EP1224296A1 - L-pantolactone-hydrolase and a method for producing d-pantolactone - Google Patents

Abstract

The invention relates to proteins which have an enzymatic activity for hydrolysing L-pantolactone. The invention also relates to nucleic acids that code for these proteins, to nucleic acid constructs, to vectors, to genetically modified micro-organisms and to a method for producing D-pantolactone.

Description

L-pantolactone hydrolase and a process for producing D-pantolactone

description

The present invention relates to proteins which have an enzymatic activity for the hydrolysis of L-pantolactone. The invention further relates to nucleic acids that code for these proteins, nucleic acid constructs, vectors, genetically modified microorganisms and a method for producing D-pantolactone.

D-pantolactone is a precursor in the chemical synthesis or biosynthesis of pantothenic acid, panthenol and pantethein and their derivatives. These are used as vitamin additives in human nutrition, in animal feed, in medicine, for example for wound healing, and in cosmetics, for example in hair cosmetics. Economic processes for the synthesis of enantiomerically pure D-pantolactone are therefore of great importance

Importance. In addition to the long-established chemical processes for the production of D-pantolactone, various biotechnological processes have also recently been worked on. An overview of pantolactone and its chemical synthesis can be found in Ullmann's Encyclopedia of Industrial Chemistry (Vol. A27, 1996, VCH Verlagsgesellschaft mbH, 69451 Weinheim, pages 559-566).

Various synthetic strategies were pursued for the biotechnological synthesis of pantolactone.

A racemate cleavage by selective hydrolysis of O-acetyl-pantolactone using lipases or esterases is described by Glänzer et al. (1988, Enzyme Microb. Technol. 10, 689-690). Such a resolution is described in the property rights

DE 40 05 150 and EP-A-0 507 278 claimed. The enantiomeric purity that can be achieved is insufficient for industrial use in this process.

Degussa describes the production of pantolactone with the enzyme oxinitrilase starting from hydroxypivalaldehyde and hydrocyanic acid via optically pure hydroxypivalaldehyde cyanohydrin (DE 41 26 580, EP-A-0 528 256, DE 41 39 987). Theoretically, 100% yield can be achieved in this reaction. The disadvantage of this reaction is the high enzyme requirement (equimolar amounts Enzyme: substrate) and the relatively low enantiomeric purity of the product (max. 82% ee).

JP 47019745 describes the synthesis of D-pantolactone using Arthrobacter, Brevibacterium, Bacillus or Corynebacterium. In the reaction, the organisms mentioned convert racemic pantolactone to D-pantolactone by metabolizing the L-pantolactone. The disadvantage of this process is that half of the starting material is metabolized and is therefore lost.

Mitsubishi Chemical Ind. And Ube Ind. Have claimed processes for the preparation of D-pantolactone from D, -pantolactone (JP 6067320, JP 62294092, JP 62294096, JP 57152895). In these methods, L-pantolactone hydrolases from the yeasts Rhodothorula, Sporidiobolus and Sterigmatomyces are described. From today's perspective (see Yamada & Shimizu, Ann.NYAcad. Sci. 672 [1992] in Enzyme Eng. XI, Clark et al., 372-386; Chimia, 47, 1993: 5-10, JP 62187426; JP 61293384; JP 61293384 ; JP 61293386; Angew. Chem. Int. Ed. Engl. 27, 1988: 622-642, Chemical Aspects of Enzyme

Biotechnology, eds. T.O. Baldwin et al. , Plenum Press, New York, 1990: 151-163) confirmed by our own work, however, the direct hydrolysis of L-pantolactone in these yeasts is doubtful. Rather, the reaction proceeds via the ketopantolactone, which is converted to the L-pantolactone by the ketopantoate oxidoreductases. In our own work, the ketopantolactone was detected as an intermediate step, which means that there is no direct hydrolysis to the L-pantolactone. The disadvantage of this process is the low pantolactone concentration, which can be implemented in the process.

This reaction via ketopantolactone and ketopantoate oxidoreductases has also been described for bacteria (e.g. Yamada & Shimizu, see above; Shimizu et al., 1988, J. Biol. Chem. 263, 12077-12084, Kataoka et al. 1992, Eur. J Biochem. 204, 799-806). Appropriate processes for the enantioselective synthesis of D-pantolactone from D, L-pantolactone via ketopantolactone and keto-pantoate have the advantage of a high yield (theoretically 100%, 90.5% achieved with Rhodococcus su), but are due to the need for cofactor (NADH or NADPH), the addition of an energy substrate (glucose), the low space-time yield and the low final concentrations (18.2-72 g / 1 D-pantolactone) are not economical. Furthermore, such a method is disadvantageous in that the two enzymes involved generally have different optics for the conversion conditions, a problem which is eliminated when using a single (hydrolytic) enzyme. The Fuji company in cooperation with the Yamada working group at Kyoto University has developed a process for the enzymatic resolution using a fungal D-pantolactone hydrolase (JP 09308-497, JP 11056356, EP-B-0 436 730, EP- B-0 504 421, 5 EP-A-0 794 251, WO 92/06182, WO 97/10341, US 5,275,949,

US 5,372,940). The enzyme can be isolated, for example, from the fungi Cylindrocarpon tonkinense, Gibberella fujikuroi and Fusarium oxysporum. D-pantolactone hydrolase is a glycosylated enzyme that consists of a 125 kDa homodimer and

10 is Ca ²⁺ -dependent (Ann. NY Acad. Sci. 1996, 799: 650-658, Enzyme Engineering). The enzyme is inhibited by Cd ²⁺ , Hg ²⁺ , Cu ²⁺ and EDTA (US 5,372,940). The purification of the D-pantolactone hydrolase was carried out by Shimizu et al. described. The purified enzyme shows specifically against a number of lactones

15 sugar lactones have hydrolase activity (Eur. J. Biochem., 209, 1992: 383-390). Its sequence shows weak homologies to gluconolactonase from Zymomonas mobilis (28.9%), human and rat paraoxonase (25.3%) and strictosidine synthase from Catharanthus roseus (15.9%; EP-A-0 794 251, Kobayashi et al.

20 1998, Proc. Natl. Acad. Be. USA, 95, 12787-12792). By Kataoko et al. describes a strong dependence of the achieved enantiomeric purity of the product on the conversion at different pH values (enzyme. Microbiol. Technol. 19: 307-310, 1996 and Appl. Microbiol. Biotechnol. 1995, 44: 333-338) , At pH values,

25 which are close to or above pH 7, lower enantiomeric purities are achieved, since the spontaneous chemical hydrolysis of L-pantolactone becomes increasingly stronger at higher pH values, thus reducing the enantiomeric purity of the product. As the optimal pH value for the production of the most enantiomerically pure D-panto-

30 lactones are given pH 5. The reaction rate of the enzyme is slowed down considerably at this pH. In order to obtain optically pure product, a subsequent crystallization is required after extraction (Yamada, H. Chimia 47, 1993: 5-10).

35

A disadvantage of the processes mentioned above is that they often only lead to products with a low optical purity and / or that they only run with low space-time yields. This leads to economically unattractive processes. Like

Before there is therefore a great need for a simple, economical biotechnological process for the production of D-pantolactone, which does not have the disadvantages mentioned above. Based on the existing chemical synthesis, this process should allow easy access to D-pantolactone in high yields and enantiomeric purities, so that no further purification of the product is required. The object was therefore to provide a simple, economical process for the preparation of D-pantolactone. This task was solved by an isolated nucleic acid sequence which codes for a polypeptide with L-pantolactone hydrolase activity, selected from the group:

a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,

b) nucleic acid sequences which, as a result of the degenerate genetic code, are derived from the nucleic acid sequence shown in SEQ ID NO: 1,

c) Derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, which code for polypeptides with the amino acid sequences shown in SEQ ID NO: 2 and have at least 50% homology at the amino acid level, without the enzymatic action of the polypeptides being significantly reduced

d) functional equivalents of the sequences mentioned under (a) to (c).

These L-pantolactone hydrolases can advantageously be found in organisms, microorganisms such as bacteria. The enzyme or the enzymes have a high enzymatic activity for the hydrolytic conversion of L-pantolactone into L-pantoic acid.

These L-pantolactone hydrolases do not convert D-pantolactone, so that the organisms, extracts or purified enzymes as well as corresponding recombinant strains or proteins can be used to produce enantiomerically pure D-pantolactone.

Derivatives of the nucleic acid sequence according to the invention with the sequence SEQ ID NO: 1 are to be understood, for example, as allele variants which have at least 50% homology at the derived amino acid level, preferably at least 60% homology, particularly preferably 70%, very particularly preferably at least 80% homology. Homology was determined using either the method of Needleman & Wunsch (J. Mol. Biol. 48, 1970: 443-453) or Smith S Waterman (Adv. Appl. Math., 2, 1981: 482-489). The homologies can advantageously be higher over partial regions of the sequences. The amino acid sequence derived from SEQ ID NO: 1 can be found in SEQ ID NO: 2. Allelic variants include in particular functional variants which can be obtained by deleting, inserting or substituting nucleotides from the sequence shown in SEQ ID NO: 1, the enzymatic Activity of the derived synthesized proteins should not be significantly reduced. Enzymes with a not significantly reduced enzymatic activity are to be understood as enzymes which have an enzymatic activity of at least 20%, preferably 50%, particularly preferably 75%, very particularly preferably 90%. The invention thus also relates to amino acid sequences which are encoded by the group of nucleic acid sequences shown above. The invention advantageously relates to amino acid sequences which are encoded by the sequence SEQ ID NO: 1.

Functional equivalents of the sequences mentioned under (a) to (c) are to be understood as nucleic acids which code for enzymes which hydrolyze L-pantolactone to the corresponding acid and which are at least 20%, preferably 50%, particularly preferably 75%, very particularly preferably Have 90% of the activity of the sequence mentioned under SEQ ID NO: 2, are not inhibited by EDTA (1 rαM solution) and are stable between pH 4 to 10. These functional equivalents also advantageously have a pH optimum between pH 7 and 8 and a temperature optimum between 70 ° C and 80 ° C.

Furthermore, derivatives are also to be understood as homologs of SEQ ID NO: 1, for example fungal or bacterial homologs, shortened sequences, single-stranded DNA or RNA of the coding and non-coding DNA sequence. Homologs of SEQ ID NO: 1 have a homology at the DNA level of at least 50%, preferably at least 60%, particularly preferably at least 70%, very particularly preferably at least 80% over the entire DNA specified in SEQ ID NO: 1 -Area.

In addition, homologs of SEQ ID NO: 1 are to be understood as derivatives such as promoter variants. The promoters which precede the specified nucleotide sequences can be changed by one or more nucleotide exchanges, by insertion (s) and / or deletion (s), but without the functionality or effectiveness of the promoters being impaired. Furthermore, the effectiveness of the promoters can be increased by changing their sequence, or completely replaced by more effective promoters, including organisms of other species.

Derivatives are also to be understood as variants whose nucleotide sequence in the range from -1 to -200 before the start codon or 0 to 1000 base pairs after the stop codon have been changed such that the gene expression and / or the protein expression is changed, preferably increased. In principle, the nucleic acid sequences according to the invention can be identified and isolated from all organisms. SEQ ID NO: 1 or its homologues can advantageously be isolated from fungi, yeasts or bacteria. Gram-negative and gram-positive bacteria are mentioned as bacteria. The nucleic acid (s) according to the invention [the plural and singular should be synonymous for the application] are preferred from gram-negative bacteria, advantageously from α-proteobacteria, β-proteobacteria or γ-proteobacteria, particularly preferably from bacteria of the Enterobacteriaceae, Pseudomonadaceae or Rhizobiaceae families , very particularly preferably isolated from bacteria of the genus Agrobacterium, Pseudomonas or Burkholderia using methods known to the person skilled in the art. The genera Beauveria or Psilocybe may be mentioned as advantageously suitable mushrooms. Advantageous yeasts can be found, for example, in the Apiotrichum genus.

SEQ ID No: 1 or its derivatives, homologs or parts of these sequences can be isolated from other fungi or bacteria, for example, using conventional hybridization methods or the PCR technique. These DNA sequences hybridize under standard conditions with the sequences according to the invention. For hybridization, it is advantageous to use short oligonucleotides of the conserved areas, for example from the active center, which can be determined by comparison with the D-pantolactone hydrolase in a manner known to the person skilled in the art (such an area is, for example, the so-called HTGT motif) used. However, longer fragments of the nucleic acids according to the invention or the complete sequences can also be used for the hybridization. These standard conditions vary depending on the nucleic acid oligonucleotide used, _Q longer fragment or complete sequence or depending on the type of nucleic acid DNA or RNA used for the hybridization. For example, the melting temperatures for DNA: DNA hybrids are approx. 10 ° C lower than those of DNA: RNA hybrids of the same length. 5

Under standard conditions, for example, depending on the nucleic acid, temperatures between 20 and 70 ° C in an aqueous buffer solution with a concentration between 0.1 to 5 x SSC (1 X SSC = 0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in the presence of 50% formamide. The hybridization conditions for DNA: DNA hybrids are advantageously 2.0 × SSC and temperatures between approximately 20 ° C. to 70 ° C., preferably between approximately 50 ° C. to 70 ° C. For DNA: RNA hybrids, the hybridization conditions are advantageously 2.0 × SSC and temperatures between approximately 20 ° C. to 60 ° C., preferably between approximately 35 ° C. to 60 ° C. These 5 specified temperatures for the hybridization are calculated melting temperature values for a nucleic acid with a length of approx. 1000 nucleotides and a G- + C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are in relevant textbooks of genetics such as Sambrook et al. , "Molecular Cloning", Cold Spring Harbor Laboratory, 1989, and can be calculated according to formulas known to the person skilled in the art, for example depending on the length of the nucleic acids, the type of hybrid or the G + C content. The person skilled in the art can obtain further information on hybridization from the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Heimes and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

Under the nucleic acid construct according to the invention are the

To understand L-pantolactone hydrolase genes with sequence SEQ ID No: 1 and its derivatives and homologues, which were advantageously functionally linked to one or more regulatory signals to increase gene expression. For example, these regulatory sequences are sequences to which inducers or repressors bind and thus regulate the expression of the nucleic acid. In addition to these new regulatory sequences, the natural regulation of these sequences may still be present before the actual structural genes and may have been genetically modified so that the natural regulation has been switched off and the expression of the genes increased. However, the nucleic acid construct can also have a simpler structure, that is to say no additional regulation signals have been inserted before the sequence SEQ ID No: 1 or its homologues and the natural promoter with its regulation has not been removed. Instead, the natural regulatory sequence is mutated so that regulation no longer takes place and gene expression is increased. The nucleic acid construct can also advantageously contain one or more so-called "enhancer sequences" functionally linked to the promoter, which enable increased expression of the nucleic acid sequence. Additional advantageous sequences, such as further regulatory elements or terminators, can also be inserted at the 3 'end of the DNA sequences. The nucleic acids according to the invention can be contained in the construct in one or more copies. The marker may also contain further markers such as antibiotic resistance or auxotrophy-complementing genes, optionally for selection on the construct.

Advantageous regulatory sequences for the method according to the invention are, for example, in promoters such as aphll (Tn5) -, tre-, cos-, tac-, trp-, lacPAI, rha, tet-, trp-tet-, lpp-, lac-, lpp- lac-, lacl ^< 3-, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, λ-P _R - or λ-Pi, -promotor contain, which are advantageously in gram-negative bacteria Find application. Further advantageous regulatory sequences are found, for example, in the gram-positive promoters such as in the constitutive or inducible Streptomyces

Promoters aphl, ermE, melC, tipA, mcrAB, gylCAB, veg, SPOl, amy and SP02, in the yeast or mushroom promoters A0X1, GAL1, ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH , In this context, the promoters of pyruvate decarboxylase and methanol oxidase from, for example, Hansenula are also advantageous. Artificial promoters for regulation can also be used.

For expression in a host organism, the nucleic acid construct is advantageously inserted into a vector such as, for example, a plasmid, a phage or other DNA, which enables optimal expression of the genes in the host. These vectors represent a further embodiment of the invention. Suitable plasmids are, for example, in E. coli pBluescript, pBAD, pQE (His-tag system), pICIC223-3, pLG338, pACYC184, pBR322, pUC18, pGEM7Z, pKK223-3, pUC19, pKC30, pRep4, pHSl, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III ¹¹³ -Bl, λgtll or pBdCI or broad-host-range plasmids such as pBBRIMCS or pRK293, in Streptomyces and other Actinomyceten pIJS304, pIJS304, pIJS301, pIJ704, pIJ703, pIJ703, or plJ361, in Bacillus pUBHO, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALSl, pIL2 or pBB116, in yeasts 2 microns, pAG-1, YE 6, YEpl3 or pEMBLYe23 or in plants pLGV23, pGHlac ^+, pBIN19, pAK2004 or pDH51. The plasmids mentioned represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018 ) can be removed.

Advantageously, the nucleic acid construct for the expression of the further genes contained additionally contains 3 'and / or 5' terminal regulatory sequences for increasing expression, which are selected for optimal expression depending on the host organism selected and gene or genes.

These regulatory sequences are intended to enable targeted expression of the genes and protein expression. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately. The regulatory sequences or factors can preferably influence the gene expression of the introduced genes positively and thereby increase. Thus, the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers". In addition, an increase in translation is also possible, for example, by improving the stability of the mRNA.

In a further embodiment of the vector, the vector containing the nucleic acid construct according to the invention or the nucleic acid according to the invention can also advantageously be introduced into the microorganisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination. This linear DNA can consist of a linearized vector such as a plasmid or only of the nucleic acid construct or the nucleic acid according to the invention.

Another object of the invention is an L-pantolactone hydrolase, characterized by the following properties:

a) conversion of L-pantolactone to the corresponding acid,

b) pH stability: L-pantolactone hydrolase is stable in a pH range from 4 to 10

c) pH optimum: 7.2 to 7.6

d) Optimum temperature: approx. 70 ° C to 75 ° C

e) no inhibition of activity by EDTA

This L-pantolactone hydrolase can be used as a free or immobilized enzyme in the process according to the invention.

For optimal expression of heterologous genes in organisms, it is advantageous to change the nucleic acid sequences according to the specific "codon usage" used in the organism. The "codon usage" can easily be determined on the basis of computer evaluations of other, known genes of the organism in question.

The expression of the genes according to the invention and of the proteins encoded by these genes in a host organism generally involves stress for these organisms. By simultaneously expressing these genes in the presence of at least one gene that encoded for a so-called stress protein or in the presence of a combination of these genes, the nucleic acids according to the invention can advantageously be expressed in the host organisms according to the invention. Stress proteins, also heat shock proteins 5 (= HSP) or molecular chaperones (English chaperone or

Gouvernante) are among the best preserved proteins in evolution in both pro- and eukaryotes and can be found universally in all organisms. They are divided according to the molecular weight in kilodaltons, e.g. HSP60, 70, 10 90 etc. These stress proteins owe their name to their ability to be induced by stress conditions such as low glucose levels, heat shock, alcohol, UV light, oxidative reagents etc.

15 Many stress proteins and constitutively formed related proteins are essential for the correct folding, assembly, stabilization and transport of proteins. By coexpression of the proteins according to the invention in the presence of at least one stress protein the nucleic acids according to the invention can

20 are advantageously expressed. A possible protein aggregation of the hydrolases can thus advantageously be prevented. The stress proteins bind to hydrophobic parts of the proteins and thus prevent incorrect folding of the proteins or enable correct folding. Already aggregated or dena-

25 turiated proteins are dissociated again and folded properly. These stress proteins often occur in the exercise of their function in cooperation with other proteins, so-called helper proteins (= cohort proteins). One therefore speaks of so-called chaperone machines, which have the advantageous effect in

30 have expression of the genes according to the invention (Frydaman et al., Nature, 370, 1994: 111-117). These chaperone machines can work with ATP consumption (= "main chaperone machines") or without ATP consumption (= "junior chaperones"). Advantageous chaperones or heat shock proteins are, for example

35 the eukaryotic genes HSP17, 5, HSP22, HSP 25, HSP27, HSP60, HSP70, HSP90, TRiC, UBI1,2,3,4 or their prokaryotic homologues such as HtpG, DnaK, DnaJ, GroES, GroEL, HtrC, ClpB, GrpE etc. Preferred chaperones are GroES, GroEL, HtpG, DnaK, DnaJ, HSP70 or HΞP27.

40

The nucleic acids according to the invention are advantageously expressed in the presence of at least one stress protein; the genes can be under the joint control of a promoter or can be read by separate promoters. Accordingly, their

45 Expression can be induced by adding one or more inductor substances simultaneously or at separate times. The nucleic acids can be on one vector or on separate ones Vectors. It is also possible to modify the host organism's stress proteins by genetic manipulation in such a way that they are overexpressed.

5 Also alternative methods for increasing the native enzyme portion such as growing the microorganisms that synthesize the protein according to the invention at low temperatures or renaturing by applying high pressures (advantageously 1 to 2 kbar) on suspensions of the protein according to the invention (with or 0 without the addition of denaturing agents Agents, for example guanidine hydrochloride) can be advantageous.

In principle, 5 all prokaryotic or eukaryotic organisms are suitable as recombinant host organisms for the nucleic acid according to the invention or the nucleic acid construct. Microorganisms such as bacteria, fungi or yeasts are advantageously used as host organisms. Gram-positive or gram-negative bacteria are advantageous, preferably bacteria from the Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaeeae or Noeardiaeeae families, yeasts such as Piehia, Saccharomyces or Hansenula or fungi such as Beauveria or Psilocybe, particularly preferably bacteria of the genus Escherichia monas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium or Rhodococcus are used. The genus and type Escherichia coli is very particularly preferred. Further advantageous bacteria can also be found in the group of oc-proteobacteria, ß-proteobacteria or γ-proteobacteria.

The host organism or organisms according to the invention

30 preferably contain at least one of the nucleic acid sequences, nucleic acid constructs or vectors described in this invention, which code for L-pantolactone hydrolases.

₅ Depending on the host organism, the organisms used in the process according to the invention are grown or cultivated in a manner known to those skilled in the art. Microorganisms are usually in a ^• liquid medium, which is a carbon source mostly in the form of sugars, a nitrogen source mostly in the form of organic

, _Q Contains nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese, magnesium salts and optionally vitamins at temperatures between 0 ° C and 100 ° C, preferably between 10 ° C and 60 ° C with gassing with oxygen. The pH of the nutrient liquid can be fixed

₄₅ value must be maintained, that is, regulated during cultivation or not. The cultivation can be batch-wise, semi-batch wise or continuous. Nutrients can start at Bfeeg Fermentation submitted or fed semi-continuously or continuously. The same applies to inducers such as isopropythiogalactoside, (IPTG), lactose, arabinose, rhamnose and antibiotics or temperature shifts, which switch on the expression of the gene according to the invention, depending on the promoter used. The racemic pantolactone can be added directly for cultivation or advantageously after cultivation. The enzymes can be isolated from the organisms by the method described in the examples or used as a crude extract for the reaction.

The host organisms advantageously contain 0.5 U / g BTM (= dry biomass) L-pantolactone hydrolase activity, preferably 4 U / g BTM, particularly preferably 20 to 150 U / g BTM, very particularly preferably 40 to 600 U / g BTM.

The process according to the invention is advantageously carried out at a temperature between 0 ° C. to 95 ° C., preferably between 10 ° C. to 85 ° C., particularly preferably between 15 ° C. to 75 ° C.

The pH in the process of the invention is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, particularly preferably between pH 6 and 8, very particularly preferably between pH 6, 5 and ¹ 7 is held; 5.

Racemic pantolactone in the process according to the invention is understood to mean pantolactone which consists of a 50:50 mixture of the two enantiomers or of any other mixture with an enrichment of one of the two enantiomers in the mixture.

In the process according to the invention, enantiomerically pure or chiral pantolactone (D or L enantiomer) is to be understood as enantiomers which show an enantiomer enrichment. Enantiomeric purities of at least 70% ee, preferably of min. 80% ee, particularly preferably of min. 90% ee, very particularly preferably min. 98% ee achieved.

Growing cells containing the nucleic acids, nucleic acid constructs or vectors according to the invention can be used for the method according to the invention. Dormant or disrupted cells can also be used. Disrupted cells are understood to mean, for example, cells which have been made permeable by treatment with, for example, solvents, or cells which have been broken up by means of enzyme treatment, by mechanical treatment (for example French press or ultrasound) or by some other method. The crude extracts obtained in this way are advantageous for the process according to the invention. suitable. Purified or purified enzymes can also be used for the process. Immobilized microorganisms or enzymes, which can advantageously be used in the reaction, are also suitable.

If free organisms or enzymes are used for the process according to the invention, these are expediently separated off before the extraction, for example by filtration or centrifugation. This is advantageously not necessary when using immobilized organisms or enzymes, but can also be done.

The D-pantolactone produced in the process according to the invention can advantageously be obtained from the aqueous reaction solution by extraction or crystallization or advantageously by extraction and crystallization. For this purpose, the aqueous reaction solution is extracted with an organic solvent. The extraction can be repeated several times to increase the yield. The solution is advantageously cooled to about 0 ° C. to 10 ° C. before extraction. Before cooling or afterwards, the aqueous solution is advantageously neutralized to about pH 6.0 to pH 7.0 in order to convert the free acid into the salt, so that it cannot be extracted under the reaction conditions. For the neutralization, for example, bicarbonate or another base such as NaOH or KOH is used. In principle, all solvents can be used as organic solvents which show a phase boundary with water, if appropriate after addition of salts, and into which the lactone can pass from the aqueous phase. Advantageous solvents are solvents which absorb only a little water, so that only a little acid passes into the solvent, such as toluene, methylene chloride, butyl acetate, diisopropyl ether, benzene, methyl tert-butyl ether, methyl isobutyl ketone, diethyl ketone or ethyl acetate.

After concentrating the organic phase, the products can usually be obtained in good chemical purities, i.e. greater than 90% chemical purity. After extraction, the organic phase with the product can also be only partially concentrated and the product crystallized out. For this purpose, the solution is advantageously cooled to a temperature of 0 ° C to 10 ° C. Crystallization can also take place directly from the organic solution or from an aqueous solution. The product which has crystallized out can be taken up again in the same or in a different solvent for recrystallization and can be crystallized again. The subsequent advantageous crystallization at least once can further increase the enantiomeric purity of the product if necessary. However, the resulting D-pantolactone can advantageously be used directly as an organic solution without crystallization.

The L-enantiomer remaining in aqueous solution can be closed to form the lactone by acidic acids, for example with sulfuric acid, and then extracted as described above. The solution is advantageously heated for lactonization. After the solvent has been stripped off in the melt, the L-lactone obtained can be racemized and recycled with catalytic amounts (about 1 to 5% mol%) of a base such as NaOH, Na pantoate or Na methylate. Due to the advantageous racemization and recycling of the undesired enantiomer, a theoretical yield of 98% can be achieved in the process according to the invention.

With the types of workup mentioned, the product of the process according to the invention can be isolated in yields of from 60 to 100%, preferably from 80 to 100%, particularly preferably from 90 to 100%, based on the racemic pantolactone used for the reaction. The isolated product is characterized by a high chemical purity of> 90%, preferably> 95%, particularly preferably> 98%. Furthermore, the products have a high enantiomeric purity, which can advantageously be further increased by crystallization if necessary.

The process according to the invention can be operated batchwise, semi-batchwise or continuously.

The products obtained in this way are suitable as starting materials for the synthesis of panthenol, pantethein and their derivatives. These substances and the enantiomerically pure pantolactone obtained can be used in combination with one another or alone for the production of pharmaceuticals, foods, animal feed or cosmetics.

The following examples illustrate the invention.

Examples:

1. L-pantolactone hydrolysis with Burkholderia caryophylli Lu681

Burkholderia caryophylli Lu681 (or other strains shown in Table la, lb) were grown for 1 to 3 days in 25 ml of complex medium (e.g. HFP = 1% peptone, 1% tryptone, 0.5% yeast extract, 0.3% NaCl), harvested, washed in buffer and resuspended (5 ml 50 mM Tris / HCl pH 7.0) and incubated for 3 h at 30 ° C. with 50 mM D, L-pantolactone. After the cells had been separated off, the concentrations of D, L-pantolactone, D, L-, D- and L-pantoic acid were determined by GC or HPLC analysis (Tab. La). Alternative was the reaction was carried out overnight under titration with 4 M NaOH (4 ml cell suspension, 50 mM D, L-pantolactone, 50 mM Tris / HCl pH 7.0 ad 20 ml distilled aqua; Tab. lb). All strains in Table 1 hydrolyzed the racemic pantolactone to L-pantoic acid. The ee value at high conversion (> 45%) and thus the enantioselectivity E of the enzyme was determined according to Straathof and Jongejan, Enzyme & Microbiol. Technology 21: 559-571, 1997.

Verification of hydrolytic activity

Burkholderia caryophylli Lu681 (or other strains from Table 1) were grown for 1 to 3 days in 25 ml of complex medium (eg HFP = 1% peptone, 1% tryptone, 0.5% yeast extract, 0.3% NaCl), harvested, in Tris-HCl buffer (50 mM, pH 7.0) washed and resuspended (5 ml 50 mM Tris / HCl pH 7.0) and incubated for 3 h at 30 ° C. with 50 mM ketopantolactone. After the cells had been separated off, the concentrations of ketopantolactone, ketopantoic acid, D, L-pantolactone, D, L-, D- and L-pantoic acid were determined by HPLC analysis (Tab. La). All strains listed except Beauveria amorpha Lu7953 were able to reduce the ketopantoic acid formed from ketopantolactone by spontaneous hydrolysis to pantoic acid. However, since D-pantoic acid was formed in all strains instead of the L-pantoic acid described in Example 1, the conversion of pantolactone to L-pantoic acid cannot result from an oxidation reduction process (via ketopantolactone and ketopantoic acid). No dependency of L-pantolactone hydrolysis on cofactors was found in the dialyzed crude extract of Lu681 or Lu5351 and when using the purified enzymes from Lu681 and Lu5351. The enzymatic activity can thus be attributed to a hydrolytic enzyme.

3. Production of D-pantolactone by hydrolysis with various wild-type strains

a. Burkholderia caryophylli Lu681

Burkholderia caryophylli Lu681 was grown in 200 ml complex medium (e.g. GYP = 1% D-glucose, 0.5% polypeptone, 0.5% yeast extract) (OD600 = 6.7, BTM = 2.97 g / 1), harvested and washed. 10 ml of the 10-fold concentrated suspension were incubated with 50 mM D, L-pantolactone in 50 mM Tris / HCl pH 7.0 (batch volume 20 ml) at 30 ° C. with titration with 4 M NaOH to pH 7.0. After 3 h and 19.5 h, the conversion c and the ee value were determined by HPLC analysis (3 h: c = 45%, ee = 95%; 19.5 h: c = 59%, ee = 89% L-pantoic acid). The latter corresponds to D-pantolactone with ee values of 73% and 100%. b. Agrobacterium radiobacter Lu5351

Agrobacterium radiobacter Lu5351 was grown in 200 ml complex medium (e.g. HFP = 1% peptone, 1% trypton, 0.5% yeast extract, 0.3% NaCl) (OD600 = 11.5, BTM = 2.90 g / 1), harvested and washed. 10 ml of the 10-fold concentrated suspension were incubated with 50 mM D, -pantolactone in 50 mM Tris / HCl pH 7.0 (batch volume 20 ml) at 30 ° C. with titration with 4 M NaOH to pH 7.0. After 3 h and 19.4 h, the conversion c and the ee value were determined by HPLC analysis (3 h: c = 20%, ee = 93%; 19.4 h: c = 53%, ee = 94% L-pantoic acid). The latter corresponds to D-pantolactone with ee values of 21% and 100%.

c. Pseudomonas diminuta Lu683

Pseudomonas dimunuta Lu683 was grown in 200 ml complex medium (eg GYP = 1% D-glucose, 0.5% polypeptone, 0.5% yeast extract) (OD600 = 7.3, BTM = 3.78 g / 1), harvested and washed. 10 ml of the 10-fold concentrated suspension were incubated with 50 mM D, L-pantolactone in 50 mM Tris / HCl pH 7.0 (batch volume 20 ml) at 30 ° C. with titration with 4 M NaOH to pH 7.0. After 3 h and 19.3 h, the conversion c and the ee value were determined by HPLC analysis (3 h: c = 48%, ee = 97%; 19.4 h: c = 69%, ee = 79% L-pantoic acid). The latter corresponds to D-pantolactone with ee values of 82% and 100%.

d. Apiotrichum humicola Lu3215

Apiotrichum humicola Lu3215 was grown in 200 ml complex medium (e.g. HFP = 1% peptone, 1% trypton, 0.5% yeast extract, 0.3% NaCl) (OD600 = 18.5, BTM = 7.34 g / 1), harvested and washed.

10 ml of the 10-fold concentrated suspension were mixed with 50 mM D, L-

Pantolactone in 50 mM Tris / HCl pH 7.0 (batch volume 20 ml)

Incubated at 30 ° C with titration with 4 M NaOH to pH 7.0. After 3 h and 19.4 h, the conversion c and the ee value were determined by HPLC analysis (3 h: c = 55%, ee = 79% with respect to L-pantoic acid).

The latter corresponds to D-pantolactone with ee values of 84%.

4. Isolation of L-pantolactone hydrolase from Burkholderia caryophylli Lu681

Burkholderia caryophylli Lu681 was grown in 14 l of complex medium (HFP = 1% peptone, 1% tryptone, 0.5% yeast extract, 0.3% NaCl) to OD600 = 10 (3 g / 1 BTM), harvested, digested and the L -Pantolactone hydrolase (approx. 200 U) purified from the crude extract. For this purpose, the cells (1128 g wet weight) were first treated in the buffer with an Ultra-Turrax rod (1.8 1, 20 mM Tris / HCl, pH 7.4) resuspended. Final volume 3 1. This solution was then freed from coarse particles on a glass suction filter through a bed of glass balls (0.1 to 0.2 mm, 200 ml). This cell suspension (3.2 l) was homogenized twice at 1500 bar in a Z04 microfluidizer. The device was rinsed with 500 ml of buffer. The combined volumes (4 l) were subjected to a first precipitation with 200 ml of an IM manganese chloride solution (final concentration 50 mM). The pH was kept at pH 7.0 by adding sodium hydroxide solution. The precipitate was centrifuged off at 6000 rpm for 30 minutes. 200 ml of a 0.2 M EDTA solution (pH 7.5) were added to the supernatant (3.1 l). With the addition, the pH dropped to 5.0. A precipitate formed which was again centrifuged off at 6000 rpm (20 minutes, Sorvall). The supernatant (3.4 l) was titrated back to pH 7.0.

Then 989 g of ammonium sulfate (corresponding to 50% saturation) were added and the mixture was stirred for 10 minutes. The turbidity was centrifuged off at 6000 rpm for 20 minutes. The supernatant obtained (3.7 l) was divided: 1.2 l were used in a phenylsepharose chromatography.

The phenyl-Sepharose column (Pharmacia, diameter 5 cm, height 25 cm, volume 490 ml) was washed with 1 1 buffer A (20 M sodium phosphate buffer, pH 7.4, 40% ammonium sulfate) and in the gradient with buffer B ( 20 mM sodium phosphate buffer, pH 7.4). At a flow of 10 ml / min, 100% buffer B was reached after 120 minutes and held for 40 minutes. Active fractions were collected and pooled (250 ml).

After dilution to <7 mS / cm, these 3 1 were chromatographed on Q-Sepharose (diameter 5 cm, height 25 cm, 430 ml,

Fast Flow, Pharmacia). The column was washed with 1 1 buffer A (20 mM sodium phosphate buffer pH 7.4 (10 ml / min). The gradient with buffer B (buffer A with 1 M NaCl) was brought to 100% B linearly in 120 minutes and for further The active fractions were collected and pooled (118 ml), this volume was concentrated (10 kD omega membrane) and dialyzed against 5 1 10 mM Tris / HCl pH 7.0, final volume 21 ml. 6 ml of this volume was applied to a Waters Q HR8, the column was previously equilibrated with buffer A (20 mM Mes, pH 6.0) and then developed with a gradient (1% per minute) after buffer B (like buffer A with 0.5 M NaCl) Active fractions were combined (3.7 ml) and dialyzed twice against 2 1 10 mM Tris / HCl pH 7.0, the dialysate became cloudy and was therefore centrifuged off (4 ml). This material was then separated by chromatography on Mono P (Pharmacia, diameter 0.5 cm, volume 5 ml). The Mono-P fractions were concentrated in 0.2 ml portions by an acetone precipitation at -20 degrees Celsius.

The pellets were then taken up in 0.005 ml of SDS sample buffer without DTT and placed on an SDS gel (Tris / Glycine Gel 12%, Novex, approx. 2.5 h 125 V, 50 mA, Laemmli, UK, 1970, Nature , 227: 680-685). After separation, the L-pantolactone hydrolase was identified by activity staining and cut out. For this purpose, the gel was briefly swirled into TBS buffer (= 50 mM Tris, 100 mM NaCl, pH 7.4) and then with 50 ml TBS + 50 ml α-naphthylacetate solution (Sigma N-8505, 0.4 g / 1 in 10% acetone) 10 min. pre-incubated. 50 ml of Fast Red TR solution (Sigma F-8764, 1 g / 1) were then added and the gel was further pivoted at RT (= approx. 23 ° C.). The L-pantolactone hydrolase was recognizable as a red-brown colored band with an apparent molecular weight of approx. 36 kDa. The protein in the cut pieces of gel was digested by trypsin and the peptides sequenced. The remaining gel was stained with Coomassie Blue. Two peptide sequences (SEQ ID NO: 3 and 4) were obtained.

5. Isolation of L-pantolactone hydrolase from Agrobacterium radiobacter Lu5351

Agrobacterium radiobacter Lu5351 was grown in 14 1 complex medium (e.g. HFP = 1% peptone, 1% trypton, 0.5% yeast extract, 0.3% NaCl) to OD600 = 10 (3 g / 1 BTM), harvested, digested and the L-pantolactone hydrolase (approx. 60 U) purified from the crude extract (Tab. 2). For this purpose, the cells (400 g wet mass) of Agrobacterium radiobacter (Lu 5351) were first resuspended by treatment with an Ultra-Turrax rod in the buffer (1.8 1, 20 mM Tris / HCl, pH 7.4) (final volume 2, 2 1). This solution was then freed from coarse particles on a glass suction filter through a bed of glass balls (0.1 to 0.2 mm, 200 ml). This cell suspension was homogenized twice at 1500 bar in the Z04 microfluidizer. The device was rinsed with 500 ml of buffer. The combined volumes (2.7 l) were subjected to a first precipitation with 135 ml of a 1 M manganese chloride solution (final concentration 50 mM). The pH was kept at pH 7.0 by adding sodium hydroxide solution and the precipitate was centrifuged off at 6000 rpm for 30 min. The supernatant (2.6 l) was mixed with 575 ml of a 0.2 M EDTA solution and the pH checked again. 711 g of ammonium sulfate (corresponding to 40% saturation) were added to these 3.15 liters and the mixture was stirred for 10 minutes. The turbidity was centrifuged off at 6000 rpm for 30 min. The supernatant obtained (3.3 l) was used in a phenylsepharose chromatography.

The phenyl-Sepharose column (Pharmacia, diameter 5 cm, height 25 cm, volume 490 ml) was washed with 1 1 buffer A (20 mM sodium phosphate buffer, pH 7.4, 40% ammonium sulfate) and in the gradient with buffer B ( 20 mM sodium phosphate buffer, pH 7.4). At a flow of 10 ml / min, 100% B was reached after 120 min and held for 40 min. Active fractions were collected and pooled (350 ml, 20.9 mS).

After dilution to 7 mS / cm (3.1 1 final volume), chromatography was carried out on Q-Sepharose (diameter 5 cm, height 25 cm, 430 ml, Fast Flow, Pharmacia). The column was washed with 1 1 buffer A (20 mM sodium phosphate buffer pH 7.4 (10 ml / min). The gradient with buffer B (buffer A with 1 M NaCl) was brought to 100% B in 120 minutes and for a further 40 The active fractions were collected and pooled (134 ml), this volume was concentrated (10 kD omega membrane) and dialyzed against 3 1 10 mM Tris / HCl pH 7.0 (final volume 19 ml). 6 ml of this volume was applied to a Waters Q HR8, the column had previously been equilibrated with buffer A (20 mM Mes, pH 6.0) and developed with a gradient (1% per minute) after buffer B (like buffer A with 0.5 M NaCl) Active fractions were combined (12.5 ml) and dialyzed against 5 1 10 mM sodium acetate buffer pH 5.0, the dialysate became cloudy and was therefore centrifuged off.

The supernatant was then separated by chromatography on Mono P (Phar acia, diameter 0.5 cm, volume 5 ml).

The Mono-P fractions were concentrated in 0.2 ml portions by acetone precipitation at -20 degrees Celsius. The pellets were then taken up in 0.005 ml SDS sample buffer without DTT and placed on an SDS gel. After separation, the L-pantolactone hydrolase was identified by an activity staining (see Example 4) and cut out. It was recognizable as a red-brown colored band with an apparent molecular weight of 36 kDa. The protein in the cut gel pieces was digested by trypsin and the peptides were sequenced. The remaining gel was stained with Coomassie Blue. Two peptide sequences (SEQ ID NO: 5 and 6) were obtained. Sequencing of SEQ ID NO: 5 revealed ambiguity for the first amino acid in the sequence. The tyrosine shown in position 1 can also be a leucine, the sequence was not clear here. 6. Substrate specificity of the purified L-pantolactone hydrolases from Lu681 and Lu5351

0.1 U / ml of a phenylsepharose peak value fraction of the purified 5 enzymes from Lu681 or Lu5351 were incubated in 150 mM pipes pH 6.8 with various esters and lactones. Samples were taken after 0, 1 and 20 h, the reaction was stopped by centrifugation through a 10 kDa filter membrane and the concentration of the substrate and the corresponding acid were determined by HPLC analysis 0. The activity is shown in Tables 3a and 3b in comparison to the activity with L-pantolactone.

For the lipase substrate 1, 2-0-dilauryl-rac-glycero-3-glutaric acid resorufine ester, an optical test was carried out for the Lu681 enzyme in the microtiter plate (Boehringer Mannheim, modified). 60 to 482 U / 1 enzyme were incubated in 45 mM KH ₂ PO pH 6.8 with 0.18 g / 1 resorufine ester (2 g / 1 in dioxane + 2% SDS + 10% H 0) at room temperature. The absorbance E was measured after 2 min and 82 min at 572 nm. Table 3c shows the extinction difference and the lipase activity calculated therefrom, which is approximately 0.05% of the L-pantolactone hydrolase activity.

7. Inhibition and activation of the purified L-pantolactone hydrolases from Lu681 and Lu5351

25

0.1 U / ml of a phenylsepharose peak value fraction of the purified proteins from Lu681 or Lu5351 were preincubated for 5 min with various effector substances in 150 mM pipes (pH 7.0). The assay was started by adding 150 mM L-pantolactone (1 h

30 30 ° C) and stopped by centrifugation through a 10 kDa filter membrane. The concentrations of D, L-, D-, and L-pantoic acid were then determined by HPLC analysis. Tables 4a and 4b show the activity in comparison to the sample without the addition of an effector substance. Overall, the purified enzymes

35 from Lu681 and Lu5351 insensitive (> 85% residual activity) to chelating substances, SH reagents, protease inhibitors, detergents (exception: Lu5351 with 74% residual activity in 1% SDS) and various cations.

40 Significant activation (+ 20%) can only be determined with HgCl ₂ (133/170%). Furthermore, competitive inhibition by D-pantolactone was detected for the recombinant 681-lactonase (E. coli cells, see example 8ff.).

45 8. Genbank + screening: cloning of L-pantolactone hydrolase from Burkholderia caryophylli Lu681

Genomic DNA from Burkholderia caryophylli Lu681 was isolated (Qiagen, Hilden), digested with EcoRI and ligated into pBluescriptKS + vector cut and dephosphorylated with EcoRI (Maniatis, T., Molecular Cloning: A laboratory manual, 1989). The ligation mixture was transformed into E. coli XLlBlue according to the instructions from Stratagene (La Jolla, Calif.). The

10 transformants were plated on LB plates with ampicillin (100 μg / ml), IPTG (= isopropyl-β-thiogalactoside, 0.2 mM) and X-Gal (80 mg / 1) and overnight at 30 or 37 ° C. incubated. The white colonies were picked on LB plates with ampicillin (100 μg / ml), IPTG (0.2 mM) and X-Gal (80 mg / 1) and again

Incubated for 15 nights. A copy of this master plate was then placed on LB-ampicillin (100 mg / ml) IPTG plates by filter contact with sterile nitrocellulose filters. After overnight incubation, the filter was subjected to an activity test on this plate (see above) with 150 mM L-pantolactone, 0.1% nitrite yellow and

20 subjected to 10 mM Tris / HCL pH 7.0 (3 h-overnight 30 ° C). A yellow colored clone (XLlBlue pKS + 681) was isolated.

9. Restriction mapping and sequencing of the EcoRI insert from E. coli XLlBlue pKS + 681

25

The plasmid DNA was isolated from E. coli XLlBlue pKS + 681 in accordance with the instructions from Qiagen (Hilden) and cut individually or in a double digest with the restriction enzymes EcoRI, BamHI, PstI and HindIII. The fragmented DNA was cut through

30 agarose gel electrophoresis analyzed in 0.8% agarose gel. The restriction map of the 7.5 kB insert shown in FIG. 3 results from the fragment sizes obtained. It has been completely sequenced (Sanger et al. 1977) and contains, among other things. the nucleotide sequence ID NO: 1. The deduced amino acid sequence

35 (SEQ ID NO: 2) in turn contains the peptides YGIEGLNNLEAL and AKEDANSTIEAED (SEQ ID NO: 3 and 4), which were found after tryptic digestion of the purified and blotted L-pantolactone hydrolase from Burkholderia caryophylli Lu681 and Agrobacterium radiobacter Lu5351 (see Examples 4 and 5).

40

Database comparisons (Genbank, EMBL, ΞwissProt status 7.5.99, [Sptrembel] and 5.1.99 [PIR]) of the nucleotide and the derived amino acid sequence showed only a slight homology to a group of hypothetical proteins and to certain ones

45 tetracycline cyclases from Streptomycetes (Tab. 5). In particular, the motif with the HTGTHVDAP consensus sequence is highly conserved in all proteins. In addition, a homology of 48 S (38% identical amino acids) found for isatin hydrolase from Pseudomonas putida WW2 (WO 94/09175), which also contains the sequence motif mentioned. Since no homology to other lactonases, esterases or lipases was found, the 5 L-pantolactone hydrolases found are a new class of enzymes. The sequence comparisons suggest a distantly related phylogenetic family, which also includes the named hypothetical proteins and tetracycline cyclases and isatin hydrolase. 10

10. L-pantolactone hydrolysis by E. coli XLlBlue pKS + 681

A full inoculation loop E. coli XLlBlue pKS + 681 was grown on an LB plate with ampicillin after growth (overnight stay = 37 ° C.)

15 (100 μg / ml), IPTG (0.2 mM) and X-Gal (80 mg / 1) resuspended in 0.5 ml Tris-HCl pH 7.0 and 50 mM D, L-pantolactone (OD600 = 2 , 5). A corresponding E. coli XLlBlue pBlue-scriptKS + sample (OD600 = 2.5) was used as a comparison. After 1 h the cells were centrifuged and D, L-pantolactone, D, L-, D- and L-pantoic acid

20 determined by HPLC analysis. Tab. 6a shows activities and ee values of the different samples. The suspension had an activity of> 90 U / L. However, no significant activity was evident in liquid culture batches (cf. Example 12).

25 11. Expression cloning of L-pantolactone hydrolase in E. coli XLlBlue pKK223-3

Using the nucleotide sequence SEQ ID NO: 1, the oligonucleotides 5 ^V - CCGGAATTCATGTGCAACAACTGC (Pl) and 5'- CCCAAGCTTCA-

30 GACCAGGGCCAGAA (P2) derived as a primer for a PCR amplification of the L-pantolactone hydrolase gene under the following conditions: 20 mM Tris / HCl pH 8, 8, 2 mM MgSO ₄ , 10 mM KC1, 10 mM ( NH ₄ ) ₂ S0 ₄ , 0.1% Triton X-100, 0.1 mg / ml BSA, 25 mM per dNTP, 0.96 μg / ml pKS + 681, 2.2 μg / ml Pl and P2, 25U / ml Pfu polymerase

35 (Stratagene, LaJolla, Calif.); The PCR parameters were as follows: 1 min 95 ° C, 1 min 55 ° C, 2.5 min 72 ° C, 30 cycles. The PCR product obtained (0.8 kB) was cut with EcoRI and HindIII and ligated into pKK223-3 (Pharmacia, Freiburg) cut and dephosphorylated with EcoRI and HindIII. The ligation mix

40 was transformed into E. coli XL1 Blue or TG1 (Stratagene, LaJolla, Calif .; DSMZ, Braunschweig, DSMZ No. 6056, Inoue et al, 1990, Gene 96: 23-28). The transformants were plated on LB-Amp plates and incubated overnight. Analogous to example 8, a filter copy and a were made from this transformation plate

45 subsequent activity tests were carried out, in which approx. 100 intensely yellow clones were identified. Ten were prepared by mini-preparation and restriction digestion (EcoRI-HindIII, EcoRI- HindIII-BamHI) of the plasmid DNA was analyzed (Maniatis, T., Molecular Cloning: A labaratory manual, 1989). They contained the plasmid pKK681 shown in FIG. 4.

5 12. L-pantolactone hydrolysis by E. coli XLlBlue pKK681

E. coli XLlBlue pKK681 was grown in 30 ml LB medium with ampicillin (100 μg / ml) and IPTG (0.5 mM) overnight at 37 ° C., harvested and in Tris-HCl buffer (50 mM, pH 7 , 0) washed 0 and resuspended (3 ml 50 mM Tris / HCl pH 7.0). 0.25 ml of

Suspension was treated with 150 mM L-pantolactone, 150 mM pipes pH 7, 0 ad 0.5 ml A. dest. added and incubated at 30 ° C for 3 h. In addition, 0.25 ml of the suspension with 50 mM D, L-pantolactone, 50 mM Tris pH 7.0 ad 0.5 ml A. dest. added and incubated for 3 h. 5 Furthermore, 2 ml of the suspension with 300 mM D, -pantolactone, 50 mM Tris pH 6.8 ad 20 ml of aqua dest. added and incubated with titration with 4 M NaOH to pH 6.8 for 3 h. After 1 h and after 3 h, samples were taken, the cells were separated and the supernatant was examined for D, pantolactone, D, L, D and L-pantoic acid 0. Tab. 6b shows activities and ee values of the different samples. The overnight culture (concentrated lx) thus has an activity of 90 to 150 U / 1.

When E. coli XLlBlue pKK681 is induced in the early 25 exponential growth phase (OD600 = 0.6, + 0.5 mM IPTG), the corresponding cells have after 5 h incubation at 37 ° C in the late exponential phase (OD600 = 4, 1) an activity of approx. 480 U / 1.

30 13. Expression cloning of L-pantolactone hydrolase in E. coli TG1 pDHE19

Using the nucleotide sequence SEQ ID No. 1, the oligonucleotides 5 -CAGGATGCCATATGTGCAACAACTGC (Pl) and 5 ^v -CCCAAGCTTCA-

35 GACCAGGGCCAGAA (P2) derived as a primer for a PCR amplification of the L-pantolactone hydrolase gene under the following conditions: 20 mM Tris-HCl pH 8, 8, 2 mM MgSO, 10 mM KC1, 10 mM (NH ₄ ) ₂ S0, 0.1% Triton X-100, 0.1 mg / ml BSA, 25 mM per dNTP, 0.96 μg / ml pKS + 681, 2.2 μg / ml Pl and P2, 25U / ml Pfu polymerase

40 (Stratagene, LaJolla, Calif.); The PCR parameters were as follows: 1 min 95 ° C, 1 min 55 ° C, 2.5 min 72 ° C, 30 cycles. The PCR product obtained (0.8 kB) was cut with Ndel and Hindlll and ligated into pDHEl9 (Prof. Mattes, Stuttgart) cut and dephosphorylated with Ndel and Hindlll. The ligation mix

45 was transformed into E. coli XLl Blue or TG1 (Stratagene, LaJolla, Calif .; DSMZ, Braunschweig, DSMZ No. 6056, Inoue et al, 1990, Gene 96: 23-28). The transformants were on LB-Ampi- cillm plates plated and incubated overnight. From this transformation plate, a filter clap on LB ampicillm (100 μg / ml) rhamnose (2 g / 1) plates (LB = Luria Broth) and a subsequent activity test were carried out, in which approx. 100 intense yellow clones were identified. Ten were analyzed by mini-preparation and restriction digestion (Ndel-Hmdlll, NdeI-HindiII-BamHI) and sequence analysis of the plasmid DNA (Maniatis, T., Molecular Cloning: A labaratory manual, 1989). They contained the plasmid pDHE681 shown in FIG. 5.

14. L-pantolactone hydrolysis by E. coli TG1 pDHE681

E. coli TG1 pDHE681 was grown in 14 l minimal medium with 40 g / l glycine and 2.5 g / l rhamnose for 6 to 7 h at 37 ° C., harvested and washed with Tris / HCl buffer (50 mM, pH 7.0) and ad 1.4 1 buffer resuspended. The activity of the single-concentrated cell suspension in the standard assay (150 mM pipes pH 7.0, 150 mM L-pantolactone, 1 h 30 ° C.) was 680 to 2700 U / 1 or 60 to 160 g / BTM.

10 ml of the 10-fold concentrated suspension were incubated with 50 mM D, L-pantolactone in 50 mM Tris / HCl pH 7.0 (batch volume 20 ml) at 30 ° C. with titration with 4 M NaOH to pH 7.0. After 0.4 h and 3 h, the conversion and the ee value were determined by HPLC analysis (0.4 h: c = 48%, ee = 92%; 3 h: c = 58%, ee = 72% with respect to L-pantomic acid). This corresponds to D-pantolactone with ee values of 84% and 100%.

15 Production of D-pantolactone by hydrolysis with E. coli TG1 pDHE681

3 g of D, L-pantolactone were dissolved in 10 ml of H ₂ O and titrated to pH 6.5 with 4 M NaOH. After adding 5 to 10 ml of cell suspension E. coli TG1 pDHE681 (Example 14) and filling up to 20 ml with H ₂ 0, the reaction was incubated at 30 ° C. with titration to pH 6.8 for 15 to 22 h. A further 0 to 2 ml of cell suspension were optionally added and the mixture was incubated for a further 3 h or 3 × 5 ml of cell suspension were added and the mixture was incubated for a further 90 h. Figure 6 shows the course based on the NaOH consumption. Depending on turnover and incubation time, ee values for D-pantolactone of 71 to 97% were achieved. / The cells were then centrifuged off from the 22 ml batches and washed with 5 ml of 50 mM Tris-HCl pH 7.0, and the supernatants were combined (20 to 25 ml) and extracted 3 times with a volume of ethyl acetate. The organic phase was dried for 1 h at room temperature after addition of 10 g Na S0 (anhydrous). The precipitate was filtered off, washed once with ethyl acetate and the filtrate was spun in at 40 ° C. for 3 h. The viscous residue was weighed and analyzed by HPLC, GC, GC-MS and H-NMR (Tab. 7). It contained pure D-pantolactone (ee 71 to 87% after 50 to 52% conversion).

5 The aqueous phase (21 ml; Na-L-Pantoat) was adjusted to pH 1 with about 5 ml of 3 MH ₂ S0 ₄ , heated at 80 ° C. for 15 minutes and mixed with 8 g of anhydrous Na ₂ S0 ₄ . The L-pantolactone obtained was extracted analogously 3 times with a volume of ethyl acetate, dried with Na ₂ SO ₄ and evaporated. For recycling into the hydrolysis, the L-pantolactone melt can be racemized by heating after adding NaOH in the presence of small amounts of Na-L-pantoate (3 h at 180 ° C.).

16. Production of D-pantolactone by hydrolysis with L-panto-5 lactone hydrolase from E. coli strains

L-pantolactone hydrolase was obtained directly from fermentation broth from E. coli (TG1 pDHE681 or preferential strains which co-express chaperones such as GroEL) by cell disruption (2 x 1000 bar in a 0 microfluidizer), cell debris separation (20 min 9000 g at 10 ° C), 10-fold concentration via cross-flow filtration (Hämoflow F60, Fresenius, membrane with exclusion of approx. 10 kDa) and heat precipitation (20 min 60 ° C, 20 min RT-10 ° C, centrifugation) and a specific activity of about 3000 U / g protein

25 enriched. This 10 x homogenate had an activity of 63-100000 U / 1 and a protein concentration of 20-30 g / 1.

The racemate cleavages of 2.3 M D, L-pantolactone (30% w / v) with heat-precipitated homogenate (16000 U / 1, 6 g / 1 protein) were performed

30 30 ° C with titration with 4 to 10 M NaOH (pH7.5) with light buffering (6 to 20 mM NaHC0 ₃ ) in a 0.75 to 1.0 1 batch approach. After incubation overnight, the enzyme was removed from the product by cross-flow filtration (Haemoflow F40, Fresenius, membrane with exclusion of approx. 10 kDa)

35 solution separated, washed 1 to 2 times with deionized water and concentrated. The enzyme was then used again under the above conditions. The durability test showed a doubling of the dwell time required for an ee value of> 90% (D-PL) from 12 to 24 h after 6 d (FIG. 7). The activity

40 losses should be reduced by using larger volumes (e.g. 10-1 approach for the F40 cartridge).

Racemate resolution batches were worked up with homogenate (50.8% conversion, 92.5% ee) by extraction with 5 x 1 45 vol. MTBE. 43% D-pantolactone with 91.4% ee and 98.2%

Purity (GCi _nt . _St. Based on 100% racemate). After heating (65 ° C / 15 min) with sulfuric acid (conc., 25 ml) and again Extraction with MTBE (5 x 1 vol) gave 53% L-pantolactone with 62.3% ee and 98.2% purity (GCi _nt .s _t .). Protein or DNA contamination was not detectable using standard procedures.

* NaOH data from day 6 not available. 50.4% conversion, 89.8% ee at 1390 min (23.17 h).

Samples from day 7 not available, therefore no analytical data

17. Production of D-pantolactone by hydrolysis with immobilized L-pantolactone hydrolase

L-pantolactone hydrolase was isolated as in Example 16 and bound to various carrier materials such as the commercially available EupergitC (Röhm GmbH, Darmstadt) or Deloxan DAPIII (Degussa, Frankfurt).

EupergitC

Eupergit is a carrier material activated by epoxy groups. As a result, the protein is mainly covalently fixed to amino groups.

Homogenate was first subjected to precipitation by heat treatment. 1.11 homogenate was incubated in amounts of 550 ml each for 30 minutes at 60 ° C. and then placed on ice for 20 minutes to cool. This solution was centrifuged (1 hour 8000 rpm GS3 rotor) to separate denatured protein. The supernatant was then concentrated on a Hemoflow F40 cartridge and re-buffered in a 20 mM HEPES buffer, pH 7.5. The amount of protein per g of Eupergite can be chosen freely. In the following example, 7.2 g of protein in 270 ml of buffer were diluted with 30 ml of IM potassium phosphate buffer pH 7.0 and salted with 17.5 g of solid NaCl. The high amount of salt promotes the binding of the protein to the carrier. The pH was adjusted to exactly pH 6.8, and 15 g of dry Eupergite were then added to this solution. The solution was agitated for 17 hours at room temperature. The reaction mixture was then suctioned off on a glass suction filter and the support was washed with water. The damp

Material weighed approx. 60 g. The storage was carried out in a 10 mM phosphate buffer, pH 7.5. Deloxan DAPIII, with and without reduction

Deloxan is a silicate modified with amino groups. These amino groups can be activated with glutardialdehyde to form a Schiff base. The excess aldehyde is then washed away and the protein is added to the activated carrier. The free amino groups of the protein react with the formation of a second Schiff's base with the still free aldehyde of the bound glutardialdehyde. This protein immobilized in this way can already be used. However, the Schiff's base tends to hydrolize, so that the protein slowly bleeds from the carrier in aqueous solution. This can be avoided by reduction with sodium borohydride. The ship's base is converted into a secondary amine. The prerequisite for this is the stability of the enzyme against reduction with sodium borohydride.

Heat precipitation of the homogenate as in the case of immobilization on EupergitC.

Activation:

140 g of Deloxan DAPIII was washed with water, then with 1.5 10, IM sodium phosphate buffer, pH 6 and 1.5 10, IM sodium phosphate buffer, pH 7.5, and resuspended. 560 ml of a 2.5% glutardialdehyde solution (pH 7.5 corrected in the same buffer) were added and the reaction was continued for 3 to 4 hours. The carrier turned orange-red. The activated carrier was then washed with 6 l of water and resuspended in 1 l of sodium phosphate buffer (pH 7.5).

For example, 40 g of activated deloxane were Given 10,000 U (approx.2.7 g protein) L-pantolactone hydrolase. The protein was incubated with the activated carrier for 18 hours at room temperature. The solution was separated from the support via a glass suction filter. The support was washed several times with water and then with 1 1 0.1M phosphate buffer, pH 7, 2 and 1 M NaCl. Half of the carrier was removed (without reduction). The other half was washed again with water and then placed in 0.1 M sodium borate buffer, pH 7.2. Reduction with sodium borohydride:

0.4 g of sodium borohydride (0.5%) was added to the carrier resuspended in 80 ml of borate buffer and agitated for 3 hours at room temperature. The carrier material became light yellow again. The carrier was sucked off through a glass suction filter and washed with 400 ml of borate buffer. The carrier was then washed with 1 liter of water and then taken up in 20 mM phosphate buffer. resolution

For racemate resolution of 2.3 MD, L-pantolactone (30% w / v) with immobilizates, these were stirred under titration with 10 M NaOH (pH 7.5) in 10 mM NaHCO ₃ in 40 ml batches at 30 ° C. until the ee value for D-pantolactone was approx. 90% (25 to 60 h). After each batch, the biocatalyst was separated from the product by filtration (suction of the reaction mixture through an HPLC solvent filter) and, if necessary, 3 x washing (deionized water) was recovered. The following figures show the NaOH curves of the service life tests in a stirred batch.

Table la

PS, pantoic acid ee values refer to the formation of (L) -pantoic acid ee, enantiomeric excess, corrected, corrected ee after deduction of those formed by chemical hydrolysis

Pantoic acid (blank)

Table lb

ee values refer to the formation of (L) -pantoic acid

ee, enantiomeric excess; E, enantioselectivity; corrected, corrected ee or E after deduction of pantoic acid (blank) formed by chemical hydrolysis

Table 2: Purification of L-pantolactone hydrolase from Lu5351

Tab.3a Substrate specificity of L-pantolactone hydrolase from Lu681

++:> 50 U / 1

+:> 5 U / 1

(+)> 0.5 U / 1 <0.5 U / 1

Tab. 3b Substrate specificity of the L-pantolactone hydrolase from Lu5351

> 50 u / i

> 5 U / 1

: +)> 0.5 U / 1 <0.5 U / 1

Tab. 3c Lipase activity of the purified L-pantolactone hydrolase from Lu681

Tab. 4a Effects of various substance additions on the activity of L-pantolactone hydrolase from Burkholderia caryophylli Lu681 in the standard assay

Tab. 4b Effects of various substance additions on the activity of L-pantolactone hydrolase from Agrobacterium radiobacter Lu5351 in the standard assay

pCMBS = p-chloromercurbiphenylsulfonic acid

DTT = dithiothreitol

SDS = sodium dodecyl sulfate

EDTA = ethylenediaminetetraacetic acid

DIFP = diisopropyl fluorophosphate

CHAPS = ([3-cholamidopropyl] dimethylammonio) -1-propanesulfonate

Table 5: Homologies of the L-pantolactone hydrolase amino acid sequence from Lu681 according to gap ⁴ or bestfit ⁵ search

Needleman & Wunsch (1970), J. Mol. Biol. 48, 443-453 Smith & Waterman (1981), Adv. Appl. Math. 2, 482-489

Table 6: Expression of L-pantolactone hydrolase in E. coli XLl Blue

a) XLl Blue pKS + 681 and pKS + (negative control)

b) XLl Blue pKK681

nb, not determined

Tab.7a

Tab. 7b

Claims

claims

1. Isolated nucleic acid sequence coding for a polypeptide with L-pantolactone hydrolase activity, selected from the group:

a) a nucleic acid sequence with the sequence shown in SEQ ID NO: 1,

b) nucleic acid sequences which, as a result of the degenerate genetic code, are derived from the nucleic acid sequence shown in SEQ ID NO: 1,

c) Derivatives of the nucleic acid sequence shown in SEQ ID NO: 1, which code for polypeptides with the amino acid sequences shown in SEQ ID NO: 2 and have at least 50% homology at the amino acid level, without the enzymatic action of the polypeptides being significantly reduced

d) functional equivalents of the sequences mentioned under (a) to (c).

2. Amino acid sequence encoded by a nucleic acid sequence according to claim 1.

3. amino acid sequence according to claim 2, coded by the sequence shown in SEQ ID NO: 1.

4. Nucleic acid construct containing a nucleic acid sequence according to claim 1, wherein the nucleic acid sequence is linked to one or more regulation signals.

5. Vector containing a nucleic acid sequence according to claim 1 or a nucleic acid construct according to claim 4.

6. Microorganism containing at least one nucleic acid sequence according to claim 1, at least one nucleic acid construct according to claim 4 or a vector according to claim 5.

7. Microorganism according to claim 6, characterized in that the microorganism is a gram-negative bacterium.

5 8. Microorganism according to claim 6 or 7, characterized in that it is a bacterium from the group of α-proteobacteria, β-proteobacteria or γ-proteobacteria in the microorganism.

10 9. Microorganism according to claims 6 to 8, characterized in that the microorganism is a bacterium from the family Enterobacteriaceae, Pseudomonadaceae or Rhizobiaceae.

15 10. Microorganism according to claims 6 to 9, characterized in that the microorganism is a bacterium of the genera Agrobacterium, Pseudomonas, Burkholderia, Salmonella or Escherichia.

20 11. L-pantolactone hydrolase, characterized by the following properties:

a) conversion of L-pantolactone to the corresponding acid,

25 b) pH stability: L-pantolactone hydrolase is stable in a pH range from 4 to 10

c) pH optimum: 7.2 to 7.6

30 d) Optimum temperature: approx. 70 ° C to 75 ° C

e) no inhibition of activity by EDTA

12. A process for the preparation of D-pantolactone, characterized in that the process comprises the following reaction steps:

a) reaction of racemic pantolactone in the presence of an L-pantolactone hydrolase according to claim 11 or a 40 L-pantolactone hydrolase with an amino acid sequence according to

Claim 2 or a microorganism according to claim 6 to D-pantolactone and L-pantoic acid

b) separation of the D-pantolactone.

45

13. The method according to claim 12, characterized in that the L-pantoic acid obtained in reaction step (b) is racemized and recycled in reaction step (a).

5 14. The method according to claim 12 or 13, characterized in that the reaction of the racemic pantolactone in the presence of an immobilized L-pantolactone hydrolase according to claim 11 or an immobilized L-pantolactone hydrolase is carried out with an amino acid sequence according to claim 2. 0

15. The method according to claim 12 or 13, characterized in that the reaction of the racemic pantolactone is carried out in the presence of a growing, dormant or digested microorganism according to claim 6. 5

16. The method according to claim 12 or 15, characterized in that the microorganism is immobilized.

17. Process according to claims 12 to 16, characterized in that the process is carried out in an aqueous reaction solution at a pH between 4 to 12.

18. The method according to claims 12 to 17, characterized in that the method at a temperature between 0 ° C.

25 to 95 ° C is carried out.

19. The method according to claim 12 or 13, characterized in that the D-pantolactone is separated off by extraction.

30 20. The method according to claims 12 to 18, characterized in that the D-pantolactone has an optical purity of at least 90% ee.

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40

45