JP4571936B2 - Pantolactone hydrolase - Google Patents

Description

  The present invention relates to an enzyme useful for optical resolution of an enantiomer of pantolactone, a gene encoding the enzyme, and use of the enzyme in a method for producing R-pantothenic acid or a salt thereof or R-pantenol.

  Enantiomers differ in their physiological effects, namely toxicological and pharmacological effects, reaction with enzymes and sensory characteristics. Only R-pantolactone is known as an intermediate in the production of R-pantothenic acid, an essential vitamin for humans and animals for growth, regeneration and normal physiological function. As a precursor of coenzyme A and as an acyl carrier protein of fatty acid synthetase, pantothenic acid has over 100 different types including carbohydrate, protein and lipid energy metabolism and synthesis of lipids, neurotransmitters, steroid hormones, porphyrins and hormones. Involved in metabolic pathways. Until recently, the widely used technique for producing R-pantolactone was the optical resolution of chemically synthesized R- and S-pantolactone. Such a method is very expensive. This is because it requires the use of expensive optical resolution agents. Furthermore, recovery of the R-enantiomer of pantolactone is more difficult. It is therefore desirable to provide a pantolactone hydrolase useful for the optical resolution of the R- and S-enantiomers of pantolactone. The use of such an enzyme that specifically degrades the S- and R-forms of pantolactone is a low-cost and easy-to-handle method for the separation of the two enantiomers of pantolactone. Examples of R-pantothenic acid salts include calcium R-pantothenate.

  In one aspect, the present invention relates to a horse liver, A. niger ATCC 9142, A.I. niger awamori ATCC 38854, or A.I. The nucleotide sequence of the gene encoding pantolactone hydrolase from niger MacRae ATCC 46951 is provided. These strains are available from American Type Culture Collection (ATCC), 10801, University Street, Manassas, 20110-2209, USA.

  In another aspect, the present invention relates to a nucleotide sequence as set forth in SEQ ID NO: 1, when derived from horse liver, the nucleotide sequence shown in SEQ ID NO: 5 when derived from niger MacRae ATCC 46951 When derived from niger ATCC 9142, the nucleotide sequence set forth in SEQ ID NO: 7 and A.I. When derived from niger awamori ATCC 38854, the nucleotide sequence of the gene encoding pantolactone hydrolase comprising the nucleotide sequence set forth in SEQ ID NO: 9 is provided.

  The nucleotide sequence can include coding and regulatory sequences for each of the indicated pantolactone hydrolases.

  A “regulatory sequence” is defined as an array of nucleic acid control sequences that direct the transcription of an operably linked nucleic acid. An example of such an expression control sequence is a promoter. A “promoter” contains the necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements that can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” is a functional linkage between a nucleic acid expression control sequence (eg, an array of promoters or transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence is linked to the second sequence. Refers to a linkage that directs transcription of the corresponding nucleic acid.

  Nucleotide sequence fragments can be used, for example, as probes in hybridization assays.

  Those skilled in the art will appreciate that, when a nucleotide sequence is inserted into a host organism, transcribed and translated to produce a functional polypeptide, multiple polynucleotide sequences will encode the same polypeptide due to codon degeneracy. You will recognize. These variants are in particular within the scope of the present invention. In addition, the invention relates to these sequences that are substantially the same as each other (determined as follows), and mutants of the wild-type polypeptide or retain the function of the polypeptide (eg, polypeptide These sequences encoding polypeptides (resulting from conservative substitutions of amino acids therein) are included. Furthermore, the mutant can be a mutant encoding the dominant negative mutant described below.

  Two nucleic acid sequences or polypeptides are said to be identical if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned with the maximum correspondence described below. With respect to two or more nucleic acid or polypeptide sequences, the term “identical” or percentage “identity” is used to compare windows (as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection). Comparison window) refers to two or more sequences or subsequences having a certain percentage of amino acid residues or nucleotides that are the same or the same when compared with the maximum correspondence. When percentage sequence identity is used with respect to a protein or peptide, residue positions that are not identical are replaced with other amino acid residues where the amino acid residues have similar chemical properties (eg, charge or hydrophobicity). It is recognized that conservative amino acid substitutions are often different, and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can be adjusted upwards to correct the conservative nature of the substitution. Typically, means for making this adjustment are well known to those skilled in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing percent sequence identity. Thus, for example, a conservative substitution is given a score between zero and 1 if the same amino acid is given a score of 1 and a non-conservative substitution is given a score of zero. Conservative substitution scoring is performed, for example, in the program PC / GENE (Intelligenetics, Mountain View, Calf., USA) according to the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17 (1998). It is calculated as follows.

  The phrase “substantially identical” with respect to two nucleic acids or polypeptides is aligned with maximum correspondence to a comparison window using one of the following sequence comparison algorithms or measured by manual alignment and visual inspection. Refers to sequences or subsequences having at least 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity. This definition also refers to a sequence in which the complement of that sequence hybridizes to a test sequence.

  For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or other parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

  The “comparison window” used in the present invention is any one segment of the number of consecutive positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, It includes pointing to a segment that can be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. Methods for alignment of sequences for comparison are well known in the art.

  “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to those nucleic acid sequences that encode the same or essentially the same amino acid sequence, or are essential if the nucleic acid does not encode an amino acid sequence. Points to the same sequence. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acid codons encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence in the present invention that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is effectively included in each described sequence.

  With respect to amino acid sequences, one skilled in the art will recognize that a single amino acid or a small percentage of amino acids in the encoded sequence (ie, less than 20%, eg, 15%, 10%, 5%, 4%, 3%, 2% or 1 %), Individual substitutions, deletions or additions to nucleic acids, or substitutions to peptide, polypeptide or protein sequences are considered “conservatively” when this change results in substitution of amino acids with chemically similar amino acids. It will be recognized that it is a “modified variant”. Conservative substitution tables giving functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for each other:
Alanine (A), serine (S), threonine (T);
Aspartic acid (D), glutamic acid (E);
Asparagine (N), glutamine (Q);
Arginine (R), lysine (K);
Isoleucine (I), leucine (L), methionine (M), valine (V); and phenylalanine (F), tyrosine (Y), tryptophan (W) (see, for example, Creighton, Proteins (1984)).

  An indication that two nucleic acid sequences or polypeptides are substantially identical indicates that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with an antibody raised against the polypeptide encoded by the second nucleic acid. It is sex. Thus, a polypeptide is typically substantially identical to a second polypeptide, eg, if the two peptides differ only by conservative substitutions. An indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions as described below.

  The phrase “specifically hybridizes” refers to a specific nucleotide sequence under stringent hybridization conditions when the sequence is present in a complex mixture (eg, whole cell or library DNA or RNA). It only refers to the binding of molecules, duplexing or hybridization.

  The phrase “stringent hybridization conditions” refers to conditions in which a probe typically hybridizes to its target sequence in a complex mixture of nucleic acid sequences, but does not hybridize to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences specifically hybridize at higher temperatures. In general, highly stringent conditions are selected to be about 5-10 ° C. below the thermal melting point (Tm) for specific sequences at a defined ionic strength and pH. Low stringency conditions are generally chosen to be about 15-30 ° C. below Tm. Tm hybridizes to the target sequence in equilibrium at 50% of the probe complementary to the target (the target sequence is present in excess, so at Tm, the temperature at which 50% of the probe is in equilibrium) (Under defined ionic strength, pH and nucleic acid concentration.) Stringent conditions include sodium ions having a salt concentration of less than about 1.0 M at pH 7.0-8.3, typically about 0.01-1.0 M sodium ion concentration (or other salt) and temperature is at least about 30 ° C. for short probes (eg, 10-50 nucleotides) and longer probes (eg, more than 50 nucleotides) ) At least about 60 ° C. Stringent conditions can also be achieved by adding a destabilizing agent such as formamide. Kill. For selective or specific hybridization, at least two times the positive signal background, preferably 10 times background hybridization.

  Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acid typically hybridizes under moderately stringent hybridization conditions.

  In the present invention, genomic DNA (gDNA) or cDNA containing the nucleic acid of the present invention can be identified in a standard Southern blot under stringent conditions using the nucleic acid sequences disclosed in the present invention. For the purposes of this disclosure, suitable stringent conditions for such hybridization include hybridization in a buffer of 40% formamide, 1M NaCl, 1% sodium dodecyl sulfate SDS at least about 50 ° C. at 37 ° C. C., usually at a temperature of about 55.degree. C. to about 60.degree. C. for 20 minutes in 0.2X SSC at least one wash or equivalent conditions. Positive hybridization is at least twice background. One skilled in the art will readily recognize that other hybridization and wash conditions can be used to provide conditions of similar stringency.

  A further indication that the polynucleotides are substantially identical is used as a probe under stringent hybridization conditions to isolate a test sequence from a cDNA or genomic library or, for example, in a Northern or Southern blot A reference sequence amplified by a pair of oligonucleotide primers from which a test sequence can be identified.

  The invention also includes an expression vector as defined herein. The expression vector contains one or more copies of each of the polynucleotide sequences described above. The expression vectors of the present invention can contain any of the polynucleotide sequences defined herein.

  Thus, in a further aspect, the invention provides an expression vector comprising a nucleotide sequence of a gene encoding pantolactone hydrolase, wherein the pantolactone hydrolase is horse liver, Bacillus subtilis, A. et al. niger ATCC 9142, A.I. niger awamori ATCC 38854 or A.I. expression vector derived from niger MacRae ATCC 46951, or pantolactone hydrolase from horse liver as shown by SEQ ID NO: 2, pantolactone hydrolase from Bacillus subtilis as shown by SEQ ID NO: 4, SEQ ID NO: A.8 shown in FIG. pantolactone hydrolase from niger ATCC 9142, A. as shown by SEQ ID NO: 10. pantolactone hydrolase from niger awamori ATCC 38854, and A. cerevisiae as shown in part by SEQ ID NO: 6. An expression vector comprising a nucleotide sequence of a gene encoding pantolactone hydrolase from niger MacRae ATCC 46951 is provided.

  The polynucleotide sequence in the expression vector may optionally be operably linked to the expression control sequences described above.

  As used herein, the phrase “expression vector” encodes a replicable vehicle having a DNA sequence encoding a polynucleotide sequence described herein and the polynucleotide sequence described herein. A replicable vehicle capable of mediating the expression of a DNA sequence. In this regard, the term “replicatable” means capable of replicating in a given type of host cell into which the vector has been introduced. Immediately upstream of the polynucleotide sequence (s) in question encodes a signal peptide, the presence of which ensures secretion of the encoded polypeptide expressed by the host harboring the vector An array can be provided. The signal sequence can be a signal sequence naturally associated with a selected polynucleotide sequence or a signal sequence of other origin.

  The vector can be any vector that can be conveniently subjected to recombinant DNA treatment, and the choice of vector will often depend on the host cell into which the vector is to be introduced. Thus, a vector can be an autonomously replicating vector, ie, a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, examples of such vectors Is a plasmid, phage, cosmid or minichromosome. Alternatively, a vector can be a vector that, when introduced into a host cell, is integrated into the host cell genome and replicated with the chromosome (s) into which the vector has been introduced. The expression vectors of the present invention can have any of the DNA sequences of the present invention and can be used for the expression of any of the polypeptides of the present invention.

  The invention also includes cultured cells or host cells that contain one or more of the polynucleotide sequences and / or one or more of the expression vectors disclosed herein. As used herein, a “cultured cell” is any cell that can grow under defined conditions and express one or more of the polypeptides encoded by the polynucleotides of the invention. Includes cells. Preferably, the cultured cells are yeast, fungi, bacteria or algae. More preferably, the cultured cells are Escherichia coli, Saccharomyces serevisiae, Pichia pastoris, Aspergillus niger or Bacillus subtilis.

  In another aspect, the present invention relates to a horse liver, A. niger ATCC 9142, A.I. niger awamori ATCC 38854, or A.I. A pantolactone hydrolase derived from niger MacRae ATCC 46951 is provided.

  In yet another aspect, the present invention provides pantolactone hydrolase, the amino acid sequence of which is represented by SEQ ID NO: 2 when the amino acid sequence is derived from horse liver, an amino acid sequence represented by SEQ ID NO: 6 when derived from niger MacRae ATCC 46951; When derived from niger ATCC 9142, the amino acid sequence represented by SEQ ID NO: 8 and A.I. A pantolactone hydrolase comprising the amino acid sequence set forth in SEQ ID NO: 10 when provided from niger awamori ATCC 38854 is provided.

  Pantolactone hydrolase from horse liver as shown by SEQ ID NO: 2 and pantolactone hydrolase from Bacillus subtilis as shown by SEQ ID NO: 4 show S-pantolactone hydrolase activity. A. As shown by SEQ ID NO: 8. pantolactone hydrolase from niger ATCC 9142, A. as shown by SEQ ID NO: 10. pantolactone hydrolase from niger awamori ATCC 38854 and A. as partially shown by SEQ ID NO: 6. Pantolactone hydrolase derived from niger MacRae ATCC 46951 exhibits R-pantolactone hydrolase activity.

  In a further aspect, the present invention includes fragments of the above amino acid sequences that have the enzymatic activity of the complete protein.

  An “amino acid sequence fragment” according to the present invention can be defined as a depleted of stretches sequence of amino acids that may be necessary for the correct folding of the protein. However, once the protein is folded, the amino acid stretch can be depleted without destroying the function of the respective protein. These stretches can be present in the protein folded as a loop or can be N- or C-terminally unrelated for the activity of the folded protein.

  Furthermore, the present invention includes derivatives of proteins comprising the enzymatic activity of said R- and S-pantolactone hydrolase. The derivatives include immobilized enzymes, such as those immobilized by inclusion, in which the enzyme is held in a membrane device, such as a hollow fiber, polymer network or microcapsule. Enzymes can also be immobilized by cross-linking or generation of enzyme crystals. A further possibility for immobilizing the enzyme is its linkage to a growing polymer, for example silica sol gel or silica graphite sol gel, or a prefabricated support such as Euergit C, Eupergit 250L, PEG, Celite ( Celite) and Amberlite XAD-7. A further technique for the immobilization of enzymes applicable to the R- and S-pantolactone hydrolases of the present invention is to disperse them in water-immiscible organic solvents. In general, enzymatically active proteins, such as the enzymes of the present invention, can be immobilized by almost any of the immobilization techniques known in the art.

  In yet another aspect of the present invention, the present invention relates to R- or S--applicable in the industrial production of the R-enantiomer of pantolactone, a precursor for the production of R-pantothenic acid and R-pantothenol. It relates to the use of isolated pantolactone hydrolase for the selective hydrolysis of pantolactone.

  Accordingly, an object of the present invention is to provide a process or method for selectively hydrolyzing R- or S-pantolactone using the isolated pantolactone hydrolase described herein. It is.

  In another aspect, the present invention provides a method for producing R-pantothenic acid or a salt thereof or R-pantenol comprising a step of selective hydrolysis of R- or S-pantolactone in the presence of the pantolactone hydrolase of the present invention. I will provide a.

  In a further aspect, the present invention provides a method for the cloning of nucleotide sequences that are immediately adjacent to a known sequence, eg, a nucleotide sequence encoding a novel pantolactone hydrolase or portion thereof.

  In particular, the present invention includes a novel PCR strategy that allows amplification of unknown sequences. The new PCR strategy involves the use of one primer for the synthesis of both the first and second DNA strands. The primer hybridizes to a known area of gDNA to be amplified. This primer shows 100% homology to this known sequence. With the help of this primer, a first PCR is performed to obtain a first single-stranded copy of genomic DNA that reaches an unknown sequence. Using a small aliquot of the first PCR reaction as a template, a DNA fragment located in the 3 'direction of the primer ("nested") used in the first PCR but logically still located in a known part of the sequence A second PCR is performed using primers that hybridize to. After random hybridization of the nested primer to the single stranded DNA produced by the first polymerase reaction, an opposite strand of the single stranded DNA is generated, which itself is then the second strand. Serves as a template for the same primers used for the second PCR to produce. Finally, with one primer that hybridizes at both ends of the new fragment, a DNA fragment that begins inside the known sequence and goes into any other sequence where the unknown flanking genomic DNA or part of the sequence is not found generate.

Thus, in another aspect, the invention provides a method for cloning a nucleotide sequence immediately adjacent to a known sequence comprising:
a) using a primer in the second PCR that randomly hybridizes to the single-stranded DNA produced in the first PCR, and using the single-stranded DNA in the first PCR as a template for the second PCR; and b) thereby Generating a counter strand that itself serves as a template for the second strand, also using the randomly hybridizing primers of step a),
A method comprising PCR amplification comprising

  The examples below provide a detailed description of the identification, characterization and expression of the gene encoding pantolactone hydrolase. These examples illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1: Purification of (S) -pantolactone hydrolysis activity from commercially available horse liver esterase acetone powder Horse liver esterase acetone powder (Fluka Holding AG, Industriestrasse 25, POBox260, CH-9471 Buchs, Switzerland) 1 g was dissolved in 40 ml of 50 mM Tris / HCl, pH 7.5 containing ₂ M ammonium sulfate, 1 mM CaCl ₂ , 1 mM MgCl ₂ and 10 μM EDTA (Buffer A). Undissolved material was separated by centrifugation and then sterile filtration through a 0.45 μM filter. The protein solution was applied to a butyl sepharose column HR26 / 10 (Amersham Pharmacia Biotech Europe GmbH, Deubendorf, Switzerland) equilibrated with buffer A. The column was washed with 100 ml of buffer A. This was followed by the application of 400 ml of linear gradient 0-100% buffer B (buffer A without ammonium sulfate). Lactonase eluted in the middle of this gradient. Fractions showing pantolactone hydrolysis activity were pooled and the buffer was exchanged with 20 mM Tris / HCl, pH 8.0, 1 mM CaCl ₂ , 1 mM MgCl ₂ and 10 μM EDTA. The pooled fractions were applied to a column HR26 / 10 (Amersham Pharmacia Biotech Europe GmbH, Deubendorf, Switzerland) pre-packed with MonoQ using a linear gradient from 0 to 350 mM NaCl. The protein eluted at the beginning of the gradient. The active fractions were again pooled, concentrated to 1 ml and applied to a Superdex 200 column (Amersham Pharmacia Biotech Europe GmbH, Deubendorf, Switzerland). The last showing a molecular size of about 31 kDa, consistent with the size shown by SDS-PAGE, using 50 mM Tris / HCl, pH 8.0, 1 mM CaCl ₂ , 1 mM MgCl ₂ and 10 μM EDTA as the elution buffer. Lactonase eluted as a peak.

The highest specific activity found was determined by incubating the enzyme in 200 mM Tris / acetate buffer, pH 9.0, 5 mM MgCl ₂ for 60 minutes at 37 ° C., 13.3 U / (mg protein) Met. The concentration of (S)-and (R) -pantolactone was determined by HPLC.

Example 2: Pre-characterization of (S) -pantolactone hydrolase from horse liver (a) Molecular size, structure and isoelectric point:
According to SDS-PAGE, the denatured protein has a molecular weight of about 35 kDa. Using the elution time of a calibrated gel filtration column, the molecular weight of the native protein was calculated to be about 31 kDa. This means that lactonase from horse liver is a 30-35 kDa monomer. Using a 2D gel, the isoelectric point was determined to be in the range of pH 5.5.

(B) N-terminal and internal amino acid sequencing:
The concentrated protein after 2D gel electrophoresis was digested with trypsin. The generated peptides were analyzed by mass spectroscopy. Peptide No. The N-terminal sequence of 66 is shown as SEQ ID NO: 11.

  This sequence, along with the MS spectrum, indicated that the protein belongs to a class of proteins known as aging marker protein-30 or reggarcine, which are found primarily in vertebrates. A regucalcin sequence from rat, mouse, human, chicken, rabbit, cow, Xenopus laevis and two less homologous sequences from Drosophila melanogaster have already been determined. However, the gene from the horse has not been isolated and sequenced yet.

(C) Metal dependence 1.3, 2.6, 5.2 or 10.4 mM Ca ²⁺ , Mg to test the effect of various divalent metal ions on the pantolactonase activity of horse liver enzymes ²⁺ or Mn ²⁺
Was added to the reaction mixture consisting of 200 mM Tris / acetate, pH 9.0 and 50 mM (R, S) -pantolactone. The reaction mixture was incubated at 37 ° C. for 60 minutes and stopped with an equal volume of methanol containing 2 mM EDTA. The decrease in (S) -pantolactone was determined by HPLC. Calcium decreased enzyme activity, while magnesium and manganese increased enzyme activity.

(D) Thermal stability The purified enzyme was incubated in 5.2 mM MgCl ₂ and 200 mM Tris / acetate, pH 9.0 at a temperature of 30-80 ° C. for 15 minutes. After 30 minutes on ice, activity was determined by a standard assay (60 minutes, 37 ° C.). The enzyme was stable up to 60 ° C. It began to inactivate at 65 ° C and lost more than 50% of its activity at 70 ° C.

Example 3: Cloning of (s) -Pantractonase from Horse Liver Kindly provided here by Roche Molecular Biochemicals, Penzberg, Germany using sequence information from all known mammalian genes (see below) Primers for PCR were designed for cDNA made from horse liver from 1-year-old “Haflinger” from Bavaria. Total RNA was prepared from liver tissue using the QIAGEN RNeasy Max Kit (Qiagen, Hilden, Germany).

  The homology of horse liver lactonase to other members of the regucalcin family is shown in Table 1. The determination was made using the program GAP in the GCG program package with standard parameters. Numbers for similarity are shown on the right side of the diagonal in the table, and numbers for identity are on the left side of the diagonal.

  An 885 bp fragment (SEQ ID NO: 1) was obtained by RT-PCR using a Titan One Tube RT-PCR Kit (Roche Molecular Biochemicals, Penzberg, Germany) and a homology primer encompassing 50 bp upstream from the start and stop codons. Nucleotides 1-885) were amplified. The reaction was performed as recommended by the supplier.

  N-terminal sequence derived from bovine (SEQ ID NO: 14), N-terminal sequence derived from rabbit (SEQ ID NO: 15), N-terminal sequence derived from human (SEQ ID NO: 16), N-terminal sequence derived from rat (SEQ ID NO: 17), N-terminal sequence derived from mouse (SEQ ID NO: 18), N-terminal sequence derived from chicken (SEQ ID NO: 19) and N-terminal sequence derived from Xenopus (SEQ ID NO: 20) and bovine Derived C-terminal sequence (SEQ ID NO: 21), C-terminal sequence derived from rabbit (SEQ ID NO: 22), C-terminal sequence derived from human (SEQ ID NO: 23), C-terminal sequence derived from rat (sequence) No. 24), C-terminal sequence derived from mouse (SEQ ID NO: 25), C-terminal sequence derived from chicken (SEQ ID NO: 26) and C-terminal sequence derived from Xenopus (SEQ ID NO: 27) Ku N- terminal 5'-primer for the first 1 PCR (SEQ ID NO: 12) and C- terminal 3'-primer (3'-CAGTTTCCTTAAGGGGGGATA-5 ') Construction of (SEQ ID NO: 13).

  The fragment was cloned into a TA cloning vector (Invitrogen, Carlsberg, CA, USA). Sequencing demonstrated that it was derived from horse liver regucalcin. The missing 5'-end was isolated by a method called 5 '/ 3'RACE using the respective kit supplied by Roche Molecular Biochemicals, Penzberg, Germany: perfect match Was used to produce single stranded DNA against horse liver cDNA isolated. Single stranded DNA was purified, a poly C tail was added, and another PCR was performed using the nested primer 5 'of the first primer and the poly G primer. A DNA fragment representing the 5'-end where the gene was not found was amplified. Using a forward primer (SEQ ID NO: 29) made according to the known sequence and one primer (SEQ ID NO: 30) starting with the last amino acid of the gene designed primarily according to the bovine and rabbit genes Was cloned. In the final PCR, the total cDNA of lactonase was isolated from the total RNA described above using the sequence information obtained for the design of 5'- and 3'-primers including the start and stop codons of the gene. . The cDNA and amino acid sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2. The gene consists of 900 bp / 299 amino acids.

  The complete gene was E. coli fused at the C-terminus to a His-tag using one of the commercially available expression vectors such as pQE60 from Qiagen. It was expressed in E. coli. Expression and purification of His-tagged protein according to a standard protocol yielded a 33 kDa protein that showed (S) -pantolactonase activity.

Example 4: A.I. Purification of (R) -pantolactone hydrolysis activity from a commercial crude preparation of niger lipase A. Identified in various commercially available enzyme products derived from niger fermentation. A commercial preparation called Lipase A “Amano” from Amano Pharmaceuticala Co. Ltd., Nagoya, Japan was used to purify each enzyme. According to the sequence data obtained from two enzymes co-purified with lactonase activity, the preparation overexpresses lipase from A. tubigensis. It appears to represent awamori lyophilized and broken cells (including fermentation broth). This material was dissolved in 20 mM Tris / HCl, pH 7.5, 2.5 mM MgCl ₂ (standard buffer). 5% ethanol was added to increase the solubility of this material. The material that did not become a solution was separated by centrifugation (30 minutes, 15000 rpm, 4 ° C.) followed by filtration through a 0.45 μm filter. The dissolved portion was first purified by ultrafiltration in an Amicon cell (50 kDa cutoff) to remove any salts and compounds with small molecular size that could interfere with the next anion exchange chromatography step. The protein was then applied to a Q-Sepharose column (Amersham Pharmacia Biotech Europe GmbH, Deubendorf, Switzerland) pre-packed with HR26 / 10. The column was washed with 200 mM standard buffer and the protein was then eluted with a linear gradient (400 ml) of 0-350 mM NaCl. Fractions containing activity were pooled. Ammonium sulfate was added to a final concentration of 1.5M and the so-treated protein solution was applied to a column pre-packed with HR26 / 10 butyl sepharose (Amersham Pharmacia Biotech Europe GmbH, Deubendorf, Switzerland). After a 200 ml wash with standard buffer containing 1.5 M ammonium sulfate, proteins were separated by a 1.5-0 decreasing ammonium sulfate gradient (400 ml). Here, the activity eluted in two peaks and these were pooled separately. The volume of both pools was reduced to less than 2 ml. The two concentrates were separated by gel filtration on a Superdex 200 column using standard buffer. The active fractions of each separation were pooled and concentrated. Pool II activity tended to elute slightly earlier than Pool I activity, indicating that the molecular weight of the corresponding protein may be slightly higher.

  80 U / ml (40 ° C.) was the highest activity determined for Pool I and Pool II, respectively. As shown in the 2D gel, the protein preparation is not completely homogeneous, which means that the true specific activity is even higher. However, since lactonase activity corresponds to the 38 kDa protein band in the elution profile of gel filtration, which is also the main protein of the final preparation, it is most likely that this band represents lactonase. According to gel filtration, native lactonase has a molecular mass of 75 kDa. This suggests a homodimeric structure of the enzyme.

Example 5: Pre-characterization of (R) -pantolactone hydrolase (a) Structure and molecular weight of native form 72 ml of Superdex 200 (Amersham Pharmacia Biotech Europe GmbH, Deubendorf, Switzerland using the conditions described in Example 4) ) To elute the enzyme. As estimated from the standard curve, this reflects a molecular mass of 75 kDa. SDS-PAGE showed a molecular mass of 38 kDa. The combination of data is as follows. It was suggested that (R) -pantolactonase from niger is a homodimer consisting of two identical subunits of 38 kDa.

(B) Thermostability 50 μl of diluted enzyme (0.1 U) was incubated for 1 hour at 0, 30, 40, 50, 55, 60, 70 ° C. in 20 mM Tris / HCl, pH 7.5. After 15 minutes on ice, residual activity was determined using standard assays. The enzyme was stable up to 50 ° C. At higher temperatures it began to lose its activity.

(C) Optimum temperature Concentrated (R) -pantolactonase (see Example 5) from a commercial source was incubated at 20, 30, 40, 50, 55 and 60 ° C. for 30 minutes under the following conditions: :
100 mM (R, S) -pantolactone,
100 mM MgCl ₂
200 mM Tris / HCl, pH 8.0
0.05 U (R) -pantolactonase in a 100 μl final volume.

  Commercially available A.M. The optimum temperature for (R) -pantolactonase from the nigert preparation was around 50 ° C. under the conditions used.

(D) Cofactor dependence and inhibition (R) -pantolactonase from niger requires Mg ²⁺ or Mn ²⁺ ions for its activity. It was completely suppressed by the addition of 1 mM EDTA.

(E) Optimal pH
The enzyme did not show a “normal” pH activity profile when the pH changed from 7.0 to 11.0. 10 mg of aminolipase A in 200 mM TEA-acetate buffer (pH 7.0-11) containing 0.1% Span 80 and 20 mM magnesium acetate was incubated at 40 ° C. for 20 minutes. Samples are quenched with an equal volume of 500 mM MES buffer, pH 5.5, containing 50 mM EDTA, centrifuged (10,000 g, 10 min), then 70:30 water-methanol, 1 ml / min at 20 ° C. Analyzed by HPLC using an eluted 0.46 × 15 cm CHIRADEX column. The highest activity was achieved around pH 10. Then, until pH 11.0, the activity did not change much. The highest selectivity was achieved around pH 9.0.

Example 6: A.1. Cultivation of niger ATCC 9142, 9029, 26875, 62863 and 10864 and evaluation of their (R) -pantolactonase activity In order to find out whether the found lactonase activity is widespread in Aspergillus sp. The niger strain was cultured under the following conditions:
Medium (amount / liter): 1.23 g NaNO ₃ , 0.5 g KH ₂ PO ₄ , 0.2 g MgSO ₄ × 7H ₂ O, 0.5 g yeast extract, 5% glucose and Vishniac and Santer, Bacteriol. Rev. 21: 195- 0.04 ml of trace metal solution described by 213 (1957).

5 × 10 ⁶ spores / ml were used to inoculate 300 ml of pre-culture at 30 ° C. in a 1 l flask. After 8 hours, the preculture was added to 1.7 l medium in a 2 l fermentor with controlled pH and dissolved oxygen. The pH was kept constant at pH 5.5 by automatic addition of 5M NaOH. Cells were grown for 14 hours under low aeration (5-10% air saturation). The dissolved oxygen level was then increased to 30-50% air saturation. After 5 hours, the mycelium was collected, filtered, washed twice with deionized water and frozen in liquid nitrogen. A portion of it was crushed by using a French Press. Media and cell lysates were examined for pantolactonase activity using standard assays.

  (R) -Pantractonase activity was measured for all A. Found primarily in niger lysates. Only very little activity was found in the medium.

Example 7: Determination of the amino acid sequence of a small peptide generated by tryptic digestion of concentrated (R) -pantolactonase from a lipase preparation The concentrated preparation of Example 5 was analyzed on a 2D gel. Two major spots of 33 and 40 kDa and pi of 4.7 and 5.6, respectively, were digested with trypsin. The generated peptide was analyzed by HPLC (instrument: Hewlett Packard 1090, Column: Vydac C18 (0.21 × 25 cm), absorbance at 210 nm, flow rate: 0.20 ml / min, buffer A: 0.075% TFA, buffer solution. B: separated by 0.065% TFA in 80% acetonitrile, gradient: 2% B (0-10 min), 2-75% B (10-120 min) Peptide selection was sequenced by Edman degradation Spot C [pI 5.6, 39 kDa (Poll C)]: Fxn42 (peptide 1) (SEQ ID NO: 81), Fxn57 (peptide 3) (SEQ ID NO: 82), Fx3 (peptide 2) (SEQ ID NO: 83), Fx74 (SEQ ID NO: 84) and Fx81 (SEQ ID NO: 85), spot C [pI 4.7, 33 kDa (PollA / B): N-terminal (SEQ ID NO: 86), Fx58 (SEQ ID NO: 87), Fx64 (arrangement) No. 88), Fx65 (SEQ ID NO: 89), Fx73 (SEQ ID NO: 90) and Fx68 (SEQ ID NO: 91), X represents an undefined amino acid, and the amino acid in parentheses could not be clearly determined It is.

Database searches using all short peptide sequences as targets did not provide reliable information that could help identify which of the two major spots represent lactonase. Therefore, we further concentrated the lactonase preparation of Example 5 by other anion exchange chromatography. Protein solutions (20 mM Tris / HCl, pH 7.5, in _2.5 mM MgCl ₂ ) were separated on a pre-packed MonoQ column HR16 / 10 (Amersham Pharmacia Biotech Europe GmbH, Deubendorf, Switzerland) on a 0-350 mM NaCl gradient. . The activity of all fractions was determined. Active fractions were analyzed by isoelectric focusing performed as described by SDS-PAGE and suppliers (Invitrogen, Carlsbad, CA, USA). Comparison of the activity data of the fractions with SDS-PAGE and IEF gels suggested that lactonase is a 38-40 kDa protein and that it has a pI around pH 5.6. This data corresponds very well to the spot PollC. Therefore, the amino acid sequence of this spot was used to design PCR primers and oligonucleotides for Southern blots. Pooling the most active fractions and analyzing again on the 2D gel showed that PollC was clearly enriched above PollA / B compared to the previous 2D gel.

Example 8: Degenerated oligonucleotides for Southern blots and A.I. Design of PCR primers for cloning of (R) -pantolactonase from niger Aspergillus codons created by the program CODONFREQUENCY from GCG program package version 10.2 using 12 different phytase sequences from different Aspergillus species Codon usage was used to reduce the number of different DNA sequence combinations possible due to the degeneracy of the genetic code. Although all possible combinations were realized at the 3 'end of the primer, some rare codons were omitted at the 5' end of the primer using Aspergillus codon usage as a determinant. In the case of oligonucleotides intended for hybridization experiments, the 3′- and 5′-ends were treated in the same way. Oligonucleotides whose name contains “S (sense)” or “AS (antisense)” were used for PCR. Oligonucleotides whose names do not contain S or AS were intended for use in hybridization experiments.

  A. The oligonucleotides used for cloning (R) -pantolactonase from niger are Fxn42 (SEQ ID NO: 31), Fxn42S (SEQ ID NO: 32), Fxn42AS (SEQ ID NO: 33), Fxn57 (SEQ ID NO: 34), Fxn57S (SEQ ID NO: 35), Fxn57AS (SEQ ID NO: 36), Fx73 (SEQ ID NO: 37), Fx73S (SEQ ID NO: 38), Fx73AS (SEQ ID NO: 39), FX74 (SEQ ID NO: 40), Fx74S (SEQ ID NO: 41) and Fx74AS (SEQ ID NO: 42).

Example 9: A.1. niger ATCC 9142, A.I. nigerNRRL3135, A.I. niger ssp. awamori (ATCC 38854) and A.I. Isolation of genomic DNA from niger MacRae (ATCC 46951) in 300 ml of YPD medium [Sherman et al., Laboratory course manual for methods in yeast genetics, Cold Spring Harbor laboratory, Cold Spring Harbor, New York (1986)] ⁶ spores / ml were inoculated. The culture was cultured overnight at 30 ° C. under vigorous shaking (200 rpm). Mycelium was collected by filtration through Whatman filter paper, washed with PBS (isotonic phosphate buffer) and frozen in liquid nitrogen. The mycelium was crushed into a fine powder in an ice-cooled nipple. The powder was resuspended in 1/3 volume of 50 mM Tris / HCl, pH 8.0, 1.0% SDS, 50 mM EDTA and incubated at 65 ° C. for 15 minutes with frequent inversion. The solution was cooled to 50 ° C. Proteinase K (100 μg / ml final concentration) was added. The solution was incubated at 50 ° C. for 1 hour. An additional 100 mg / ml proteinase K was added and the incubation continued for a further 3 hours. One third volume of phenol / chloroform / isoamyl alcohol (50/49/1) was added. After complete but gentle mixing, the emulsion was centrifuged (12000 g, 20 min, 4 ° C.). The aqueous phase was removed and extracted again. After a further centrifugation step (12000 g, 10 min, 4 ° C.), the aqueous phase was extracted with chloroform. 0.7 volume of isopropanol was added to the aqueous phase and the solution was gently mixed for 15 minutes. The precipitated DNA was collected by centrifugation (10000 g, 30 minutes, 4 ° C.). 1/10 volume of 3M KCl, pH 5.2 was added to the remaining supernatant and incubated for 15 minutes with gentle shaking. After a further centrifugation step, the two pellets were washed twice with 70% ethanol, dried on air and dissolved in 0.5 ml of water. Finally, DNA was treated with RNase to remove residual RNA.

Example 10: A.1. Cloning of (R) -pantolactonase from niger ATCC 9142 Niger ATCC 9142 genomic DNA (gDNA) was prepared as described in Example 6. As PCR machines, Robotar Infinity from Stratagene (Stratagene, La Jolla, CA, USA) and TAQ polymerase Kit from Roche Molecular Biochemicals (Penzberg, Germany) were used for PCRs:
1 μl of gDNA (diluted 1/10) Step 1: 5 min-95 ° C.
1 μl of nucleotide mixture Step 2: 30 sec-95 ° C.
5 μl of PCR standard buffer with Mg ²⁺ Step 3: 30 sec-50 ° C.
Primer S (100 pM / μl) 1 μl Step 4: 1 min-72 ° C.
Primer AS (100 pM / μl) 1 μl
1 μl of TAQ polymerase from Roche Molecular Biochemicals
40 μl of H ₂ O
Steps 2-4 were repeated 35 times.

  All primers were checked for self-priming. Fxn57AS was the only primer that generated a significant amount of a self-priming product around 300-320 bp. Using a hybridization temperature of 55 ° C., PCRs 5, 6, 8, 10, and 11 still produced one or more products. At 60 ° C., 30 sec amplification and 30 cycles, PCRs 5, 6, 8, 10 and 11 still generate one or more PCR products, which correspond to products produced at lower hybridization temperatures.

The weak 840 bp product of PCR reaction 12 was isolated from an agarose gel using the QIAEXII gel extraction kit from Qiagen (Qiagen, Hilden, Germany) and dissolved in 40 μl of H ₂ O.
1 μl of 840 bp fragment (diluted 1/10) Step 1: 5 min-95 ° C.
1 μl of nucleotide mixture Step 2: 30 sec-95 ° C.
5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec-55 ° C.
Primer S10 pMol / μl) 1 μl Step 4: 30 sec-72 ° C.
Primer AS10pMol / μl) 1μl
1 μl of TAQ polymerase from Roche Molecular Biochemicals
40 μl of H ₂ O
Steps 2-4 were repeated 30 times.

  The primer combinations of reactions 2, 5, 10, 11 and 135 were used. In addition to reaction 2, all other reactions resulted in PCR products. This means that the sequences of primers 42S / AS, 57S / AS, 73S / AS and 74S / AS are part of the 840 bp fragment. The close proximity of these four primers strongly indicates that the 840 bp fragment is part of the lactonase gene in question. This assumption was supported by sequencing. The amino acid sequence of peptide Fx57 (SEQ ID NO: 90) was confirmed in the 840 bp fragment, but the sequence of peptide Fxn42 (SEQ ID NO: 45) was only partially verified.

  A. It was also possible to isolate the corresponding DNA fragment of niger MacRae. A. In the case of awamori, only the products of primers Fxn57S and Fx73AS could be isolated by this approach. These Aspergilli sequences are described in A.P. It has approximately 90% identity at the DNA level to the sequence of niger ATCC 9142.

The following approach was used to isolate the 5′- and 3′-ends of the lactonase gene:
(A) Isolation of the 3′-end niger ATCC 9142 gDNA 1 μl Step 1: 5 min-95 ° C.
1 μl of nucleotide mixture Step 2: 30 sec-95 ° C.
5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec-55 ° C.
Primer Oal2AS or Oal3S Step 4: 30 sec-72 ° C
(10 pMol / μl) 1 μl
1 μl of TAQ polymerase from Roche Molecular Biochemicals
40 μl of H ₂ O
Steps 2-4 were repeated 30 times.

The PCR mixture was extracted twice with an equal volume of phenol / chloroform and once with chloroform alone. The DNA was then precipitated by adding 1/10 volume of 3M potassium acetate, pH 5.32 and 2 volumes of ethanol. After 15 minutes centrifugation (14000 rpm in an Eppendorf table centrifuge), the pellet was washed with 70% ethanol, air dried and finally dissolved in 20 μl of sterile water.
Second PCR
4 μl of DNA from the first PCR Step 1: 5 min-95 ° C.
1 μl of nucleotide mixture Step 2: 30 sec-95 ° C.
5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec-58 ° C.
Primer Oal4AS or Oal1AS Step 4: 1 min-72 ° C
(10 pMol / μl) 1 μl
From Roche Molecular Biochemicals
High Fidelity Mix 1μl
38 μl of H ₂ O
Steps 2-4 were repeated 35 times.

  Oal4S primer was used to generate a 900 bp DNA molecule. Sequencing showed that it encoded the 3'-end of the gene.

(B) 5′-end isolation:
Using DIG-labeled oligonucleotides Fxn42, Fxn57, Fx73 and Fx74 as probes and the DIG system (Roche Molecular Biochemicals) with chemiluminescent detection on X-ray films, Hybridization experiments were performed on niger ATCC 9142 genomic DNA. Hybridization and detection were performed as recommended by the supplier.

10 μg of genomic DNA was digested with BamHI, EcoRI, HindIII, PstI, SacI and XhoI. Hybridization was performed overnight at 42 ° C. using Roche Molecular Biochemicals hybridization solution. After two washing steps at room temperature in 2 × SSC, 0.1% SDS, washing was performed twice at 50 ° C. in 0.5 × SSC, 0.1% SDS. CSPD (2-chloro-5- (4-methoxyspiro {1,2-dioxetane-3,2 ′-(5′-chloro) tricyclo [3.3.1.1 ^3,7 ] decane as a substrate for alkaline phosphatase } -4-yl) After the detection treatment with 1-phenyl disodium phosphate), the X-ray film was exposed to the filter for 1 hour (room temperature). There was no signal available for the film.

  Therefore, the random primed DIG labeled PCR products of reactions 5 and 11 (see Example 11) were used as probes. Hybridization was performed in the same way, just washing conditions were slightly changed from 2 x 5 minutes at room temperature and 2 x 15 minutes at 55 ° C. The following bands appeared on the film.

In order to clone the entire gene or at least its lacking 5′-end, The chromosomal DNA of niger ATCC 9142 is digested with HindIII, thereby generating an approximately 4 kb fragment containing the lactonase gene according to the hybridization experiment:
Chromosomal DNA 20μl
10 x buffer 20 μl
HindIII (10 U / μl) 5 μl
175 μl of H ₂ O
16 hours incubation at 37 ° C

Digested DNA was purified by PCR purification kit from Qiagen (Qiagen, Hilden, Germany) and eluted in 100 μl. The following ligation was performed overnight at 16 ° C. The purified DNA was diluted to 2 ml with water. Ligase and ligase buffer were added according to the manufacturer's recommendations (Roche Molecular Biochemicals). The completed ligation was used as a template for different PCRs using partially isolated lactonase gene internal primers.
Ligation solution 5 μl Step 1: 5 min-95 ° C.
1 μl of nucleotide mixture Step 2: 30 sec-95 ° C.
5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec-55 ° C.
Sense primer (10 pMol / μl) 1 μl Step 4: 3 min-72 ° C.
Antisense primer (10pMol / μl) 1μl
1 μl High Fidelity polymerase mixture from Roche Molecular Biochemicals
40 μl of H ₂ O
Steps 2-4 were repeated 32 times.

  The four primer combinations Oal3S / Fxn57AS, Oal4S / Fxn57AS, Oal3S / Oal8AS and Oal4S / Oal8AS all resulted in a single 4 kb DNA fragment. All primer pairs were directed outside the gene. Thus, the lactonase gene must be located on a DNA molecule that has been circularized as a self-ligated HindIII fragment.

  Fragments were isolated from the gel using the QIAEX II gel extraction kit from Qiagen and sequenced by ABI310 Genetic Analyzer using the dideoxy method recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA). The isolated fragment is A. It was confirmed to contain both ends of the (R) -pantolactonase gene from niger ATCC 9142.

Containing the exact amino acid sequences of all peptides sequenced using primers Oal10S and Oal12AS; niger ssp. It was also possible to isolate awamori homologous genes. The only exception is the sequence of peptide Fxn42. Fxn42 was obtained from two peptide sequencing at that time. Perhaps the two different peptides eluted at the same time in an HPLC experiment separating the tryptic digested enzyme fragments. However, sequencing initially showed only the sequence of the major peptide with a strong background. After finishing the first peptide, the sequence of the second peptide was the remaining sequence generated for the other four amino acids:
a) K-P-F-A-H-Q-V-K (SEQ ID NO: 43) and b) R-H-H-N-A-P-A-P-T-P-E-D-P -E-R-R (SEQ ID NO: 44) gives Fxn42, K-P-F-A-H-Q-V-K-T-X-E-D (SEQ ID NO: 45).

This also explains why oligonucleotides Fxn42S and Fxn42AS based on peptide Fxn42 do not give usable results. According to the amino acid sequence, the protein from strain ATCC 9142 has a molecular mass of 36573 Da and an estimated absorbance at 280 nm of 1.83 (1 mg / ml protein solution). niger ssp. The amino acid sequence from awamori has a calculated molecular mass of 36547 Da and an estimated E _{280 nm} of 1.831 for a 1 mg / ml protein solution (Pace et al., 1995).

Example 11: A.1. Cloning and expression of the (R) -pantolactonase gene from niger ATCC 9142 Compare and assemble the sequences obtained by all three methods described in Example 10 to generate a 2443 bp continuous sequence encompassing the entire lactonase gene I let you. In order to determine the start and stop codons of genes and possible introns, the available amino acid sequences of peptides obtained from purified genes from commercial preparations are determined from the two GCG program package releases 10.2. Used together with the results of the programs TESTCODE and CODONPREFERENCE. The stop codon and one intron were determined by both programs in the same way, but the start codon was not specifically available for other sequenced peptides at the N-terminus of peptide Fx74 (SEQ ID NO: 84) It was not clear. Comparison of all possible translations to homologous amino acid sequences found during database searches also did not help. This is because the N-terminal portion of the protein appears to be quite heterogeneous. Therefore, two methionine residues found upstream of the position of peptide Fx74 but in the same reading frame were chosen and both were used for the construction of the respective expression cassette. A CT rich region was found between base pairs 666-742 of SEQ ID NO: 7. However, the location of this CT-rich region was actually less preferred than one Met et al. As the start codon. This is because the distance between this region and the start codon usually varies considerably in fungi. A possible polyadenylation site was also identified downstream of the gene.

  A. In the case of a 72 bp long intron of the gene from niger ATCC 9142, the DNA stretch from nucleotides 976 to 981 most likely represents the 5′-splice site, and the stretch from nucleotides 1029 to 1035 or 1016 to 1022 Represents the putative internal consensus sequence and the DNA stretch from 1045-1047 represents the 3'-splice site (SEQ ID NO: 7). A. The corresponding sequence of the niger MacRae gene is located at bp 32-37, 66-72 and 80-82 in a 51 bp long intron (SEQ ID NO: 79). A. niger ssp. In the case of a 50 bp long intron of the awamori oal gene, the 5'-splice site starts at nucleotide 203 (GTGCCC), the internal consensus region starts at nucleotide 236 (AACTAAC), and the 3'-splice site. Starts at nucleotide 250 (CAG) (SEQ ID NO: 9).

  The consensus sequence for the 5'-splice site is GTPuNGPy. None of the listed sites have a G at position 5. The internal consensus yeast sequence for lariat formation is TACTAAC. The 3'-splice site has the consensus sequence PyAG [Unkles, Gene organization in industrial filamenous fungi. In Applied molecular genetics of filamentous fungi (Kinghorn, ed), pp. 28-53. Blackie Academic & Professional, Wester Cleddens Road, Bishopbriggs , Glasgow G64 2NZ, UK, Glasgow (1992)].

The following cDNA was generated:
-E. oal for expression in E. coli (SEQ ID NO: 98)
OalEco (SEQ ID NO: 94) for expression in Saccharomyces cerevisiae
OalESshort for expression in S. cervisiae (a shorter version of 31 amino acids using the second possible Met at position 32 of SEQ ID NO: 95)
-E. oalS for expression in E. coli (oalS was created because of the misreading sequence of peptide Fxn42, which was finally identified as an artifact)
-S. oalSEco for expression in Cervisiae
-E. oalhis containing a C-terminal his-tag for expression in E. coli
OalEhis for expression in S. cervisiae
-E. oalNhis containing an N-terminal his-tag for expression in E. coli (SEQ ID NO: 92)
OalENhis (SEQ ID NO: 96) for expression in S. cervisiae (construct oalsec containing the signal peptide of A. terreus cbs phytase for expression and secretion in S. cervisiae)

General Procedure First, the predicted coding sequence (cDNA) of the oaal gene was isolated in two PCRs. Two PCR products were assembled by the third PCR. The following primers were used:
Oal1AS (SEQ ID NO: 46), Oal2AS (SEQ ID NO: 47), Oal3S (SEQ ID NO: 48), Oal4S (SEQ ID NO: 49), Oal5S (SEQ ID NO: 50), Oal6AS (SEQ ID NO: 51), Oal7AS (SEQ ID NO: 52), Oal8AS (SEQ ID NO: 53), Oal9AS (SEQ ID NO: 54), Oal10S (SEQ ID NO: 55), Oal10SEco (SEQ ID NO: 56), OalENhis (SEQ ID NO: 57), Oal10SHis (SEQ ID NO: 58), Oal11S (SEQ ID NO: 59), Oal12AS ( SEQ ID NO: 60), Oal12ASEco (SEQ ID NO: 61), Oal12AHis (SEQ ID NO: 62), Oal2ASHisEco (SEQ ID NO: 63), Oal13S (SEQ ID NO: 64), Oal13AS (SEQ ID NO: 65), Oal14S (SEQ ID NO: 66), Oal14AS (SEQ ID NO: 67), Oal15S (SEQ ID NO: 68), Oal15AS (SEQ ID NO: 69), Oal16S (SEQ ID NO: 70) (second Met), OalsecS (SEQ ID NO: 71), OalsecAS (SEQ ID NO: 72), pQE80EcoS (SEQ ID NO: 73), pQE80EcoNhisS (SEQ ID NO: 74), pQE80BamAS (SEQ ID NO: 75), pQE80BamhisAS (SEQ ID NO: 76), pQE80EcoSshort (SEQ ID NO: 77) and CP-a (SEQ ID NO: 78).

  Oal1S begins immediately before the upstream upstream codon, while Oal9AS begins several nucleotides downstream of the predicted stop codon. To allow assembly of the two generated PCR products in the third PCR reaction without introns, Oal14S and Oal14AS are complementary and contain sequences from the ends of exon I and exon II.

1 μl of gDNA (diluted 1/10) Step 1: 5 min-95 ° C.
1 μl of nucleotide mixture Step 2: 30 sec-95 ° C.
5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec-55 ° C.
Oal11S (10 pMol / μl) 1 μl Step 4: 1 min-72 ° C.
Oal14AS (10 pMol / μl) 1 μl
1 μl of High Fidelity polymerase mixture from Roche Molecular Biochemicals
40 μl of H ₂ O
Steps 2-4 were repeated 35 times.

1 μl of gDNA (diluted 1/10) Step 1: 5 min-95 ° C.
1 μl of nucleotide mixture Step 2: 30 sec-95 ° C.
5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec-55 ° C.
Oal14S (10 pMol / μl) 1 μl Step 4: 1 min-72 ° C.
Oal9AS (10pMol / μl) 1μl
1 μl High Fidelity polymerase mixture from Roche Molecular Biochemicals
40 μl of H ₂ O
Steps 2-4 were repeated 35 times.

  A. PCR on the niger ATCC 9142 gDNA showed a PCR product of the expected size. The PCR product was purified by agarose gel electrophoresis. They were extracted from the gel using a QIAEXII kit from Qiagen (Qiagen, Hilden, Germany) and used for the third PCR:

0.5 μl each of PCR products 1 and 2 Step 1: 5 min-95 ° C.
1 μl of nucleotide mixture Step 2: 30 sec-95 ° C.
5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec-55 ° C.
Oal12S (10 pMol / μl) 1 μl Step 4: 1 min-72 ° C.
Oal10AS (10 pMol / μl) 1 μl
1 μl High Fidelity polymerase mixture from Roche Molecular Biochemicals
40 μl of H ₂ O
Steps 2-4 were repeated 30 times.

  The final product was purified by agarose gel electrophoresis, extracted from the gel and cloned into a TA vector for direct cloning of PCR products supplied by Invitrogen (Carlsbad, CA, USA). All other necessary cloning steps were performed as described in Sambrock et al. (1989). A plasmid containing the correct insert (oal1) checked by sequencing was used as a template for all upcoming construction steps.

(A) E.E. Constructs for expression in E. coli and Saccharomyces cerevisiae oa and oal Eco:
PCR using primers pQE80EcoS and pQE80BamAS was performed on oal1 to generate the construct Eco-oal-Bam using the following protocol:

1 μl of plasmid containing oal1 as insert (10 ng)
1 μl of nucleotide mixture
5 μl of PCR standard buffer with Mg ²⁺
1 μl of pQE80EcoS (10 pMol / μl)
1 μl of pQE80BamAS (10 pMol / μl)
1 μl High Fidelity polymerase mixture from Roche Molecular Biochemicals
40 μl of H ₂ O
Stage 1: 5 minutes -95 ° C
Stage 2: 30 sec-95 ° C
Stage 3: 30 seconds -55 ° C
Step 4: 1 min-72 ° C
Steps 2-4 were repeated 30 times.

  The PCR product is purified by agarose gel electrophoresis, the fragment in question is extracted from the gel (QIAEXII, Qiagen), digested with EcoRI and BamHI, purified again by agarose gel electrophoresis, ligated into the vector pQE80 from Qiagen, And E. E. coli Top10 was transformed. Four clones of plasmid were isolated and the insert sequenced. Plasmids containing the correct construct were coli M15 or equivalent E. coli. E. coli strain was transformed. All molecular treatments are known to those skilled in the art as standard treatments.

  For yeast expression constructs, the primers used were replaced with Oal10EcoS and Oal12ASEco, the PCR product was digested with EcoRI alone, and the final product was compared to pYES2 or the equivalent for S. cervisiae using the EcoRI restriction sites of the vector. Cloned into an expression vector. S. cervisiae strain, eg INVSc1 (Invitrogen, Carlsbad, CA, USA) transformation is supplied according to or together with Hinnnen et al., Proc. Natl. Acad. Sci. USA 75: 1929-1933 (1978) Was done according to the manual.

(B) E.E. The construct pQE80oalS for expression in E. coli (31 amino acid shortened version using a second possible Met:
Primers pQE80EcoSshort and pQE80BamAS were used against the same template using the same PCR protocol as described above. The completed PCR reaction was performed as described in (a).

(C) S.M. Constructs for expression in Cervisiae ealS:
Primers Oal16S and Oal12ASEco were used. All others were processed as described in (a).

(D) E.E. A construct containing a C-terminal his-tag for expression in E. coli pQE80oalhis:
Primers pQE80EcoS and pQE80BamhisAS were used for PCR. See (a).

(E) S.M. Constructs for expression in Cervisiae: oalhis:
Primers Oal10Seco and Oal12ASHisEco were used for PCR. See (a).

(F) E.E. The construct pQE80oalNhis containing an N-terminal His-tag for expression in E. coli:
Primers pQE80EcoNhisS and pQE80BamAS were used for PCR (see a)

(G) S.M. Constructs for expression in Cervisiae oalENhis:
Primers OalENhis and Oal12ASEco were used for PCR (see a).

(H) S.M. A. for expression and secretion in C. cerevisiae. Constructs containing signal peptide of terreus cbs phytase oalsec:
The construct was prepared using three independent PCRs. First, the signal sequence was isolated by PCR on the consensus phytase gene [Lehmann et al., Protein Eng. 13: 49-57 (2000)].

1.0 μl of plasmid containing the consensus phytase gene as an insert (10 ng)
Nucleotide mixture 1.0 μl
5.0 μl PCR standard buffer with Mg ²⁺
CP-a (10 pMol / μl) 1.0 μl
pOalsecAS (10 pMol / μl) 1.0 μl
1.0 μl High Fidelity Polymerase Mixture from Roche Molecular Biochemicals
40 μl of H ₂ O
Stage 1: 5 minutes -95 ° C
Stage 2: 30 sec-95 ° C
Stage 3: 30 seconds -55 ° C
Step 4: 1 min-72 ° C
Steps 2-4 were repeated 30 times.

1.0 μl of plasmid containing oaal gene (10 ng)
Nucleotide mixture 1.0 μl
5.0 μl PCR standard buffer with Mg ²⁺
Oalsec S (10 pMol / μl) 1.0 μl
Oal12ASEco (10 pMol / μl) 1.0 μl
1.0 μl High Fidelity Polymerase Mixture from Roche Molecular Biochemicals
40 μl of H ₂ O
Stage 1: 5 minutes -95 ° C
Stage 2: 30 sec-95 ° C
Stage 3: 30 seconds -55 ° C
Stage 4: 45 seconds -72 ° C
Steps 2-4 were repeated 30 times.

The PCR product was purified by agarose gel electrophoresis on a 1.5% agarose gel, extracted from the gel (QIAEXII, Qiagen) and used for the third PCR:
PCR1 product 0.5 μl
PCR2 product 0.5 μl
Nucleotide mixture 1.0 μl
5.0 μl PCR standard buffer with Mg ²⁺
CP-a (10 pMol / μl) 1.0 μl
Oal12ASEco (10 pMol / μl) 1.0 μl
1.0 μl of High Fidelity polymerase mixture from Roche Molecular Biochemicals
40 μl of H ₂ O
Stage 1: 5 minutes -95 ° C
Stage 2: 30 sec-95 ° C
Stage 3: 30 seconds -55 ° C
Step 4: 1 min-72 ° C
Steps 2-4 were repeated 30 times.

  The PCR product was purified by agarose gel electrophoresis, extracted from the gel, digested with EcoRI, purified again by agarose gel electrophoresis, and ligated to the S. cervisiae expression vector described above.

Example 12: Expression of oa (a) E. The constructs pQE80oal, pQE80oalS, pQE80oalhis and pQE80oalNhis in E. coli were transformed into E. coli containing pREP4 containing a repressor of the lac promoter. Overnight cultures expressed in E. coli M15 (see Qiagen, Hilden, Germany expression manual) were diluted to 0.1 OD at _{600 nm} and grown at 30 ° C. to an OD _{600 nm} of 1.5. The culture was then induced with 0.5 mM IPTG and incubated at 30 ° C. for an additional 6 hours. Cells were harvested by centrifugation (5000 g, 20 minutes) and frozen at -80 ° C. They were dissolved using B-PER (Pierce, Rockford, IL, USA) according to the manufacturer's rotocol. After a further centrifugation step, the supernatant was used for hydrolysis of (R) -pantolactone.

  In transformants containing the constructs pQE80oalS and pQE80oalHis, no activity was found in the supernatant or cell lysate, whereas the constructs pQE80oal and pQE80oalNhis were used for the soluble protein. Nearly 5% was active (R) -pantolactonase.

  The construct containing the N-terminal His-tag was expressed in the same way as the gene without the His-tag. Purification of His-tagged proteins was performed in native form from cell lysates using the protocol described in “QIAexpressionist” from Qiagen (hilden, Germany).

(B) S.M. The constructs oalEco, oalES, oalEhis, oalENhis and oalsec in S. cervisiae It was expressed in C. cerevisiae INVScl or comparable strains. After transformation and SD ^ura- medium ([Sherman et al., Laboratory course manual for methods in yeast genetics, Cold Spring Harbor laboratory, Cold Spring Harbor, New York (1986)] Incubate for 4 days, crush the grown colonies and transfer to 2 ml of SD ^ura- liquid medium and incubate with vigorous shaking for 3 days at 30 ° C. These cultures are precultured for 25 ml of SD ^ura- medium After another 3 days of cultivation at 30 ° C. with vigorous shaking, 500 ml of YPD medium was inoculated with the culture prepared above (Sherman et al., Supra) in a 2 liter flask. After a further 3 days of culturing, the cells were separated from the medium by centrifugation and lysed using Y-PER (Pierce, Rockford, IL).

  E. As in the case of E. coli expression, only the constructs ealEco and oaLENhis resulted in the expression of active (R) -pantolactonase found only in cell lysates. It was possible to produce 300 U of (R) -pantolactonase using 75 ml of YPD culture. The corresponding lysate showed a specific activity of about 5 U / mg total protein.

  Using the sequence data of the intron positions indicated above, A. oal gene from Awamori and A. The oal gene from niger ATCC 46951 can be constructed and expressed in a comparable manner.

Example 13: E.I. coli or S. coli. Immobilization of heterologously expressed Oal in Cervisiae and its application to hydrolytic resolution of (RS) -pantolactone For multiple use in bioreactors, A. (R) -pantolactonase from niger was immobilized. E. coli or S. coli. After expression in C. cerevisiae, the cells can be transformed by chemical means such as B-PER (E. coli) or Y-PER (S. cervisiae) (Pierce, Rockford, IL, USA) or a machine such as sonication or high pressure. Disrupted by specific means. The lysate was clarified by centrifugation. This preparation was used directly for immobilization. Alternatively, additional purification steps such as ammonium sulfate precipitation were included prior to immobilization to further increase specific Oal activity.

The biocatalyst was immobilized as follows:
(1) Immobilization of Oal in Silica-Sol-Gel Poly (glyceryl silicate) -1.0 (PGS-1.0) is described in the literature [Gill and Ballesteros, J. Am. Chem. Soc. 120: 8587-8598 ( 1998)] and dissolved in half of its weight in ice-cold water. A 100 or 200 mg portion of this is thoroughly mixed with 50 or 100 mg of an ice-cold preparation of biocatalyst stock (soluble enzyme from various preparations) in a 2 or 5 ml vial in 50 mM phosphate, pH 7.0. The vial was gently rotated for 2 minutes to form a thin coating of hydrogel on the vial wall. The vial was then left on ice for 20 minutes and then transferred to the refrigerator (opened). The hydrogel was aged at 5 ° C. for 48-72 hours to form a xerogel. Xerogel-coated vials are washed with 2 × 1 or 2 × 4 ml of 50 mM phosphate buffer, pH 7, by shaking at 100 rpm, 5 ° C., followed by 2 × 1 or 2 containing 10 mM magnesium acetate. Washed with 4 ml of 50 mM TEA-acetate, pH 7.

(2) Immobilization of Oal in sol-gel silica-polyvinyl alcohol The treatment was mixed with an appropriate amount of polyvinyl alcohol (PVA from Aldrich, 85K 13% w / w in water) before combining the DGS solution with the lactonase enzyme stock. Except for this, it was the same as the treatment in (1). Further processing was the same as in (1).

(3) Immobilization of Oal on supported polyethyleneimine activated with glutaraldehyde:
Polyethyleneimine (1 g of anhydrous high molecular weight branched polymer from Aldrich), CA (0.2 g, 36% Ac) and cellulose acetate butyrate (0.2 g, 48% acetate and butyrate content from Aldrich) in dichloromethane Dissolve in 5 mL and use the solution to coat 7 g of Fullers Earth (30-60 mesh, from Aldrich). The wet coated material was dried in air for 0.5 hours at room temperature to give about 9 g of coated fuller earth. This was then suspended in an ice-cold mixture of glutaraldehyde (50% w / w, 8 mL, from Aldrich) and phosphate buffer (50 mM, pH 8, 50 mL) with stirring at 200 rpm for 2 hours. The activated support was washed with water (4 × 10 mL), then with phosphate buffer (20 mM, pH 8, 4 × 10 mL) and then drained. The wet support was mixed with an ice-cold solution of lactonase enzyme stock (phosphate, 50 mM, pH 8) and the suspension was stirred at 200 rpm for 20-30 hours. The liquid was decanted from the immobilized enzyme and the wet immobilizate was washed with phosphate (3 × 5 mL), treated with ethanolamine (20 mM, pH 8, 20 mL) and again phosphate (20 mM, Washed with pH 7, 2 × 5 mL) and then drained.

(4) Reaction conditions for hydrolysis resolution of (RS) -pantolactone:
Reactions were performed in 2 or 4 mL vials using 50 or 100 mg of biocatalyst and 1 or 2 mL of 0.5 racemic lactone (0.75 M triethylamine acetate containing 20 mM magnesium acetate, pH 8.5). The vial was incubated for the required time at 40 ° C., 200 rpm, the solution drained, analyzed by chiral HPLC, and the catalyst washed with fresh substrate solution (2 × 1 or 2 mL) before starting the next cycle. Samples for analysis were quenched with an equal volume of 500 mM MES buffer containing 50 mM EDTA, pH 5.5, centrifuged (10,000 g, 10 min) and then using a 0.46 × 15 cm CHIRADEX column. , 70:30 water-ethanol, 1 mL / min, analyzed by HPLC eluting at 20 ° C. Initial rates were measured at 15 minutes and E values were determined at the end of each cycle.

  The performance of selected immobilized enzyme preparations in batch splitting of (RS) -pantolactone is summarized in Table 2. In both sol-gel and covalent immobilization protocols, the catalyst left 62-72% of their initial activity even after 24 cycles of 1 hour batch reaction and enantiomeric excess and enantiomer of (R) -pantolactone It proved to be effective with a selectivity of 89-98% and 71-88% respectively. Furthermore, when tested in extended batch tests, ie when each batch reaction is allowed to proceed for one day instead of one hour, the catalyst performs in a very similar manner, providing good long-term operational stability. Show.

Example 14: Immobilized recombinant E. coli. Application of E. coli expressed Oal to continuous resolution of (RS) -pantolactone Sol-gel-PVA immobilized recombinant lactonase biocatalyst (E) prepared as described above in Example 14 (2) Was used for continuous resolution of racemic lactones in a packed bed reactor: immobilized biocatalyst (1.1 g, 0.21 kUg ⁻¹ , 0.231 kU total) And packed in a 0.46 × 15 cm Omnifit jacketed glass column fitted with a Teflon end piece. This was connected to a 1 × 10 cm Omnifit jacketed glass column packed with 1-2 mm cellulose beads supplied by two syringe pumps equipped with 50 mL glass / Teflon syringes. The column was connected to a circulating water bath and maintained at 40 ° C. during the split experiment. Feed tris-acetate buffer (100 mM containing 100 mM magnesium stearate, pH 7) at a rate of 5 ml / min for 30 minutes at room temperature, then racemic lactone (1.5 M in water) and tris-acetate buffer ( The reactor was conditioned by feeding together a solution of 2.5 M, pH 8.25) containing 50 mM macnesium acetate at a volume ratio of 1: 1 and a total flow rate of 2 mL / min for 30 minutes at room temperature. The flow rate was then reduced to 1.2 mL / min, the column was heated to 40 ° C. and the system was allowed to equilibrate for 15 minutes. The reactor is thus operated for about 45 minutes and the eluate is collected in an ice bath, under which conditions a total of 52.7 mL of feed (corresponding to 5.14 g of RSPL) lasts 46-48%. It was processed at the conversion rate. The pH of the eluate is adjusted to pH 6.5 with sulfuric acid (10% v / v / aqueous), the solution is extracted with dichloromethane (10 × 75 mL) and the organic layer is dried over anhydrous magnesium sulfate and rotary evaporated. Yielded concentrated (S) -pantolactone (2.67 g, 98% of theory) consisting of 10% RPL and 90% SPL (analyzed by chiral HPLC). The aqueous phase is acidified with sulfuric acid (40% w / w) to pH 1.5, heated to 70 ° C. for 1 hour, cooled in ice, saturated with sodium chloride, then extracted with dichloromethane (10 × 75 mL), The organic layer was dried over anhydrous magnesium sulfate and rotary evaporated to give concentrated (R) -pantolactone with a enantiomeric purity of 93% (2.23 g, 92% of the theoretical yield). The combined recovery of (R)-/ (S) -lactone was 95%.

  The results of Examples (13) and (14) show that the recombinant lactonase is an effective biocatalyst for the hydrolysis resolution of racemic pantolactone and that the catalyst can be effectively immobilized and It clearly shows that it can be applied to the production of highly concentrated (R) -pantolactone with an enantiomeric excess of 90-98% using both batch and continuous operations.

Example 15: Cloning and expression of (S) -pantolactone-specific lactonase from B. subtilis Using the amino acid sequences of lactonase from bovine and horse liver, we have limited homology to these sequences. Several other sequences shown were found. Among these, the yvre gene from B. subtilis (SMP-30 / CGR1 FAMILY / YVRE BATSU annotated as a member of the virtual 33.2 kDa protein) is the necessary restriction site for the latter cloning into the expression vector. It was cloned by PCR from genomic DNA using its 5′-end and 3′-end primers, including (EcoRI and PstI). PCR conditions were: The PCR conditions were the same as those used for isolation of the oal gene from niger genomic DNA (see Example 8). PCR products and vectors (PET41a from Stratagene (La Jolla, CA, USA)) were digested with EcoRI and PstI, cleaned by agarose gel electrophoresis, ligated and E. coli. E. coli Top10 cells (Stratagene, La Jolla, CA, USA) were transformed. Using this strategy, the gene is transformed into E. coli. Fused in frame to the gene for the GST protein from E. coli.

Expression and purification was performed according to the manufacturer's protocol. Additionally 5 mM CaCl ₂
The activity of the purified protein was determined at 40 ° C. using a standard assay containing The enzyme showed very high selectivity for (S) -pantolactone. The preparation had a specific activity of the concentrated fusion protein of 1-2 U / mg. The enzyme was only active in the presence of Mg ²⁺ or Ca ²⁺ ions.

Claims (7)

(I) the nucleotide sequence set forth in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9, or
(Ii) a nucleotide sequence encoding a protein having at least 95% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 and having pantolactone hydrolase activity
DNA consisting of (I) the nucleotide sequence set forth in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9, or
(Ii) a nucleotide sequence encoding a protein having at least 95% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 and having pantolactone hydrolase activity
An expression vector comprising DNA consisting of A host cell transformed with the expression vector according to claim 2 . (I) a protein comprising the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, or
(Ii) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, and having pantolactone hydrolase activity
Pantolactone hydrolase. For selective hydrolysis of R- or S-pantolactone, one or several amino acids are deleted, substituted or added in the protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 or the amino acid set forth in SEQ ID NO: 4. Use of pantolactone hydrolase consisting of a protein having an amino acid sequence and having a pantolactone hydrolase activity. Use of the pantolactone hydrolase according to claim 4 for the selective hydrolysis of R- or S-pantolactone. The pantolactone hydrolase according to claim 4, a protein comprising the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid set forth in SEQ ID NO: And a process for selectively hydrolyzing R- or S-pantolactone in the presence of pantolactone hydrolase comprising a protein having pantolactone hydrolase activity, and a method for producing R-pantothenic acid or a salt thereof or R-pantenol .