CN118956813A - Modified esterase and its use - Google Patents

Abstract

The present invention addresses the problem of providing a modified esterase which can be expected to have high reaction efficiency and can be used for various purposes. The present invention provides a modified esterase having an amino acid sequence comprising 1 or more amino acid substitutions selected from the group consisting of K7P, F, Y, V, 107, M, L, 222, M, V, 229I and S235T in the amino acid sequence of SEQ ID NO.1 or an amino acid sequence having 91% or more homology with the amino acid sequence, wherein 1 or more properties selected from the group consisting of specific activity, high temperature stability, high temperature reactivity and pH stability are improved.

Description

Modified esterases and their use

The present application is a divisional application of the following application.

The invention name is as follows: modified esterases and their use

Filing date: 2019, 4 and 10 days

Application number: 201980029176.0 (PCT/JP 2019/015709)

Technical Field

The present application relates to modified esterases. Specifically, provided are modified esterases having improved properties and uses thereof. The present application claims priority based on patent application publication No.2018-088943 filed in japan at 5/2 of 2018, the entire contents of which are hereby incorporated by reference.

Background

Esterases are enzymes that hydrolyze esters, and many esterases are isolated from a variety of organisms. Esterases from Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) having unique and broad substrate specificity are industrially useful for the synthesis of organic compounds used in, for example, the fields of pharmaceuticals, fine chemicals, etc.; the decomposition of harmful compounds in foods can be used for various applications (for example, refer to non-patent document 1).

[ Prior Art literature ]

[ Non-patent literature ]

Non-patent document 1 applies microbiology and biotechnology (Appl Microbial Biotechnol) (2002) 60:288-292

Disclosure of Invention

[ Problem ] to be solved by the invention

However, since esterases are proteins composed of amino acids, the use conditions (strong acid and alkali, high temperature, high concentration organic solvents, etc.) cause denaturation of proteins, and thus the use of esterases cannot function as a catalyst, and thus the application range may be limited. For example, the synthesis reaction of a pharmaceutical intermediate is often carried out under severe conditions (e.g., a reaction at a high temperature, a reaction in an organic solvent, a reaction in an acidic pH region), and stability is particularly problematic. Therefore, depending on the reaction conditions, sufficient enzyme activity cannot be obtained, and the reaction efficiency (yield) is lowered.

Accordingly, the present invention aims to provide: by improving the characteristics of the esterase derived from Acinetobacter calcoaceticus, the esterase can exert a wide range of substrate specificities to the maximum under various conditions of use, thereby improving the usefulness or utility value of the esterase. In the conventional applications, improvement of the characteristics can bring about improvement of the reaction efficiency and the like, and on the other hand, new applications (i.e., expansion of the applications) can be created.

Means for solving the problems

In order to solve the above problems, the present inventors have attempted to change esterases derived from Acinetobacter calcoaceticus. Through repeated experiments, mutation points (amino acid residues) effective for improving the characteristics (specific activity, high temperature stability, high temperature reactivity and pH stability) of the mutant are successfully determined, and the mutant (modified esterase) with high usefulness is obtained. This result is also important in providing information and means for designing and obtaining mutants that can achieve the object of improving the characteristics.

Thus, there is often experienced a case where the possibility of producing an additive effect or a synergistic effect by combining the effective 2 mutations is high. In addition, the same enzymes have a high similarity in structure (primary structure, steric structure), and the possibility that the same mutation produces the same effect is high, and in view of such technical knowledge, if useful mutations found in an esterase derived from Acinetobacter calcoaceticus having the amino acid sequence of SEQ ID NO. 1 are applied to other esterases having a high structural similarity to the esterase, the possibility that the same effect is exhibited is high, and further, those skilled in the art can recognize that such application is effective.

The following invention is based on the above achievements and observations.

[1] A modified esterase having an amino acid sequence comprising 1 or more amino acid substitutions selected from the group consisting of K7P, F Y, V107M, L222M, V229I and S235T in the amino acid sequence of sequence No. 1, or an amino acid sequence having 91% or more homology with the amino acid sequence, wherein the modified esterase has an improved specific activity, high temperature stability, high temperature reactivity and pH stability as compared to an esterase comprising the amino acid sequence of sequence No. 1.

[2] The modified esterase according to [1], wherein the amino acid contained in the amino acid sequence of the modified esterase is substituted with S235T, and the specific activity is improved.

[3] The modified esterase according to [1], wherein the amino acid contained in the amino acid sequence of the modified esterase is substituted with K7P, F, Y, L M or V229I, and the high-temperature stability is improved.

[4] The modified esterase according to [1], wherein the amino acid contained in the amino acid sequence of the modified esterase is substituted with V229I or S235T, and the high-temperature reactivity is improved.

[5] The modified esterase according to [1], wherein the amino acid sequence of the modified esterase contains amino acid substitutions F13Y, V107,107, 107M, L222, 222M, V229,229I and S235T, and the pH stability is improved.

[6] A modified esterase having an amino acid sequence of any of SEQ ID NO. 2 to 7 or an amino acid sequence having a homology of 91% or more with the amino acid sequence (wherein, when the reference of homology is the amino acid sequence of SEQ ID NO. 2, the amino acid sequences differ at positions other than proline at position 7; in the case of the amino acid sequence of SEQ ID NO. 3 as a reference for homology, the amino acid sequence differs at positions other than tyrosine 13, in the case of the amino acid sequence of SEQ ID NO. 4 as a reference for homology, the amino acid sequence differs at positions other than methionine 107, in the case of the amino acid sequence of SEQ ID NO. 5 as a reference for homology, the amino acid sequence differs at positions other than methionine 222, in the case of the amino acid sequence of SEQ ID NO. 6 as a reference for homology, the amino acid sequence differs at positions other than isoleucine 229, in the case of the amino acid sequence of SEQ ID NO. 7 as a reference for homology, and in the case of the amino acid sequence other than threonine 235), the modified esterase has improved specific activity, high-temperature stability, high-temperature reactivity and pH stability as compared with the esterase comprising the amino acid sequence of SEQ ID NO. 1.

[7] A gene encoding the modified esterase according to any one of [1] to [6 ].

[8] The gene according to [7], wherein the gene comprises any one of the base sequences of SEQ ID NOS.8 to 13.

[9] A recombinant DNA comprising the gene of [7] or [8 ].

[10] A microorganism which retains the recombinant DNA of [9 ].

[11] The microorganism according to [10], wherein the host is: coli (ESCHERICHIA COLI), bacillus subtilis (Bacillus subtilis), bacillus licheniformis (Bacillus licheniformis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and Brevibacillus choshinensis (Brevibacillus chosinensis).

[12] An enzyme preparation comprising the modified esterase according to any of [1] to [6 ].

[13] A process for the preparation of a modified esterase comprising the following steps (I) to (III):

(I) A step of preparing a nucleic acid encoding any one of the amino acid sequences of SEQ ID NOS.2 to 7;

(II) a step of expressing the above nucleic acid, and

(III) recovering the expression product.

Drawings

Specific Activity of mutants [ FIG. 1 ]. The specific activity of the mutant was compared with that of the wild-type enzyme.

FIG. 2 shows the stability of the mutants at high temperatures. The residual rate of activity after 30 minutes of treatment at 80℃was compared with that of the wild-type enzyme.

FIG. 3 shows the reactivity of the mutants at high temperatures. The relative activity (ratio of enzyme activity at 60℃to enzyme activity at 30 ℃) was compared with the wild-type enzyme.

FIG. 4 shows the pH stability of the mutants. The residual rate of activity after 30 minutes of treatment at pH4.0 was compared with that of the wild-type enzyme.

Detailed Description

For convenience of description, the following definitions are given for terms used in part in connection with the present invention.

(Terminology)

The term "modified esterase" refers to an enzyme obtained by modifying or mutating a reference esterase (hereinafter referred to as "reference esterase"). Typically, the reference esterase is an esterase derived from Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) having the amino acid sequence of SEQ ID NO. 1.

The term "esterase derived from Acinetobacter calcoaceticus" refers to an esterase derived from Acinetobacter calcoaceticus, and includes an esterase produced by Acinetobacter calcoaceticus or an esterase expressed by other microorganisms or the like using genetic information of the esterase.

In the present invention, "amino acid substitution" is performed as modification or mutation. Thus, if the modified esterase and the reference esterase are compared, a difference in the presence of a part of the amino acid residues is found. In this specification, a modified esterase is also referred to as a modified enzyme or a mutant.

In this specification, each amino acid is expressed by 1 letter according to a conventional practice, as shown below.

Methionine: m, serine: s, alanine: a, threonine: t, valine: v, tyrosine: y, leucine: l, asparagine: n, isoleucine: i, glutamine: q, proline: p, aspartic acid: d, phenylalanine: f, glutamic acid: e, tryptophan: w, lysine: k, cysteine: c, arginine: r, glycine: g, histidine: h

In the present specification, the position of the mutation point is determined by the number attached from the N-terminus to the C-terminus, with the 1 st amino acid residue at the N-terminus.

Conventionally, amino acid residues, i.e. "mutation points", where amino acid substitutions are carried out are expressed with a combination of 1 letter representing the amino acid class and a number representing the amino acid position. The "mutation" caused by the amino acid substitution is indicated by adding 1 letter of the substituted amino acid type to the right side of the display showing the mutation point. Thus, for example, when lysine at position 7 is a mutation point, it is denoted as "K7", and when lysine at position 7 is substituted with proline, it is denoted as "K7P".

1. Modified esterases

The 1 st aspect of the present invention relates to a modified esterase (modified enzyme). Typically, the modified enzyme of the present invention has an amino acid sequence comprising 1 or 2 or more amino acid substitutions selected from the group consisting of K7P, F, Y, V, 107, M, L, 222, M, V, 229I and S235T in the amino acid sequence of SEQ ID NO. 1. According to this feature, the specific activity, high temperature stability, high temperature reactivity, pH stability, or 2 or more of them are improved as compared with the esterase comprising the amino acid sequence of SEQ ID NO. 1. The amino acid sequence of SEQ ID NO.1 is a sequence of an esterase derived from Acinetobacter calcoaceticus.

In order to facilitate understanding, judgment and determination of the high temperature stability and high temperature reactivity of the modified enzyme, the high temperature in the high temperature stability is "75 to 85 ℃ and the high temperature in the high temperature reactivity is" 55 to 65 ℃. Further, pH stability refers to stability in the acidic pH region. In order to facilitate understanding, judgment and determination of pH stability, the acidic pH range is defined as 3 to 5.

For example, the activity may be evaluated based on the activity calculated by the measurement method shown in examples described later. The modified enzyme having an increased specific activity has a higher specific activity than the reference esterase (i.e., an esterase comprising the amino acid sequence of SEQ ID NO: 1). The specific activity of the modified enzyme is, for example, 1.1 to 1.3 times the specific activity of the reference esterase.

For example, the high temperature stability can be evaluated based on the residual activity after 30 minutes of treatment at 80 ℃. The modified enzyme having improved high-temperature stability has an activity which is higher than that of the reference esterase (i.e., an esterase comprising the amino acid sequence of SEQ ID NO: 1). The residual rate of the activity of the modified enzyme is, for example, 2 to 10 times the residual rate of the activity of the reference esterase. Preferably 3 to 10 times. Further, the activity residual rate was calculated as follows.

Activity remaining ratio = (enzyme activity after heat treatment/enzyme activity before heat treatment) ×100 (%)

For example, the high temperature reactivity can be evaluated based on the ratio of the enzyme activity at 60℃to the enzyme activity at 30 ℃ (relative activity at 60℃based on the activity at 30 ℃). The modified enzyme having improved high-temperature reactivity has a relative activity higher than that of the reference esterase (i.e., an esterase comprising the amino acid sequence of SEQ ID NO: 1). The relative activity of the modified enzyme is, for example, 1.2 to 2 times the relative activity of the reference esterase. Preferably 1.5 to 2 times. Further, the relative activity was calculated as follows.

Relative activity= (enzyme activity at 60 ℃ C./enzyme activity at 30 ℃ C.) ×100 (%)

For example, the pH stability can be evaluated based on the residual activity after 30 minutes of treatment at pH 4. The modified enzyme having improved pH stability has an activity which is higher than that of the reference esterase (i.e., an esterase comprising the amino acid sequence of SEQ ID NO: 1). The residual rate of the activity of the modified enzyme is, for example, 1.08 to 1.3 times the residual rate of the activity of the reference esterase. Preferably 1.15 to 3 times. Further, the activity residual rate was calculated as follows.

Activity remaining ratio= (enzyme activity after treatment/enzyme activity before treatment) ×100 (%)

In the present specification, "comprising an amino acid substitution" means that a mutation point (i.e., a position of an amino acid residue at which a specific amino acid substitution is produced) is changed to a substituted amino acid. Therefore, when an amino acid sequence (mutant amino acid sequence) containing an amino acid substitution is compared with an amino acid sequence (reference amino acid sequence) of SEQ ID NO. 1 containing no amino acid substitution, a difference in amino acid residues can be confirmed at the position of the amino acid substitution.

K7P represents a mutation in which the 7 th amino acid (lysine) in the amino acid sequence of SEQ ID NO. 1 is substituted with proline. F13Y represents a mutation in which the 13 th amino acid (phenylalanine) in the amino acid sequence of SEQ ID NO. 1 is substituted with tyrosine. V107M represents a mutation in which the 107 th amino acid (valine) in the amino acid sequence of SEQ ID NO. 1 is substituted with methionine. L222M represents a mutation in which the 222 th amino acid (leucine) in the amino acid sequence of SEQ ID NO. 1 is substituted with methionine. V229I represents a mutation in which the 229 th amino acid (valine) in the amino acid sequence of SEQ ID NO. 1 is substituted with isoleucine. S235T represents a mutation in which the 235 th amino acid (serine) in the amino acid sequence of SEQ ID NO. 1 is substituted with threonine.

Specific examples of the modified enzyme of the present invention include esterases composed of any one of the amino acid sequences of SEQ ID Nos. 2 to 7 (corresponding to mutant 1 (K7P), mutant 2 (F13Y), mutant 3 (V107M), mutant 4 (L222M), mutant 5 (V229I) and mutant 6 (S235T) in this order). As shown in the examples described below, it can be confirmed that: the esterase (mutant 1 (K7P)) comprising the amino acid sequence of SEQ ID NO. 2 has improved high-temperature stability; the esterase (mutant 2 (F13Y)) comprising the amino acid sequence of SEQ ID NO. 3 has improved high-temperature stability and pH stability; the pH stability of the esterase (mutant 3 (V107M)) consisting of the amino acid sequence of SEQ ID NO. 4 was improved; the esterase (mutant 4 (L222M)) comprising the amino acid sequence of SEQ ID NO. 5 has improved high-temperature stability and pH stability; the esterase (mutant 5 (V229I)) comprising the amino acid sequence of SEQ ID NO. 6 has improved high-temperature stability, high-temperature reactivity and pH stability; the esterase (mutant 6 (S235T)) comprising the amino acid sequence of SEQ ID NO. 7 has improved specific activity, high-temperature reactivity and pH stability.

In general, when a part of the amino acid sequence of a certain protein is mutated, the mutated protein may have the same function as the protein before mutation. That is, mutation of the amino acid sequence does not substantially affect the function of the protein, and the function of the protein is maintained before and after the mutation. On the other hand, when the homology of the amino acid sequences of 2 proteins is high, the possibility that both show the same property is high. Considering these technical general knowledge, it is considered that the modified enzyme has an amino acid sequence which is not completely identical (i.e., 100% homology) but shows high homology to the amino acid sequence of the modified enzyme, namely, "the amino acid sequence of SEQ ID NO. 1 contains 1 or more amino acid substitutions selected from the group consisting of K7P, F13Y, V107M, E P, S155P, L222M, V I and S235T (the specific example of the amino acid sequence is the amino acid sequence of SEQ ID NO. 2 to 7)", and that the enzyme is substantially identical to the modified enzyme (substantially identical esterase) if the desired properties are improved. the homology is preferably 90% or more, more preferably 91% or more, still more preferably 92% or more, still more preferably 93% or more, still more preferably 95% or more, still more preferably 98% or more, and most preferably 99% or more. If the modified enzyme is compared to substantially the same esterase, a slight difference in amino acid sequence is seen. Wherein the difference in the set amino acid sequence is generated at a position other than the position at which the amino acid substitution is performed. Therefore, when the reference of homology is the amino acid sequence of SEQ ID NO. 2, the amino acid sequence differs at positions other than proline at position 7; when the criterion of homology is the amino acid sequence of SEQ ID NO. 3, the amino acid sequences are different at positions other than tyrosine 13; When the reference of homology is the amino acid sequence of SEQ ID NO.4, the amino acid sequence differs at a position other than methionine at position 107; when the reference of homology is the amino acid sequence of SEQ ID NO. 5, the amino acid sequence differs at positions other than methionine at position 222; when the reference of homology is the amino acid sequence of SEQ ID NO. 6, the amino acid sequence differs at positions other than isoleucine 229; when the reference of homology is the amino acid sequence of SEQ ID NO. 7, the amino acid sequence differs at positions other than threonine at position 235. In other words, in the amino acid sequence showing 90% or more homology with the amino acid sequence of SEQ ID NO. 2, the 7 th amino acid is proline. Similarly, in the amino acid sequence showing homology of 90% or more with the amino acid sequence of SEQ ID NO. 3, amino acid at position 13 is tyrosine, in the amino acid sequence showing homology of 90% or more with the amino acid sequence of SEQ ID NO. 4, amino acid at position 107 is methionine, in the amino acid sequence showing homology of 91% or more with the amino acid sequence of SEQ ID NO. 5, amino acid at position 222 is methionine, in the amino acid sequence showing homology of 91% or more with the amino acid sequence of SEQ ID NO. 6, amino acid at position 229 is isoleucine, in the amino acid sequence showing homology of 90% or more with the amino acid sequence of SEQ ID NO. 7, The amino acid at position 235 is threonine.

The "slight difference in amino acid sequence" herein is produced by deletion, substitution, addition, insertion of amino acids, or a combination thereof. Typically, it means that mutation (change) is generated in an amino acid sequence by deletion, substitution of 1 to several (upper limit of, for example, 3, 5, 7, 10) amino acids constituting the amino acid sequence, or addition, insertion, or a combination of 1 to several (upper limit of, for example, 3, 5, 7, 10) amino acids. "slight differences in amino acid sequence" preferably result from conservative amino acid substitutions. "conservative amino acid substitution" as used herein refers to the substitution of an amino acid residue for one having a side chain with the same properties. Amino acid residues are classified according to their side chains into several families of basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions are preferably substitutions between amino acid residues within a homologous family. It is also known that the amino acid residues constituting the active center of the esterase (SEQ ID NO: 1) derived from Acinetobacter calcoaceticus are serine 97, aspartic acid 226 and histidine 256, and therefore it is preferable that these amino acid residues are not affected when mutation is performed. Examples of the functions of the protein that are maintained before and after mutation without substantially affecting the functions of the protein due to slight differences in amino acid sequences include: an amino acid sequence in which amino acid 132 (glutamic acid) in the amino acid sequence of SEQ ID NO. 1 is proline; the 192 st amino acid (glycine) in the amino acid sequence of SEQ ID NO. 1 is serine or the 244 rd amino acid (leucine) in the amino acid sequence of SEQ ID NO. 1 is proline. As a result of comparing the specific activity with the high temperature stability of esterases having these amino acid sequences, they were equivalent to SEQ ID No. 1.

However, the homology (%) of 2 amino acid sequences or 2 base sequences (hereinafter, "2 sequences" are used as terms including them) can be determined, for example, in the following order. First, the two sequences are aligned to enable optimal comparison (e.g., gaps can be introduced in the first sequence to optimize its alignment with the second sequence). When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is identical to a molecule at a corresponding position in the second sequence, the molecules at that position can be said to be identical. Homology of 2 sequences is a function of the number of identical positions in common in the 2 sequences (i.e. homology (%) = number of identical positions/total number of positions x 100), preferably also taking into account the number and size of gaps required for optimization of the alignment.

The comparison of 2 sequences and the determination of homology can be accomplished using mathematical algorithms. Specific examples of mathematical algorithms that can be used for sequence comparison are those described in Karlin and Altschul (1990) proc.Natl. Acad. Sci. USA 87:2264-68 and described in Karlin and Altschul (1993) Proc.Natl. Acad.Sci.USA 90:5873-77, but is not limited thereto. Such algorithms are programmed in Altschul et al (1990) J.mol.biol.215:403-10, and XBLAST programs (version 2.0). In order to obtain an equivalent nucleotide sequence, for example, BLAST nucleotide search may be performed using the NBLAST program with score=100 and wordlength =12. In order to obtain an equivalent amino acid sequence, for example, BLAST polypeptide search may be performed using the XBLAST program with score=50 and wordlength =3. To obtain a gap alignment for comparison, altschul et al (1997) Amino ACIDS RESEARCH (17) can be used: 3389-3402 Gapped BLAST. With BLAST and Gapped BLAST, default parameters for the corresponding programs (e.g., XBLAST and NBLAST) can be used. See http for details: and/www.ncbi.nlm.nih.gov. As examples of other mathematical algorithms that can be utilized for sequence comparison are Myers and Miller (1988) Comput Appl biosci.4: 11-17. Such algorithms are for example programmed into an ALIGN program that can be utilized in GENESTREAM web servers (IGH Montpellier, france) or ISREC servers. When the ALIGN program is used for the comparison of amino acid sequences, for example, PAM120 residue matrix scale may be used, and gap length penalty=12 and gap penalty=4 may be set.

The GAP program of the GCG software package can be used to determine homology of two amino acid sequences using the Blossom 62 matrix or PAM250 matrix, set to a GAP weight=12, 10, 8, 6 or 4, and a GAP length weight=2, 3 or 4. Further, the homology of two base sequences can be determined using the GAP program of the GCG package (available through http:// www.gcg.com) with a GAP weight=50 and a GAP length weight=3.

Typically, the modified enzyme of the present invention is obtained by mutating an esterase comprising the amino acid sequence of SEQ ID NO. 1, namely, an esterase derived from Acinetobacter calcoaceticus (a combination of 1 or 2 or more of K7P, F13Y, V107M, L M, V229I and S235T described above). For substantially the same esterases described above, they can be obtained by: further mutating the enzyme after the esterase mutation constituted by the amino acid sequence of SEQ ID NO. 1 (caused by a combination of 1 or more than 2 of the above-mentioned K7P, F13Y, V107M, L222M, V229I and S235T); An esterase derived from the same genus as that of the Acinetobacter calcoaceticus strain producing the esterase comprising the amino acid sequence of SEQ ID NO. 1, such as an esterase comprising an amino acid sequence having high homology with the amino acid sequence of SEQ ID NO. 1, is subjected to an equivalent mutation, or an esterase obtained by the mutation is further subjected to a mutation. In the "equivalent mutation" herein, in the amino acid sequence having high homology with the amino acid sequence of SEQ ID NO. 1, an amino acid residue corresponding to the amino acid residue at the mutation point (position 7, 13, 107, 222, 229 or 235 of the amino acid sequence of SEQ ID NO. 1) in the present invention is substituted. Examples of esterases each having an amino acid sequence having high homology with the amino acid sequence of SEQ ID NO. 1 include: an esterase derived from acinetobacter giganteum (Acinetobacter guillouiae) and having the amino acid sequence of seq id No. 15 (homology of 98% in amino acid sequence); an esterase derived from Acinetobacter baumannii ABNIH (Acinetobacter baumannii ABNIH 3) and having the amino acid sequence of SEQ ID NO. 16 (homology 97% of amino acid sequence); an esterase derived from Acinetobacter saikokii (Acinetobacter seifertii) and having the amino acid sequence of SEQ ID NO. 17 (homology of 90% in amino acid sequence); An esterase derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa) and having the amino acid sequence of SEQ ID No. 18 (homology of 80% in amino acid sequence); an esterase derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa) and having the amino acid sequence of SEQ ID NO. 19 (homology of 80% in amino acid sequence); an esterase derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa) and having the amino acid sequence of SEQ ID No. 20 (homology of 80% in amino acid sequence); an esterase from Wu Menba g (Burkholderia ubonensis) having the amino acid sequence of SEQ ID NO. 21 (homology of 81% in amino acid sequence); Esterase from P.paraburkholderia (Paraburkholderia ferrariae) having the amino acid sequence of SEQ ID NO. 22 (homology of 81% in amino acid sequence); an esterase derived from Burkholderia pseudokohlii (Burkholderia pseudomultivorans) and having the amino acid sequence of SEQ ID No. 23 (homology of 81% in amino acid sequence); an esterase derived from Acinetobacter sp.NBRC 110496 (Acinetobacter sp.NBRC 110496) and having the amino acid sequence of SEQ ID No. 24 (homology of amino acid sequence 81%); An esterase derived from Acinetobacter saikokii (Acinetobacter seifertii) and having the amino acid sequence of SEQ ID NO. 25 (homology of 90% in amino acid sequence); an esterase derived from Acinetobacter sp.NIPH809 (Acinetobacter sp.NIPH809) and having the amino acid sequence of SEQ ID No. 26 (homology of amino acid sequence 80%); an esterase from Pseudomonas fluorescens A506 (A506 Pseudomonas fluorescens A506) having the amino acid sequence of SEQ ID NO. 27 (homology 80% of amino acid sequence); Esterases from Pseudomonas sp.ABAC61 (Pseudomonas sp.ABAC 61) and having the amino acid sequence of SEQ ID No. 28 (homology of 79% of amino acid sequence); esterases from pseudomonas putida IFO12996 (Pseudomonas putida IFO 12996) and having the amino acid sequence of seq id No. 29 (homology of 76% in amino acid sequence); an esterase derived from Pseudomonas putida MR2068 (Pseudomonas putida MR 2068) and having the amino acid sequence of SEQ ID NO. 30 (homology of 76% in amino acid sequence); An esterase derived from Pseudomonas aeruginosa (Pseudomonas aeruginosa) and having the amino acid sequence of SEQ ID NO. 31 (homology of 80% in amino acid sequence).

In the present specification, the term "equivalent" when used with respect to amino acid residues means that the proteins (enzymes) to be compared contribute equally to the function thereof. For example, when the amino acid sequences to be compared are aligned with respect to the amino acid sequence of the reference esterase (the amino acid sequence of SEQ ID NO. 1) so that the optimal comparison can be performed while taking into consideration partial identity of the primary structure (amino acid sequence), gaps may be introduced as needed to optimize the alignment, and the amino acid at the position corresponding to the specific amino acid in the reference amino acid sequence may be specified as the "equivalent amino acid". Instead of or in addition to the comparison between primary structures, it is also possible to specify "comparable amino acids" by comparison between the three-dimensional structures. By using the stereo structure information, a highly reliable comparison result can be obtained. In this case, a method of comparing the atomic coordinates of the three-dimensional structures of a plurality of enzymes may be employed. The steric structure information of the mutant enzyme can be obtained, for example, by Protein Data Bank (http:// www.pdbj.org/index_j. Html).

An example of a method for determining a protein stereo structure by X-ray crystal structure analysis is shown below.

(1) The protein is crystallized. Crystallization is indispensable for determining a three-dimensional structure, and is industrially useful as a purification method of a protein with high purity and a storage method with high density and stability. In this case, the protein to which the substrate or the like is bound as a ligand may be crystallized.

(2) Diffraction data were collected by irradiating the produced crystals with X-rays. In addition, protein crystals are often damaged by X-ray irradiation, which results in deterioration of diffraction ability. In this case, recently, a low temperature measurement technique has been spread in which a crystal is rapidly cooled to about-173 ℃ and diffraction data is collected in this state. In addition, finally, in order to collect high resolution data for structural determination, a synchrotron radiation light source of high brightness is used.

(3) For the crystal structure analysis, phase information is required in addition to diffraction data. In the case where the crystal structure of the target protein is unknown, it is impossible to determine the structure by using a molecular substitution method, and the phase problem must be solved by a heavy atom isomorphous substitution method. The heavy atom isomorphous substitution method is a method of introducing metal atoms having a large atomic number such as mercury and platinum into a crystal and obtaining phase information by utilizing the contribution of the large X-ray scattering ability of the metal atoms to X-ray diffraction data. The determined phase can be improved by smoothing the electron density of the solvent region in the crystal. Since the fluctuation of water molecules in the solvent region is large, the electron density is hardly observed, and thus by making the electron density in this region approximately 0, the true electron density can be approximated and even the phase can be improved. In addition, when a plurality of molecules are contained in the asymmetric unit, the electron density of these molecules is averaged, whereby the phase can be improved more greatly. The protein model is fitted in an electron density map using the phase calculation thus improved. This process is performed on a computer map using a program such as QUANTA from MSI company (usa). Then, the structure is refined by using a program such as X-PLOR from MSI company, and the structure analysis is completed. In the case where the crystal structure of the target protein is known, the atomic coordinates of the known protein can be used to determine the target protein by a molecular substitution method. Molecular substitution and structural refinement can be performed using the program cns_solve ver.11, etc.

2. Nucleic acids encoding modified esterases and the like

In accordance with the 2 nd aspect of the present invention, there is provided a nucleic acid related to the modified enzyme of the present invention. That is, a gene encoding a modified enzyme, a nucleic acid that can be used as a probe for identifying a nucleic acid encoding a modified enzyme, a nucleic acid that can be used as a primer for amplifying or mutating a nucleic acid encoding a modified enzyme, or the like can be provided.

Genes encoding modified enzymes are typically used for the preparation of modified enzymes. According to the genetic engineering production method using the gene encoding the modified enzyme, the modified enzyme in a more homogeneous state can be obtained. In addition, this method can also be referred to as a preferred method in the case of preparing a large amount of modified enzymes. Furthermore, the use of the gene encoding the modified enzyme is not limited to the preparation of the modified enzyme. For example, the nucleic acid can be used as an experimental tool for the purpose of elucidating the mechanism of action of the modified enzyme or as a tool for designing or producing a further modified form of the enzyme.

In the present specification, the term "gene encoding a modified enzyme" refers to a nucleic acid which, when expressed, gives the modified enzyme, and naturally includes a nucleic acid having a nucleotide sequence corresponding to the amino acid sequence of the modified enzyme, and naturally includes a nucleic acid in which a sequence not encoding the amino acid sequence is added to such a nucleic acid. In addition, degeneracy of codons is also contemplated.

Examples of sequences (base sequences) of genes encoding modified enzymes are shown in SEQ ID Nos. 8 to 13. These sequences code for mutants as shown in the examples described below.

Sequence number 8: mutant 1 (K7P)

Sequence number 9: mutant 2 (F13Y)

Sequence number 10: mutant 3 (V107M)

Sequence number 11: mutant 4 (L222M)

Sequence number 12: mutant 5 (V229I)

Sequence number 13: mutant 6 (S235T)

The enzyme of the present invention does not have a signal sequence and is not normally secreted outside the cell, but when it is not secreted outside the cell, it can be introduced into a host using a gene construct in which a sequence (signal sequence) encoding a signal peptide is added to the 5' -terminal side of the above sequence (any of SEQ ID Nos. 8 to 13). The signal sequence may be selected according to the host. The present invention can be used as long as it is a signal sequence capable of expressing the mutant of interest. As a signal sequence that can be used, there can be exemplified: sequences encoding signal peptides for the alpha-factor (Protein Engineering,1996, vol9, p.1055-1061); a sequence encoding a signal peptide of an alpha-factor receptor; a sequence encoding a signal peptide of the SUC2 protein; a sequence encoding a signal peptide of the PHO5 protein; a sequence encoding a signal peptide of BGL2 protein; a sequence encoding a signal peptide of the AGA2 protein; a sequence encoding a signal peptide of TorA (trimethylamine-N-oxide reductase); a sequence encoding a signal peptide from the PhoD (phosphatase) of bacillus subtilis; a sequence encoding a signal peptide of LipA (esterase) from bacillus subtilis; a sequence encoding a signal peptide of a peak amylase derived from Aspergillus oryzae (Aspergillus oryzae) (Japanese patent application laid-open No. 2009-60804); sequences encoding signal peptides from the alpha-amylase of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) (European journal of biochemistry (Eur. J. Biochem.)) 155,577-581 (1986)); sequences encoding signal peptides from neutral proteases of Bacillus subtilis (applied and environmental microbiology (APPLIED AND ENVIRONMENTAL MICROBIOLOGY), apr.1995, p.1610-1613Vol.61, no. 4); a sequence encoding a signal peptide of a cellulase derived from a bacterium belonging to the genus Bacillus (Japanese patent laid-open No. 2007-130012).

The nucleic acid of the present invention can be prepared into a separated state by using standard genetic engineering methods, molecular biology methods, biochemical methods, chemical synthesis, etc., with reference to the sequence information disclosed in the present specification or the attached sequence listing.

In another embodiment of the present invention, a nucleic acid having a function equivalent to that of a protein encoded by the modified enzyme of the present invention when compared with the nucleotide sequence of the gene encoding the modified enzyme of the present invention, but having a different nucleotide sequence in a part thereof (hereinafter, also referred to as "identical nucleic acid". Furthermore, the nucleotide sequence defining the identical nucleic acid is also referred to as "identical nucleotide sequence"). Examples of the same nucleic acid include: DNA which is composed of a base sequence comprising 1 or more base substitutions, deletions, insertions, additions or inversions based on the base sequence of a nucleic acid encoding the modified enzyme of the present invention and which encodes a protein having an enzyme activity (i.e., esterase activity) characteristic of the modified enzyme. Substitution, deletion, etc. of bases can be generated at a plurality of positions. The term "plurality" as used herein varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein encoding the nucleic acid, and is, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases.

The same nucleic acid has, for example, 60% or more, preferably 70% or more, more preferably 80% or more, still more preferably 85% or more, still more preferably about 90% or more, still more preferably 95% or more, and most preferably 99% or more homology to the base sequence as a reference.

For example, the same nucleic acid as described above can be obtained by treatment with restriction enzymes, treatment with exonucleases, DNA ligases or the like, introduction of mutations using the site-specific mutagenesis method (Molecular Cloning, third Edition, chapter 13,Cold Spring Harbor Laboratory Press,New York), the random mutagenesis method (Molecular Cloning, third Edition, chapter 13,Cold Spring Harbor Laboratory Press,New York) or the like. The same nucleic acid can be obtained by other methods such as ultraviolet irradiation.

Another embodiment of the present invention relates to a nucleic acid having a base sequence complementary to a base sequence of a gene encoding the modified enzyme of the present invention. In still another embodiment of the present invention, there is provided a nucleic acid having a nucleotide sequence at least about 60%, 70%, 80%, 90%, 95%, 99%, 99.9% homologous to a nucleotide sequence of a gene encoding the modified enzyme of the present invention or a nucleotide sequence complementary thereto.

A further aspect of the present invention relates to a device comprising: nucleic acid of a base sequence which hybridizes under stringent conditions to a base sequence of a gene encoding the modified enzyme of the present invention or a base sequence complementary to the same base sequence. "stringent conditions" herein means conditions under which so-called specific hybridization is formed and non-specific hybridization is not formed. Such stringent conditions are well known to those skilled in the art and can be set, for example, by reference to molecular cloning (Molecular Cloning) (third edition, cold spring harbor laboratory Press, N.Y.), modern guidelines for molecular biology experiments (Current protocols in molecular biology) (F.M. Osbert et al, 1987). Examples of stringent conditions include the following conditions: hybridization solution (50% formamide, 10 XSSC (0.15M NaCl,15mM sodium citrate, pH 7.0), 5 XDenhardt's solution, 1% SDS, 10% dextran sulfate, 10. Mu.g/ml denatured salmon sperm DNA, 50mM phosphate buffer (pH 7.5)) was used, followed by incubation at about 42℃to about 50℃and washing at about 65℃to about 70℃using 0.1 XSSC, 0.1% SDS. More preferable stringent conditions include, for example, conditions in which 50% formamide, 5 XSSC (0.15M NaCl,15mM sodium citrate, pH 7.0), 1 XDenhardt's solution, 1% SDS, 10% dextran sulfate, 10. Mu.g/ml denatured salmon sperm DNA, and 50mM phosphate buffer (pH 7.5) are used as the hybridization solution.

In another embodiment of the present invention, a nucleic acid (nucleic acid fragment) having a part of a nucleotide sequence of a gene encoding the modified enzyme of the present invention or a nucleotide sequence complementary thereto is provided. Such a nucleic acid fragment can be used for detection, identification, amplification, and/or the like of a nucleic acid having a base sequence of a gene encoding the modified enzyme of the present invention. The nucleic acid fragment is designed to contain, for example, at least a portion that hybridizes with a continuous nucleotide portion (for example, about 10 to about 100 bases long, preferably about 20 to about 100 bases long, more preferably about 30 to about 100 bases long) in the base sequence of the gene encoding the modified enzyme of the present invention. When used as a probe, the nucleic acid fragment may be labeled. For example, fluorescent substances, enzymes, and radioisotopes can be used for labeling.

Still another aspect of the present invention relates to a recombinant DNA comprising the gene of the present invention (a gene encoding a modified enzyme). The recombinant DNA of the present invention may be provided in the form of a vector, for example. In the present specification, the term "vector" refers to a nucleic acid molecule capable of transporting a nucleic acid inserted therein into a target such as a cell.

Depending on the purpose of use (cloning, expression of proteins), an appropriate vector is selected in consideration of the type of host cell. Examples of vectors using E.coli as a host include M13 phage or a modified form thereof, lambda phage or a modified form thereof, pBR322 or a modified form thereof (pB 325, pAT153, pUC8, etc.), pET21, etc., examples of vectors using yeast as a host include pYepSec, pMFa, pYES2, pPICC 3.5K, etc., examples of vectors using insect cells as a host include pAc and pVL, and examples of vectors using mammalian cells as a host include pCDM8 and pMT2 PC.

The vector of the present invention is preferably an expression vector. "expression vector" refers to a vector into which a nucleic acid inserted can be introduced into a target cell (host cell) and which enables its expression in the cell. Expression vectors typically contain promoter sequences required for expression of the inserted nucleic acid, enhancer sequences to facilitate expression, and the like. Expression vectors comprising selectable markers may also be used. When the expression vector is used, the presence or absence (and the extent of the introduction of the expression vector can be confirmed by using a selection marker.

The insertion of the nucleic acid of the present invention into a vector, the insertion of a selectable marker gene (if necessary), the insertion of a promoter (if necessary), etc. can be performed using standard recombinant DNA techniques (for example, refer to Molecular Cloning, the well-known method using restriction enzymes and DNA ligases by Third Edition,1.84,Cold Spring Harbor Laboratory Press,New York).

As the host cell, from the viewpoint of ease of handling, a microorganism such as Aspergillus (for example, aspergillus oryzae), bacillus (for example, bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens), brevibacterium (for example, brevibacterium chondrium), escherichia coli (ESCHERICHIA COLI), and Bacillus (Saccharomyces cerevisiae) can be used, but a host cell in which recombinant DNA is replicable and the gene of the modified enzyme can be expressed can be used. Preferably, E.coli (ESCHERICHIA KOLI), and budding yeast (Saccharomyces cerevisiae) may be used. Microorganisms of the genus Acinetobacter (e.g., acinetobacter calcoaceticus) may also be used as hosts. In addition, as an example of the Escherichia coli, escherichia coli BL21 (DE 3) may be mentioned when the T7-based promoter is used, and Escherichia coli JM109 may be mentioned when the T7-based promoter is not used. Examples of budding yeasts include budding yeast SHY2, budding yeast AH22, and budding yeast INVSc1 (Invitrogen).

Another aspect of the present invention relates to a microorganism (i.e., transformant) harboring the recombinant DNA of the present invention. The microorganism of the present invention can be obtained by transfection or transformation using the vector of the present invention described above. For example, the method of calcium chloride (journal of molecular biology (Journal of Molecular Biology) (J.mol.biol.), volume 53, page 159 (1970)), hanahan method (journal of molecular biology, volume 166, page 557 (1983)), SEM method (Gene, volume 96, page 23 (1990)), chung et al (journal of national academy of sciences (Proceedings of the National Academyof Sciences of the USA), volume 86, page 2172 (1989)), calcium phosphate coprecipitation method, electroporation (Potter, H.et al., proc.Natl.Acad.Sci.U.S.A.81,7161-7165 (1984)), lipofection (Felgner, P.L.etal., proc.Natl.Acad.Sci.U.S.A.84,7413-7417 (1984)), and the like can be used. In addition, the microorganism of the present invention can be used for producing the modified enzyme of the present invention.

3. Enzyme preparation comprising modified esterases

The modified enzyme of the present invention may be provided in the form of an enzyme preparation, for example. The enzyme preparation may contain an excipient, a buffer, a suspension, a stabilizer, a preservative, physiological saline, and the like, in addition to the active ingredient (the modified enzyme of the present invention). As the excipient, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, white sugar, glycerol, etc. can be used. As the buffer, phosphates, citrates, acetates, and the like can be used. As the stabilizer, propylene glycol, ascorbic acid, and the like can be used. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl parahydroxybenzoate, and the like can be used. As the preservative, ethanol, benzalkonium chloride, p-hydroxybenzoic acid, chlorobutanol and the like can be used.

4. Use of modified esterases

The present invention also relates to uses of the modified enzyme and enzyme preparation, and various reactions using the modified enzyme or enzyme preparation of the present invention, production methods and production methods of various compounds using the same, and the like can be provided. Specifically, the modified enzyme or enzyme preparation of the present invention can be used for (1) production of an organic compound used for pharmaceutical products and the like; (2) The production of ester compounds used in the field of fine chemicals is considered; (3) hydrolysis of the lactone compound; etc. Examples of (1) include the production of captopril, which is one of angiotensin converting enzyme inhibitors (ACE inhibitors), by enantioselective hydrolysis of DL-MATI (DL-beta-acetylthioisobutyrate (DL-beta-acetylthioisobutyrate)) which is DAT (D-beta-acetylthioisobutyric acid (D-beta-Acetylthioisobutyric acid)) which is a pharmaceutical intermediate of Alazepril (see Jounal of Molecular Catalysis B: enzymatic 38 (2006) 163-170). On the other hand, examples of (2) include the production of linear ester compounds ((R) 3-bromo-2-methylpropanoic acid Methyl ester (Methyl (R) -3-bromo-2-methylpropionate), cetrimate hydrochloride (Methyl cetraxate hydrochloride) and the like) having various substituents on the side chains (see Appl Microbiol Biotechnol (2002) 60:288-292). Further, as an example of (3), there may be mentioned: decomposition of aldehyde lactones (D-galactose-gamma-lactone, L-mannose-gamma-lactone, etc.), aromatic lactones (3, 4-dihydrocoumarin, homogentic acid gamma-lactone, etc.) (see Eur. J. Biochem.267,3-10 (2000)).

5. Process for preparing modified esterase

A further aspect of the invention relates to a method for preparing the modified enzyme. In one embodiment of the production method of the present invention, the inventors have succeeded in producing a modified enzyme by a genetic engineering method. In this embodiment, a nucleic acid encoding any one of the amino acid sequences of SEQ ID NOS 2 to 7 is prepared (step (I)). The term "nucleic acid encoding a specific amino acid sequence" as used herein refers to a nucleic acid which, when expressed, gives a polypeptide having the amino acid sequence, and a nucleic acid consisting of a base sequence corresponding to the amino acid sequence may be used, or an extra sequence (which may be a sequence encoding an amino acid sequence or a sequence not encoding an amino acid sequence) may be added to such a nucleic acid. In addition, degeneracy of codons is also contemplated. The "nucleic acid encoding any one of the amino acid sequences of SEQ ID Nos. 2 to 7" can be prepared in an isolated state by using standard genetic engineering methods, molecular biology methods, biochemical methods, and the like, with reference to the sequence information disclosed in the present specification or the appended sequence Listing. Here, the amino acid sequences of SEQ ID Nos. 2 to 7 are each obtained by mutating the amino acid sequence of an esterase derived from Acinetobacter calcoaceticus. Thus, even if a necessary mutation is applied to a gene encoding an esterase derived from Acinetobacter calcoaceticus, a nucleic acid (gene) encoding any one of the amino acid sequences 2 to 7 can be obtained. Many methods for position-specific base sequence substitution are known in the art (for example, refer to Molecular Cloning, third Edition, cold Spring Harbor Laboratory Press, new York), and suitable methods may be selected for use. As the site-specific mutagenesis method, a site-specific amino acid saturation mutagenesis method can be used. The site-specific amino acid saturation mutation method is based on the steric structure of a protein, and is to estimate a position related to a desired function, and introduce an amino acid saturation mutation "Semi-rational, semi-random: semi-rational semi-random (J.mol. Biol.331,585-592 (2003)). For example, site-specific amino acid saturation mutations can be introduced by using a kit such as Quick change (Stratagene Co.) or overlap extension (over lap extention) PCR (Nucleic acid Res.16,7351-7367 (1988)). As the DNA polymerase used for PCR, taq polymerase or the like can be used. Among them, DNA polymerases having high precision such as KOD-PLUS (Toyobo Co., ltd.) and Pfu turbo (Stratagene Co.) are preferably used.

Next, step (I) is performed to express the prepared nucleic acid (step (II)). For example, an expression vector into which the above nucleic acid is inserted is first prepared, and the host cell is transformed with the vector.

Next, the transformant is cultured under conditions that produce the modified enzyme as an expression product. The transformant may be cultured according to a usual method. The carbon source used in the medium may be any carbon compound that can be assimilated, and for example, glucose, sucrose, lactose, maltose, molasses, pyruvic acid, and the like can be used. The nitrogen source may be any available nitrogen compound, and for example, peptone, meat extract, yeast extract, casein hydrolysate, soybean meal alkali extract and the like can be used. In addition, salts such as phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, zinc, etc. can be used as needed; a specific amino acid; specific vitamins, etc.

On the other hand, the culture temperature may be set in the range of 30℃to 40℃and preferably around 33℃to 37 ℃. The culture time may be set in consideration of the growth characteristics of the transformant to be cultured, the production characteristics of the modified enzyme, and the like. The pH of the medium is adjusted within the range where the transformant grows and the enzyme is produced. The pH of the medium is preferably adjusted to about 6.0 to 9.0 (preferably, pH7.0 or thereabout).

Next, the expression product (modified enzyme) is recovered (step (III)). The culture solution containing the cultured cells may be used as an enzyme solution directly or after concentration, removal of impurities, or the like, but in general, the expression product is temporarily recovered from the culture solution or the cells. If the expression product is a secreted protein, it can be recovered from the culture medium, and if the expression product is otherwise recovered from the cell body. In the case of recovering from the culture solution, for example, the culture supernatant is filtered, centrifuged to remove insoluble matters, and then concentrated under reduced pressure by combination; concentrating by a membrane; salting out with ammonium sulfate and sodium sulfate; fractional precipitation using methanol, ethanol, or acetone; dialyzing; heating treatment; isoelectric point treatment; gel filtration is performed by using various chromatographic methods such as adsorption chromatography, ion exchange chromatography, and affinity chromatography (for example, gel filtration using dextran (Sephadex) gel (GE HEALTHCARE Bio-sciences) or the like, DEAE sepharose CL-6B (GE HEALTHCARE Bio-sciences), octyl sepharose CL-6B (GE HEALTHCARE Bio-sciences), CM sepharose CL-6B (GE HEALTHCARE Bio-sciences)), or the like, and purification is performed, whereby purified products of the modified enzyme can be obtained. On the other hand, in the case of recovering from the cell body, the cell body is obtained by subjecting the culture solution to filtration, centrifugation or the like, then the cell body is disrupted by a mechanical method such as pressure treatment, ultrasonic treatment, physical pulverization treatment or the like or an enzymatic method based on lysozyme or the like, and then the purified product of the modified enzyme can be obtained by separation and purification in the same manner as described above.

The purified enzyme obtained as described above may be provided by powdering the enzyme by, for example, freeze drying, vacuum drying, spray drying, or the like. In this case, the purified enzyme may be dissolved in advance in phosphate buffer, triethanolamine buffer, tris-HCl buffer, or GOOD buffer. Preferably, phosphate buffer and triethanolamine buffer can be used. Here, examples of the buffer for GOOD include PIPES, MES and MOPS.

In general, the expression of the gene to the recovery of the expression product (modified enzyme) is carried out as described above using an appropriate host-vector system, but a cell-free synthesis system may also be used. Herein, "cell-free synthesis system (cell-free transcription system, cell-free transcription/translation system)" means that a ribosome derived from a living cell (or obtained by a genetic engineering method) is used instead of a living cell; transcription and translation factors, etc., and the mRNA and protein encoded by the transcription and translation factors are synthesized in vitro from nucleic acids (DNA and mRNA) serving as templates. In general, a cell extract obtained by purifying a cell disruption solution as needed is used in a cell-free synthesis system. The cell extract usually contains various factors such as ribosomes and initiation factors, and various enzymes such as tRNA, which are required for protein synthesis. In the synthesis of protein, various amino acids, energy sources such as ATP and GTP, and other substances necessary for the synthesis of protein such as creatine phosphate are added to the cell extract. Naturally, when synthesizing a protein, it is also possible to supplement, if necessary, a ribosome, various factors, various enzymes, and the like which are prepared separately.

The development of transcription/translation systems to reconstruct the individual molecules (factors) required for protein synthesis has also been reported in the literature (Shimizu, y.et al.: nature biotech.,19,751-755,2001). In this synthesis system, genes of 31 factors consisting of 3 initiation factors, 3 elongation factors, 4 factors related to termination, 20 aminoacyl-tRNA synthetases that bind each amino acid to tRNA, and methionyl tRNA formyl transferase, which constitute the protein synthesis system of bacteria, were amplified from E.coli genome, and the protein synthesis system was reconstituted in vitro using them. Such a reconstituted synthetic system may also be utilized in the present invention.

The term "cell-free transcription/translation system" can be used interchangeably with cell-free protein synthesis system, in vitro translation system or in vitro transcription/translation system. In an in vitro translation system, RNA is used as a template to synthesize proteins. As the template RNA, total RNA, mRNA, in vitro transcription products, and the like can be used. In another in vitro transcription/translation system, DNA is used as a template. The template DNA should preferably comprise a ribosome binding region and comprise a suitable termination sequence. In addition, in the in vitro transcription/translation system, conditions for adding factors necessary for each reaction are set so that the transcription reaction and the translation reaction proceed continuously.

[ Example ]

< Search for mutation Point effective for improvement of Properties >

The objective was to improve the characteristics of the esterase derived from Acinetobacter calcoaceticus (SEQ ID NO: 1), select mutation points (substituted amino acid residues) from the viewpoints of enhancement of hydrogen bond, enhancement of hydrophobicity, enhancement of high density (stacking) and enhancement of loops, and design 15 mutants (modified esterases). Each of the designed mutants was prepared by the following method, and the characteristics thereof were evaluated. The amino acid sequence of SEQ ID NO. 1 is a mature body (without signal peptide) of an esterase derived from Acinetobacter calcoaceticus.

1. Acquisition of mutants (modified esterases)

(1) Introduction and transformation of mutations

In order to introduce random mutations into each mutation introduction point, PCR (PRIMESTAR GXL DNA polymerase (Takara Bio Inc.) was performed using random primers with the plasmid (pET 21 (Esterase)) into which the esterase gene was inserted as a template. After PCR, a Ligation reaction (16℃overnight) using Dpn I (Takara Bio Inc.) and T4 kinase (Toyo Kabushiki Kaisha) and a High-efficiency Ligation reagent (Ligation High) was performed (16℃overnight), followed by transformation to E.coli BL21 (DE 3), thereby obtaining random mutants in which random mutations were introduced into each mutation introduction point.

< PCR conditions >

Composition of the reaction solution: mu.l of 5X PRIMESTAR GXL buffer, 2. Mu.l of dNTP mix (2.5 mM each), 0.5. Mu.l of forward primer, 0.5. Mu.l of reverse primer, 0.25. Mu.l of template, 1. Mu.l of PRIMESTAR GXL DNA polymerase (Takara Bio Inc.) (adjusted to a total of 25. Mu.l with sterile distilled water)

Reaction conditions: 15 cycles at 98℃for 10 seconds, at 60℃for 15 seconds and at 68℃for 2 minutes

(2) Culture of transformant and acquisition of enzyme solution (bacterial cell extract)

The constructed esterase gene recombinant E.coli expression strain (host: E.coli BL21 (DE 3)) was used to attempt to obtain a culture broth (cultured cells). The culture solution (cultured cell) of each esterase gene recombinant E.coli expression strain was obtained by 2-stage culture. First, each esterase gene recombinant E.coli expression strain was inoculated into 5mL LBroth (Invitrogen) (Amp: 100. Mu.g/mL), cultured for 16 hours with a shaking incubator (150 rpm,37 ℃), and then 0.5mL of the strain was inoculated into 50mL Teriffic Broth (Invitrogen) (Amp: 100. Mu.g/mL). Then, the cells were cultured at 200rpm and 33℃for 48 hours, and 0.1mM IPTG was added to the culture solution from the start of the culture to 24 hours, thereby inducing the expression of the enzyme. After 50mL of the culture broth was centrifuged (7,000g. Times.10 min, 4 ℃) and the supernatant was removed to recover the cultured cells.

After each of the obtained cultured cells was suspended in 25mL of 100mM phosphate buffer (pH 7.0), 10g of beads (0.1 mM) was added (Anjing machine), and the cells were physically disrupted using a bead disrupter (2,500 rpm, on:60 seconds, off:30 seconds, 10 cycles, 4 ℃ C.) (Anjing machine). After the physical disruption solution was centrifuged (7,000g. Times.10 minutes, 4 ℃ C.), the supernatant was recovered as a cell extract.

(3) Obtaining of purified enzyme

The bacterial cell extract was filtered through a filter, and the amount of the bacterial cell extract was made up to 20mL. Then, ammonium sulfate precipitation (40 to 80% fraction recovery) was performed according to the following procedure. First, ammonium sulfate was added to reach 40% saturation. After centrifugation and recovery of the supernatant, ammonium sulfate was added to reach 80% saturation. And (5) centrifugal separation is carried out again, and centrifugal precipitation is recovered. The recovered pellet was suspended in 10mL of 10mM phosphate buffer (pH 7.0).

The enzyme solution after the thiamine precipitation was desalted and concentrated by ultrafiltration to reduce the conductivity to about 2mS/cm or less. Subsequently, the sample was purified by the following 2-stage chromatography (DEAE-Sepharose column chromatography, phenyl-Sepharose column chromatography). In addition, after NaCl was added to the fraction recovered by DEAE-Sepharose column chromatography to reach a concentration of 2M and the conductivity was adjusted, it was subjected to phenyl-Sepharose column chromatography.

(I) DEAE-Sepharose column chromatography

The chromatographic column used: deae-Sepharose FF 1mL (GE Healthcare)

Buffer solution: 10mM phosphate buffer (pH 7.0)

Eluting: elution of the target enzyme by a linear gradient of NaCl (0.fwdarw.0.3M)

Sample injection amount: 5mL (total desalted concentrated sample)

Flow rate: 1 mL/min

(Ii) Phenyl-sepharose column chromatography

The chromatographic column used: phenyl-HP 1mL (GE Healthcare)

Buffer solution: 10mM phosphate buffer (pH 7.0)

Eluting: elution of the target enzyme by a linear gradient of NaCl (2 M.fwdarw.0M)

Sample injection amount: 10mL (total amount of DEAE purification fraction)

Flow rate: 1 mL/min

The eluted fraction of the phenyl-sepharose column chromatography was recovered as a purified enzyme sample.

2. Characterization of mutants

< Activity assay >

2.1ML of 50mM phosphate buffer (pH 7.0), 0.3mL of 5mM 3, 4-dihydrocoumarin (Tokyo formation (TCI)) solution (50 mM phosphate buffer (pH 7.0) (containing 40% ethanol)), and 0.6mL of enzyme solution were mixed and reacted. In the present assay, the change in absorbance of the product (3- (2-hydroxypropyl) propionic acid) produced by the reaction was subjected to kinetic measurement (absolute value 270nm,30 ℃ C. For 5 minutes), whereby the amount of enzyme producing 1nmol of the product (3- (2-hydroxypropyl) propionic acid) in 1 minute was defined as 1 unit (U). In the present measurement, the Δod at which an accurate measurement value is obtained is in the range of Δod=0.01 to 0.05/2 minutes (270 nm absolute), and if necessary, the enzyme solution is diluted with 50mM phosphate buffer at ph 7.0.

(1) Evaluation of specific Activity

Specific activity was evaluated using purified enzyme samples. The enzyme activity of the purified enzyme of each mutant was measured by the above-mentioned activity measurement method, and the specific activity was calculated. The protein concentration was measured by using the absorbance at a wavelength of 280nm, and the protein concentration was calculated by setting the absorbance at A280nm to 1.0 to 1 mg/ml. The specific activities of the mutants are shown in FIG. 1. It was confirmed that the specific activity of mutant 6 (S235T) was improved.

(2) Evaluation of high temperature stability

High temperature stability was evaluated using purified enzyme samples. First, the purified enzyme of each mutant was subjected to heat treatment (80 ℃ C., 30 minutes). The enzyme activity was measured by the above-mentioned activity measurement method after diluting the heat-treated sample and the untreated sample to an appropriate concentration with a diluent (50 mM phosphate buffer (pH 7.0)). The residual activity was calculated from the measured value (enzyme activity) as follows, and the high-temperature stability was evaluated.

Activity remaining ratio = (enzyme activity after heat treatment/enzyme activity before heat treatment) ×100 (%)

The evaluation results are shown in FIG. 2. Improvement in high temperature stability was confirmed in 4 mutants (mutant 1 (K7P), mutant 2 (F13Y), mutant 4 (L222M), mutant 5 (V229I)). In particular, mutant 1 (K7P), mutant 4 (L222M) and mutant 5 (V229I) have high temperature stability.

(3) Evaluation of high temperature reactivity

High temperature reactivity was evaluated using purified enzyme samples. First, the purified enzyme of each mutant was diluted to an appropriate concentration with a diluent (50 mM phosphate buffer (pH 7.0)), and then the enzyme activity at each temperature (30 ℃ C., 60 ℃ C.) was measured. The high temperature reactivity was evaluated using the relative activity (ratio of enzyme activity at high temperature (60 ℃) to enzyme activity at 30 ℃) calculated by the following calculation formula.

Relative activity= (enzyme activity at high temperature (60 ℃)/enzyme activity at 30 ℃) x 100 (%)

The evaluation results are shown in FIG. 3. The improvement of the high temperature reactivity was confirmed in 2 mutants (mutant 5 (V229I), mutant 6 (S235T)). In particular mutant 5 (V229I) has high temperature reactivity.

(4) Evaluation of pH stability

The pH stability was evaluated using purified enzyme samples. First, the purified enzyme of each mutant was subjected to low pH treatment (pH 4.0, 30 minutes). The enzyme activity was measured by the above-mentioned activity assay method after diluting the treated sample and the untreated sample to an appropriate concentration with a diluent (50 mM phosphate buffer (pH 7.0)). The residual activity was calculated from the measured value (enzyme activity) as follows, and the high-temperature stability was evaluated.

Activity remaining ratio = (enzyme activity after low pH treatment/enzyme activity before treatment) ×100 (%)

The evaluation results are shown in FIG. 4. The improvement in pH stability was confirmed in 7 mutants (mutant 2 (F13Y), mutant 3 (V107M), mutant 4 (L222M), mutant 5 (V229I), and mutant 6 (S235T)). In particular, mutant 2 (F13Y), mutant 3 (V107M), mutant 4 (L222M) and mutant 5 (V229I) have high pH stability.

As above, 8 mutants with improved properties were successfully obtained. The amino acid sequences of the respective mutants are shown below.

Mutant 1 (K7P): sequence number 2

Mutant 2 (F13Y): sequence number 3

Mutant 3 (V107M): sequence No. 4

Mutant 4 (L222M): sequence number 5

Mutant 5 (V229I): sequence number 6

Mutant 6 (S235T): sequence number 7

[ Industry ] usability

The modified esterase of the invention has improved properties (specific activity, high-temperature reactivity, high-temperature stability, and pH stability). Therefore, the use or application range thereof is wide, and improvement of the reaction efficiency can be expected.

The present invention is not limited by the description of the embodiments and examples of the invention described above. Various modifications are also included in the present invention within the scope that can be easily conceived by those skilled in the art without departing from the description of the claims. The disclosures of papers, published patent publications, and patent publications identified in this specification are incorporated herein by reference in their entirety.

Claims (11)

1. A modified esterase having an amino acid sequence comprising 1 or more amino acid substitutions selected from the group consisting of K7P, F Y, V M and L222M in the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having 91% or more homology with the amino acid sequence, wherein the modified esterase has an improved specific activity, high-temperature stability, high-temperature reactivity and pH stability as compared with an esterase comprising the amino acid sequence of SEQ ID NO. 1.

2. The modified esterase according to claim 1, wherein the amino acid sequence of the modified esterase comprises an amino acid substitution of K7P, F to Y, V M or L222M, and the high-temperature stability is improved.

3. The modified esterase according to claim 1, wherein the amino acid sequence of the modified esterase comprises the amino acid substitutions K7P, F to Y, V M and L222M, and the pH stability is improved.

4. A modified esterase having an amino acid sequence of any of SEQ ID NO. 2 to 5 or an amino acid sequence having a homology of 91% or more with the amino acid sequence,

Wherein, when the reference of homology is the amino acid sequence of SEQ ID NO. 2, the amino acid sequence differs at a position other than proline at position 7; when the criterion of homology is the amino acid sequence of SEQ ID NO. 3, the amino acid sequence differs at positions other than tyrosine 13; when the homology is based on the amino acid sequence of SEQ ID NO. 4, the amino acid sequence differs at a position other than methionine at position 107; when the homology is based on the amino acid sequence of SEQ ID NO. 5, the amino acid sequence differs at positions other than methionine 222,

The modified esterase has improved properties of 1 or more selected from the group consisting of specific activity, high-temperature stability, high-temperature reactivity and pH stability, as compared with the esterase comprising the amino acid sequence of SEQ ID NO. 1.

5. A gene encoding the modified esterase according to any of claims 1 to 4.

6. The gene according to claim 7, wherein the gene comprises any one of the base sequences of SEQ ID Nos. 8 to 11.

7. A recombinant DNA comprising the gene of claim 5 or 6.

8. A microorganism harboring the recombinant DNA according to claim 7.

9. The microorganism according to claim 8, wherein the host is E.coli, B.subtilis, B.licheniformis, B.amyloliquefaciens or B.desmodium.

10. An enzyme preparation comprising the modified esterase according to any of claims 1 to 4.

11. A process for the preparation of a modified esterase comprising the following steps (I) to (III):

(I) A step of preparing a nucleic acid encoding any one of the amino acid sequences of SEQ ID NOS.2 to 5;

(II) a step of expressing the nucleic acid, and

(III) recovering the expression product.