WO2004085651A2

WO2004085651A2 - Pantolactone hydrolase

Info

Publication number: WO2004085651A2
Application number: PCT/EP2004/002902
Authority: WO
Inventors: Iqbal Gill; Dahai Guo; Martin Lehmann; Dermot P. Moynagh; Rao Hanumantha Valivety
Original assignee: Dsm Ip Assets B.V.
Priority date: 2003-03-28
Filing date: 2004-03-19
Publication date: 2004-10-07
Also published as: GB0516602D0; GB2413329A; WO2004085651A3; JP2006521103A; KR20050119151A; KR101156882B1; JP4571936B2; CN1768141A; GB2413329B; CN1768141B

Abstract

The present invention relates to nucleotides sequences of a gene coding for a pantolactone hydrolase, as well as vectors and host cells containing such nucleotides sequences and pantolactone hydrolases encoded by such nucleotides sequences. Processes for making and using pantolactone hydrolases are also provided.

Description

Novel Enzymes

The present invention relates to enzymes useful for optical resolution of enantiomers of pantolactone, to genes encoding said enzymes, and to the use of said enzymes in a process for the preparation of R-pantothenic acid or a salt thereof or R-panthenol.

Enantiomers differ in their physiological, i.e. toxicological and pharmacological effects, their reaction with enzymes and their sensorial characteristics. Only R-pantolactone is known as an intermediate in the preparation of R-pantothenic acid, a vitamin, which is essential for humans and animals for growth, reproduction, and normal physiological functions. As precursor of coenzyme A and as acyl carrier protein of fatty acid synthetase, pantothenic acid is involved in more than hundred different metabolic pathways including energy metabolism of carbohydrates, proteins and lipids as well as the synthesis of lipids, neurotransmitters, steroid hormones, porphyrins and hormones. Until recently, a widely used technique for the preparation of R-pantolactone has been the optical resolution of chemically synthesized R- and S-pantolactone. Such a process is very cost intensive because it requires the use of expensive optical resolving agents. Moreover, the recovery of the R-enantiomer of pantolactone is rather difficult. Therefore, it is desirable to provide pantolactone hydrolases useful for the optical resolution of R- and S-enantiomers of pantolactone. The use of such enzymes, specifically hydrolyzing either the S- or the R-form of pantolactone, is a less expensive and easier to handle method for the separation of the two enantiomers of pantolactone. Examples of a salt of R-pantothenic acid include calcium R-pantothenate.

In one embodiment the present invention provides a nucleotide sequence of a gene coding for a pantolactone hydrolase, said pantolactone hydrolase being derived from horse liver, A. niger ATCC 9142, A. niger awamori ATCC 38854 or A. niger MacRae ATCC 46951. These strains are publicly available from the American Type Culture Collection (ATCC), 10801 University Boulvard, Manassas, VA 20110-2209 USA.

In another embodiment the invention provides a nucleotide sequence of a gene coding for a pantolactone hydrolase which comprises, when derived from horse liver, the nucleotide sequence as illustrated in SEQ ID NO: 1, when derived from A. niger MacRae ATCC 46951 the nucleotide sequence as illustrated in SEQ ID NO: 5, when derived from A. niger ATCC 9142 the nucleotide sequence as illustrated in SEQ ID NO: 7, and when

NS/08.03.2004 derived from A. niger awamoή ATCC 38854 the nucleotide sequence as illustrated in SEQ ID NO: 9.

The nucleotide sequences may comprise the coding sequences as well as the regulatory sequences of the respective pantolactone hydrolases as indicated.

A "regulatory sequence" is defined as an array of nucleic acid control sequences that direct transcription of an operably linked nucleic acid. An example of such an expression control sequence is a promoter. A "promoter" includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which 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" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

Fragments of the nucleotide sequences maybe used, e.g. as probes in hybridization assays.

In the case where the nucleotide sequence is inserted, transcribed and translated in a host organism to produce a functional polypeptide, one of skill will recognize that because of codon degeneracy a number of polynucleotide sequences will encode the same polypeptide. These variants are specifically within the scope of the present invention. In addition, the present invention specifically includes those sequences that are substantially identical (determined as described below) to each other and that encode polypeptides that are either mutants of wild type polypeptides or retain the function of the polypeptide (e.g., resulting from conservative substitutions of amino acids in the polypeptide). In addition, variants can be those that encode dominant negative mutants as 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 for maximum correspondence as described below. The terms "identical" or percent "identity", in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988), e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).

The phrase "substantially identical", in the context of two nucleic acids or polypeptides, refers to sequences or subsequences that have at least 60%, preferably 80%, most preferably 90-95%, nucleotide or amino acid residue identity when aligned for maximum correspondence over a comparison window as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to a sequence of which the complement of that sequence hybridizes to the 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 alternative 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.

A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, in which a sequence maybe compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acid codons encode any given protein. For instance, 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 altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily 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 implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, or substitutions to a peptide, polypeptide, or protein sequence which alter a single amino acid or a small percentage of amino acids (i.e. less than 20%, such as 15%, 10%, 5%, 4%, 3%, 2% or 1%) in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

The following six groups each contain amino acids that are conservative substitutions for one another:

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, e.g., Creighton, Proteins (1984)).

An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another 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 to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).

The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target sequence, typically in a complex mixture of nucleic acid sequences, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, highly stringent conditions are selected to be about 5- 10°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. Low stringency conditions are generally selected to be about 15- 30°C below the T_m. The T_m is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., more than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times 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 that 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 acids typically hybridize under moderately stringent hybridization conditions.

In the present invention, genomic DNA (gDNA) or cDNA containing nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. For the purposes of this disclosure, suitable stringent conditions for such hybridizations are those which include hybridization in a buffer of 40% formamide, 1 M NaCl, 1% sodium dodecyl sulfate (SDS) at 37°C, and at least one wash in 0.2X SSC at a temperature of at least about 50°C, usually about 55°C to about 60°C, for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.

A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, that can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot.

The present invention also includes expression vectors as defined herein. The expression vectors include one or more copies of each of the polynucleotide sequences set forth above. The expression vectors of the present invention may contain any of the polynucleotide sequences defined herein.

Therefore, in a further embodiment the present invention provides an expression vector comprising a nucleotide sequence of a gene coding for a pantolactone hydrolase, said pantolactone hydrolase being derived from horse liver, Bacillus subtilis, A. niger ATCC 9142, A niger awamori ATCC 38854 or A. niger MacRae ATCC 46951, or an expression vector comprising a nucleotide sequence of a gene coding for a pantolactone hydrolase derived from horse liver as illustrated by SEQ ID NO: 2, from Bacillus subtilis as illustrated by SEQ ID NO: 4, from A. niger ATCC 9142 as illustrated by SEQ ID NO: 8, from A. niger awamori ATCC 38854 as illustrated by SEQ ID NO: 10, and from A. niger MacRae ATCC 46951 as partially illustrated by SEQ ID NO: 6.

The polynucleotide sequences in the expression vectors may optionally be operably linked to an expression control sequence as defined above.

As used herein, the phrase "expression vector" is a replicatable vehicle that carries, and is capable of mediating the expression of, a DNA sequence encoding the polynucleotide sequences set forth herein. In the present context, the term "replicatable" means that the vector is able to replicate in a given type of host cell into which it has been introduced.

Immediately upstream of the polynucleotide sequence(s) of interest, there maybe provided a sequence coding for a signal peptide, the presence of which ensures secretion of the encoded polypeptide expressed by host cells harboring the vector. The signal sequence may be the one naturally associated with the selected polynucleotide sequence or of another origin.

The vector may be any vector that may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication; examples of such a vector are a plasmid, phage, cosmid or mini-chromosome. Alternatively, the vector ma be one which, when introduced in a host cell, is integrated in the host cell genome and is replicated together with the chromosome(s) into which it has been integrated. The expression vector of the invention may carry any of the DNA sequences of the invention and be used for the expression of any of the polypeptides of the invention.

The present invention also includes cultured cells or host cells containing one or more of the polynucleotide sequences and/or one or more of the expression vectors disclosed herein. As used herein, a "cultured cell" includes any cell capable of growing under defined conditions and expressing one or more of polypeptides encoded by a polynucleotide of the present invention. Preferably, the cultured cell is a yeast, fungus, bacterium, or alga. More preferably, the cultured cell is Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Aspergillus niger or Bacillus subtilis.

In another embodiment the present invention provides a pantolactone hydrolase derived from horse liver, A. niger ATCC 9142, A. niger awamori ATCC 38854 or A. niger MacRae ATCC 46951.

In yet another embodiment the present invention provides a pantolactone hydrolase the amino acid sequence thereof comprising an amino acid sequence being illustrated by SEQ ID NO:2 when derived from horse liver, by SEQ ID NO: 6 when derived from A. niger MacRae ATCC 46951, by SEQ ID NO: 8, when derived from A. niger ATCC 9142 and by SEQ ID NO: 10, when derived from A. niger awamori ATCC 38854.

The pantolactone hydrolases derived from horse liver as illustrated by SEQ ID NO: 2 and from Bacillus subtilis as illustrated by SEQ ID NO: 4 exhibit an S-pantolactone hydrolase activity. The pantolactone hydrolases derived from A. niger ATCC 9142 as illustrated by SEQ ID NO: 8, from A. niger awamori ATCC 38854 as illustrated by SEQ ID NO: 10 and from A. niger MacRae ATCC 46951 as partially illustrated by SEQ ID NO: 6 exhibit an R- pantolactone hydrolase activity.

In a further embodiment the present invention comprises fragments of the above amino acid sequences, said fragments having the enzymatic activity of the complete protein.

"Fragments of an amino acid sequence" according to the present invention can be defined as sequences depleted of stretches of amino acids, which maybe necessary for a proper folding of a protein. However, once the protein is folded the amino acid stretches can be deleted without destroying the function of the respective protein. These stretches may be present in the folded protein as loops, or may be N- or C- terminal sequences not being of direct relevance for the activity of the folded protein.

Moreover, the present invention encompasses derivatives of the proteins comprising the enzyme activity of said R- and S-pantolactone hydrolases. Said derivatives include immobilized enzymes, e.g. immobilized by inclusion, wherein the enzyme is retained within a membrane device such as a hollow fiber, a polymeric network, or a microcapsule. The enzyme may also be immobilized by cross-linking or by the generation of crystals of the enzyme. A further possibility to immobilize the enzyme is its linkage to a growing polymer such as silica sol gels or silica graphite sol gels or by binding the enzyme to a prefabricated carrier such as EupergitC, Eupergit 250L, PEG, Celite and Amberlite XAD-7. A further technique for the immobilization of enzymes applicable for the R- and S-pantolactone hydrolases of the present invention is their dispersion in water immiscible organic solvents. In general, enzymatically active proteins such as the enzyme of the present invention can be immobilized by almost all of the immobilizing techniques known in the art.

In yet another embodiment the present invention is concerned with the use of the isolated pantolactone hydrolases for a selective hydrolyzation of either R- or S- pantolactone applicable in the industrial production of the R-enantiomer of pantolactone which is a precursor for the production of R-pantothenic acid and R- pantothenol.

Thus, it is an object of the present invention to provide a process or a method for a selective hydrolyzation of either R- or S-pantolactone using the isolated pantolactone hydrolases described herein.

In another embodiment the present invention provides a process for the production of R-pantothenic acid or a salt thereof or of R-pantothenol comprising the step of selective hydrolyzation of either R- or S-pantolactone in the presence of the pantolactone hydrolase of the invention.

In a further embodiment the invention provides a process for the cloning of a nucleotide sequence directly adjacent to a known sequence, e.g. a nucleotide sequence encoding a novel pantolactone hydrolase or parts thereof.

In particular, the present invention comprises a new PCR strategy enabling the amplification of unknown sequences. The new PCR strategy comprises the use of one primer for both the synthesis of the first and second DNA strand. The primer hybridizes to an already known area of the gDNA to be amplified. The primer shows 100% homology to the Icnown sequence. With the help of this primer a first PCR is carried out providing a first single strand copy of the genomic DNA, reaching into the unknown sequence. Using a small aliquot of the first PCR reaction as template a second PCR is carried out using a primer which hybridizes to a DNA fragment located in the 3' direction of the primer ("nested") used in the first PCR but logically still in the known part of the sequence. After random hybridization of this nested primer to single- stranded DNA, which was produced in the first polymerase reaction, the opposite strand of this single-stranded DNA is generated, which itself then serves as a template for the same primer as used for the second PCR producing the second strand. At the end, a DNA fragment that starts inside the known sequence and proceeds into the unknown adjacent genomic DNA or any other DNA for which part of the sequence is missing is generated by one primer that hybridizes on both ends of the new fragment.

Therefore, in another embodiment the present invention provides a process for the cloning of a nucleotide sequence directly adjacent to a known sequence the process comprising a PCR amplification comprising: a) the use of a single-stranded DNA of a first PCR as template for a second PCR, using a primer in said second PCR that randomly hybridizes to the single-stranded DNA produced in the first PCR; and b) thereby generating the opposite strand which itself serves as template for the second strand still using the randomly hybridizing primer of step a).

The following examples give a detailed description of the identification, characterization and expression of the genes coding for pantolactone hydrolases. The examples are illustrative only and are not intended to limit the scope of the invention in any way.

Example 1: Purification of the (S) -pantolactone hydrolyzing activity from a commercially available horse liver esterase acetone powder

1 g horse liver esterase acetone powder (Fluka Holding AG, Industriestrasse 25, P.O. Box 260, CH-9471 Buchs, Switzerland) was dissolved in 40 ml 50 mM Tris/HCl, pH 7.5 containing 2 M ammonium sulfate, 1 mM CaCl₂, 1 mM MgCl₂₎ and 10 μM EDTA (buffer A). Undissolved material was separated by centrifugation and, after that, by sterile filtration through a 0.45 μM filter. The protein solution was applied onto a butyl sepharose column HR 26/10 (Amersham Pharmacia Biotech Europe GmbH, Dϋbendorf, Switzerland) equilibrated with buffer A. The column was washed with 100 ml buffer A. After this a 400 ml linear gradient from 0 to 100% buffer B (buffer A without ammonium sulfate) was applied. The lactonase eluted in the middle of the gradient. Fractions showing pantolactone hydrolyzing activity were pooled, the buffer was exchanged to 20 mM Tris/HCl, pH 8.0, 1 mM CaCl₂₎ 1 mM MgCl₂, and 10 μM EDTA. The pooled fractions were applied onto a MonoQ pre-packed column HR 26/10 (Amersham Pharmacia Biotech Europe GmbH, Dύbendorf, Switzerland) using a linear gradient from 0 to 350 mM NaCl. The protein eluted in the beginning of the gradient. Again, active fractions were pooled, concentrated to 1 ml and applied onto a Superdex 200 column (Amersham Pharmacia Biotech Europe GmbH, Dϋbendorf, Switzerland). With 50 mM Tris/HCl, pH 8.0, 1 mM CaCl₂, 1 mM MgCl₂, and 10 μM EDTA as elution buffer, the lactonase eluted as the last peak indicating a molecular size of about 31 kDa, which is in line with the size as indicated by a SDS-PAGE.

The highest specific activity found was 13.3 U/(mg protein) determined by incubating the enzyme at 37°C for 60 min in 200 mM Tris/acetate buffer, pH 9.0, 5 mM MgCl₂. The concentrations of (S)- and (R) -pantolactone were determined by HPLC.

Example 2: Preliminary characterization of the (S)-pantolactone hydrolyzing enzyme from horse liver

(a) Molecular size, structure, and iso-electric point:

According to the SDS-PAGE, the denatured protein has a molecular mass of about 35 kDa. Using the elution time of a calibrated gel filtration column, the molecular mass of the native protein was calculated to be about 31 kDa. This means that the lactonase from horse liver is a monomer of 30 to 35 kDa. Using a 2D gel, the iso-electric point was determined to be in the range of pH 5.5.

(b) N-terminal and internal amino acid sequencing: The enriched protein after 2D gel electrophoresis was digested by trypsin. The generated peptides were analyzed by mass spectrometry. The N-terminal sequence of peptide No. 66 is illustrated as SEQ ID NO: 11.

This sequence together with the MS-spectrum indicated that the protein belongs to the class of proteins known as senescence marker proteins-30 or regucalcins, which have been mainly found in vertebrates. The regucalcin sequences from rat, mouse, man, chicken, rabbit, cow, Xenopus laevis, and two less homologous sequences from Drosophila melanogaster have been already determined. However, the gene from horse has not been isolated and sequenced yet.

(c) Metal dependency: To test the effect of different divalent metal ions on the pantolactonase activity of the horse liver enzyme, 1.3, 2.6, 5.2, or 10.4 mM of Ca²⁺, Mg²⁺, or Mn²⁺ 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 for 60 min at 37°C and stopped with an equal volume of methanol containing 2 mM EDTA. The decrease of (S) -pantolactone was determined by HPLC. While calcium reduced the enzyme activity, magnesium and manganese increased the activity of the enzyme.

(d) Thermostability:

Purified enzyme was incubated in 5.2 mM MgCl₂ and 200 mM Tris/acetate, pH 9.0, for 15 min at temperatures between 30 and 80°C. After 30 min on ice, the activity was determined with the standard assay (60 min, 37°C). The enzyme was stable up to 60°C. It started to inactivate at 65°C and lost more than 50% of its activity at 70°C.

Example 3: Cloning of the (S)-pantolactonase from horse liver

The sequence information from all known mammalian genes (see below) was used to design primers for PCR on cDNA made from horse liver from a one-year-old "Hafflinger" from Bavaria, kindly provided by Roche Molecular Biochemicals, Penzberg, Germany. The QIAGEN RNeasy Maxi Kit (Qiagen, Hilden, Germany) was used to prepare total RNA from the liver tissue.

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

Table 1: Amino acid sequence similarity and identity among the regucalcin family.

Chick: Chicken; Xlaev: Xenopus laevis; D.mela: Drosophila melanogaster

A 885 bp fragment (nucleotides 1-885 of SEQ ID NO: 1) was amplified by RT-PCR using the Titan One Tube RT-PCR Kit (Roche Molecular Biochemicals, Penzberg, Germany) and homology primers covering the start codon and a sequence 50 bp upstream from the stop codon. The reaction was performed as recommended by the supplier. Construction of the N-terminal 5'-primer (SEQ ID NO: 12) and C-terminal 3'-primer (3'-CAGTTTCCTTAAGGGGGGATA-5') (SEQ ID NO: 13) for the first PCR based on N- terminal sequences derived from bovine (SEQ ID NO: 14), rabbit (SEQ ID NO: 15), human (SEQ ID NO: 16), rat (SEQ ID NO: 17), mouse (SEQ ID NO: 18), chicken (SEQ ID NO:19) and Xenopus (SEQ ID NO: 20), and C-terminal sequences derived from bovine (SEQ ID NO: 21), rabbit (SEQ ID NO: 22), human (SEQ ID NO: 23), rat (SEQ ID NO: 24), mouse (SEQ ID NO: 25), chicken (SEQ ID NO: 26) and Xenopus (SEQ ID NO: 27).

The fragment was cloned into a TA cloning vector (Invitrogen, Carlsberg, CA, USA). Sequencing proved that it stemmed from the horse liver regucalcin. The missing 5'-end was isolated by a method called 573' RACE using the respective kit supplied by Roche Molecular Biochemicals, Penzberg, Germany: a perfectly matching primer (SEQ ID NO: 28) was used to produce single stranded DNA on isolated horse liver cDNA. The single stranded DNA was purified, a polyC tail was added and another PCR was performed using a nested primer 5' of the first primer and a polyG primer. A DNA fragment was amplified that represented the missing 5'-end of the gene. The 3'-end was cloned using a forward primer (SEQ ID NO: 29) made according to the already known sequence and one primer (SEQ ID NO: 30) starting with the last amino acid of the gene, which was designed mainly according to the bovine and rabbit gene. In a last PCR, using the obtained sequence information for the design of 5'- and 3'-primer covering the start and the stop codon of the gene, the entire cDNA of the lactonase was isolated from the total RNA as described above. 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 expressed in E. coli fused C-terminally to a His-tag using one of the commercially available expression vectors like the pQE60 from Qiagen. Expression and purification of the His-tagged protein according to standard protocols yielded a protein of 33 kDa that showed (S) -pantolactonase activity.

Example 4: Purification of the (- )-pantolactone hydrolyzing activity from commercial crude preparations of an A. niger lipase A

An activity specifically hydrolyzing (jR)-pantolactone was identified in different commercially available enzyme products stemming from A. niger fermentations. For purification of the respective enzyme, the commercial preparation called Lipase A "Amano" from Amano Pharmaceuticals Co. Ltd., Nagoya, Japan, was used. According to the sequence data which were obtained from two enzymes that were co-purified together with the lactonase activity, it appeared that the preparation represents the lyophilized and broken cells (including the fermentation broth) of A. awamori over- expressing the lipase from A. tubigensis. The material was dissolved in 20 mM Tris/HCl, pH 7.5, 2.5 mM MgCl₂ (standard buffer). 5% ethanol was added to increase the solubility of the material. Material, which did not go into solution was separated by centrifugation (30 min, 15000 rpm, 4°C) followed by a filtration through a 0.45 μm filter. The dissolved part was first purified by ultrafiltration in an Amicon cell (50 kDa cut-off) to remove any salts and compounds with a small molecular size, which could disturb the following anionic exchange chromatography step. Then the protein was applied onto a HR 26/10 prepacked Q-sepharose column (Amersham Pharmacia Biotech Europe GmbH, Dύbendorf, Switzerland). The column was washed with 200 ml standard buffer, and then the proteins were eluted by a linear gradient (400 ml) from 0 to 350 M NaCl. Fractions containing activity were pooled. Ammonium sulfate was added to a final concentration of 1.5 M and the so treated protein solution was applied onto a HR 26/10 butyl sepharose pre-packed column (Amersham Pharmacia Biotech Europe GmbH, Dϋbendorf, Switzerland). After 200 ml of washing with standard buffer containing 1.5 M ammonium sulfate, the proteins were separated by 1.5 to 0 M decreasing ammonium sulfate gradient (400 ml). Here the activity eluted in two peaks, which 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 the standard buffer. The active fractions of each separation were pooled and concentrated. The activity of pool II tended to elute a little earlier than that of pool I, which indicates that the molecular weight of the corresponding protein might be slightly higher.

80 U/ml (40°C) was the highest activity yet determined for pool I and pool II, respectively. As shown on the 2D gels, the protein preparation is not fully homogenous meaning that the real specific activity is even higher. However, because the lactonase activity corresponded to a protein band of 38 kDa in the elution profile of the gel filtration, which was also the dominating protein of the final preparation, this band represented most probably the lactonase. According to the gel filtration, the native lactonase has a molecular mass of 75 kDa. This suggests homodimeric structure of the enzyme.

Example 5: Preliminary characterization of the (j )-pantolactone-hydrolyzing enzyme

(a) Structure and molecular weight of the native form:

The enzyme eluted on a Superdex 200 (Amersham Pharmacia Biotech Europe GmbH, Dϋbendorf, Switzerland) at 72 ml using the conditions as described in Example 4. As deduced from a standard curve, this reflected a molecular mass of 75 kDa. SDS-PAGE indicated a molecular mass of 38 kDa. The combination of the data suggested that the (R) -pantolactonase from A. niger is a homodimer consisting of two identical subunits of 38 kDa.

(b) Thermostability:

50 μl diluted enzyme (0.1 U) was incubated at 0, 30, 40, 50, 55, 60, 70°C in 20 mM Tris/HCl, pH 7.5 for one hour. After 15 min on ice, the residual activity was determined using the standard assay. The enzyme was stable up to 50°C. At higher temperatures, it started to loose its activity.

(c) Temperature optimum:

The strongly enriched (R) -pantolactonase from the commercial source (see Example 5) was incubated for 30 min at 20, 30, 40, 50, 55, and 60°C under the following conditions: 100 mM (R, S) -pantolactone 100 mM MgCl₂ 200 mM Tris/HCl, pH 8.0 0.05 U (i?)-pantolactonase in 100 μl final volume.

The temperature optimum of the (J?) -pantolactonase from the commercial A. niger preparation was around 50°C under the conditions used.

(d) Cofactor dependence and inhibition:

The (R) -pantolactonase from A. niger needs Mg²⁺ or Mn²⁺ ions for its activity. It was totally inhibited by the addition of 1 mM EDTA.

(e) pH-optimum:

The enzyme did not show a "normal" pH-activity profile, when the pH was varied from 7.0 to 11.0. 10 mg Amano Lipase A in 200 mM TEA- acetate buffer (pH 7.0 to 11) containing 0.1% Span 80, and 20 mM of magnesium acetate were incubated for 20 min at 40°C. Samples were quenched with an equal volume of 500 mM MES buffer, pH 5.5, containing 50 mM EDTA, centrifuged (10,000 g, 10 min), then analyzed by HPLC using a 0.46x15 cm CHIRADEX column eluted with 70:30 water-methanol, 1 ml/min, 20°C. The highest activity is reached around pH 10. From there to pH 11.0, the activity did not change considerably. The highest selectivity was reached around pH 9.0.

Example 6: Cultivation of A. niger ATCC 9142, 9029, 26875, 62863, and 10864 and evaluation of its (_J )-pantolactonase activity

In order to find out whether the lactonase activity found is wide-spread in the genus Aspergillus, five A. niger strains were cultivated under the following conditions: Medium (amounts per liter): 1.2 g NaNO₃, 0.5 g KH₂PO₄, 0.2 g MgSO4x7 H₂O, 0.5 g yeast extract, 5% glucose and 0.04 ml of the trace metal solution as described by Vishniac and Santer, Bacteriol. Rev. 21:195-213 (1957).

5xl0⁶ spores per ml were used to inoculate a 300 ml pre-culture in a 1-1 flask at 30°C. After 8 h, the pre-culture was added to 1.71 medium in a 21 fermentor with control of pH and dissolved oxygen. The pH was held constant at pH 5.5 by automatic addition of 5 M NaOH. The cells were grown for 14 h under low aeration (5-10% air saturation). Afterwards the dissolved oxygen level was increased to 30-50% air saturation. 5 h later the mycelium was harvested, filtered, washed twice with de-mineralized water and frozen in liquid nitrogen. A part of it was directly disrupted by using a French Press. Medium and cell lysate were investigated for pantolactonase activity using the standard assay.

(R) -pantolactonase activity was mainly found in the lysate of all A. niger strains tested. Only a very small activity was found in the medium.

Example 7: Determination of the amino acid sequence of small peptides generated by trypsin digestion of the enriched (j )-pantolactonase from the lipase preparation

The enriched preparation of Example 5 was analyzed on a 2D gel. The two major spots at 33 and 40 kDa and pi's of 4.7 and 5.6, respectively, were digested with trypsin. The generated peptides were separated by HPLC (Instrument: Hewlett Packard 1090, Column: Vydac C18 (0.21 x 25 cm), absorption at 210 nm, Flow: 0.20 ml/min, Buffer A: 0.075% TFA, Buffer B: 0.065% TFA in 80% acetonitrile, Gradient: 2% B (0-lOmin), 2- 75% B (10-120 min). A selection of the peptides was sequenced by Edman degradation, spot C [pi 5.6, 39 kDa (Poll C)]:Fxn 42 (peptide 1)(SEQ ID NO: 81), Fxn 57 (peptide 3) (SEQ ID NO: 82), Fx 73 (peptide 2) (SEQ ID NO: 83), Fx 74(SEQ ID NO: 84) and Fx 81(SEQ ID NO: 85), spot C [pi 4.7, 33 kDa (Poll A/B)]: N-terminus(SEQ ID NO: 86), Fx 58(SEQ ID NO: 87), Fx 64(SEQ ID NO: 88), Fx 65(SEQ ID NO: 89), Fx 73(SEQ ID NO: 90) and Fx 68(SEQ ID NO: 91). X stands for an undefined amino acid, while an amino ' acid in parenthesis is one that could not be unequivocally determined.

A database search using all short peptide sequences as targets resulted in no reliable information that could help to identify which of the two major spots represents the lactonase. Therefore, we enriched the lactonase preparation of Example 5 even more by another anionic exchange chromatography. The protein solution (in 20 mM Tris/HCl, pH 7.5, 2.5 mM MgCl₂) was separated on a pre-packed MonoQ column HR 16/10 (Amersham Pharmacia Biotech Europe GmbH, Dϋbendorf, Switzerland) in a 0 to 350 mM NaCl gradient. The activity of all fractions was determined. Active fractions were analyzed by SDS-PAGE and by iso-electric focusing performed as described by the supplier (Invitrogen, Carlsbad, CA, USA). Comparing the activity data of the fractions with the gels of the SDS-PAGE and the IEF suggested that the lactonase is a protein of 38 to 40 kDa and it has a pi around pH 5.6. This data corresponded very well to spot PollC. Therefore, the amino acid sequences of this spot were used for designing PCR primers and oligonucleotides for Southern blots. The most active fractions were pooled and analyzed again on a 2D gel, which showed that PollC was clearly enriched over PolLA/B in comparison to the former 2D gel.

Example 8: Design of degenerated oligonucleotides for Southern blots and PCR primers for cloning of the (_J )-pantolactonase from A. niger

The codon usage of Aspergillus generated by the program CODONFREQUENCY from the GCG program package version 10.2 using 12 different phytase sequences from different Aspergillus species was used to reduce the number of different DNA sequence combinations possible because of the degeneration of the genetic code. While in the 3'- end of the primers every possible combination was realized, some rare codons were omitted in the 5-'end of the primer using the Aspergillus codon usage as decisive factor. In the case of the oligonucleotides intended for hybridization experiments, the 3'- and 5'- end were treated the same way. Oligonucleotides, the name of which contains an "S (sense)" or an "AS (anti-sense)", were used for PCR. Oligonucleotides the name of which does not contain an S or AS were intended for use in hybridization experiments.

Oligonucleotides used for cloning of the (J?) -pantolactonase from A. 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: Isolation of genomic DNA from A. niger ATCC 9142, A. niger NRRL

3135, A. niger ssp. awamori (ATCC 38854) and A niger MacRae (ATCC 46951)

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)] was inoculated with lxlO⁶ spores/ml. The cultures were cultivated overnight at 30°C under vigorous shaking (200 rpm). The mycelium was collected by filtration through Whatman filter paper, washed with PBS (isotonic phosphate buffer) and frozen in liquid nitrogen. The mycelium was ground to a fine powder in an ice-cold mortar. The powder was re-suspended in 1/3 volume of 50 mM Tris/HCl, pH 8.0, 1.0% SDS, 50 mM EDTA and was incubated for 15 min at 65°C 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 h. Another 100 mg/ml proteinase K was added and the incubation was continued for another 3 h. 1/3 volume of phenol/chloroform/isoamylalcohol (50/49/1) was added. After thorough but gentle mixing, the emulsion was centrifuged (12000 g, 20 min, 4°C). The aqueous phase was removed and extracted again. After another centrifugation step (12000 g, 10 min, 4°C), the aqueous phase was extracted with chloroform. 0.7 volumes isopropanol was added to the aqueous phase and the solution was mixed gently for 15 min. The precipitated DNA was collected by centrifugation (10000 g, 30 min, 4°C). 1 /10 volume of 3 M KCl, pH 5.2 was added to the remaining supernatant and incubated under gentle shaking for 15 min. After another centrifugation step, the two pellets were washed twice with 70% ethanol, dried on air, and dissolved in 0.5 ml water. Finally, the DNA was treated with RNase to remove residual RNA.

Example 10: Cloning of the (R)-pantolactonase from A. niger ATCC 9142

Genomic DNA (gDNA) of A. niger ATCC 9142 was prepared as described in Example 6. The Robocycler Infinity from Stratagene (Stratagene, La Jolla, CA, USA) as PCR machine, and the TAQ polymerase Kit from Roche Molecular Biochemicals (Penzberg, Germany) were used for the PCRs:

1 μl gDNA (1/10 diluted) step 1 5 min - - 95°C

1 μl nucleotide mix step 2 30 sec - - 95°C

5 μl PCR standard buffer with Mg²⁺ step 3 30 sec - - 50°C

1 μl primer S (100 pM/μl) step 4 1 min - - 72°C

1 μl primer AS (100 pM/μl) 1 μl TAQ polymerase from Roche Molecular Biochemicals

40 μl H₂O

Steps 2 to 4 were repeated 35-times. Primer combinations:

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

The faint 840 bp product of PCR reaction 12 was isolated from the agarose gel using the QIAEX II gel extraction l t from Qiagen (Qiagen, Hilden, Germany) and dissolved in 40 μl H₂0.

1 μl 840 bp fragment (1/10 diluted) step l: 5 min - 95°C 1 μl nucleotide mix step 2: 30 sec - 95°C 5 μl PCR standard buffer with Mg²⁺ step 3: 30 sec - 55°C 1 μl primer S (10 pMol/μl) step 4: 30 sec - 72°C 1 μl primer AS (10 pMol/μl)

1 μl TAQ polymerase from Roche Molecular Biochemicals 40 μl H₂O Steps 2 to 4 were repeated 30-times.

Primer combinations of reactions 2, 5, 10, 11, and 12 were used. Beside reaction 2, every other reaction resulted in a PCR product. 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 of interest. This assumption was supported by sequencing. While the amino acid sequence of peptide Fx 57 (SEQ ID NO: 90) was confirmed in the 840 bp fragment, the sequence of peptide Fxn 42 (SEQ ID NO: 45) could only partly be confirmed.

It was also possible to isolate the corresponding DNA fragment of A. niger MacRae. In the case of A. awamori only the product of primers Fxn57S and Fx73AS could be isolated by this approach. The sequences of these Aspergϊlli have around 90% identity on DNA level to the sequence of A niger ATCC 9142.

To isolate the 5'- and 3'-end of the lactonase gene, the following approaches were used:

(a) Isolation of the 3'-end:

1 μl gDNA of A niger ATCC 9142 step 1: 5 min - 95°C

1 μl nucleotide mix step 2: 30 sec - 95°C

5 μl PCR standard buffer with Mg²⁺ step 3: 30 sec - 55°C

1 μl primer Oal2AS or Oal3S (10 pMol/μl) step 4: 30 sec - 72°C 1 μl TAQ polymerase from Roche Molecular Biochemicals 41 μl H₂O

Steps 2 to 4 were repeated 30-times.

The PCR mixtures were extracted two times with an equal volume of phenol/ chloroform and one time with chloroform alone. Then the DNA was precipitated by adding 1/10 volume of 3 M potassium acetate, pH 5.2 and 2 volumes of ethanol. After 15 min of centrifugation (14000 rpm in a Eppendorf table centrifuge), the pellet was washed with 70% ethanol, air dried and finally dissolved in 20 μl sterile water.

Second PCR:

4 μl DNA from first PCR step 1 : 5 min - 95°C 1 μl nucleotide mix step 2: 30 sec - 95°C

5 μl PCR standard buffer with Mg²⁺ step 3: 30 sec - 58°C 1 μl primer Oal4S or OallAS (10 pMol/μl) step 4: 1 min - 72°C 1 μl High Fidelity Mix from Roche Molecular Biochemicals

38 μl H₂O Steps 2 to 4 were repeated 35-times.

Using the Oal4S primer, a DNA molecule of 900 bp was generated. Sequencing revealed that it codes for the 3'-end of the gene.

(b) Isolation of the 5'-end: Hybridization experiments were done on the genomic DNA of A. niger ATCC 9142 using the DIG-labeled oligonucleotides Fxn42, Fxn57, Fx73, and Fx74 as probes and the DIG- system with chemiluminescent detection on X-ray films (Roche Molecular Biochemicals). Hybridization and detection was done as recommended by the supplier.

10 μg genomic DNA was digested with BαraHI, EcoRI, Hindlll, Pstl, Sad, and Xhol. Hybridization was done overnight at 42°C using the Roche Molecular Biochemicals hybridization solution. After two washing steps at room temperature in 2x SSC, 0.1% SDS, two washing steps at 50°C in 0.5x SSC, 0.1% SDS followed. After the detection procedure with CSPD (Disodium 2-chloro-5-(4-methoxyspiro{l,2-dioxetane-3,2'-(5'- chloro)tricyclo[3.3.1.1 '⁷] decan}-4-yl)-l-phenyl phospate) as substrate of the alkaline phosphatase, an X-ray film was exposed for 1 h (room temperature) to the filter. There was no usable signal on the films.

Therefore, the random-primed DIG-labeled PCR products of reactions 5 and 11 (see Example 11) were used as probes. The hybridization was done in the same way, just the washing conditions were slightly changed to 2 x 5 min at RT, and 2 x 15 min at 55°C. The following bands appeared on the film:

To clone the entire gene or at least the missing 5'-end of it, the chromosomal DNA of A niger ATCC 9142 was digested with Hindlll which generates a ca. 4 kb fragment that contained the lactonase gene according to the hybridization experiments:

20 μl chromosomal DNA 20 μl 10 x buffer 5 μl HmdIII (10 U/μl) 175 μl H₂O

Incubation for 16 h at 37°C.

The digested DNA was purified with the PCR purification kit from Qiagen (Qiagen, Hilden, Germany) and eluted in 100 μl. The following ligation was done overnight at 16°C. The purified DNA was diluted to 2 ml with water. Ligase and ligase buffer was added according to the recommendation of the manufacturer (Roche Molecular Biochemicals). The completed ligation was used as template for different PCRs using internal primers of the partly isolated lactonase gene:

5 μl ligation solution step 1: 5 min - 95°C 1 μl nucleotide mix step 2: 30 sec - 95°C

5 μl PCR standard buffer with Mg²⁺ step 3: 30 sec - 55°C

1 μl sense primer (10 pMol/μl) step 4: 3 min - 72°C

1 μl anti-sense primer (10 pMol/μl)

1 μl High Fidelity polymerase mix from Roche Molecular Biochemicals 40 μl H₂O

Steps 2 to 4 were repeated 32-times.

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

The fragments were isolated from the gel using the QIAEX II gel extraction kit from Qiagen and sequenced with an ABI 310 Genetic Analyzer using the dideoxy method as recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA). The obtained sequence confirmed that the isolated fragment contained both ends of the (R)- pantolactonase gene from A. niger ATCC 9142.

Using primers OallOS and Oall2AS, it was also possible to isolate the homologous gene of A. niger ssp. awamori, which contained the exact amino acid sequence of all peptides sequenced. The only exception is the sequence of peptide Fxn 42. Fxn 42 resulted from sequencing of two peptides at the time. Most probably two different peptides eluted at the same time in the HPLC runs separating the trypsin digested enzyme fragments. The sequencing first revealed only the sequence of the major peptide with, however, a strong background. After finishing the first peptide, the sequence of the second peptide was the remaining one, which went on for another 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 K-P-F-A-H-Q-V-K-T-x-E-D (SEQ ID NO: 45), which is Fxn 42

This also explains why oligonucleotides Fxn42S and Fxn42AS, which were based on peptide Fxn 42, gave no 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), while the amino acid sequence from A. niger ssp. awamori has a calculated molecular mass of 36547 Da and an estimated E_280nm of 1.831 for a 1 mg/ml protein solution (Pace et al., 1995).

Example 11: Cloning and expressing the (J?)-pantolactonase gene from A. niger ATCC 9142

The sequences obtained by all three methods described in Example 10 were compared and assembled to generate a continuous sequence of 2443 bp covering the entire lactonase gene. In order to determine the start and the stop codon and possible introns of the gene, the available amino acid sequences of the peptides obtained from the purified gene from the commercial preparation were used together with the results of the two programs TESTCODE and CODONPREFERENCE of the GCG program package release 10.2. While the stop codon and one intron were determined by both programs in the same way, the start codon was not obvious, in particular because there was no other sequenced peptide available N-terminally of peptide Fx 74 (SEQ ID NO: 84). Also a comparison of all possible translations to homologous amino acid sequences found during a database search did not help, because the N-terminal part of the proteins appeared to be quite heterogeneous. Therefore, two methionine residues, which were found upstream of the location of peptide Fx 74 but in the same reading frame, were selected and both were used for the construction of respective expression cassettes. A CT-rich region was found between base pairs 666-742 of SEQ ID NO: 7. However, the location of this CT-rich region did not really favor one Met over the other as the start codon, because the distance between this region and the start codon usually varies quite a bit in fungi. Also, a possible polyadenylation site was identified downstream of the gene.

In the case of the 72 bp long intron of the gene from A. niger ATCC 9142, the DNA stretch from nucleotide 976 to 981 represents most probably the 5'-splice site, the stretch from nucleotide 1029 tol035 or from 1016 to 1022 the putative internal consensus sequence, and the DNA stretch from 1045 to 1047 the 3'-splice site (SEQ ID NO: 7). The corresponding sequences of the A. niger MacRae gene are located from bp 32 to 37, 66 to 72, and 80 to 82 in the 51 bp long intron (SEQ ID NO: 79). In the case of the 50 bp long intron of the oal gene of A niger ssp. awamori, the 5'-splicing site starts at nucleotide 203 (GTGCCC), the internal consensus region at nucleotide 236 (AACTAAC), and the 3'- splicing site at nucleotide 250 (CAG) (SEQ ID NO: 9).

The consensus sequence for the 5'-splice site is GTPuNGPy. None of the listed sites has a G on position 5. The yeast consensus sequence of the internal site for lariat formation is TACTAAC. The 3'-splice site has the consensus sequence PyAG [Unkles, Gene organization in industrial filamentous fungi. In Applied molecular genetics of filamentous fungi (Kinghorn, ed.), pp. 28-53. Blackie Academic & Professional, Wester Cleddens Road, Bishopbriggs, Glasgow G642NZ, UK, Glasgow (1992)].

The following cDNAs were generated:

- oal (SEQ ID NO: 98) for expression in E. coli

- oalEco (SEQ ID NO: 94) for expression in Saccharomyces cerevisiae

- oalEShort (a 31 amino acids shorter version using the second possible Met at position

32 of SEQ ID NO: 95) for expression in S. cerevisiae - oalS for expression in E. coli. (oalS was made because of the misleading sequence of peptide Fxn 42, which was finally identified as an artifact)

- oalSEco for expression in S. cerevisiae

- oalhis containing a C-terminal his-tag for expression in E. coli

- oalEhis for expression in S. cerevisiae - oalNhis (SEQ ID NO: 92) containing an N-terminal his-tag for expression in E. coli

- oalENhis (SEQ ID NO: 96) for expression in S. cerevisiae (construct oalsec containing the signal peptide of the phytase of A. terreus cbs for expression and secretion in S. cerevisiae)

General procedure: First, the assumed coding sequence (cDNA) of the oal gene was isolated in two PCRs. The two PCR-products were assembled by a third PCR. The following primers were used:

OallAS (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), OalδAS (SEQ ID NO: 53), Oal9AS (SEQ ID NO: 54), OallOS (SEQ ID NO: 55), OallOSEco (SEQ ID NO: 56), OalENhis (SEQ ID NO: 57), OallOSShis (SEQ ID NO: 58), OalllS (SEQ ID NO: 59), Oall2AS (SEQ ID NO: 60), Oall2ASEco (SEQ ID NO: 61), Oall2AShis (SEQ ID NO: 62), Oall2ASHisEco (SEQ ID NO: 63), Oall3S (SEQ ID NO: 64), Oall3AS (SEQ ID NO: 65), Oall4S (SEQ ID NO: 66), Oall4AS (SEQ ID NO: 67), Oall5S (SEQ ID NO: 68), Oall5AS (SEQ ID NO: 69), Oall6S (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).

OalllS starts directly in front of the more upstream start codon, while Oal9AS begins a few nucleotides downstream of the assumed stop codon. Oall4S and Oall4AS are complementary and contain sequences from the end of exon I and the start of exon II, in order to enable the assembling of the two generated PCR products in a third PCR reaction without the intron.

1 μl gDNA (1/10 diluted) step 1: 5 min - 95°C 1 μl nucleotide mix step 2: 30 sec - 95°C

5 μl PCR standard buffer with Mg²⁺ step 3: 30 sec - 55°C

1 μl OalllS (10 pMol/μl) step 4: 1 min - 72°C l μl Oall4AS (10 pMol/μl)

Steps 2 to 4 were repeated 35-times.

1 μl gDNA (1/10 diluted) step 1 5 min - 95°C

1 μl nucleotide mix step 2: 30 sec - 95°C

5 μl PCR standard buffer with Mg²⁺ step 3: 30 sec - 55°C 1 μl Oall4S (10 pMol/μl) step 4: 1 min - 72°C l μl Oal9AS (10 pMol/μl)

Steps 2 to 4 were repeated 35-times.

The PCR on the gDNA of A niger ATCC 9142 revealed PCR products of the expected size. The PCR products were purified by agarose gel electrophoresis. They were extracted from the gel using the QIAEX II Kit from Qiagen (Qiagen, Hilden, Germany) and used for the third PCR:

0.5 μl of each of PCR products 1 and 2 step 1: 5 min - 95° 1 μl nucleotide mix step 2: 30 sec - 95°C

5 μl PCR standard buffer with Mg²⁺ step 3: 30 sec - 55°C

1 μl Oall2S (10 pMol/μl) step 4: 1 min - 72°C l μl Oall0AS (10 pMol/μl) 1 μl High Fildelity polymerase mix from Roche Molecular Biochemicals 40 μl H₂O

Steps 2 to 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 as supplied by Invitrogen (Carlsbad, CA, USA). All other necessary cloning steps were performed as described in Sambrook et al. (1989). A plasmid containing the correct insert (oall), which was checked by sequencing, was used as template for all upcoming construction steps.

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

lμl of a plasmid containing oall as an insert (10 ng) 1 μl nucleotide mix 5 μl PCR standard buffer with Mg²⁺ 1 μl pQE80EcoS (10 pMol/μl) 1 μl pQESOBamAS (10 pMol/μl)

step l: 5 min - 95°C step 2: 30 sec - 95°C step 3: 30 sec - 55°C step 4: 1 min - 72°C

Steps 2 to 4 were repeated 30-times.

The PCR product was purified by agarose gel electrophoresis, the fragment of interest was extracted out of the gel (QIAEX II, Qiagen), digested by £coRI and BamHI, again purified by agarose gel electrophoresis, ligated into the vector pQE80 from Qiagen, and transformed into E. coli ToplO. The plasmids of 4 clones were isolated and the inserts sequenced. A plasmid containing the correct construct was transformed into E. coli Ml 5 or an equivalent E. coli strain. All molecular procedures are known to the skilled artisan as standard procedures. For the yeast expression construct, the primers used were replaced by OallOEcoS and Oall2ASEco, the PCR product was digested with EcoRI alone and the final product was cloned into pYES2 or an equivalent expression vector for S. cerevisiae using the EcoRI restriction site of the vector. Transformation of S. cerevisiae strains, e.g. INVScl (Invitrogen, Carlsbad, CA, USA), was done according to Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929-1933 (197δ) or to the manual, which was supplied together with the strain.

(b) Construct pQEδOoalS (a 31 amino acids shortened version using the second possible Met) for expression in E. coli: Using the same PCR protocol as mentioned above, primers pQEδOEcoSshort and pQEδOBamAS were used on the same template. The completed PCR reaction was treated as described in (a).

(c) Construct oalS for expression in S. cerevisiae:

Primers Oall6S and Oall2ASEco were used. Everything else was done as described in (a).

(d) Construct pQEδOoalhis containing a C-terminal his-tag for expression in E. coli: Primers pQEδOEcoS and pQEδOBamhisAS were used for the PCR, see (a).

(e) Construct oalhis for expression in S. cerevisiae:

Primers OallOSeco and Oall2ASHisEco were used for the PCR, see (a).

(f) Construct pQEδOoalNhis containing a N-terminal His-tag for expression in E. coli: Primers pQE80EcoNhisS and pQEδOBamAS were used for the PCR (see a).

(g) Construct oalENhis for expression in S. cerevisiae:

Primers OalENhis and Oall2ASEco were used for the PCR (see a).

(h) Construct oalsec containing the signal peptide of the phytase of A. terreus cbs for expression and secretion in S. cerevisiae:

The construct was prepared using three independent PCRs. Firstly, the signal sequence was isolated by a PCR on the gene of consensus phytase [Lehmann et al., Protein Eng. 13:49-57(2000)].

1.0 μl of a plasmid containing the gene of consensus phytase as an insert (10 ng) 1.0 μl nucleotide mix

5.0 μl PCR standard buffer with Mg²⁺

1.0 μl CP-a (10 pMol/μl)

1.0 μl OalsecAS (10 pMol/μl) 1.0 μl High Fidelity polymerase mix from Roche Molecular Biochemicals 40 μl H₂O

step 1:5 min -95°C step 2: 30 sec - 95°C step3:30sec-55°C step 4: 30 sec - 72°C

Steps 2 to 4 were repeated 30-times.

1.0 μl of a plasmid containing the oall gene (10 ng) 1.0 μl nucleotide mix 5.0 μl PCR standard buffer with Mg²⁺ 1.0 μl OalsecS (10 pMol/μl) 1.0 μl Oall2ASEco (10 pMol/μl)

1.0 μl High Fidelity polymerase mix from Roche Molecular Biochemicals 40 μl H₂O

step 1 5 min- -95°C step 2 30 sec - -95°C step 3 -55°C step 4 45 sec - -72°C

Steps 2 to 4 were repeated 30-times.

The PCR products were purified by agarose gel electrophoresis on a 1.5% agarose gel, extracted from the gel (QIAEX II, Qiagen) and used for a third PCR:

0.5 μl product of PCR 1 0.5 μl product of PCR 2 1.0 μl nucleotide mix 5.0 μl PCR standard buffer with Mg²⁺ 1.0μlCP-a(10pMol/μl) 1.0 μl Oall2ASEco (10 pMol/μl)

step 1:5 min- 95°C step2:30sec-95°C step 3: 30 sec - 55°C step 4: 1 min - 72°C Steps 2 to 4 were repeated 30-times.

The PCR product was purified by agarose gel electrophoresis, extracted out of the gel, digested by EcoRI, purified by agarose gel electrophoresis again, and ligated into the S. cerevisiae expression vector as described above.

Example 12: Expression of oal

(a) In E. coli:

The constructs pQEδOoal, pQEδOoalS, pQEδOoalhis, and pQEδOoalNhis were expressed in E. coli Ml 5 containing pREP4 harboring the repressor of the lac promoter (see the expression manual of Qiagen, Hilden, Germany). An overnight culture was diluted to an OD of 0.1 at 600 nm and grown at 30°C to an OD₆₀₀nm of 1.5. Then the cultures were induced with 0.5 mM IPTG and cultured for another 6 h at 30°C. The cells were harvested by centrifugation (5000 g, 20 min) and frozen at -80°C. They were lysed using B-PER (Pierce, Rockford, IL, USA) following the protocol of the manufacturer. After another centrifugation step, the supernatant was used for hydrolysis of (i^)-pantolactone.

In transformants containing the constructs pQEδOoalS and pQEδOoalhis, activity was neither found in the supernatant nor in the cell lysate, whereas using the constructs pQEδOoal and pQESOoalNhis, around 5% of the soluble protein was active (R)- pantolactonase.

The construct containing a N-terminal His-tag was expressed in the same way as the gene without the His-tag. The purification of the His-tagged protein was done in native form from the cell lysate using the protocol as described in the "QIAexpressionist" from Qiagen (Hilden, Germany).

(b) In S. cerevisiae:

The constructs oalEco, oalES, oalEhis, oalENhis, and oalsec were expressed in S. cerevisiae INVScl or a comparable strain. After transformation and plating on SD^ura" medium [Sherman et al., Laboratory course manual for methods in yeast genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1986)], the plates were incubated for 3 to 4 days at 30°C, grown colonies were picked and transferred into 2 ml SD^ura" liquid medium and cultivated under vigorous shaking at 30°C for 3 days. These cultures were used as pre-cultures for 25 ml SD^{ur "} medium. After another three days of cultivation at 30°C under vigorous shaking 500 ml YPD medium was inoculated with the prepared cultures [Sherman et al., above] in a 2-1 flask. After another 3 days of culturing under identical conditions, the cells were separated from the medium by centrifugation and lysed using Y-PER (Pierce, Rockford, IL). As in the case of the E. coli expression, only the constructs oalEco and oalENhis resulted in expression of active (R) -pantolactonase, which was found only in the cell lysate. Using a 75 ml YPD culture, it was possible to produce 300 U of (R) -pantolactonase. The corresponding lysate showed a specific activity of around 5 U/mg of total protein.

Using the sequence data of the position of the intron as shown above, also the oal gene from A. awamori as well as the oal gene from A. niger ATCC 46951 can be constructed and expressed in comparable ways.

Example 13: Immobilization of Oal heterologously expressed in E. coli or 5. cerevisiae and its application to the hydrolytic resolution of (RS)-pantolactone

For multiple use in a bioreactor, the (R) -pantolactonase from A niger was immobilized. After expression in E. coli or S. cerevisiae the cells were disrupted by chemical means like B-PER (E. coli) or Y-PER (S. cerevisiae) (Pierce, Rockford, IL, USA) or by mechanical means like sonication or high pressure. The lysate was cleared by centrifugation. This preparation was directly used for immobilization. Alternatively, before immobilization, another purification step like ammonium sulfate precipitation was included to further increase the specific Oal activity.

The biocatalysts were immobilized as follows:

(1) Immobilization of Oal in Silica Sol-Gels:

Poly(glyceryl silicate)- 1.0 (PGS-1.0) was prepared according to the literature [Gill and Ballesteros, J. Am. Chem. Soc. 120:δ5δ7-δ59δ (1998)], and dissolved in half its weight of ice-cold water. A 100 or 200 mg portion of this was thoroughly mixed with 50 or 100 mg of an ice-cold preparation of biocatalyst stock (soluble enzyme from various preparations) in 50 mM phosphate, pH 7.0, in a 2 ml or 5 ml vial, and the vial gently rotated over a period of 2 min as to form a thin coating of hydrogel on the walls of the vial. The vial was then left on ice for 20 min, then transferred (open) to a refrigerator. The hydrogel was allowed to age at 5°C for 48-72 h to form the xerogel. The xerogel- coated vial was washed with 2 x 1 or 2 x 4 ml of 50 mM phosphate buffer, pH 7, followed by 2 x 1 or 2 x 4 ml of 50 mM TEA- Acetate, pH 7, containing 10 mM magnesium acetate, by shaking at 100 rpm, 5°C.

(2) Immobilization of Oal in Sol-Gel Silica-Polyvinyl Alcohol:

The procedure was similar to that in (1), except that the DGS solution was mixed with the appropriate quantity of polyvinyl alcohol (PVA, from Aldrich, 13% w/w of 85 K in water) prior to combining with the lactonase enzyme stock. Further processing was as for (l). (3) Immobilization of Oal onto glutar aldehyde- activated, supported polyethyleneimine: Polyethyleneimine (1 g, 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) were dissolved in 5 mL of dichloromethane, and the solution used to coat 7 g of Fullers Earth (30-60 mesh, from Aldrich). The wet coated material was dried under air at room temperature for 0.5 h to give ca. 9 g of coated Fullers Earth. This was then suspended in an ice-cold mixture of glutaraldehyde (8 mL of 50% w/w, from Aldrich) and phosphate buffer (50 mL of 50 mM, pH 8), with stirring at 200 rpm, for 2 h. The activated support was washed with water (4 x 10 mL), then phosphate buffer (4 x 10 mL of 20 mM, pH δ), then drained. The wet support was mixed with an ice-cold solution of lactonase enzyme stock (in phosphate, 50 mM, pH δ) and the suspension stirred at 200 rpm for 20-30 h. The liquid was decanted from the immobilized enzyme and the wet immobilizate was washed with phosphate (3 x 5 mL), treated with ethanolamine (20 mL of 20 mM, pH δ), washed again with phosphate (2 x 5 mL of 20 mM, pH 7), then drained.

(4) Reaction Conditions for hydrolytic 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 M racemic lactone (in 0.75 M triethylamine- acetate, pH δ.5, containing 20 mM magnesium acetate). Vials were incubated at 40°C, 200 rpm for the requisite time period, the solution drained off and analyzed by chiral HPLC, and the catalyst washed with fresh substrate solution (2 x 1 or 2 mL) before commencing the next cycle. Samples for analysis were quenched with an equal volume of 500 mM MES buffer, pH 5.5, containing 50 mM EDTA, centrifuged (10,000 g, 10 min), then analyzed by HPLC using a 0.46x15 cm CHIRADEX column eluted with 70:30 water-methanol, 1 mL/min, 20°C. Initial rates were measured at 15 min and the E values were determined at the end of each cycle.

The performances of selected immobilized enzyme preparations in the batch resolution of (RS) -pantolactone are summarized in Table 2. Both sol-gel and covalent immobilization protocols proved effective, with the catalysts retaining 62-72% of their initial activity and displaying enantiomeric excesses and enantioselectivities for (R)- pantolactone of δ9-9δ% and 71-88 respectively, even after 24 cycles of lh batch reactions. Furthermore, the catalysts performed in a very similar fashion when tested in extended batch runs, that is when each batch reaction was allowed to progress for one day rather than 1 h, indicating good long-term operational stabilities. Table 2: Batch resolution of (jRS)-pantolactone by immobilized lactonase preparations

Catalyst*: Biocatalyst/Prod. Organism (Recomb. Activity)

Example 14: Application of immobilized recombinant E. coZi-expressed Oal to the continuous resolution of (RS)-pantolactone.

Sol-gel-PVA-immobilized recombinant lactonase biocatalyst (expressed in E. coli), prepared as described above in Example 14(2) was used to perform the continuous resolution of racemic lactone in a packed-bed reactor: immobilized biocatalyst (1.1 g, 0.21 kU g^"1, total of 0.231 kU) was dry packed into a 0.46 x 15 cm Omnifit jacketed glass column fitted with Teflon endpieces. This was connected in turn to a 1 x 10 cm Omnifit jacketed glass column packed with 1-2 mm cellulose beads, which was fed by two syringe pumps fitted with 50 mL glass/Teflon syringes. The columns were connected to a circulating water bath and maintained at 40°C during the resolution run. The reactor was conditioned by feeding Tris-acetate buffer (100 mM, pH 7, containing 100 mM magnesium acetate) at a rate of 5 mL min^"1, room temperature for 30 min, followed by the combined feeding of solutions of racemic lactone (1.5 M in water) and Tris-acetate buffer (2.5 M, pH 8.25, containing 50 mM magnesium acetate) at a volumetric ratio of 1:1 and a total flow rate of 2 mL min^"1 for 30 min, room temperature. The flow rate was then dropped to 1.2 mL min^"1, the columns heated to 40°C, and the system allowed to equilibrate for 15 min. The reactor was thus operated for ca. 45 min, the eluate being collected in an ice bath, under which conditions a total of 52.7 mL of feed (corresponding to 5.14 g of RSPL) had been processed at a sustained conversion of 46-48%. The pH of the eluate was adjusted to pH 6.5 with sulphuric acid (10% v/v aqueous), the solution extracted with dichloromethane (10 x 75 mL) and the organic layer dried over anhydrous magnesium sulphate and rotary evaporated to yield enriched (S) -pantolactone (2.67 g, 98% of theoretical) consisting of 10% RPL and 90% SPL (analyzed by chiral HPLC). The aqueous phase was acidified with sulphuric acid (40% w/w) to pH 1.5, heated to 70°C for 1 h, cooled in ice, saturated with sodium chloride, then extracted with dichloromethane (10 x 75 mL), the organic layer dried over anhydrous magnesium sulphate and rotary evaporated to yield enriched (R) -pantolactone (2.23 g, 92% of theoretical yield) with an enantiomeric purity of 93%. The combined recovery of the ( )-/(S)-lactones was 95%.

The results of Examples (13) and (14) clearly demonstrate that the recombinant lactonases are efficient biocatalysts for the hydrolytic resolution of racemic pantolactone, and that the catalysts can be effectively immobilized and the immobilizates therein applied to the production of highly enriched (R) -pantolactone with an enantiomeric excess of 90-98%, using both batch and continuous operations.

Example 15: Cloning and expressing a (S)-pantolactone specific lactonase from B. subtilis

Using the amino acid sequences of the lactonases from bovine and horse liver, we found several other sequences that showed a limited homology to these sequences. Among those, the yvre gene from B. subtilis (YVRE BACSU, annotated as a member of the SMP- 30 / CGR1 FAMILY/ hypothetical 33.2 kDa protein) was cloned by PCR from genomic DNA using primers of its 5' and 3'-end which also contained the required restriction sites (EcoRl and Pstl) for latter cloning into an expression vector. The PCR conditions were identical to those used for isolation of the oal gene from the genomic DNA of A niger (see Example 8). PCR product and vector (PET41a from Stratagene (La Jolla, CA, USA)) were digested with EcoRI and Pstl, cleaned by agarose gel electrophoresis, ligated, and transformed into E. coli ToplO cells (Stratagene, La Jolla, CA, USA). Using this strategy, the gene was fused in frame to the gene of the GST protein from £. coli.

Expression and purification was done according to the protocol of the manufacturer. The activity of the purified protein was determined at 40°C using the standard assay additionally containing 5 mM CaCl₂. The enzyme showed a very high selectivity for (S)- pantolactone. The preparation had a specific activity of 1-2 U per mg enriched fusion protein. The enzyme was only active in the presence of Mg²⁺ or Ca ⁺ ions.

Claims

1. A nucleotide sequence of a gene coding for a pantolactone hydrolase, said pantolactone hydrolase being derived from horse liver, A. niger ATCC 9142, A. niger awamori ATCC 38854 or A niger MacRae ATCC 46951.

2. A nucleotide sequence according to claim 1 which comprises, when derived from horse liver, the nucleotide sequence as illustrated in SEQ ID NO: 1, when derived from A. niger MacRae ATCC 46951 the nucleotide sequence as illustrated in SEQ ID NO: 5, when derived from A. niger ATCC 9142 the nucleotide sequence as illustrated in SEQ ID NO: 7, and when derived from A. niger awamori ATCC 38854 the nucleotide sequence as illustrated in SEQ ID NO: 9.

3. An expression vector comprising a nucleotide sequence of a gene coding for a pantolactone hydrolase, said pantolactone hydrolase being derived from horse liver, Bacillus subtilis, A. niger ATCC 9142, A. niger awamori ATCC 38854 or A. niger MacRae ATCC 46951.

4. A host cell transformed with the expression vector according to claim 3.

5. A pantolactone hydrolase derived from horse liver, A. niger ATCC 9142, A. niger awamori ATCC 38δ54 or A niger MacRae ATCC 46951.

6. A pantolactone hydrolase the amino acid sequence thereof comprising an amino acid sequence being illustrated by SEQ ID NO: 2 when derived from horse liver, by SEQ ID NO: 6 when derived from A. niger MacRae ATCC 46951, by SEQ ID NO: δ, when derived from A niger ATCC 9142 and by SEQ ID NO: 10, when derived from A. niger awamori ATCC 3δδ54.

7. The pantolactone hydrolase according to claims 5 or 6 wherein the pantolactone hydrolase is immobilized.

8. The use of a pantolactone hydrolase according to claim 6 or a pantolactone hydrolase derived from Bacillus subtilis as illustrated by SEQ ID NO: 4 for a selective hydrolyzation of either R- or S-pantolactone.

9. A process for the production of R-pantothenic acid or a salt thereof or of R- pantothenol comprising the step of selective hydrolyzation of either R- or S-pantolactone in the presence of a pantolactone hydrolase according to claim 6 or a pantolactone hydrolase derived from Bacillus subtilis as illustrated by SEQ ID NO: 4.

10. A process for the cloning of a nucleotide sequence directly adjacent to a known sequence the process comprising a PCR amplification comprising: a) the use of a single-stranded DNA of a first PCR as template for a second PCR, using a primer in said second PCR that randomly hybridizes to the single-stranded DNA produced in the first PCR; and b) thereby generating the opposite strand which itself serves as template for the second strand still using the randomly hybridizing primer of step a).