A179T, which has tl / 2-75 of 3.6 in contrast, to that of PSac-D3 which is 9.3 minutes at 75 ° C. Figure 5 shows a reference sequence of PS4 (SEQ ID: 1), derived from the amino acid sequence of the maltotetrahydrolase from Pseudomonas saccharophila. Figure 6 shows the sequence of a variant of PS4 (SEQ ID: 2); the amino acid sequence of Pseudomonas saccharophila maltotetrahydrolase with the substitutions G134R, A141P, I157L, G223A, H307L, S334P,? 33Y, D34 ?, L178F and A179T. Figure 7 shows the sequence of the variant of PS4 (SEQ ID: 3); the amino acid sequence of maltotetrahydrolase of. Pseudomonas saccharophila with the substitutions G134R, A141P, I157L, G223A, H307L, S334P,? 33Y, D34 ?, L178F, A179T and G121D. Figure 8A shows the sequence of the variant of PS4 (SEQ ID: 4); the amino acid sequence of the maltotetrahydrolase from Pseudomonas saccharophila with the substitutions G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, L178F, A179T, G121D and G87S. Figure 8B shows the sequence of PSac-D20 (SEQ ID No. 4a); the amino acid sequence of Pseudomonas saccharophila maltotetrahydrolase with 13 substitutions and suppression of the starch binding domain. Figure 8C shows the sequence of PSac-D14 (SEQ ID No. 4b); the amino acid sequence of Pseudomonas saccharophila maltotetrahydrolase with 14 substitutions and suppression of the starch binding domain. Figure 8D shows the sequence Psac-D34; the amino acid sequence of maltotetrahydrolase from Pseudomonas saccharophila with 11 substitutions and suppression of the starch binding domain. Figure 9 shows an amino acid sequence of the maltotetrahydrolase from Pseud nonas saccharophila (SEQ ID No: 5). The precursor of glucan-1-alpha, maltotetrahydrolase from Pseudamanas saccharophila (EC 3.2.1.60) (G4-amylase) (maltotetraose-forming amylase) (Exo-maltotetrahydrolase) (exo-amylase-forming maltotetraose). SWISS-PROT access number P22963. Figures 10A and 10B show a nucleic acid sequence of the maltotetrahydrolase from Pseudomonas saccharophila (SEQ ID No: 6). The mta gene of P. saccharophila coding for maltotetrahydrolase (EC number = 3 .2 .1 .60). GenBank access number X16732.

Figure 11 shows an amino acid sequence of the maltotetrahydrolase from Pseudomonas stutzeri (SEQ ID No. 7). Figure 12 shows the sequence of PStu-D34 (SEQ ID No: 8); the amino acid sequence of Pseudomonas stutzeri maltotetrahydrolase with substitutions G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N. Figure 13 shows the sequence of PStu-D20 (SEQ ID No: 9); the amino acid sequence of maltotetrahydrolase from Pseudomonas stutzeri with G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N and G121D. Figure 14 shows the sequence of PStu-D14 (SEQ ID No.: 10); the amino acid sequence of maltotetrahydrolase from Pseudomonas stutzeri with G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, G121D and G87S. Figure 15 shows the sequence of Pseudomonas stutzeri (Pseudomonas perf ec take iná) (SEQ ID No: 11). The precursor of glucan-1, 4-alpha-maltotetrahydrolase (EC 3. 2.1.60) (G4-amylase) (maltotetraose-forming amylase) (exo-maltotetrahydrolase) (maltotetraose-forming exo-amylase). SWISS-PROT access number P13507. Figure 16 shows the nucleic acid sequence of maltotetrahydrolase from Pseudomonas stutzeri. The maltotetraose amylase-forming gene (amyP) of P. stutzeri, cds. complete. GenBank access number M24516 (SEQ ID No: 12).

DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention will employ, unless otherwise indicated, the conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of experience ordinary in the art. Such techniques are explained in the literature.

See for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements, Current Protocols in Molecular Biology, ch 9,13, and 16, John Wiley &Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O.D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855, Lars-Inge Larsson "Immunocytochernistry: Theory and Practice", CRC Press Inc., Baca Mouse, Florida, 1988, ISBN 0-8493-6078-1, John D. Psund (ed); "Immunochemical Protocols, vol 80", in ^ 5 _the_ series: "Methods in_ Molecular. Biology", Humana Press, Totowa, New Jersey, 1998, ISBN 0-89603-493-3, Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, NY, Marcel Dekker, ISBN 0-8247- 0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited by Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3. Each of these general texts is incorporated by reference in the present. As used herein, "PS4" will refer to the family members related to or having the sequence functional homology with the non-maltogenic exoamylase of Pseudomonas saccharophila, such as the exoamylase having the amino acid sequence described in SEQ ID No: SEQ ID No: 5 or the exoamylase not maltogenic of Pseudomonas stutzeri, such as a polypeptide having the amino acid sequence described in SEQ ID No: 7 or SEQ ID No.11. Other members of the family are described in Table 1. The numbering of the position with respect to the PS4 variants derived from Pseudomonas saccharophila exoamylase of be with respect to SEQ ID No: 1: 1 DQAGKSPAGV RYHGGDEIIL QGFHWNWRE APND AND ILR Q.QASTIAADG FSAIWMPVPW 61 RDFSSWTDGG KSGGG GYFW HDFNKNGRYG SDAQLRQAAG ALGGAGVKVL YDWPNHMNR 121 GYPDKEINLP AGQGFWRNDC ADPGNYPNDC DDGDRFIGGE SDLNTGHPQI YGMFRDEL f? 181 LRSGYGAGGF RFDFVRGYAP ERVDS MSDS ADSSFCVGEL KGPSEYPS DWRNTASWQQ "" 241 IIKDWSDRAK CPVFDFALKE "RMQNGSVADW KHGLNGNPDP RWREVAVTFV D HDTGYSPG 301 QNGGQHHWAL QDGLIRQAYA YILTSPGTPV VYWSHMYDWG YGDFIRQLIQ VRRTAGVRAD 361 SAISFHSGYS GLVATVSGSQ QTLWALNSD LANPGQVASG SFSEAVNASN GQVRVRSGS 421 GDGGGNDGGE GGLVNVNFRC DNGVTQ GDS VYAVGNVSQL GNSPASAVR LTDTSSYPTW 0 481 KGSIALPDGQ NV? WKCLIR EADATLVRQ QSGGNNQVQA AAGASTSGSF The reference sequence is derived from the sequence of Pseudomonas saccharophila which has SWISS-PROT accession number P22963, but without the signal sequence MSHILRAAVLAAVLLPFPALA. 5 The nutnexac system, although it can use a specific sequence as a base reference point, is also applicable to all relevant homologous sequences. For example, the numbering of the position can be applied to homologous sequences from other Pseudomonas species, or homologous sequences from other bacteria. Preferably, such homologs have 60% or more of homology, for example 70% or more, 80% or more, 90% or more or 95% or more of homology, with the reference sequence SEQ ID No: 1. Sequential homology between proteins can be ascertained using well-known alignment programs and hybridization techniques described herein.

The numbering of the position with respect to PS4 variants derived from a Pseudomonas stutzeri shall be with respect to SEQ ID No: 7: 1 DQAGKSPNAV RYHGGDEIIL QGFHWNVVRE APNDWYNILR QQAATIAADG FSAIW PVPW 61__RDFSSWSDGS KSGGG GYFW HDFNKNGRYG SDAQLRQAAS ALGGAGVKVL YDWPNHMNR 121 GYPDKEINLP AGQGFWRNDC ADPGNYPNDC DDGDRFIGGD ADLNTGHPQV YGMFRDEFTN 181 LRSQYGAGGF RFDFVRGYAP ERVNSWMTDS ADNSFCVGEL WKGPSEYPNW DWRNTASWQQ 241 IIKDWSDRAK CPVFDFALKE RMQNGSIADW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHHWAL QDGLIRQAYA Y? LTSPGTPV VYWSHMYDWG YGDFIRQLIQ VRRAAGVRAD 361 SAISFHSGYS GLVATVSGSQ QTLWALNSD LGNPGQVASG SFSEAVNASN GQVRVWRSGT 421 GSGGGEPGAL VSVSFRCDNG ATQMGDSVYA VGNVSQLGNW SPAAALRLTD TSGYPTWKGS 481 IALPAGQNEE WKCLIRNEAN ATQVRQWQGG ANNSLTPSEG ATTVGRL As used herein, "variant PS4 nucleic acids" will refer to nucleic acids encoding PS4 polypeptides that are variants of members of the PS4 family. As used herein, "variant PS4 polypeptides" or "PS4 variant" will refer to polypeptides that are variants of the members of the PS4 family. As used herein, "progenitor enzymes", "progenitor sequence", "progenitor polypeptide" and "progenitor polypeptides" will mean the enzymes and polypeptides upon which the variant PS4 polypeptides are based. The progenitor enzyme can be a precursor enzyme (for example, the enzyme that is effectively mutated) or it can be prepared de novo. The progenitor enzyme can be a wild-type enzyme. As used herein, "variant" will mean a molecule that is derivable from a molecule that is progenitor. The variants will include the polypeptides as well as the nucleic acids. The variants will include substitutions, insertions, transversions and investments, among other things, in one or more sites. Variants will include truncations. The variants will include homologs and functional derivatives of the progenitor molecules. The variants will include sequences that are complementary to the sequences that are capable of hybridizing to the nucleotide sequences presented herein. For example, a variant sequence is complementary to sequences capable of hybridizing under severe conditions (eg, 50 ° C and 0.2 x SSC { 1 x SSC = 0.15 M sodium chloride, 0.015 Na3 citrate, pH 7.0.} .) to the nucleotide sequence presented here. More preferably, the term variant encompasses sequences that are complementary to sequences that are capable of hybridizing under conditions of high stringency or severity (eg, 65 ° C and 0.1 x SSC { Lx SSC = 0.15 M sodium chloride, Na3 citrate 0.015 M, pH 7.0.).) to the nucleotide sequences presented herein. As used in this, "precursor" will mean an enzyme used to produce a modified enzyme. The precursor can be a modified enzyme by mutagenesis. Similarly, the precursor can be a wild-type enzyme, a variant wild-type enzyme or an already mutated enzyme. As used herein, "functional equivalent" in relation to a progenitor enzyme will mean a molecule having similar or identical function to a progenitor molecule. The progenitor molecule can be a non-maltogenic exoamylase of Pseudomonas saccharophila or a non-maltogenic exoamylase of Pseudomonas stutzeri, or a polypeptide obtained through other sources. The functionally equivalent enzyme may have a different amino acid sequence, but it will have non-maltogenic exoamylase activity. Examples of assays for determining functionality are described herein, and are known to one of skill in the art. As used herein, "isolated" will mean that the sequence is at least substantially free of at least one other component with which the sequence is naturally associated and found in nature. As used herein, "purified" will mean that the sequence is in a relatively pure state, eg, about 90% pure, or at least about 95% pure, or at least about 98% pure.

As used herein, "amylase" will mean an enzyme that is, among other things, capable of catalyzing the degradation of starch. Amylases are hydrolases that break the α-D- (1-4) -O-glycosidic bonds in the starch. In general, a-amylases (EC 3.2.1.1, aD- (1-V4) -glucan-glucanohydrolase) are defined as endo-action enzymes that break the aD- (1-> 4) -glucosidic bonds within the starch molecule in a random way. In contrast, exo-action amylolytic enzymes, such as ß-amylases (EC 3.2.1.2, aD- (1- »4) -glucan-maltohydrolase) and some product-specific amylases such as maltogenic α-amylase (EC 3.2). .1.133) break the starch molecule from the non-reducing end of the substrate. The β-amylases, the a-glucosidases (EC 3.2.1.20, aD-glucoside-glucohydrolase), glucoamylase (EC 3.2.1.3, aD- (1- >) -glucan-glucohydrolase), and product specific amylases can produce malto-oligosaccharides of a specific length from the starch. As used herein, "non-maltogenic exoamylase enzyme" will mean an enzyme that does not initially degrade the starch to substantial amounts of maltose. Tests for making such determinations are provided herein. As used herein, "linear malto-oligosaccharide" will mean 2 -20 units of an α-D-glucopyranose linked by an α- (1-4) bond.

As used herein, "thermostable" refers to the ability of the enzyme to maintain activity after exposure to elevated temperatures. The thermostability of an enzyme such as a non-maltogenic exoamylase is measured by its half-life. The half-life (tl / 2) is the time in minutes during which half of the activity of the enzyme is inactivated under defined conditions. The half-life value is calculated by measuring the residual amylase activity. The half-life assays are conducted in more detail in the Examples. As used herein, "pH stable" refers to the ability of the enzyme to retain activity over a wide range of pHs. The pH tests are conducted as described in the Examples. As used herein, "exo-specific (a) a" refers to an improved "exo-specificity index", eg, increased, as compared to an exo-specificity ratio of an unsubstituted exoamylase. . As used herein, "exo-specificity index" will mean the ratio of the total amylase activity to the total endoamylase activity. Assays for measuring the activity of amylase and endoamylase are provided herein.

As used herein, "food" will include prepared food, as well as an ingredient for a food such as flour. As used herein, "food ingredient" will include a formulation, which is or can be added to functional foods or food products, and includes formulations used at low levels in a wide variety of products that require, for example, acidification or emulsification. The food ingredient may be in the form of a solution or as a solid - depending on the use and / or the mode of application and / or the mode of administration. As used herein, "functional food" means a food capable of providing not only a nutritional effect and / or a taste satisfaction, but is also capable of distributing an additional beneficial effect to the consumer. As used herein, "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein." In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme". As used herein, "peptoid form" will refer to the variant amino acid residues wherein the carbon a substituent group is on the nitrogen atom of the residue rather than on the a carbon atom. The processes for preparing the peptides in the peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89 (20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13 (4), 132-134. As used herein, "nucleotide sequence" or "nucleotide sequence" or "nucleic acid sequence" refers to an oligonucleotide sequence or polynucleotide sequence, and variants, homologs, fragments, and derivatives thereof (such as portions of the same) . The nucleotide sequence may be of a genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded, representing either the sense or antisense strand. As used herein, the term nucleotide sequence includes genomic DNA, cDNA, synthetic DNA and RNA. Preferably, this means DNA and RNA, more preferably, the cDNA sequence encoding a variant polypeptide of PS4. As used herein, "starch" will mean the starch per se or a component thereof, especially amylopectin. The term "starch medium" means any suitable medium comprising starch. The term "starch product" means any product that contains or is based on or is derived from starch.

Preferably, the starch product contains or is based on or is derived from the starch obtained from wheat flour. As used herein, "flour" shall mean the finely ground grain of wheat or other grain. For example, flour can be obtained from wheat per se and not from another grain. Wheat flour may refer to wheat flour per se, as well as wheat flour when it is present in a medium, such as dough. As used herein, "baked bread product" means any baked product based on mass obtainable by mixing the flour, water and a fermentation agent or yeast, under mass forming conditions. Additional components can be added to the dough mixture. As used herein, "homologue" and "homology" will mean an entity having some degree of identity with the amino acid sequence of interest and the nucleotide sequences of interest. A homologous sequence is taken to include at least 75, 80, 85 or 90% identical amino acid sequence, preferably at least 95, 96, 97, 98 or 99% identical to the sequence of interest. Typically, homologs will comprise the same active sites as the amino acid sequence of interest. As used herein, "hybridization" will include the process by which a strand of nucleic acid binds with a complementary strand through the use of base pairing as well as the amplification process as carried out in the polymerase chain reaction. The PS4 nucleic acid can exist as single-stranded or double-stranded DNA or RNA, an RNA / DNA heteroduplex or an RNA / DNA copolymer. As used herein, "copolymer" refers to a single nucleic acid strand comprising ribonucleotides and deoxyribonucleotides. The PS4 nucleic acid can even be optimized at the codon to further increase expression. As used herein, "synthetic" will refer to that which is produced by chemical or enzymatic synthesis in vi tro. This includes, but is not limited to, the PS4 nucleic acids made with optimal use of the codon for host organisms such as methylotrophic yeasts of Pi chi a and Hansenul a. As used herein, "transformed cells" will include cells that have been transformed by the use of recombinant DNA techniques. The transformation typically occurs by the insertion of one or more nucleotide sequences within a cell. The inserted nucleotide sequence can be a heterologous nucleotide sequence (for example, it is a sequence that is not natural for the cell to be transformed.) In addition, or in the alternative, the inserted nucleotide sequence can be a homologous nucleotide sequence, for example , is a sequence that is natural for the cell to be transformed) - so that the cell receives one or more extra copies of a nucleotide sequence already present in it. As used herein, "operably linked" will mean that the described components are in a relationship that allows them to function in their intended manner. A regulatory sequence operably linked to a coding sequence is ligated in a manner such that expression of the coding sequence is achieved under the condition compatible with the control sequences. As used in the present, "biologically active (o)" shall refer to a sequence that has a similar structural function (but not necessarily to the same degree), and / or similar regulatory function (but not necessarily to the same degree) and / or similar biological function (but not necessarily to the same degree) to the sequence of natural origin. I. Detailed Description of the Polypeptides of the Invention In a first aspect, the invention provides a polypeptide comprising a variant of PS4, the PS4 variant is derivable from a progenitor polypeptide. The progenitor enzyme may preferably be a non-maltogenic exoamylase, preferably a non-maltogenic bacterial exoamylase enzyme. The progenitor enzyme may preferably be a polypeptide that displays non-maltogenic exoamylase. The progenitor enzyme can be a non-maltogenic exoamylase of Pseudomonas saccharophila such as the exoamylase described in SEQ ID No .: 1 or SEQ ID No .: 5. The progenitor enzyme can be a non-maltogenic exoamylase of Pseudomonas stutzeri, such as a polypeptide as described in SEQ ID No: 7 or SEQ ID No.11. Other members of the PS4 family can be used as progenitor enzymes as described in Table 1 below. Preferably, members of the PS4 family will generally be similar to, homologous to or functionally equivalent to the exoamylases described in SEQ ID No: 1, SEQ ID No: 5, SEQ ID No: 7 or SEQ ID No: 11, and they can be identified by standard methods such as the selection of hybridization of a suitable library using probes, or by analysis of the genomic sequence. The methods of identification are as described below.

Table 1: Progenitor sequences (members of the PS4 family). The described sequences differ from the sequence of Pseudomonas saccharophila in the positions shown at the top of the table, by the inclusion of substitutions consisting of the amino acid residues described. For example, pS4ccl-S161A is a variant of non-maltogenic exoamylase from wild-type Pseudomonas, and thus can be used as a progenitor enzyme.

In addition, non-maltogenic exoamylases from other strains of Pseudomonas spp, such as ATCC17686, can also be used as a progenitor polypeptide. The polypeptide residues of the PS4 variant can be inserted into any of these progenitor sequences to generate the polypeptide sequences of PS4 variants. The variant polypeptide of PS4 varies from the progenitor sequence by the inclusion of a number of mutations comprising amino acid substitutions. In preferred embodiments, the parent polypeptide is a non-maltogenic exoamylase from the non-maltogenic exoamylase of Pseudomonas saccharophila having a sequence described in SEQ ID No: as described in SEQ ID No: 5. In other preferred embodiments, the parent polypeptide comprises a non-maltogenic exoamylase of Pseudomonas stutzeri having a sequence shown as described in SEQ ID No: 7 or described in SEQ ID No: 11. In a preferred embodiment, the PS4 variant differs from the parent polypeptide by the inclusion of amino acid substitutions, the substitutions are located in a position comprising at least one position selected from the group consisting of: 4, 9, 13, 33, 34, 42, 70, 71, 87, 99, 100, 108, 113, 121, 131, 134, 135, 141, 153, 157, 158, 160, 161, 166, 170, 171, 178, 179, 184, 188, 198, 199, 221, 223, 238, 270, 277, 290, 307, 315, 334, 335, 342, 343, 372, 392, 398, 399, 405, 415, 425, where the reference to the numbering of the position is with respect to a sequence of exoamylase from Pseudomonas saccharophila shown as SEQ ID No: 1. Preferably, the position is at least one position selected from the group consisting of: 33, 34, 87, 121, 134, 141, 157, 178, 179, 223, 307 and 334. In still another embodiment, the variant further comprises a substitution selected from the group consisting of 71, 108, 158, 171, 188, and 343. In yet another embodiment, the variant further comprises a substitution selected from the group consisting of 113, 198, and 290. Preferably, the PS4 variant comprises al. minus one substitution selected from the group consisting of: N33Y, J334N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P. Preferably, the PS4 variant comprises the following substitutions: N33Y, D34N, G134R, A141P, I157L, G223A, H307L and S334P with at least one additional substitution of L178F or A179T. Preferably, the PS4 variant comprises one of the following substitutions: N33Y, D34N, I157L, L178F, A179T, G223A or H307L. Preferably, the PS4 variant comprises one of the following substitutions: G87S, G134R, A141P, or S334P. In yet another embodiment, the PS4 variant comprises one of the following substitutions: K71R, K108R, G158D, Y171S, G188A, and D343E. In yet another embodiment, the PS4 variant comprises one of the following substitutions: V131I, Y198F, Y198W, and V290I. While not wishing to be bound by any theory, the inventors propose that the three-dimensional crystal structure of Pseudomonas amylase, in conjunction with a proposed model binding site, (indicated by the software program Molecular Operating Environment [MOE] ] available from Chemical Computing Group, Inc., Montreal Canada) indicates the positions of residues 121, 157, 223, and 307 that could be in close proximity to the binding site to the substrate. Since the data as shown in the examples demonstrate that G121D improves the stability of the enzyme and the exo-specificity and G223A improves the thermostability of the enzyme, given the improvements already observed, and the positions in close proximity to the binding site to the substrate, further improvement can be obtained by elaborating all possible amino acid replacements at each such position. In one embodiment, close proximity refers to particular positions that are within 10.0 angstroms of the substrate binding site. In another embodiment, close proximity refers to the particular positions that are within 7.5 angstroms of the substrate binding site. In one embodiment, close proximity refers to particular positions that are within 6.0 angstroms of the substrate binding site, eg, C-alpha from G121 to the substrate approximately 5.9 angstroms; C-alpha from G223 to the substrate approximately 5.82 angstroms. In one embodiment, the variant of PS4 comprises a combination selected from the following groups: G134R, A141P, I157L, G223A, H307L, S334P, D343E, and G121D; G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, and G121D; G134R, A141P, I157L, G223A, H307L, S334P, D343E, and N33Y; G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F, A179T, G87S, G121D, S214 ?, and T375A; G134R, A141P, I157L, G223A, H307L, S334P, D343E,? 33Y, D34 ?, K71R, L178F, A179T, G121D; Y171S, G188A, and? 138D; G13_4R, ^ A141P, I157L, G223A, H307L, S334P, D343E,? 33Y, D34 ?, K71R, L178F, A179T, and G121D; G87S, G121D, G134R, A141P, I157L, G223A, H307L, S334P, D343E,? 33Y, D34 ?, K71R, L178F, and A179T; G87S, G121D, G134R, A141P, I157L, G223A, H307L, S334P, D343E,? 33Y, D34 ?, K71R, L178F, A179T, and G188A; G134R, A141P, I157L, G223A, H307L, S334P, K71R, L178F, and A179T; G134R, A141P, I157L, G223A, H307L, S334P, L178F, and A179T; G134R, A141P, I157L, G223A, H307L, S334P,? 33Y, D34 ?, L178F, and A179T; G134R, A141P, I157L, G223A, H307L, S334P, L178F, A179T, G87S, and G121D; G134R, A141P, I157L, G223A, H307L, S334P, L178F, A179T, and G121D; G134R, A141P, I157L, G223A, H307L, S334P,? 33Y, D34 ?, K71R, L178F, A179T, and G121D; G87S G121D G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T; G87S G121D G134R, A141P I157L G223A H307L S334P K71R L178F A179T; G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D; and G134R, A141P I157L G223A H307L S334P K71R L178F A179T G121D. In another embodiment, at least one substitution comprises a combination selected from the group of: A141P, I157L, G223A, H307L and S334P; G121D, G134R, A141P, I157L, G223A, H307L and S334; G87S, G121D, G134R, A141P, I157L, G223A, H307L and S334P; G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; ? 33Y, D34 ?, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; and? 33Y, D34 ?, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P. In another embodiment, the exo-amylase comprising at least one substitution comprises a combination selected from the group of the following:? 33Y, D34 ?, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; ? 33Y, D34 ?, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; and N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P. In another embodiment, the exo-amylase comprising at least one substitution comprises a combination selected from the group of the following: G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; 113F, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; V113I, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; A199V, D343E, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, and S334P; V113I, A141P, I157L, Y198F, G223A, V290I, S334P, and D343E; V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; V113I, A141P, Y198F, G223A, V290I, and H307L; V113I, A141P, Y198F, G223A, V290I, S334P, and D343 ?; V113I, A141P, Y198F, G223A, A268P, V290I, and S399P V113I, A141P, Y198F, G223A, V290I, and S399P; V113I, A141P, Y198W, G223A, and V290I; V113I, A141P, Y198F, G223A, and V290I; Y198F, G223A, and V290I; Y198W, G223A, and V290I; V113I, A141P, I157L, Y198F, G223A, and V290I; V113M; V113A; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, 1315V, S334P, and D343E; D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; V113I, G134R, A141P, I157L, G188S, Y198F, G223A, V290I, H307L, S334P, and D343E; K71R, V113I, G134R, A141P, I157L, L178L, Y198F, G223A, V290I, H307L, G313G, S334P, and D343E; D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, G313G, S334P, and D343E; V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L, S334P, and D343E; V113I, G134R, A141P, I157L, 11701, Y198F, G223A, V290I, H307L, G313G, S334P, and D343E V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L, G313G, S334P, and D343E; G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, and A405E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, and A405V; A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; V113I, G134R, A141P, I157L, Y198L, G223A, V290I, H307L, S334P, and D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; K71R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; K108R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; D34G, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; G4D, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, and D343E; A141P, G134R, G223A, H307L, I157L, V113I, V290I, Y198F, and G188A; In preferred embodiments, the PS4 variant is an amino acid sequence comprising any of SEQ ID No: 2; SEQ ID No: 3; SEQ ID No: 4; SEQ ID No. 4a, SEQ ID No. 4b, SEQ ID No. 4c, SEQ ID No: 8, SEQ ID No: 9 or SEQ ID: 10. The PS4 variant polypeptide may comprise one or more mutations in addition to those previously described. Other mutations, such as deletions, insertions, substitutions, transversions, transitions, and inversions, may also be included in one or more other sites. Similarly, the polypeptide may be lacking at least one of the substitutions described above. The PS4 variant may also comprise a conservative substitution that may appear as a similar substitution by similar (eg, basic by basic, acid by acid, polar by polar, etc.). Non-conservative substitution may also occur, for example, from one kind of residue to another, or alternatively involving the inclusion of non-natural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter designated as B), norleucine-ornithine (hereinafter referred to as 0), pyrilalanine, thienylalanine, naphthylalanine and phenylglycine. The sequences may also have deletions, insertions or substitutions of amino acid residues that produce a silent change and result in a functionally equivalent substance. The deliberate amino acid substitutions can be elaborated based on the similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues) and is therefore useful for grouping amino acids together in functional groups. The amino acids can be grouped together based on the properties of their side chain only. However, it is more useful to also include the mutation data. The amino acid groups derived in this way are likely to be conserved for structural reasons. These groups can be described in the form of a Venn diagram (Livingstone C D. and Barton GJ (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation" Compu t Appl Bi osci. 9: 745- 756) (Taylor WR (1986) "The classification of amino acid conservation" J.

Theo: Bi ol. 119; 205-218). Conservative substitutions can be made, for example, according to the following table which describes a generally accepted Venn diagram that groups the amino acids.

The amino acid sequences of the variant may also include suitable spacer groups, inserted between any two amino acid residues in the sequence, including alkyl groups, such as the methyl, ethyl or propyl groups in addition to the amino acid spacers such as the residues of glycine or ß-alanine. An additional form of variation involves the presence of one or more amino acid residues in peptoid form. The variant of PS4 may also comprise a homologous sequence. A homologous sequence comprises a nucleotide sequence of at least 7580, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to a nucleotide sequence coding for a variant of PS4. Typically, the homologs will comprise the same sequences that code for the active sites as the sequence of interest. Homology comparisons can be conducted visually, or more usually, with the help of readily available sequence comparison programs. These commercially available computer programs can calculate% homology between two or more sequences. The percentage of homology can be calculated on contiguous sequences, for example, one sequence is aligned with the other sequence and each amino acid in a sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called an alignment "without empty spaces". Typically, such alignments without empty spaces are made only on a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in a pair of otherwise identical sequences, an insertion or deletion will cause the following amino acid residues to be put out of alignment, giving as thus potentially resulting in a large reduction in the percentage of homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the full homology score. This is achieved through the insertion of "empty spaces" in the sequential alignment, to try to maximize the local homology. However, these more complex methods assign "penalties for empty space" to each empty space that appears in the alignment so that, for the same number of identical amino acids, a sequential alignment with as few empty spaces as possible -reflecting a higher ratio between the two compared sequences- will achieve a higher rating than one with many empty spaces. The "affine empty space costs" are typically used, which charge a relatively high cost for the existence of an empty space, and a smaller penalty for each subsequent waste in the empty space. This is the most commonly used empty space qualification system. The high penalties for empty space, of course, will produce optimized alignments with less empty space. Most alignment programs allow penalties for empty space to be modified. However, it is preferred to use the default values when such software is used for sequential comparisons. For example, when the GCG Wisconsin Bestfit package is used, the default empty space penalty for the amino acid sequences is -12 for an empty space and -4 for each extension. The calculation of the maximum percentage of homology therefore requires first the production of an optimal alignment, taking into account penalties for empty space. A suitable computer program to carry out such alignment is the GCG Wisconsin package Bestfit (Devereux et al., 1984 Nuc.Aids Research 12, p.387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4th Ed., Chapter 18), FASTA (Altschul et al. , 1990 J. Mol. Biol. 403-410) and the GENEWORKS comparison toolkit. BLAST and FASTA are available for offline and online search (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. BLAST 2 Sequences is also available for comparison of the protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174 (2): 247-50; FEMS Microbiol Lett 1999 177 (1): 187-8 and tatiana@ncbi.nlm .nih.gov). Although the final homology percentage can be measured in terms of identity, the alignment process itself is typically not based on a comparison of all or none. Rather, it is generally used - a scaled similarity rating matrix, which assigns ratings for each pairwise comparison, based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix-the default matrix for the BLAST program set. GCG Wisconsin programs generally use either public default values or a custom symbol comparison table if provided (see user manual for additional details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. Alternatively, the percentage homologies can be calculated using the multiple alignment feature in DNASIS "(Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG &Sharp PM (1988), Gene 73 (1), 237- 244).

Once the software has produced an optimal alignment, it is possible to calculate the percentage of homology, preferably the percentage of sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. Preferred embodiments also include functional equivalents. The PS4 variant polypeptides described herein are derivatives of, or are variants of, the polypeptides that preferably exhibit non-maltogenic exoamylase activity. Preferably, these progenitor enzymes are non-maltogenic exoamylases by themselves. The PS4 variant polypeptides themselves in the preferred embodiments also show non-maltogenic exoamylase activity. The PS4 variants described herein will preferably have exospecificity, for example measured by exo-specificity indices, as described above, consistent with their exoamylases. In addition, they preferably have greater or increased exospecificity when compared to the parent enzymes or polypeptides from which they are derived. Thus, for example, the PS4 variant polypeptides can have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or higher. exo-specificity when compared to their parent polypeptides, preferably under identical conditions.

These may have 1.5x or greater, 2x or greater, 5x or greater lOx or greater, 50x or greater, lOOx or greater, when compared to their parent polypeptides, preferably under identical conditions. In preferred embodiments, the functional equivalents will have sequential homology to at least one of the members of the PS4 family. Functional equivalents will have sequential homology to either the non-maltogenic exoamylases of Pseudomonas saccharophila and Pseudomonas stutzeri mentioned above, preferably both. The functional equivalent may also have sequential homology with any of the sequences described as SEQ ID NOs: 1 to 12, preferably SEQ ID NO: 1 or SEQ ID NO: 7, or both. The sequence homology between such sequences is preferably at least 60%, preferably 65% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, preferably 90% or more, preferably 95% or more. In other embodiments, functional equivalents will be capable of specifically hybridizing to any of the sequences described above. Methods for determining whether one sequence is capable of hybridizing to another are known in the art, and are for example described in Sambrook et al. (supra) and Ausubel, F.M. et al. (supra). In highly preferred embodiments, the functional equivalents will be capable of hybridizing under stringent conditions, for example 65 ° C and 0. lxSSC. { lxSSC = 0.15 M NaCl, 0.015 M Na3 citrate, pH 7.0} . The amino acid sequence may be prepared / isolated from a suitable source, or it may be prepared synthetically, or it may be prepared by the use of recombinant DNA techniques. Several methods are described below. 2. Detailed Description of the Nucleic Acids of the Invention In a second aspect, the invention provides a nucleic acid, the nucleic acid encoding a polypeptide comprising a variant of PS4 that is derivable from a progenitor polypeptide, as described above. A person skilled in the art will be aware of the relationship between the nucleic acid sequence and the polypeptide sequence, in particular, the genetic code and the degeneracy of this code, and will be able to construct such PS4 nucleic acids without difficulty. For example, a person skilled in the art will be aware that for each amino acid substitution in the sequence of the PS4 variant polypeptide, one or more codons encoding the substitute amino acid may exist. Accordingly, it will be apparent that, depending on the degeneracy of the genetic code with respect to that particular amino acid residue, one or more PS4 nucleic acid sequences, corresponding to that polypeptide sequence variant of PS4, can be generated. Thus, for example, a nucleic acid sequence of the PS4 variant can be derived from a progenitor sequence encoding a polypeptide having, wherein the PS4 variant nucleic acid codes for the amino acid substitutions at the following positions: G134, A141, 1157, G223, H307, S334, N33 and D34, together with one or both of L178 and A179. Mutations in the amino acid sequence without the nucleic acid sequence can be performed by any of a number of techniques, as are known in the art. In particularly preferred embodiments, mutations are introduced into progenitor sequences by PCR (polymerase chain reaction) using appropriate primers, as illustrated in the Examples. The progenitor enzymes can be modified at the level of the amino acids or at the level of the nucleic acid, to generate the variant sequences of PS4 described herein. Therefore, a preferred embodiment of this aspect of the invention provides for the generation of variant PS4 polypeptides by introducing one or more corresponding codon changes in the nucleotide sequence encoding a non-maltogenic exoamylase polypeptide. It will be appreciated that the above codon changes can be made in any nucleic acid sequence of the PS4 family. For example, changes in sequence can be made to a nucleic acid sequence of the non-maltogenic exoamylase from Pseudomonas saccharophila or Pseudomonas stutzeri (eg, X16732, SEQ ID NO: 6 or M24516, SEQ ID NO: 12). The progenitor enzyme may comprise the "complete" enzyme, for example, at its full length as it appears in nature (or as mutated), or may comprise a truncated form thereof. The variant of PS4 derived from it may consequently also be truncated, or be "full length". The truncation may be at the N-terminus or at the C-terminus. The progenitor enzyme or the PS4 variant may lack one or more portions, such as sub-sequences, signal sequences, domains or portions, whether active or not. For example, the progenitor enzyme of the variant polypeptide of PS4 may lack a signal sequence, as described above. Alternatively, or in addition, the progenitor enzyme or the variant of. PS4 may lack one or more catalytic or binding domains.

In highly preferred embodiments, the progenitor enzyme or the PS4 variant may lack one or more of the domains present in the non-maltogenic exoamylases, such as the starch binding domain. For example, the PS4 polypeptides may have only the sequence up to position 429, relative to the numbering of a non-maltogenic exoamylase from Pseudomonas saccharophila, as shown in SEQ ID NO: 1. For example, this is the case for the PS4 variants: pSac-D34, pSac-D20 and pSac-D14 or the other variants described in the Examples. Typically, the nucleotide sequence of the PS4 variant is prepared using recombinant DNA techniques. However, in an alternative embodiment, the nucleotide sequence could be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc Acids Res Symp Ser 225-232). A nucleotide sequence that encodes either an enzyme having the specific properties as defined herein, or for an enzyme that is suitable for modification, such as a progenitor enzyme, can be identified and / or isolated and / or purified of any cell or organism that produces said enzyme. Various methods within the art are known for the identification and / or isolation and / or purification of nucleotide sequences. By way of example, PCR amplification techniques for preparing more than one sequence can be used once a suitable sequence has been identified and / or isolated and / or purified. As a further example, a genomic DNA and / or cDNA library can be constructed using chromosomal DNA or messenger RNA, from the organism that produces the enzyme. If the amino acid sequence of the enzyme or a part of the amino acid sequence of the enzyme is known, labeled oligonucleotide probes can be synthesized, and used to identify the clones encoding the enzyme from the genomic library prepared from the organism. Alternatively, a labeled oligonucleotide probe containing the sequences homologous to another known enzyme gene could be used to identify the clones encoding the enzyme. In the latter case, hybridization and washing conditions of lesser demand or severity are used. Alternatively, clones encoding enzymes could be identified by inserting genomic DNA fragments into an expression vector, such as a plasmid, by transforming the negative bacteria to the enzyme with the resulting genomic DNA library, and then planting the bacteria transformed on agar plates containing a substrate for the enzyme (for example, maltose), thereby allowing the clones to express the enzyme to be identified. In a further alternative, the nucleotide sequence encoding the enzyme can be prepared synthetically by established standard methods, for example the phosphoramidite method described by Beucage S.L. et al., (1981) Tetrahedron Letters 22, p. 1859-1869 or the method described by Matthes et al., (1984) EMBO JX 3, p. 801-805. In the phosphoramidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, then purified, annealed, ligated and cloned into appropriate vectors. The nucleotide sequence may be of mixed genomic and synthetic origin, synthetic origin and of mixed cDNA or of genomic origin and mixed cDNA, prepared by ligation of fragments of synthetic, genomic or cDNA origin according to standard techniques. Each ligated fragment corresponds to the various parts of the complete nucleotide sequence. The DNA sequence can also be prepared by the polymerase chain reaction (PCR) using specific primers, for example as described in U.S. Patent No. 4,683,202 or Saiki RK et al., (Science (1988)). 239, pp. 487-491).

The nucleotide sequences described herein, and suitable for use in the methods and compositions described herein, may include synthetic or modified nucleotides therein. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and / or the addition of acridine or polylysine chains at the 3 'and / or 5' ends of the molecule. For the purposes of this document, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications can be carried out in order to increase the in vivo activity or the lifetime of the nucleotide sequences. A preferred embodiment of the invention provides the nucleotide sequences and the use of nucleotide sequences that are complementary to the sequences presented herein, or any derivative or fragment thereof. If the sequence is complementary to a fragment of the same, then that sequence can be used as a probe to identify similar coding sequences in other organisms, etc. Polynucleotides that are not 100% homologous to the variant sequences of PS4 can be obtained in a number of ways. Other variants of the sequences described herein may be obtained for example by probing the DNA libraries made from a range of individuals, eg, individuals from different populations. In addition, other homologs can be obtained, and such homologs and fragments of the same in general will be able to selectively hybridize to the sequences shown in the sequence sequence of the present. Such sequences can be obtained by probing cDNA libraries made from libraries of genomic DNA from other species, or probing such libraries with probes comprising all or part of any of the sequences in the lists of annexed sequences under conditions of medium to high demand. Similar considerations apply for obtaining homologs of species and allelic variants of the polypeptide or nucleotide sequences described herein. Strain / species variants and homologs can also be obtained using degenerate PCR that will use primers designed to target the sequences within the variants and the homologues that code for the conserved amino acid sequences. The primers used in the degenerate PCR will contain one or more degenerate positions and will be used at less demanding conditions than those used for the cloning sequences with the single sequence primers against known sequences. Conserved sequences can be predicted, for example, by alignment of amino acid sequences from various variants / homologs. The sequence alignments can be made using computer software known in the art as described herein. Alternatively, such polynucleotides can be obtained by site-directed mutagenesis of characterized sequences. This may be useful where, for example, silent changes in the codon sequence are required, to optimize the codon sequences for a particular host cell in which the polynucleotide sequences being expressed are. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides. The polynucleotides can be used to produce a primer, for example, a PCR primer, a primer for an alternative amplification reaction, a probe, for example, labeled with a revealing label by conventional means using radioactive or non-radioactive labels -o. the polynucleotides can be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by __ the term polynucleotides. Polynucleotides such as DNA polynucleotides and probes can be produced recombinantly, synthetically or by any means available to those of skill in the art. These can also be cloned using standard techniques. In general, the primers will be produced by synthetic means, involving a gradual fabrication of the desired nucleic acid sequence, one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art. Longer polynucleotides will generally be produced using recombinant means, for example using PCR cloning techniques (polymerase chain reaction). The primers can be designed to contain suitable restriction enzyme recognition sites, so that the amplified DNA can be cloned into a suitable cloning vector. Preferably, the variant sequences are at least as biologically active as the sequences presented herein.

A preferred embodiment of the invention includes the sequences that are complementary to the nucleic acid sequences of the PS4 variants or the sequences that are capable of hybridizing to any of the nucleotide sequences of the PS4 variants (including the sequences complementary to those presented herein), as well as the nucleotide sequences that are complementary to the sequences that can hybridize to the nucleotide sequences of the PSR variants (including sequences complementary to those presented here). A preferred embodiment provides polynucleotide sequences that are capable of hybridizing to the nucleotide sequences presented herein, under conditions of intermediate to maximum requirement. A preferred embodiment includes the nucleotide sequences that can hybridize to the nucleotide sequence of a PS4 variant nucleic acid, or the complement thereof, under stringent conditions (eg, 50 ° C and 0.2xSSC). More preferably, the nucleotide sequences can hybridize to the nucleotide sequences of a variant of PS4, or the complement thereof, under conditions of high demand (eg 65 ° C and O.lxSSC). Once a nucleotide sequence encoding the enzyme has been isolated, or a nucleotide sequence encoding the putative enzyme has been identified, it may be desirable to mutate the sequence in order to prepare an enzyme. Accordingly, a sequence of the PS4 variant can be prepared from a progenitor sequence. Mutations can be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites. A suitable method is described in Morinaga et al., (Biotechnology (1984) 2, p.646-649). Another method to introduce mutations within the nucleotide sequences that encode the enzyme is described in Nelson and Long (Analytical Biochemistry (1989), 180, pp. 147-151). An additional method is described in Sarkar and Sommer (Biotechniques (1990), 8, pp. 404-407 - "The megaprimer method of site directed mutagenesis"). In a preferred embodiment, the sequence for use in the methods and compositions described herein is a recombinant sequence - for example a sequence that has been prepared using recombinant DNA techniques. Such techniques are explained, for example, in the literature, for example, J. Sambrook, E.F. Fritsch and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press. 3. Detailed Description of the Compositions of the Invention A third aspect of the invention provides the compositions comprising the polypeptides which are variants of the polypeptides having non-maltogenic exoamylase activity, as well as the uses of such variant polypeptides and compositions. The compositions include the polypeptide variants together with another component. A preferred embodiment of the invention comprises a polypeptide variant of PS4, optionally together with an additional ingredient or an additional enzyme, or both. In addition to the PS4 variant polypeptides, one or more enzymes may be added, for example added to the food, to the dough preparation, to the food product or to the starch composition. Additional enzymes that can be added to the dough include oxidoreductases, hydrolases, such as lipases (e.g., lipase (EC 3.1.1) capable of hydrolyzing the carboxylic ester linkages to free carboxylate or such as triacylglycerol lipase (EC 3.1. 1.3), galactolipase (EC 3.1.1.26), phospholipase Al (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4) and lipoprotein-lipase A2 (EC 3.1.1.34)) and esterases as well as glucosidases such as α-amylase , pullulanase and xylanase. Oxidoreductases, such as the enzyme of oxidation to maltose, a glucose oxidase (EC 1.1.3.4), carbohydrate oxidase, glycerol oxidase, pyranose oxidase, galactose oxidase (EC 1.1.3.10) and hexose oxidase ( EC 1. 1.3.5), can be used for the reinforcement of the dough and to control the volume of the baked products, and the xylanases and other hemicellulases can be added to improve the handling properties of the dough, the softness of the crumb and the volume of bread. Lipases are useful as dough reinforcers and as crumb softeners, and a-amylases and other amylolytic enzymes can be incorporated into the dough to control the volume of the bread and to further reduce the firmness of the crumb. Additional enzymes that can be used can be selected from the group consisting of a cellulase, a hemicellulase, a starch degradation enzyme, a protease, a lipoxygenase. In a preferred embodiment, a polypeptide variant of PS4 can be combined with amylases, in particular, maltogenic amylases. Maltogenic α-amylase (glucan-1,4-maltohydrolase, E.C. 3.2.1.133) is capable of hydrolyzing amylose and amylopectin to maltose in the alpha configuration. A maltogenic alpha-amylase from Bacillus (EP 120 693) is commercially available (Novo Nordisk A / S, Denmark) and is widely used in the bread industry as an anti-tearing agent due to its ability to reduce retrogradation of the starch (see, for example, WO91 / 04669). Maltogenic amylase alf shares several characteristics with cyclodextrin glucanotransferases (CGTases), including secuencial_ homology (Henrissat B, Bairoch A; Biochem. J., 316, 695-696 (1996)) and formation transglucosylation products (Christophersen , C, et al., 1997, Starch, Vol. 50, No. 1, 39-45). A preferred embodiment includes combinations comprising variant PS4 polypeptides, together with alpha-amylase or any of its variants. Such combinations are useful for the production of foods such as baked goods. Variants, homologs, and mutants of the variants described in U.S. Patent No. 6,162,628, the disclosure of which is incorporated by reference herein, may be used in combination with the variant polypeptides of PS4 described herein . In particular, any of the polypeptides described in that document, specifically the variants of SEQ ID NO: 1 of U.S. Patent No. 6,162,628 in one or more positions corresponding to Q13, 116, D17, N26, can be used. N28, P29, A30, S32, Y33, G34, L35, K40, M45, P73, V74, D76 N77, D79, N86, R95, N99, 1100, H103, Q119, N120, N131, S141, T142, A148, N152 , A163, H169, N171, G172, 1174, N176, N187, F188, A192, Q201, N203, H220, N234, G236, Q247, K249, D261, N266, L268, R272, N275, N276, V279, N280, V281 , D285, N287, F297, Q299, N305, K316,? 320, L321,? 327, A341,? 342, A348, Q365,? 371,? 375, M378, G397, A381, F389,? 401, A403, K425,? 436, S442,? 454,? 468,? 474, S479, A483, A486, V487, S493, T494, S495, A496, S497, _A498, Q500,? 507, 1510,? 513, K520, Q526, A555, A564, S573,? 575 , Q581, S583, F586, K589,? 595, G618,? 621, Q624, A629, F636, K645,? 664 and / or T681. The additional enzyme can be added together with any dough ingredient including flour, water or other optional ingredients or additives, or the dough-improving composition. The additional enzyme can be added before or after the flour, water and optionally other ingredients and additives, or the dough-improving composition. The additional enzyme may conveniently be a liquid preparation or be in the form of a dry composition. Some enzymes of the dough-improving composition are capable of interacting with one another under the conditions of the dough, to a degree where the effect on the improvement of the rheological and / or machinability properties of a flour dough and / or The quality of the product made from the mass by the enzymes is not only additive, but the effect is synergistic. In relation to the improvement of the product made from the dough (finished product), it can be found that the combination results in a substantial synergistic effect with respect to the structure of the migajon. Also, with respect to the specific volume of the baked product a synergistic effect can be found. 4. _. Vectors, Cells and Methods. Expression of a PS4 Polypeptide A fourth aspect provides the vectors comprising a polypeptide variant of PS4, the cells comprising a polypeptide variant of PS4 and the methods for expressing a polypeptide variant of PS4. The nucleotide sequence for use in the methods and compositions described herein can be incorporated into a recombinant replicable vector. The vector can be used to replicate and express the nucleotide sequence, in the form of an enzyme, and / or from a compatible host cell. The expression can be controlled using control sequences, for example regulatory sequences. The enzyme produced by a recombinant cell of the host by the expression of the nucleotide sequence, can be secreted or can be contained intracellularly, depending on the sequence and / or the vector used. The coding sequences can be designed with signal sequences which direct the secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.

The polynucleotides can be incorporated into a recombinant replicable vector. The vector can be used to replicate the nucleic acid in a compatible host cell. The vector comprising the p-nucleotide sequence can be transformed into a suitable host cell. Suitable hosts can include bacterial, yeast, insect and fungal cells. Polypeptides and polynucleotides of the PS4 variant can be expressed by introducing a polynucleotide into a replicable vector, introducing the vector into a compatible host cell and developing the host cell under conditions that give rise to replication of the vector. The vector can be recovered from the host cell. The PS4 nucleic acid can be operatively linked to transcriptional and translational regulatory elements, active in a host cell of interest. The PS4 nucleic acid can also encode a fusion protein comprising the signal sequences, such as, for example, those derived from the glucoamylase gene of Schwanniomyces occidentali s, a gene coupling to the a factor from Saccharomyces cerevisiae and TAKA amylase from Aspergillus oryzae. Alternatively, the PS4 nucleic acid can encode a fusion protein comprising a membrane binding domain. The PS4 variant can be expressed at the desired levels in a host organism using an expression vector. An expression vector comprising a PS4 nucleic acid can be any vector capable of expressing the gene encoding the PS4 nucleic acid in the selected host organism, and the choice of vector will depend on the host cell within which it is going to be introduced. Thus, the vector can be a self-replicating vector, for example a vector that exists as an episomal entity, the replication of which is independent of chromosomal replication, such as, for example, a plasmid, a bacteriophage or an element episomal, a minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome of the host cell and replicated together with the chromosome. The expression vector typically includes the components of a cloning vector, such as, for example, an element that allows autonomous replication of the vector in the selected host organism, and one or more phenotypically detectable markers for selection purposes. The expression vector typically comprises the control nucleotide sequences that encode for a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activating genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the PS4 variant to an organelle of the host cell, such as a peroxisome or a particular compartment of the host cell. Such an address sequence to the object includes, but is not limited to, the SKL sequence. For low expression or the direction of the control sequences, the nucleic acid sequence of the PS4 variant is operably linked to the control sequences in an appropriate manner with respect to the expression. Preferably, a polynucleotide in a vector is operably linked to a control sequence that is capable of providing expression of the coding sequence by the host cell, eg the vector is an expression vector. The control sequences can be modified, for example by the addition of transcriptional regulatory elements to make the level of transcription directed by the control sequences, more responsive to the transcriptional modulators. The control sequences can in particular comprise promoters. In the vector, the nucleic acid sequence encoding the variant PS4 polypeptide is operably combined with a suitable promoter sequence. The promoter can be any DNA sequence having the transcription activity in the host organism of choice, and can be derived from genes that are homologous or heterologous to the host organism. Examples of suitable promoters for directing the transcription of the modified nucleotide sequence, such as the PS4 nucleic acids, in a bacterial host, include the promoter of the E. coli lac operon, the dagA promoters of the Streptomyces coelicolor agarose gene, the promoters of the a-amylase gene of Bacillus licheniformis (amyL), the aprE promoter of Bacillus subtilis, the promoters of the maltogenic amylase gene (amyM) of Bacillus stearothermophilus, the promoters of the a-amylase gene (amyQ) of Bacillus amyloliguefaciens, the promoters of the xylA and xylB genes of Bacillus subtilis and a promoter derived from a promoter derived from Lactococcus sp, including the P170 promoter. When the gene encoding the polypeptide of the PS4 variant is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a lambda phage promoter. For the transcription of a fungus species, examples of useful promoters are those derived from the genes encoding the TAKA amylase from Aspergillus oryzae, the Rhizomucor miehei aspartic proteinase, the neutral Aspergillus N-ger amylase, the -amylase stable to the acid of A. niger, the glucoamylase of A. niger, the lipase of Rhizomucor miehei, the alkaline protease of Aspergillus oryzae, the triosyphosphate isomerase of Aspergillus oryzae or the acetamidase of Aspergillus nidulans. The examples of Suitable promoters for expression in a yeast species include, but are not limited to, the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the A0X1 or AOX2 promoters of Pichia pas tori s. Examples of suitable bacterial host organisms are gram-positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearother ophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium and Bacillus thuringiensis, Streptomyces species such as Streptomyces murinus, bacterial species producing lactic acid including Lactobacillus reuteri, Leuconostoc spp. , Pediococcus spp. and Streptococcus spp. alternatively, strains of gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or Pseuodc? onadaceae can be selected as the host organism. A suitable yeast host organism can be selected from biotechnologically relevant yeast species such as, but not limited to, yeast species such as Pichia sp. , Hansenula sp or Kluyveromyces, Yarrow ± pia species or a species of Saccharopyces, including Saccharcmyces cerevisiae or a species belonging to Schizosaccharcmyces such as, for example, S. Pombe species. Preferably, a strain of the methylotrophic yeast species Pichia pastoris is used as the host organism. Preferably, the host organism is a species of Hansenula. Suitable host organisms among the filamentous fungi include Aspergillus species, for example, Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori or Aspergillus nidulans. Alternatively, strains of a Fusarium species, for example Fusarium oxysporum or a Rizhomucor species such as Rhizomucor miehei, can be used as the host organism. Other suitable strains include Termomyces and Mucor species. Host cells comprising polynucleotides can be used to express polypeptides, such as polypeptides of the PS4 variant, fragments, homologs, variants or derivatives thereof. The host cells can be cultured under suitable conditions that allow the expression of the proteins. The expression of the polypeptides can be constitutive, such that they are continuously produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, the addition of an inducing substance to the culture medium, for example dexamethasone or IPTG. The polypeptides can be extracted from the host cells by a variety of techniques known in the art, including enzymatic, chemical and / or osmotic lysis and physical disintegration. The polypeptides can also be produced recombinantly in a cell-free system, such as the TnT rabbit reticulocyte system (Promega) 5. Use (s) In the following description and examples, unless the context indicates otherwise, doses of variant PS4 polypeptides are given in parts per million (micrograms per gram) of flour., "1 D34" as used in Table 2 indicates 1 part per million of pSac-D34. The PS4 substitution mutants described herein can be used for any purpose for which the progenitor enzyme is suitable. In particular, these can be used in any application for which exo-maltotetrahydrolase is used. In highly preferred embodiments, these have the added advantage of increased thermostability, or increased exoamylase activity or greater pH stability, or any combination. Examples of suitable uses for variant PS4 polypeptides and nucleic acids include food production, in particular baking, such as the production of food products; the additional examples are described in more detail below. The following system is used to characterize polypeptides having non-maltogenic exoamylase activity, which are suitable for use according to the methods and compositions described herein. These systems can, for example, be used to characterize the progenitor polypeptides or PS4 variants described herein. As an initial background information, waxy corn amylopectin (obtainable from WAXILYS 200 from Roquette, France) is a starch with a very high amylopectin content (above 90%). 20 mg / ml waxy corn starch is heated to boiling for 3 minutes. In a buffer of MES 50 irM (2- (N-morpholino) ethanesulfonic acid), 2 mM calcium chloride, pH 6.0 and subsequently incubated at 50 ° C and used within half an hour.

One unit of non-maltogenic exoamylase is defined as the amount of enzyme that releases the equivulent hydrolysis products at 1 μmol of reducing sugar per minute, when incubated at 50 ° C in a test tube with 4 ml of waxy corn starch at 10 mg / ml in 50 mM MES, 2 mM calcium chloride, pH 6.0 prepared as described above. Reducing sugars are measured using maltose as the standard and using the dinitrosalicylic acid method of Bernfeld, Methods Enzymol. , (1954), 1, 149-158 or other method known in the art for quantifying reducing sugars. The pattern of the hydrolysis product of the non-maltogenic exoamylase is determined by the incubation of 0.7 units of the non-maltogenic exoamylase for 15 or 300 minutes at 50 ° C in a test tube with 4 ml of waxy corn starch at 10 mg / ml. ml, in the buffer prepared as described above. The reaction is stopped by immersing the test tube for 3 minutes in a boiling water bath. The hydrolysis products are analyzed and quantified by anion exchange HPLC using a Dionex PA 100 column with sodium acetate, sodium hydroxide and water as eluents, with pulsed amperometric detection and with known linear maltooligosaccharides from glucose to maltoheptase as standards. The response factor used for maltoctane to maltodecase is the response factor found for maltoheptaose. Preferably, the polypeptides of the parent of PS4 (and polypeptides of the PS4 variant) have non-maltogenic exoamylase activity such that if an amount of 0.7 units of said non-maltogenic exoamylase were incubated for 15 minutes at a temperature of 50 ° C at pH 6.0 in 4 ml of a 10 mg aqueous solution of boiling preheated waxy corn starch, per ml of buffer solution containing 50 mM 2- (N-morpholino) ethanesulfonic acid and 2 mM calcium chloride, then the enzyme would produce one or more hydrolysis products which could consist of one or more linear malto-oligosaccharides of two to ten units of D-glucopyranosyl and optionally glucose; such that at least 60%, at least 70%, at least 80% and / or at least 85% by weight of the hydrolysis products consisting of two to ten units of D-glucopyranosyl and optionally glucose, would consist of linear maltooligosaccharides of three to ten units of D-glucopyranosyl, preferably linear maltooligosaccharides consisting of four to eight D-glucopyranosyl units. For ease of reference, and for the present purposes, the incubation characteristic of an amount of 0.7 units of non-maltogenic exoamylase for 15 minutes at a temperature of 50 ° C at pH 6.0 in 4 ml of an aqueous solution of 10 mg of boiling preheated waxy corn starch, per ml of buffer solution containing 50 mM 2- (N-morpholino) ethanesulfonic acid and 2 mM calcium chloride, may be referred to as the "Waxy Maize Starch Incubation Test". Thus, alternatively expressed, a preferred non-maltogenic exoamylase is characterized as possessing the ability in the incubation test of waxy maize starch, to produce one or more hydrolysis products which could consist of one or more linear maltooligosaccharides of two to ten D-glucopyranosyl units and optionally glucose; such that at least 60%, preferably at least 70%, more preferably at least 80% and most preferably at least 85% by weight of the hydrolysis product (s) could consist of linear maltooligosaccharides of three to ten D-glucopyranosyl units, preferably linear maltooligosaccharides consisting of four to eight D-glucopyranosyl units. The hydrolysis products in the waxy maize starch incubation test include one or more linear malto-oligosaccharides of two to ten D-glucopyranosyl units and optionally glucose. The hydrolysis products in the incubation test of waxy maize starch may also include other hydrolytic products. However, the percentage amounts by weight of the linear igosaccharide maltool of three to ten D-glucopyranosyl units are based on the amount of the hydrolysis product consisting of one or more maltool lignosaccharides from two to ten D-glucopyranosyl units and optionally glucose. . In other words, the percentage by weight of the ionic maltool igosaccharides I of three to ten D-glucopyranosyl units are not based on the amount of hydrolytic products other than one or more maltool. a die z D-glucopyranosyl units and glucose. The hydrolysis products can be analyzed by any suitable means. For example, the hydrolysis products can be analyzed by anion exchange HPLC using a Dionex PA 100 column with pulsed amperometric detection and, for example, with known linear maltooligosaccharides of glucose to maltoheptase as standards. Preferably, the PS variants described herein are active during baking and hydrolysis of the starch during and after the gelatinization of the starch granules, which starts at temperatures of about 55 ° C. The more thermostable the non-maltogenic exoamylase is, the longer this may be active, and thus the latter will provide a greater anti-alienating effect. However, during baking above temperatures of about 85 ° C, inactivation of the enzyme may take place. If this happens, the non-maltogenic exoamylase can be gradually inactivated, so that there is substantially no activity after the baking process in the final bread. Therefore, preferably non-maltogenic exoamylases suitable for use as described, have an optimum temperature above 50 ° C and below 98 ° C. The exo-specificity can be usefully measured by determining the ratio of the total amylase activity to the total endoamylase activity. This ratio is referred to in this document as an "index of exo-specificity". In preferred embodiments, an enzyme is considered an exoamylase if it has an exospecificity index of 20 or more, for example its total amylase activity (including exo-amylase activity) is 20 times or more, greater than its endoamylase activity . In highly preferred embodiments, the exo-specificity index of exoamylases is 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more. In highly preferred embodiments, the exo-specificity index is 150 or more, 200 or more, 300 or more, or 400 or more.

Total amylase activity and total endoamylase activity can be measured by any means known in the art. For example, the total amylase activity can be measured by testing the total number of reducing ends released from a starch substrate. Alternatively, the use of a Betamyl assay is described in greater detail in the Examples, and for convenience, the amylase activity as evaluated in the Examples, is described in terms of the "Betamyl Units" in the Tables and Figures. The endoamylase activity can be evaluated by using a Phadebas Equipment (Pharmacia and Upjohn). This makes use of a cross-linked starch marked with blue (marked with an azo dye); Only internal cuts in the starch molecule release the marker, while external cuts do not. The release of the dye can be measured by spectrophotometry. Accordingly, the Phadebas Team measures the endoamylase activity, and for convenience, the results of such an assay (described in the Examples) are referred to herein as "Phadebas units". In a preferred embodiment, therefore, the exo-specificity index is expressed in terms of Betamyl Units / Phadebas Units.

The exo-specificity can also be evaluated according to the methods described in the prior art, for example, in Publication Number WO99 / 50399. This measures the exo-specificity by means of a ratio between the activity of endoamylase to the exoamylase activity. Thus, in a preferred aspect, the PS4 variants described herein will have less than 0.5 units of endoamylase (EAU) per unit of exoamylase activity. Preferably, the non-maltogenic exoamylases which are suitable for use according to the present invention have less than 0.05 EAU per unit of exoamylase activity, and more preferably less than 0.01 EAU per unit of exoamylase activity. Variant PS4 polypeptides, nucleic acids, host cells, expression vectors, etc., can be used in any application for which an amylase can be used. In particular, these can be used to replace any non-maltogenic exoamylase. These can be used to supplement the activity of non-maltogenic amylase or exoamylase, either alone or in combination with other non-maltogenic amylases or exoamylases. The variant sequences of PS4 can be used here in various applications in the food industry such as in baked goods and in beverages, these can be used in other applications such as a pharmaceutical composition, or even in the chemical industry. In particular, the PS4 variant polypeptides and the nucleic acids thereof are useful for various industrial applications, including baking (as described in WO99 / 50399) and flour standardization (increase or improvement of volume). These can be used to produce maltotetraose from starch and other substrates. Variant polypeptides of PS4 can be used to increase the volume of bakery products such as bread. Thus, food products that comprise or are treated with the variant PS4 polypeptides are expanded in volume when compared to products that have not been treated as such, or treated with the parent polypeptides. In other words, food products have a greater volume of air per volume of food product. Alternatively, or in addition, the food products treated with the variant polypeptides of PS4 have a lower density, or weight (or mass) per volumetric ratio. In particularly preferred embodiments, the variant PS4 polypeptides are used for the bread volume. The increase or expansion of the volume is beneficial because this reduces the gumminess or the starchy feeling of the food. Light foods are preferred by consumers, and the customer experience is improved. In preferred embodiments, the use of the variant PS4 polypeptides increases the volume by 10%, 20%, 30%, 40%, 50% or more. The variant polypeptides of PS4 and the nucleic acids described herein can be used as -o in the preparation of a food. In particular, these can be added to a food, for example, as a food additive. In a preferred aspect, the food is for human consumption. The food may be in the form of a solution or as a solid, depending on the use and / or the mode of application and / or the mode of administration. The polypeptides and nucleic acids of the PS4 variant can be used as a food ingredient. The polypeptides and nucleic acids of the PS4 variant described herein can be -or can be added to-food supplements. The polypeptides and nucleic acids of the PS4 variant described herein can be -or can be added to-functional foods. The variant polypeptides of PS4 can also be used in the manufacture of a food product or article. Typical food products include dairy products, poultry products, fish products and dough products. The dough product may be any processed dough product, including fried doughs, deep-fried, roasted, baked, boiled and boiled, such as steamed bread and rice cakes. In highly preferred embodiments, the food product is a bakery product. Preferably, the food product is a bakery product. The typical bakery products (baked) include bread - such as loaves, rolls, buns, pizza bases, etc., pastas, pretzels, tortillas, cakes, cookies, biscuits, etc. The variant proteins of ps4 are capable of retarding the stripping of starch media, such as starch gels. The variant polypeptides of PS4 are especially capable of retarding the damaging degeneration of starch. Accordingly, the use of the variant polypeptides of PS4 as described herein, when added to the starch at any stage of its processing into a food product, for example, before, during or after baking in bread, can retard or prevent or encourage retrogradation. Such use is described in more detail below. For the evaluation of the antiarritorial effect of the polypeptides of the PS4 variants having non-maltogenic exoamylase activity described here, the firmness of the migajon can be measured 1, 3 and 7 days after baking by means of a Universal Food Texture Analyzer Instron 4301 or similar equipment known in the art. Yet another method, traditionally used in the art and which is used to evaluate the effect of starch degeneration of a variant PS4 polypeptide having non-maltogenic exoamylase activity, which is based on differential scanning calorimetry (DSC). in English) . Here, the enthalpy of fusion of the amylopectin degenerated in the bread crumb or the crumb coming from a mass of the model system, baked with or without enzymes (control), is measured. The DSC equipment applied in the described examples is a Mettler-Toledo DSC 820 run with a temperature gradient of 10 ° C per minute, from 20 to 95 ° C. For the preparation of the samples, 10 to 20 mg of migajon are weighed and transferred to Mettler-Toledo aluminum trays which are then hermetically sealed. The masses of the model system used in the described examples contain standard wheat flour and optimum amounts of water and buffer with or without the non-maltogenic PS4 variant exoamylase. These are mixed in a 50 g Brabender Farinograph for 6 or 7 minutes, respectively. The samples of the masses are placed in glass test tubes (15 x 0.8 cm) with a lid. These test tubes are subjected to a baking process in a water bath, starting with 30 minutes of incubation at 33 ° C, followed by heating from 33 to 95 ° C with a gradient of 1.1 ° C per minute, and finally a incubation for 5 minutes at 95 ° C. Subsequently, the tubes are stored in a thermostat at 20 ° C before the DSC analysis. In preferred embodiments, the PS4 variants described herein have a reduced melting enthalpy, as compared to the control. In highly preferred embodiments, the PS4 variants have a melting enthalpy reduced by 10% or more. Preferably, they have a melting enthalpy reduced by 20% q more, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, when compared to the control. Table 2 Table 2 above shows the DSC values of the model mass systems prepared with different doses of PSac-D34 after 7 days of storage. Tested 0. 5, 1 and 2 parts per million (or microgram per gram) of flour.

The variant polypeptides of PS4 can be used in the preparation of food products, in particular, starch products. The method comprises the formation of the starch product by the addition of such an xeno-maltogenic exoamylase enzyme. as a polypeptide variant of PS4, to a starch medium. If the starch medium is a dough, then the dough is prepared by mixing the flour, the water, the non-maltogenic exoamylase which is a variant PS4 polypeptide, and optionally other ingredients and possible additives. A preferred flour is wheat flour or rye flour, or mixtures of wheat flour and rye flour. However, the dough comprising flour derived from other types of cereals, such as for example rice, corn, barley and durra are also contemplated. Preferably, the starch product is a bakery product. More preferably, the starch product is a bread product. Even more preferably, the starch product is a baked farinaceous bread product. Thus, if the starch product is a baked farinaceous bread product, then the process involves mixing - in any suitable order - flour, water and a fermentation agent, under mass forming conditions and adding in addition a polypeptide variant of PS4, optionally in the form of a premix.

The fermentation agent can be a chemical fermentation agent such as sodium bicarbonate or any strain of Saccharomyces cerevisiae (Baker's Yeast). The non-maltogenic exoamylase of the PS4 variant can be added together with any dough ingredient including the water or the dough ingredients mixture or with any additive or mixture of additives. The dough can be prepared by any conventional method of dough preparation, common in the bakery industry, or in any other industry that produces products based on dough flour. A preferred embodiment is a process for making a bread product comprising (a) the provision of a starch medium; (b) adding to the starch medium, a polypeptide variant of PS4 as described herein; and (c) applying heat to the starch medium during or after step (b) to produce a bread product. Another preferred embodiment is a process for making a bread product comprising adding a polypeptide variant of PS4 to a starch medium as described. The PS4 variant polypeptide of non-maltogenic exoamylase can be added as a liquid preparation or as a dry powdery composition, comprising either the enzyme as the sole active component or in admixture with one or more additional dough ingredients, or dough additives. .

Another preferred embodiment is the use of such bread or dough-improving compositions in baking.

A further embodiment provides a baked product or baked dough obtained from the bread improving composition or the dough improving composition. Yet another embodiment provides a baked product or dough obtained from the use of a bread-improving composition or a dough-improving composition. A dough can be prepared by mixing flour, water, a dough improving composition comprising the variant polypeptide of PS4 (as described above) and optionally other ingredients and additives. The dough improving composition can be added together with any dough ingredient including flour, water or other ingredients or optional additives. The dough-improving composition can be added before flour or water or other ingredients and optional additives. The dough-improving composition can be added after flour or water, or other ingredients and optional additives. The dough can be prepared by any conventional dough preparation method, common in the bakery industry or in any other industry that makes products based on flour dough. The dough-improving composition may be added as a liquid preparation or in the form of a dry powder composition comprising either the composition as the sole active component or in admixture with one or more other ingredients or dough additives. The amount of the non-maltogenic exoamylase of the variant PS4 polypeptide, which is added, is usually an amount that results in the presence in the finished dough, from 50 to 10,000 units per kg of flour, preferably 100 to 50,000 units per kg of flour. Preferably, the amount is in the range of 200 to 20,000 units per kg of flour. In the present context, one unit of the non-maltogenic exoamylase is defined as the amount of enzyme released by the hydrolysis products, equivalent to 1 μmol of reducing sugar per minute, when incubated at 50 ° C in a test tube with 4 ml of waxy corn starch at 10 mg / ml in 50 mM MES, 2 mM calcium chloride, pH 6.0 as described hereinafter. The dough as described herein, generally comprises ground wheat grain and wheat flour and / or other types of ground grain, flour or starch such as corn flour, corn starch, corn flour, rice flour, grain ground rye, rye flour, oatmeal, ground oat grain, soybean meal, ground sorghum grain, sorghum flour, potato flour, or potato starch. The dough can be fresh, frozen, or partially baked.

The dough can be a fermented dough or a dough that is going to be subjected to fermentation. The dough can be fermented in various ways, such as by the addition of chemical fermentation agents, for example, sodium bicarbonate or by the addition of a yeast (fermentation flour), but it is preferred to ferment the dough by adding a suitable yeast culture, such as the culture of Saccharcmyces cerevisiae (baker's yeast) for example a commercially available strain of S. cerevisiae. The dough may comprise fat such as granulated fat or shortening (butter to make the dough more friable). The dough may further comprise an additional emulsifier such as mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, polyoxyethylene stearates, or lysolecithin. Yet another embodiment is a premix comprising flour together with the combination as described herein. The premix may contain other dough-improving additives and / or bread improvers, for example, any of the additives, including the enzymes mentioned herein. In order to further improve the properties of the baked product and impart distinctive qualities to the baked product, additional dough ingredients and / or dough additives may be incorporated into the dough. Typically, such additional aggregate components may include dough ingredients such as salt, grains, fats and oils, sugar and sweetener, dietary fibers, protein sources such as milk powder, soy gluten or eggs, and dough additives such as emulsifiers. , other enzymes, hydrocolloids, flavoring agents, oxidizing agents, minerals and vitamins. Emulsifiers are useful as dough reinforcers and as softeners of the migajon. As dough reinforcers, the emulsifiers can provide tolerance with respect to resting time and shock tolerance during the test. In addition, dough reinforcers will improve the tolerance of a given mass to variations in fermentation time. Most dough reinforcers also improve the elasticity in the oven, which means the increase in the volume of the items tested to the baked goods. In the end, the reinforcers will emulsify any masses present in the recipe mixture. Suitable emulsifiers include lecithin, polyoxyethylene stearate, mono- and diglycerides of edible fatty acids, acetic acid esters of mono- and diglycerides of edible fatty acids, lactic acid esters of mono- and diglycerides of edible fatty acids, acid esters citric of mono- and diglycerides of edible fatty acids, esters of diacetyltartaric acid of mono- and diglycerides of edible fatty acids, sucrose esters of edible fatty acids, sodium stearoyl-2-lactylate and calcium stearoyl-2-lactylate. The additive or additional dough ingredient can be added together with any dough ingredient including flour, water or other optional ingredients or additives, or the dough improving composition. The additive or additional dough ingredient can be added before the flour, water, other ingredients and optional additives or the dough-improving composition. The additive or additional dough ingredient can be added after the flour, water, other ingredients or optional additives or the dough-improving composition. The additional dough additive or ingredient may conveniently be a liquid preparation. However, the additive or additional dough ingredient may conveniently be in the form of a dry composition. Preferably, the additional dough additive or the additional dough ingredient is at least 1% by weight of the farinaceous component of the dough. More preferably, the additional dough additive or ingredient is at least 2%, preferably at least 3%, preferably at least 4%, preferably at least 5%, preferably at least 6%. If the additive is a fat, then typically the fat can be present in an amount of 1 to 5%, typically 1 to 3%, more typically about 2%. Other uses may be found in lawyer case numbers 674510-2007 and GC807P, which are incorporated by reference herein, including any drawings, references or figures. EXAMPLES Example 1. Cloning of PS4 Pseudomonas saccharophila are developed overnight on LB medium and the chromosomal DNA is isolated by standard methods (Sambrook J, 1989). A fragment of 2190 base pairs containing the "open reading structure of PS4 (Zhou et al., 1989) is amplified from the chromosomal DNA of P. Saccharophila by PCR using primers Pl and P2 (see Table 3). The resulting fragment is used as a template in a PCR nested with primers P3 and P4, amplifying the open reading structure of PS4 without its signal sequence, and introducing an Ncol site at the 5 'end of the gene, and a BamHl site At the 3 'end, a codon for an N-terminal methionine, allowing the intracellular expression of PS4, is introduced along with the Ncol site.The 1605 base pair fragment is cloned in, from pCRBLUNT TOPO (Invitrogen) and the integrity of The construction analyzed by sequencing The shuttle vector pDP66K of E. coli or Bacillus (Penninga et al., 1996) is modified to allow the expression of PS4 lowered the control of the P32 promoter and the ctgasa signal sequence. Rabbit, pCSmta is transformed into B. subtilis. A second expression construct is elaborated, in which the starch binding domain of PS4 is removed. In a PCR with primers P3 and P6 (Table 3) on pCSmta, a truncated version of the mta gene is generated. The full-length mta gene in pCSmata is exchanged with the truncated version which resulted in the plasmid pCSmta-SBD. Example 2. Mutagenesis Directed to the Site of PS4 Mutations are introduced into the mta gene by 2 methods. Either by a method based on 2 steps of PCR, or by a Rapid Exchange method (Quick Exchange QE). For convenience, the mta gene is divided into 3 parts. A Pvul-Fspl fragment, an Fspl-Pstl fragment and a PstI-AspI fragment, additionally referred to as fragments 1, 2 and 3 respectively. In the 2-step PCR-based method, mutations are introduced using the Pfu DNA polymerase (Stratagene). A first PCR is carried out with a mutagenesis primer (Table 4) for the coding strand plus a downstream primer on the lower strand (either 2R or 3R in Table 3). The reaction product is used as a primer in a second PCR, together with a primer upstream (5 ') on the coding strand. The product of the last reaction is cloned into pCRBLUNT topo (Invitrogen) and after sequencing, the fragment is exchanged with the corresponding fragment in pCSmta, using the Rapid Exchange method (Stratagene), mutations are introduced using two complementary primers in a PCR on a plasmid containing the mta gene, or part of the mta gene. For this purpose a convenient group of plasmids is constructed, comprising 3 plasmids of SDM and 3 plasmids of pCS ?. The SDM plasmids each possess 1 of the fragments of the mta gene as mentioned above, in which the desired mutation is introduced by QE. After verification by sequencing, the fragments are cloned into the plasmid pCS? corresponding to the container. The plasmids pCS? they are inactive derivatives of pCSmta. The activity is restored by cloning the corresponding fragment from the SDM plasmid, making easy selection possible.

Table 3. Primers used in the cloning of the mta gene, and standard primers used in the construction of site-directed mutants with the 2-step PCR method.

Table 4. Primers used to introduce mutations directed to the site in mta Table 5. Characteristics of the plasmids SDM and pCS ?.

Example 3. Multi SDM The PS4 variants were generated using a Multi Site Directed Mutagenesis Team QuickChange (Stratagene) according to the manufacturer's protocol with some modifications as described. Step 1: Mutant Strain Synthesis Reaction (PCR) Inoculate 3 ml of LB (22 g / 1 of Lennox L Broth Base, Sigma) + antibiotics (0.05 μg / ml Kanamycin, Sigma) in a 10 ml Falcon tube Incubate at 37 ° C, approximately 200 rpm - - Submit the cells to centrifugation (5000 rpm / 5 minutes) - Empty the medium Prepare the ds-DNA template using the QIAGEN mini plasmid purification protocol 1. The mutant strand synthesis reaction for the thermal cycle was prepared as follows: PCR mixture: 2.5 μl multi-reaction buffer 10X QuickChange® 0.75 μl QuickSolution X μl Primers (primer length 28-35 bp -> 10 pmol length of the primer 24-27 bp - »7 pmol primer length 20-23 bp - 5 pmol 1 μl of the dNTP mixture X μl of the ds-DNA template (200 ng) 1 μl of the QuickChangel® multiple enzymatic mixture) ( 2.5 U / μl) (PfuTurbom DNA polymerase) X μl of, H20 (to a final volume of 25 μl) Mix all the components by pipetting and centrifuging briefly the reaction mixtures 2. Cycle the reactions using the following parameters: 35 cycles of denaturation (96 ° C / 1 minute) annealing with primer (62.8 ° C / 1 minute) elongation (65 ° C / 15 minutes) then keep at 4 ° C Preheat the cap the PCR machine at 105 ° C and the plate at 95 ° C before the PCR tubes are placed in the machine (Eppendorf thermal cycler).

Step 2: Digestion with Dpn I 1. Add 2 μl of the restriction enzyme Dpn (10 U / μl) to each amplification reaction, mix with pipette and centrifuge the mixture. 2. Incubate at 37 ° C for ~ 3 hours.

Step 3: Transformation of ultracompetent XLIO-Gold® cells 1. Thaw XLIO-Gold cells on ice. Take aliquots of 45 μl of cells by mutagenesis reaction to pre-cooled Falcon tubes. 2. Turn on the water bath (42 ° C) and place a tube with the NZY + broth in the bath to preheat. 3. Add 2 μl of ß-mercaptoethanol mixture to each tube.

Shake and tap gently and incubate 10 minutes on ice, stirring in a whirlpool every 2 minutes. 4. Add 1.5 μl of DNA treated with Dpn I to each aliquot of cells, vortex to mix and incubate on ice for 30 minutes. 5. Apply heat pulses to the tubes in the water bath at 42 ° C for 30 seconds and place on ice for 2 minutes. 6. Add 0.5 ml of pre-warmed NZY + broth to each tube and incubate at 37 ° C for 1 hour with shaking at 225-250 rpm. 7. Sow 200 μl of each transformation reaction on LB plates (33.6 g / l of Lennox L agar, Sigma) containing 1% starch and 0.05 μg / ml Kanamycin. 8. Incubate the transformation plates at 37 ° C overnight.

Table 6. Table for primers of pPD77dl4: Table Table for primers of pPD77d20: Table 8. Table for primers of pPD77d34: Vector system based on pPD77 The vector system used for pPD77 is based on pCRbluntTOPOII (Invitrogen). The zeocin resistance cassette has been removed by pmll, the fragment of 393 base pairs removed. The expression cassette from the pCC vector (P32-ssCGTase-PS4-tt) has then been inserted into the vector. Ligation of the PS4 variant within pCCMini The plasmid that contains the relevant mutations (created by MSDM) is cut with the restriction enzyme Ncol and HindIII (Biolabs): 3 μg of plasmid DNA, X μl of buffer 2, units of Ncol, 20 units of HindIII, Incubation for 2 hours at 37 ° C. The digestion is run on a 1% agarose gel. Fragments with a size of 1293 base pairs (PS4 gene) are cut out of the gel and purified using the gel purification equipment. Qiagen. The pCCMini vector is then cut with restriction enzymes in Ncol and HindIII, and the digestion is then run on a 1% agarose gel. The 3569 base pair size fragment is cut out of the gel and purified using the Qiagen gel purification kit. Ligation: use the rapid DNA ligation equipment (Roche) Use the double amount of the insert compared to the vector for example 2 μl of insert (PS4 gene) 1 μl of vector 5 μl of DNA ligation buffer T4 2xconc 1 μl of dH20 1 μl of DNA-ligase-T4 Ligate at 5 min at room temperature. Transform the ligature into competent cells One Shot TOPO according to the manufacturer's protocol (Invitrogen). Use 5 μl of ligature for transformation. Sow 50 μl of the transformation mixture on plates LB (33.6 g / l of Lennox L agar, Sigma) containing 1% starch and 0.05 μg / ml kanamycin.

The vectors containing the insert (variants of PS4) can be recognized by halo formation on the starch plates.

Example 4, Transformation in Bacillus subtilis (Protoplast transformation) Bacillus subtilis (strain DB104A; Smith et al., 1988; Gene 70, 351-361) is transformed with the mutated pCS plasmids according to the following protocol.

A. Means for protoplastif ication and transformation 2 x SMM per liter: 342 g of sucrose (1 M); 4.72 g of sodium maleate (0.04 M); 8.12 g of magnesium chloride hexahydrate (0.04 M); pH 6.5 with concentrated sodium hydroxide. Distribute in 50 ml portions and heat in an autoclave for 10 minutes. 4 x YT 2 g of yeast extract + 3.2 g of (1/2 NaCl) tryptone + 0.5 g of sodium chloride per 100 ml. SMMP Mix equal volumes of 2 x SMM and 4 x YT.

PEG 10 g of polyethylene glycol 6000 (BDH) or 8000 (Sigma) in 25 ml 1 x SMM (autoclave for 10 minutes).

B. Means for plating / regeneration Agar Minimum Difco agar at 04%. Heat in autoclave for 15 minutes Succinate 270 g / 1 (1 M), pH 7.3 with HCl. Heat in sodium autoclave for 15 minutes Shock absorber 3.5 g K2HP04 + 1.5 g KH2P04 per 100 ml. of phosphate Heat in autoclave for 15 minutes MgCl2 20.3 g MgCl2, 6H20 per 100 ml (1 M). Casamino acids 5% solution (w / v). Autoclave for 15 minutes extract of 10 g per 100 ml, autoclave for 15 minutes yeast glucose solution of 20% (w / v). Autoclave for 10 minutes Regeneration medium DM3: mix at 60 ° C (water bath, 500 ml bottle): 250 ml of sodium succinate 50 ml of casamino acids 25 ml of yeast extract 50 ml of phosphate buffer 15 ml of glucose 10 ml of MgCl2 100 ml of molten agar Add the appropriate antibiotics: chloramphenicol and tetracycline, 5 μg / ml; erythromycin, 1 μg / ml. Selection on kanamycin is problematic in the DM3 medium: concentrations of 250 μg / ml may be required. C. Preparation of Pro toplast 1. Use plastic or glassware free of detergent, throughout. 2. Inoculate 10 ml of 2 x YT medium in a 100 ml flask from a single colony. Develop an overnight culture at 25-30 ° C on an agitator (200 rpm). 3. Dilute overnight culture 20 times in 100 ml of 2 x YT medium (250 ml flask) and develop to an optical density D0600 = 0.4 - 0.5 (about 2 hours) at 37 ° C on a shaker (200-250 rpm). 4. Harvest the cells by centrifugation (9000 g, 20 minutes, 4 ° C). 5. Remove the supernatant with a pipette, resuspend the cells in 5 ml of SMMP + 5 mg of lysozyme, sterilize by filtration. 6. Incubate at 37 ° C on a shaker with a water bath (100 rpm). After 30 minutes and from this at intervals to 15 minutes, examine samples of 25 μl by microscopy. Continue incubation until 99% of the cells are converted into protoplasts (globular appearance). Harvest the protoplasts by centrifugation (4000 g, 20 minutes, room temperature) and pipette the supernatant. Resuspend the button gently in 1-2 ml of SMMP.

The protoplasts are now ready for use. (Portions (eg, 0.15 ml) can be frozen at -80 ° C for future use (no addition of glycerol is required.) Although this may result in some reduction in the transformability, 106 transformants per μg of DNA may be obtained with the frozen protoplasts). D. Tran s formation 1. Transfer 450 μl of PEG to a microtube. 2. Mix 1-10 μl of DNA (0.2 μg) with 150 μl of protoplasts and add the mixture to the microtube with PEG.

Mix it immediately, but gently. 3. Leave for 2 minutes at room temperature, and then add 1.5 ml of SMMP and mix. 4. Harvest the protoplasts by microcentrifugation (10 minutes, 13,000 rpm (10-12,000 g)) and empty the supernatant. Remove the remaining droplets with gauze. Add 300 μl of SMMP (do not vortex) and incubate for 60-90 minutes at 37 ° C in a shaker with a water bath (100 rpm) to allow the expression of antibiotic resistance markers. (The protoplasts are resuspended enough through the stirring action of the water bath). Make the appropriate dilutions in 1 x SSM and sow 0.1 ml on DM3 plates.

Example 5. Fermentation of the PS4 variants in shake flasks The shake flask substrate is prepared as follows: The substrate is adjusted to pH 6.8 with 4N sulfuric acid or sodium hydroxide before heating in an autoclave. Place 100 ml of the substrate in a 500 ml flask with a buffer screen and autoclave for 30 minutes. Subsequently, 6 ml of sterile dextrose syrup is added. Dextrose syrup is prepared by mixing a volume of 50% w / v dextrose with a volume of water, followed by heating in an autoclave for 20 minutes. The shake flask is inoculated with the variants and incubated for 24 hours at 35 ° C / 180 rpm in an incubator. After incubation, the cells are separated from the broth by centrifugation (10,000 x g in 10 minutes) and finally, the supernatant is released from the cells by microfiltration at 0.2 μm. The cell-free supernatant is used for the tests and application tests.

Example 6. Amylase assays Beta test One unit of betamyl is defined as the activity that degrades 0.0351 mmol per 1 minute of the maltopentase coupled to PNP, so that 0.0351 mmol of PNP per 1 minute can be released by the excess a-glucosidase in the test mixture. The assay mixture contains 50 μl of 50 mM sodium citrate, 5 mM calcium chloride, pH 6.5 with 25 μl of the enzyme sample and 25 μl of the betamyl substrate (Glc5-PNP and α-glucosidase) from Megazyme, Ireland (1 bottle dissolved in 10 ml of water). The test mixture is incubated for 30 minutes at 40 ° C and then capped by the addition of 150 μl of 4% Tris. The absorbance at 420 nm is measured using an ELISA vector and the activity of betamyl is calculated based on the activity = A420 * d in betamyl units / ml of the enzyme sample evaluated. Endo-amylase assay The endo-amylase assay is identical to the Phadebas assay run according to the manufacturer (Pharmacia &Upjohn Diagnostics AB). Exo-specificity The ratio of exo-amylase activity to Phadebas activity was used to evaluate the exo-specificity. Specific activity For PSac-D14, PSac-D20 and PSac-D34 variants, an average specific activity of 10 units of betamyl per microgram of purified protein was found, measured according to Bradford (1976; Anal. Biochem. 72, 248). This specific activity is used based on the activity to calculate the doses used in the application tests. Example 7. Determination of the half-life Tl / 2 is defined as the time (in minutes) during which half of the enzymatic activity is inactivated under the defined heat conditions. In order to determine the half-life of the enzyme, the sample is heated for 1-10 minutes at constant temperatures of 60 ° C to 90 ° C. The half-life is calculated based on the residual betamyl test. Procedure: In an Eppendorf bottle, 1000 μl of the buffer is preheated for at least 10 minutes at 60 ° C or more. The heat treatment of the sample is initiated after the addition of 100 μl of the sample to the preheated buffer under continuous mixing (800 rpm) of the Eppendorf bottle in a thermal incubator (Termomixer comfort from Eppendorf). After 0, 2, 4, 6, 8 and 9 minutes of incubation, the treatment defined by transfer of 45 μl of the sample to 1000 μl of the buffer balanced at 20 ° C and incubating for one minute at 1500 rpm and at 20 ° C. Residual activity is measured with the betamyl assay.

Calculation: The calculation of tl / 2 is based on the loglO slope (the logarithm in base 10) of the residual betamyl activity versus the incubation time. Tl / 2 is calculated as the slope / 0.301 = tl / 2.

Example 8. Results Table 9. Biochemical properties of the Psac variants compared to wild type PSac-ccl.

Table 10 Table 11 Table 12 Table 12 Table 13 Table 14 Table 15 Example 9. Baking tests model The masses are elaborated in the Farinograph a . 0 ° C. 10.00 g of reformed flour are weighed and added in the Farinograph; after 1 minute, the reference / sample is mixed (reference = buffer or water, sample = enzyme plus buffer or water) and added with a sterile pipette through the holes of the mixing container. After 30 seconds, the flour is scraped off the edges, also through the holes of the kneading containers. The sample is kneaded for 7 minutes. A damper or water test is performed on the Farinograph before the final reference is run. FU must be 400 above the reference, if not, it must be adjusted with, for example, the amount of liquid. The reference / sample is removed with a spatula and placed in the hand (with a disposable glove on it), before it is filled into small glass tubes (approximately 4.5 cm in length) that are placed in NMR tubes and covered . 7 tubes per mass are made. When all the samples have been prepared, the tubes are placed in a water bath (programmable) at 33 ° C (without plugs) for 25 minutes, and after that the water bath is separated to stand for 5 minutes at 33 ° C, then heated to 98 ° C. ° C in 56 minutes (1.1 ° C per minute) and finally rest for 5 minutes at 96 ° C. The tubes were stored at 20.0 ° C in a thermal cupboard. The content of the solid of the migajon was measured by MR? of protons using an MRI analyzer? Bruker? MS 120 Minispec in the days 1, 3 and 7, as shown for the crumb samples prepared with 0, 0.05, 1 and 2 ppm of PSacD34 in Figure 2. The smallest increase in solid content over time represents the reduction in amylopectin degeneration. After 7 days of storage at 20.0 ° C in a thermal cupboard, 10-20 mg samples of migajon are weighed and placed in standard 40 μl aluminum DSC capsules and kept at 20 ° C. The capsules are used for differential scanning calorimetry on a MettIer Toledo DSC 820 instrument. As parameters . a heating cycle of 20-95 ° C with 10 ° C per minute of heating and gas flow: N2 / 80 ml per minute are used. The results are analyzed and the enthalpy for the fusion of the degenerated amylopectin is calculated in J / g. Example 10. Anti-tearing effects Bread model lozenges are prepared and measured according to Example 8. As shown in Table 2, the PS4 variants show a strong reduction of the degeneration of amylopectin 5 after baking, as it is measured by differential scanning calorimetry compared to the control. The PS4 variants show a clear dosage effect. Example 11. Firming effects in baking tests The baking tests were carried out with either a standard white bread sponge and the US toasted bread dough recipe. The sponge mass is prepared from 1600 g of "Classical for all purpose of Siseo Mills, United States" flour, 950 g of water, 40 g of soybean oil and 32 g of dry yeast. The sponge is mixed for 1 minute at low speed and consequently 3 minutes at speed 2 in a Hobart spiral mixer. The sponge is subsequently fermented for 2.5 hours at 35 ° C, 85% relative humidity, followed by 0.5 hours at 5 ° C. After this, 400 g of flour, 4 g of dry yeast, 40 g of salt, 2.4 g of calcium propionate, 240 g of high fructose corn syrup (Isosweet), 5 g of PANODAN emulsifier 205.5 g of flour enzyme-active soybean, 30 g of non-active soybean meal, 220 g of water and 30 g of an ascorbic acid solution (prepared from 4 g of ascorbic acid solubilized in 500 g of water) are added to the sponge. The resulting mass is mixed for 1 minute at low speed and then for 6 minutes at speed 2 in a Diosna mixer. After this, the dough is rested for 5 minutes at room temperature, and then pieces of dough of 550 g are cut, resting for 5 minutes, and then rolled on a Glimek laminator with the settings 1: 4, 2: 4, 3:15, 4:12 and 10 on each side, and transferred to a baking dish. After 60 minutes, testing at 43 ° C at 90% relative humidity, the masses are baked for 29 minutes at 218 ° C. Firmness and elasticity were measured with a TA-XT 2 texture analyzer. Softness, cohesiveness and elasticity are determined by analysis of bread slices by texture profile analysis using a Stable Micro Systems texture analyzer , United Kingdom. The following adjustments were used: Speed before the test: 2 mm / seconds Speed of the test: 2 mm / seconds Speed after the test: 10 mm / seconds. Distance from. Break test: 1% Distance: 40% Force: 0. 098 N Time: 5.00 seconds Count: 5 Load cell: 5 kg Trigger type: Auto-0.01 N The results are shown in figures 3 and 4. Example 12 Control of the volume of Danish buns. Danish buns are prepared from a mass based on 2000 g of Danish reform flour (from Cerealia), 120 g of compressed yeast, 32 g of salt, and 32 g of sucrose.

Water is added to the mass according to the previous water optimization. The dough is mixed in a Diosna mixer (2 minutes at low speed and 5 minutes at high speed). The temperature of the dough after mixing is maintained at 26 ° C. 1350 g of mass are weighed and rested for 10 minutes in a heating cabinet at 30 ° C. The rolls are molded in Fortuna moulder and tested for 45 minutes at 34 ° C and at 85% relative humidity. Subsequently, the buns are baked in a Bago 2 oven for 18 minutes at 250 ° C with steam in the first 13 seconds. After baking the buns are cooled for 25 minutes, before weighing and measuring the volume. The buns are evaluated with respect to the appearance of the bark, the homogeneity of the crumb, the coverage of the bark, and the specific volume (measuring the volume with the rape seed displacement method). Based on these criteria, it is found that the PS4 variants increase the specific volume and improve the quality parameters of the Danish buns. In this way, the PS4 variants are able to control the volume of the baked products. Each of the applications and patents mentioned in this document, and each document cited or referred to in each of the previous applications and patents, including during the prosecution of each of the applications and patents ("documents cited in the application") and any instructions or catalogs of the manufacturer, for any products cited or mentioned in each of the applications and patents, and in any of the documents cited in the application, are incorporated by reference herein. In addition, all documents cited in this text, and all documents cited or referred to in the documents cited in this text, and any instructions or catalogs of manufacturer for any products cited and mentioned in this text, are incorporated by reference herein. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art, without departing from the scope and spirit of the invention. Although the invention has been described in connection with the specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Rather, the various modifications of the modes described to carry out the invention, which are obvious to those skilled in molecular biology or related fields, are intended to be within the scope of the claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An exo-specific non-maltogenic exoamylase, characterized in that it comprises an amino acid sequence, at least 75% identical with SEQ ID Nos. 1, 5, 7 or 11 and at least one substitution selected from the group consisting of 4, 33, 34 , 70, 71, 87, 99, 108, 113, 121, 134, 141, 157, 158, 171, 178, 179, 188, 198, 199, 223, 290, 307, 315, 334, 343, 399 and 405

2. The exoamylase according to claim 1, characterized in that the amino acid sequence is at least 75% identical with SEQ ID No. : 1. The exoamylase according to claim 1, characterized in that the amino acid sequence is at least 75% identical with SEQ ID No. 5. The exoamylase according to claim 1, characterized in that the amino acid sequence is at least 75% identical with SEQ ID No. 7. The exoamylase according to claim 1, characterized in that the amino acid sequence is at least 75% identical with SEQ ID No. 11. 11. The exoamylase according to claim 1, characterized in that the sequence of amino acids comprises at least 75% amino acid sequence identical to SEQ ID Nos. 2-4b or 8-10. The exoamylase according to claim 1, characterized in that at least one substitution is selected from G4D, N33Y, D34N, G70D, K71R, G87S, A99V, K108R, V113I, G121D, G134R, A141P, I157L, G158D, Y171S, L178F, A179T, G188A, Y198F, Y198F, Y198L, A199V, G223A, V290I, H307L, I315V, S334P, D343E, S399P, A405F and A405E. 8. The exoamylase according to claim 1, characterized in that at least one position is selected from 33, 34, 71, 87, 121, 134, 141, 157, 178, 179, 223, 207, 334 and 34

3. 9. The exoamylase according to claim 8, characterized in that at least one substitution is selected from N33Y, D34N, K71R, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L, S334P, and D343E. The exoamylase according to claim 8, characterized in that it also comprises at least one additional substitution at a selected position of 108, 158, 171 and 188. 11. The exoamylase according to claim 10, characterized in that at least one substitution additional is selected from K108R, G158D, Y171S, and G188A. 12. The exoamylase according to claim 1, characterized in that at least one substitution comprises a combination selected from the following: G134R, A141P I157L G223A H307L S334P D343E G121D; G134R A141P I157L G223A H307L S334P D343E N33Y G121D; G134R A141P I157L G223A H307L S334P D343E? 33Y; G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T G87S G121D S214? T375A; G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T G121D Y171S G188A? 138D; G134R, A141P I157L G223A H307L S334P D343E? 33Y D34? K71R L178F A179T G121D; G87S G121D G134R, A141P I157L G223A H307L S334P D343E ? 33Y D34N K71R L178F A179T; G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G188A; G134R, A141P I157L G223A H307L S334P K71R L178F A179T; G134R, A141P I157L G223A H307L S334P L178F A179T; G134R, A141P I157L G223A. H307L S334P N33Y D34N L178F A179T; G134R, A141P I157L G223A H307L S334P L178F A179T G87S G121D; G134R, A141P I157L G223A H307L S334P L178F A179T G121D; G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D E343D; G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T; G87S G121D G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T Y33N N34D E343D; G134R, A141P I157L G223A H307L S334P D343E N33Y D34N K71R L178F A179T G121D; G134R, A141P I157L G223A H307L S334P K71R L178F A179T G121D; G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; 113F, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; A99V, V1131, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, I157L, Y198F, G223A, V290I, H307L, S334P, D343E V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; A199V, D343E, V1131, A141P, I157L, Y198F, G223A, V290I, H307L, S334P; V113I, A141P, I157L, Y198F, G223A, V290I, S334P, D343E; V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G121D, G134R,. A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, A141P, Y198F, G223A, V290I, H307L; V113I, A141P, Y198F, G223A, V290I, S334P, D343E; V113I, A141P, Y198F, G223A, A268P, V290I, S399P V113I, A141P, Y198F, G223A, V290I, S399P; V113I, A141P, Y198, G223A, V290I; V113I, A141P, Y198F, G223A, V290I; Y198F, G223A, V290I; Y198, G223A, V290I; V113I, A141P, I157L, Y198F, G223A, V290I; V113M; V113A; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, 1315V, S334P, D343E; D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, G188S, Y198F, G223A, V290I, H307L, S334P, D343E; K71R, V113I, G134R, A141P, I157L, L178L, Y198F, G223A, V290I, H307L, G313G, S334P, D343E; D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, G313G, S334P, D343E; V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, 11701, Y198F, G223A, V290I, H307L, G313G, S334P, D343E V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L, G313G, S334P, D343E; G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, A405V; A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198L, G223A, V290I, H307L, S334P, D343E; V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; K71R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; K108R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; D34G, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; G4D, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E; and A141P, G134R, G223A, H307L ,. I157L, V113I, V290I, Y198F, G188A. The exoamylase according to claim 3, characterized in that at least one substitution comprises a combination selected from the following: G134R, A141P, I157L, G223A, H307L and S334P; G121D, G134R, A141P, I157L, G223A, H307L and S334; G87S, G121D, G134R, A141P, I157L, G223A, H307L and S334P; G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; and N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P. 1

4. The exoamylase according to claim 3, characterized in that at least one substitution comprises a combination selected from the following: N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; and N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P. 1

5. The exoamylase according to claim 1, characterized in that the amylase comprises at least the amino acid selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 4a, SEQ ID NO: 4b, SEQ ID No. 4c, SEQ ID No: 8, SEQ ID No: 9, and SEQ ID No: 10. 1

6. The exoamylase according to claim 1, characterized in that the exoamylase is derived from a Pseudomonas sp. 1

7. The exoamylase according to claim 15, characterized in that the Pseudomonas sp. it is selected from Pseudomonas saccharophi la and Pseudomonas s tu tzeri. 1

8. A nucleic acid sequence, characterized in that it is at least 75% identical to the nucleic acid sequence encoding the exoamylase according to claim 1. 1

9. A vector, characterized in that it comprises the nucleic acid sequence according to Claim 18. 20. A host cell, characterized in that it comprises the nucleic acid sequence according to claim 19.