CN1768141B - Pantolactone hydrolase - Google Patents

Abstract

The present invention relates to nucleotide sequences of genes encoding pantolactone hydrolase, as well as vectors and host cells comprising the above nucleotide sequences, and pantolactone hydrolase encoded by the above nucleotide sequences. The invention also provides methods of making and using pantolactone hydrolases.

Description

pantolactone hydrolase

The present invention relates to the enzyme that can be used for the photoresolution (optical resolution) of pantolactone (pantolactone) enantiomer, relates to the gene of encoding said enzyme, also relates to said enzyme in the preparation of R-pantothenic acid or its salt or R - Use of R-panthenol in a method.

Enantiomers differ in their physiological effects, ie toxicological and pharmacological effects, reactions with enzymes, and in their sensorial characteristics. Only R-pantothenic acid lactone is known as an intermediate in the preparation of R-pantothenic acid, a vitamin which is essential for growth, reproduction and normal physiological functions of humans and animals. As the precursor of coenzyme A and as the acyl carrier of fatty acid synthase, pantothenic acid is involved in more than one hundred different metabolic pathways, including energy metabolism of carbohydrates, proteins and lipids, as well as lipids, neurotransmitters, steroids Hormones, porphyrins, and hormone synthesis. Until recently, the widely used method for the preparation of R-pantolactones was the photoresolution of chemically synthesized R- and S-pantolactones. This method is very expensive as it requires the use of expensive photoresolution reagents. In addition, the recovery of the R-enantiomer of pantolactone is also quite difficult. Accordingly, it would be desirable to provide pantolactone hydrolases useful for the photoresolution of the R- and S-enantiomers of pantolactone. The use of this enzyme, especially to hydrolyze the S- or R-form of pantothenolactone, is a more economical and more manipulative method for the separation of the two enantiomers of pantothenolactone. Examples of salts of R-pantothenic acid include calcium R-pantothenate.

In one embodiment, the present invention provides the nucleotide sequence of the gene encoding pantothenate lactone hydrolase, which is obtained from horse liver, A. niger ATCC 9142, A. nigerawamori ATCC 38854 or A. Obtained in .niger MacRae ATCC 46951. The public can obtain the above strains from American Type Culture Collection (ATCC), 10801 University Boulvard, Manassas, VA 20110-2209USA.

In another embodiment, the present invention provides a nucleotide sequence encoding pantothenate hydrolase, said sequence comprising: when the pantothenate hydrolase is from horse liver, the nucleus shown in SEQ ID NO: 1 Nucleotide sequence; When the pantothenate lactone hydrolase is from A.niger MacRae ATCC 46951, the nucleotide sequence shown in SEQ ID NO: 5; when the pantothenate lactone hydrolase is from A.nigerATCC9142, such as SEQ ID NO: The sequence shown in 7; and when the pantothenate lactone hydrolase is from A. niger awamori ATCC38854, the sequence shown in SEQ ID NO: 9.

The nucleotide sequence may comprise the coding sequence and the regulatory sequence of each pantothenate hydrolase mentioned above.

A "regulatory sequence" is defined herein as an arrangement 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" includes the necessary nucleic acid sequences near the site where transcription begins. A promoter also optionally includes distal enhancer or repressor elements, which can be positioned as many as several thousand base pairs after the start site of transcription. A "constitutive" promoter is one 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 an arrangement of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs a sequence corresponding to the second Transcription of a sequence of nucleic acids.

Fragments of nucleic acid sequences can be used, for example, as probes in hybridization assays.

Where a nucleotide sequence is inserted into a host organism, transcribed and translated to produce a functional polypeptide, the skilled artisan will recognize that, due to codon degeneracy, many polynucleotide sequences will encode the same polypeptide. Such variants are also expressly included within the scope of the present invention. In addition, the present invention expressly includes sequences that are substantially identical to one another (determined as described below) that encode polypeptides that are mutants of wild-type polypeptides or polypeptides that retain polypeptide function (e.g. resulting from conservative substitutions of polypeptide amino acids). In addition, variants may be those encoding dominant negative mutants described below.

Two nucleic acid sequences or polypeptides are said to be "identical" if their respective nucleotide sequences or amino acid residues are identical when aligned for maximum identity as described below . The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to: when comparing and aligning for maximum identity over a comparison window, the sequence Two or more sequences or subsequences are identical, or have a specified percentage of amino acid residues or nucleotides that are identical, as measured by one of the comparison algorithms or by manual alignment and visual inspection. When percentages of sequence identity are used with respect to proteins or peptides, it is recognized that residue positions that are not identical typically differ by conservative amino acid substitutions, in which amino acid residues are replaced by others with similar chemical properties (e.g. , charge or hydrophobicity) amino acid residues and thus do not alter the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitutions. Methods for making such adjustments are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thus increasing the percent sequence identity. So, for example, give a score between 0 and 1 for conservative substitutions where identical amino acids are given 1 point and non-conservative substitutions are given 0 points. Conservative substitutions are calculated according to, for example, the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4: 11-17 (1988), for example, implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA). give points.

The phrase "substantially identical" in the context of two nucleic acids or polypeptides means that when aligned for maximum identity over the comparison window, they are identified using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Where measured, a sequence or subsequence having at least 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity. This definition also refers to the complement of a sequence that 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 other parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

As used herein, "comparison window" includes: refers to a fragment whose number of consecutive positions is selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, wherein the sequence can be Comparisons are made to a reference sequence having the same number of consecutive positions, after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known in the art.

"Conservatively modified variants" applies to amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to nucleic acids that encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, there are multiple functionally identical nucleic acid codons that encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where alanine is defined by a codon, the codon can be changed to any of the corresponding codons described above without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. The skilled artisan will recognize that every codon in a nucleic acid (except AUG, which is usually the only codon encoding methionine) can be modified to produce a functionally identical molecule. Accordingly, every silent variation of a nucleic acid which encodes a polypeptide is implicit in every described sequence.

With respect to amino acid sequences, the skilled artisan will recognize that individual substitutions, deletions or additions to nucleic acids, or to peptide, polypeptide or protein sequences, change single amino acids or small percentages (i.e., less than 20%) in the encoded sequence , such as 15%, 10%, 5%, 4%, 3%, 2%, or 1%) of amino acid substitutions are "conservatively modified variants" in which the amino acid is replaced by a chemically similar amino acid replace. Conservative substitution tables providing functionally similar amino acids are well known in the art.

Each of the following six groups contains amino acids that may be conservatively substituted 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, eg, Creighton, Proteins (1984)).

The sign that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid can perform immunological cross-reaction with the antibody raised against the polypeptide encoded by the second nucleic acid. Thus, for example, when two peptides differ only by conservative substitutions, then typically one of the polypeptides is substantially identical to the second polypeptide. 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 "hybridizes specifically to" means that, under stringent hybridization conditions, a molecule only binds to a specific nucleotide sequence when the sequence is present in a complex mixture (e.g., total cellular DNA or RNA, or library DNA or RNA) Combine, form double strands or hybridize.

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, high stringency conditions are selected to be about 5-10°C lower than the melting temperature ( _Tm ) for the specific sequence at a given ionic strength and pH. Low stringency conditions are typically selected to be about 15-30°C lower than the _Tm . _Tm is: (at a given ionic strength, pH and nucleic acid concentration,) at equilibrium, the temperature at which 50% of the probes complementary to the target hybridize to the target sequence (when the target sequence is present in excess, at Tm, at equilibrium 50% of the probes were bound). Stringent conditions will be the following: a salt concentration of less than about 1.0 M sodium ion, typically a sodium ion concentration (or other salt) of about 0.01 to 1.0 M, pH 7.0 to 8.3, for short probes (e.g., 10 to 50 nucleotides), the temperature is at least about 30°C, and for long probes (eg, greater than 50 nucleotides), the temperature is at least about 60°C. Stringent conditions can also be achieved by 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 also substantially identical if the polypeptides encoded by such nucleic acids are substantially identical. This would occur, for example, if a copy of the nucleic acid was created with the maximum codon degeneracy permitted by the genetic code. In such cases, typically, the nucleic acids hybridize under moderately stringent hybridization conditions.

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

Another indication that two polynucleotides are identical is that a reference sequence amplified by a pair of oligonucleotide primers can be used as a probe under stringent hybridization conditions to isolate cDNA or genomic libraries. The sequence to be tested, or the sequence to be tested was identified in a northern or Southern blot.

The invention also includes expression vectors as defined herein. Expression vectors include one or more copies of any of the polynucleotide sequences listed above. An expression vector of the invention may contain any polynucleotide sequence as defined herein.

Therefore, in another embodiment, the present invention provides an expression vector comprising the nucleotide sequence of a gene encoding pantothenate hydrolase from horse liver, Bacillus subtilis , A.niger ATCC 9142, A.niger awamori ATCC38854 or A.niger MacRae ATCC46951; or the vector contains the nucleotide sequence of a gene encoding pantothenate hydrolase derived from horse liver, such as As shown in SEQ ID NO: 2, from Bacillus subtilis, as shown in SEQ ID NO: 4, from A. niger ATCC9142, as shown in SEQ ID NO: 8, from A. niger awamori ATCC38854, as shown in SEQ ID NO: 10 , and from A. niger MacRae ATCC46951, shown in part in SEQ ID NO:6.

Alternatively, the polynucleotide sequence in the expression vector may be operably linked to the expression control sequences defined above.

The phrase "expression vector" as used herein is a replicatable vector carrying DNA sequences encoding the polynucleotide sequences set forth herein and capable of regulating the expression of these DNA sequences. In the present context, the term "replicable" means that the vector can be replicated in a given type of host cell into which the vector is introduced. Near the polynucleotide sequence upstream of the polynucleotide sequence of interest, a sequence encoding a signal peptide may be provided, the presence of which ensures secretion of the encoded polypeptide expressed by the host cell carrying the vector. The signal sequence may be naturally associated with the selected polynucleotide sequence, or may be derived from another source.

The vector can be any vector conventionally used in recombinant DNA techniques, the choice of which will generally depend on the host cell into which it is to be introduced. Thus, a vector may be an autonomously replicating vector, ie a vector that exists as extrachromosomal material that replicates independently of chromosomal replication; examples of such vectors are plasmids, phages, cosmids or minichromosomes. In addition, the vector may be a vector which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the host cell genome into which it has been integrated. The expression vector of the present invention can carry any DNA sequence of the present invention, and can be used to express any polypeptide of the present invention.

The invention also includes cultured cells or host cells containing one or more polynucleotide sequences disclosed herein and/or one or more expression vectors disclosed herein. "Cultured cells" as used herein include any cells capable of growing under given conditions and expressing one or more polypeptides encoded by the polynucleotides of the present invention. Preferably, the cultured cells are yeast, fungi, bacteria or algae. More preferably, the cultured cells are Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Aspergillus niger or Bacillus subtilis.

In another embodiment, the present invention provides pantothenate lactone hydrolase obtained 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 pantothenate lactone hydrolase, the amino acid sequence of which comprises: when derived from horse liver, the amino acid sequence shown in SEQ ID NO: 2; when derived from A. niger MacRae ATCC 46951 When from A. niger ATCC 9142, the amino acid sequence shown in SEQ ID NO: 8; when from A.niger awamori ATCC 38854, the amino acid sequence shown in SEQ ID NO: 10 amino acid sequence.

Pantolactone hydrolase from horse liver as shown in SEQ ID NO: 2 and from Bacillus subtilis as shown in SEQ ID NO: 4 exhibits S-pantolactone hydrolase activity. Pantothenic acid from A. niger ATCC 9142 as shown in SEQ ID NO: 8, from A. niger awamori ATCC 38854 as shown in SEQ ID NO: 10 and partially from A. nigerMacRae ATCC 46951 as shown in SEQ ID NO: 6 The lactone hydrolase exhibits R-pantolactone hydrolase activity.

In another embodiment, the present invention comprises a fragment of the above amino acid sequence, which fragment has the enzymatic activity of the complete protein.

A "fragment of an amino acid sequence" according to the present invention may be defined as a sequence lacking a stretch of amino acids which may be essential for the correct folding of a protein. However, once the protein is folded, this stretch of amino acids can be deleted without disrupting the individual protein's function. These segments may be present in the folded protein, such as loops, or may be N- or C-terminal sequences not directly related to the activity of the folded protein.

Furthermore, the present invention includes derivatives of proteins comprising said R- and S-pantolactone hydrolase activity. Said derivatives include immobilized enzymes, for example by inclusion, wherein said enzymes are retained on membrane devices, such as hollow fibers, polymeric networks or microcapsules. It is also possible to immobilize the enzyme by cross-linking or by creating enzyme crystals. Another possibility for the immobilization of enzymes is to bind them to growing polymers, such as silica sol-gels or silica-graphite sol-gels, or by binding enzymes to prefabricated supports such as Eupergit C, Eupergit 250L, PEG, Celite, and Amberlite XAD-7. Another enzyme immobilization technique suitable for the R- and S-pantolactone hydrolases of the present invention is to disperse them in a water-immiscible organic solvent. In general, proteins with enzymatic activity, such as the enzymes of the invention, can be immobilized by almost any immobilization technique known in the art.

In yet another embodiment, the present invention relates to the use of the isolated pantothenolactone hydrolase for the selective hydrolysis of R- or S-pantolactone hydrolase, which can be used for the R-enantiomerization of pantolactone The R-enantiomer of pantolactone is the precursor for the production of R-pantothenic acid and R-panthenol.

Accordingly, it is an object of the present invention to provide a method or route for the selective hydrolysis of R- or S-pantolactone using the isolated pantolactone hydrolase described herein.

In another embodiment, the present invention provides a method for producing R-pantothenic acid or a salt thereof or R-panthenol, which comprises degrading R- or S-pantothenolactone in the presence of the pantothenolactone hydrolase of the present invention A step of selective hydrolysis is carried out.

In another embodiment, the present invention provides a method for cloning a nucleotide sequence immediately adjacent to a known sequence, such as a nucleotide sequence encoding a novel pantolactone hydrolase or a portion thereof.

Specifically, the present invention includes novel PCR strategies capable of amplifying unknown sequences. The new PCR strategy involves using the same primers for the synthesis of the first and second DNA strands. The primers hybridize to already known regions of the gDNA to be amplified. This primer showed 100% homology to the known sequence. With the help of this primer, the first PCR is performed, providing the first single-stranded copy of the genomic DNA, reaching the unknown sequence. Using a portion of the product of the first PCR reaction as a template to perform a second PCR using (nested) primers that hybridize to DNA fragments positioned 3' to the primers used in the first PCR, but logically Still in a known part of the sequence. After random hybridization of this nested primer with the single-stranded DNA produced by the first polymerase reaction, the antisense strand of this single-stranded DNA is generated, which itself serves as the same primer used in the second PCR to generate the second strand template. In the end, DNA fragments starting within the known sequence, extending to adjacent genomic DNA or to any other DNA with missing parts of the sequence, are produced with a primer that hybridizes to both ends of the new fragment.

Accordingly, in another embodiment, the present invention provides a method of cloning a nucleotide sequence immediately adjacent to a known sequence, said method comprising PCR amplification comprising:

a) using the single-stranded DNA of the first PCR as a template to perform a second PCR in which primers capable of randomly hybridizing to the single-stranded DNA produced by the first PCR are used; and

b) Thus, still using the random hybridization primers from step a), an antisense strand is generated which itself serves as a template for the second strand.

The following examples describe in detail the identification, characterization and expression of genes encoding pantolactone hydrolase. These examples are illustrative only and are not intended to limit the scope of the invention in any way.

Example 1 Purification of (S)-pantolactone hydrolytic activity from commercially available horse liver esterase acetone powder

1 g of horse liver esterase acetone powder (Fluka Holding AG, Industriestrasse 25, POBox 260, CH-9471 Buchs, Switzerland) 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, after which filter sterilization was performed with a 0.45 μM filter. The protein solution was loaded onto a Butyl Sepharose column HR26/10 (Amersham Pharmacia Biotech Europe GmbH, Dübendorf, Switzerland) equilibrated with buffer A. The column was washed with 100 ml of buffer A. Then 400 ml of a linear gradient from 0 to 100% of buffer B (buffer A without ammonium sulfate) was applied. Lactonase was eluted in the middle of the gradient. Fragments showing pantolactone hydrolytic activity were collected, 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 fragments were loaded onto a MonoQ-pre-packed column HR 26/10 (Amersham Pharmacia Biotech Europe GmbH, Dübendorf, Switzerland) applying a linear gradient of 0 to 350 mM NaCl. Proteins were eluted at the beginning of the gradient. The active fragments were collected again, concentrated to 1 ml, and loaded onto a Superdex 200 column (Amersham Pharmacia Biotech Europe GmbH, Dübendorf, Switzerland). 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 was eluted as the last peak, which showed a molecular size of about 31 kDa, which was consistent with that of SDS-PAGE. match the dimensions shown.

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

Example 2 Preliminary characterization of (S)-pantolactone hydrolase from horse liver

(a) Molecular size, structure and isoelectric point:

According to SDA-PAGE, the denatured protein has a molecular weight of approximately 35 kDa. Using corrected elution times from the gel filtration column, the molecular weight of the native protein was calculated to be approximately 31 kDa. This means that lactonase from horse liver is a 30 to 35 kDa monomer. With 2D gels, the isoelectric point was determined to be in the range of pH 5.5.

(b) N-terminal and internal amino acid sequencing:

Proteins enriched after 2D gel electrophoresis were digested with trypsin. The resulting peptides were analyzed by mass spectrometry. The N-terminal sequence of peptide No. 66 is shown in SEQ NO: 11.

The sequence, together with the MS-spectrum, revealed that the protein belongs to a family of proteins known as senescence marker protein-30 or regucalcins, which are mainly found in vertebrates. Calmodulin sequences from rat, mouse, human, chicken, rabbit, bovine, Xenopus laevis, and two less homologous sequences from Drosophila melanogaster have been determined. However, this gene from horses has not been isolated and sequenced.

(c) Metal dependence:

To determine the effect of different divalent metals on the pantothenate lactonase activity of horse liver enzymes, 1.3, 2.6, 5.2 or 10.4 mM Ca ²⁺ , Mg ²⁺ or Mn ²⁺ were added to 200 mM Tris/ Acetate and 50mM (R, S)-pantothenolactone in the reaction mixture. The reaction mixture was incubated at 37°C for 60 minutes and terminated with an equal volume of methanol containing 2 mM EDTA. The reduction of (S)-pantolactone was determined by HPLC. Calcium reduces enzyme activity, while magnesium and manganese increase 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 to 80° C. for 15 minutes. After 30 minutes on ice, activity was determined using standard assay methods (60 minutes, 37°C). The enzyme remains stable up to 60°C. It starts to inactivate at 65°C and loses more than 50% of its activity at 70°C.

Example 3 Cloning of (S)-pantothenate lactonase from horse liver

Sequence information from all known mammalian genes (see below) was used to design primers for PCR against cDNA prepared from a one-year-old "Hafflinger" horse liver from Bavaria supplied by Roche Molecular Biochemicals, Courtesy of Penzberg, Germany. Total RNA from liver tissue was prepared with the QIAGEN RNeasy Maxi Kit (Qiagen, Hilden, Germany).

The homology between horse liver lactonase and other members of the calmodulin family is shown in Table 1. Assays were performed by the GAP program in the GCG package, using standard parameters. Numbers for similarity are shown to the right of the diagonal in the table, and numbers for identity are shown to the left of the diagonal.

Table 1: Amino acid sequence similarities and identities within the calmodulin family

horse ox rabbit people rat mouse chicken X.laev D.mela horse - 92.6 92.0 90.6 88.3 88.3 84.3 77.9 48.8 ox 91.0 - 90.3 93.0 90.3 90.0 83.6 76.6 48.9 rabbit 90.6 89.3 - 91.0 87.6 87.6 82.6 77.6 47.7 people 89.6 91.3 89.3 - 90.0 90.6 82.3 76.9 48.4 rat 86.0 87.3 85.0 88.3 - 95.7 80.9 77.9 49.3 mouse 86.3 87.3 85.3 88.6 94.0 - 81.6 77.3 50.0 chicken 77.9 77.9 76.6 77.9 75.9 75.9 - 81.9 48.9 X.laev 70.9 69.9 71.2 70.2 71.6 70.2 73.9 - 47.5 D.mela 36.8 37.0 37.2 37.5 37.0 38.4 36.3 37.0 -

Chicken: Chicken; X.laev: Xenopus laevis; D.mela: Drosophila melanogaster

Using Titan One Tube RT-PCT Kit (Roche Molecular Biochemicals, Penzberg, Germany) and homologous primers covering the upstream 50bp sequence of the start codon and stop codon, a fragment of 885bp was amplified by RT-PCT (SEQ ID NO : 1-885 nucleotides of 1). The reaction was carried out according to the method recommended by the manufacturer.

The N-terminal 5' primer (SEQ ID NO: 12) and the C-terminal 3'-primer (3'-CAGTTTCCTTAAGGGGGGATA-5') (SEQ ID NO: 13) for the first PCR were constructed based on 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 the N-terminal sequence of Xenopus (SEQ ID NO: 20) and 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 the C-terminal sequence of Xenopus (SEQ ID NO: 27).

This fragment was cloned into the TA cloning vector (Invitrogen, Carlsberg, CA, USA). Sequencing proved it to be from horse liver calmodulin. The missing 5'-end was isolated by a method called 5'/3'RACE with a separate kit provided by Roche Molecular Biochemicals, Penzberg, Germany: with a perfectly matched primer (SEQ ID NO: 28) from Single-stranded DNA was produced from isolated horse liver cDNA. The single-stranded DNA is purified, polyC tails are added, and another PCR is performed using the nested primer at the 5' end of the first primer, the polyG primer. A DNA fragment representing the deleted 5'-end of the gene is amplified. The 3'- end. In the final PCR, the obtained sequence information was used to design 5'- and 3'-primers covering the start and stop codons of the gene, and the full-length cDNA of 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 900bp/299 amino acids.

A commercially available expression vector, eg pQE60 from Qiagen, was used to express the complete gene in E. coli with a His-tag fused at its C-terminus. Expression and purification of the His-tagged protein following standard procedures yielded a 33 kDa protein exhibiting (S)-pantolactonase activity.

Example 4 Purification of (R)-pantolactone hydrolysis activity by commercially available A.niger lipase A crude preparation sex

Among the different commercially available enzyme products obtained from the fermentation of A. niger, specific hydrolysis of (R)-pantothenolactone was identified. For purification of specific enzymes, a commercial preparation known as Lipase A "Amano" from Amano Pharmaceuticals Co. Ltd., Nagoya, Japan was used. The preparation was obtained from lyophilized and disrupted A. awamori cells overexpressing lipase from A. tubigensis based on sequence data obtained from two enzymes co-purified with lactonase. This material was dissolved in 20 mM Tris/HCl, pH 7.5, 2.5 mM _MgCl2 (standard buffer). Add 5% ethanol to increase the solubility of the material. Material that did not go into solution was separated by centrifugation (30 min, 15000 rpm, 4°C) followed by filtration (with a 0.45 μm filter). The dissolved fraction was first purified by ultrafiltration on an Amicon cell ultrafilter (50kDa cut point) to remove any salts and small molecules that would disturb the subsequent anion exchange chromatography step. Proteins were then loaded onto HR26/10 prepacked Q-Sepharose columns (Amersham Pharmacia Biotech Europe GmbH, Dübendorf, Switzerland). The column was washed with 200 ml of standard buffer, and then the protein was eluted by a linear gradient (400 ml) of 0 to 350 mM NaCl. Fragments containing activity are pooled. Ammonium sulfate was added to a final concentration of 1.5M, and the treated protein solution was loaded onto a HR 26/10 butyl sepharose prepacked column (Amersham Pharmacia Biotech Europe GmbH, Dübendorf, Switzerland). Proteins were separated by washing with 200 ml of standard buffer containing 1.5 M ammonium sulfate and a gradient of decreasing ammonium sulfate from 1.5 to 0 M (400 ml). At this point the activity elutes in two peaks which are collected separately. The volume of the two pools was reduced to less than 2ml. The two concentrates were separated by gel filtration on a Superdex 200 column with standard buffer. Active fragments from each isolate were pooled and concentrated. The activity of pool II tended to elute slightly earlier than pool I, suggesting that the corresponding protein may be of slightly higher molecular weight.

80 U/ml (40°C) is the highest activity that has been measured for pool I and pool II respectively. As shown by the 2D gels, the protein preparations were not completely homogeneous, suggesting that the true specific activity could be even higher. However, since lactonase activity corresponds to the 38 kDa protein band in the gel filtration elution profile and is the predominant protein in the final preparation, this band is most likely lactonase. According to the results of gel filtration, the natural lactonase has a molecular weight of 75 kDa. This suggests a homodimeric structure of the enzyme.

Example 5 Preliminary characterization of (R)-pantolactone hydrolase

(a) Structure and molecular weight of native form:

The enzyme was eluted with Superdex 200 (AmershamPharmacia Biotech Europe GmbH, Dübendorf, Switzerland) at 72 ml using conditions as described in Example 4. Extrapolated from the standard curve, this reflects a molecular weight of 75 kDa. SDS-PAGE showed a molecular weight of 38 kDa. The combination of the above data suggests that the (R)-pantolactonase from A. niger is a homodimer composed of two identical 38 kDa subunits.

(b) thermal stability:

50 μl of diluted enzyme (0.1 U) were incubated for one 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 assay methods. The enzyme is stable up to 50°C. At higher temperatures, it begins to deactivate.

(c) Optimum temperature:

Strongly enriched (R)-pantolactonase from a commercial source (see Example 5) was incubated for 30 minutes at 20, 30, 40, 50, 55 and 60°C using the following conditions:

100mM (R, S)-pantothenate

100mM _MgCl2

200mM Tris/HCl, pH 8.0

0.05 U(R)-pantolactonase in a final volume of 10 μl.

The temperature optimum for (R)-pantolactonase from a commercial A. niger preparation was about 50°C under the conditions used.

(d) Cofactor dependence and inhibition:

The activity of (R)-pantolactonase from A. niger requires Mg ²⁺ or Mn ²⁺ . Adding 1mM EDTA will completely inhibit its activity.

(e) optimal pH:

The enzyme did not show a "normal" pH-activity profile when the pH was varied between 7.0 and 11.0. 10 mg Amano Lipase A in 200 mM TEA-acetate buffer (pH 7.0 to 11.0) containing 0.1% Span 80, 20 mM magnesium acetate was incubated for 20 minutes at 40°C. Add an equal volume of 500mM MES buffer to stop the reaction of the sample, the pH of the buffer is 5.5, containing 50mM EDTA; the sample is centrifuged (10,000g, 10 minutes), and then analyzed by HPLC, wherein, with 0.46 × 15cm CHIRADEX column, eluted with 70:30 water-methanol, 1ml/min, 20°C. The highest activity is reached at a pH of about 10. There was no appreciable change in activity from this position to pH 11.0. The highest selectivity is achieved at about pH 9.0.

Embodiment 6 cultivates A.niger ATCC9142, 9029, 26875, 62863 and 10864, and to Its (R)-pantothenic acid lactonase activity was evaluated

To see if lactonase activity is widespread in the Aspergillus genus, five A. niger strains were grown under the following conditions:

Medium (relative to volume per liter): 1.2g NaNO ₃ , 0.5g KH ₂ PO ₄ , 0.2g MgSO ₄ x 7H ₂ O, 0.5g yeast extract, 5% glucose and 0.04ml trace metal solution, all Such solutions are described by Vishniac and Santer, Bacteriol. Rev. 21:195-213 (1957).

A 300 ml preculture in 11 flasks was inoculated at 30°C with 5 x 10 ⁶ spores per ml. After 8 hours, the preculture was added to 1.71 medium in a 21 fermenter with pH and dissolved oxygen control. The pH was maintained at 5.5 by automatic addition of 5M NaOH. Cells were incubated for 14 hours under hypoaerobic conditions (5-10% air saturation). The dissolved oxygen level was then increased to 30-50% air saturation. After 5 hours, the mycelia were harvested, filtered, washed twice with demineralized water, and frozen in liquid nitrogen. A part of them was directly lysed with a French Press. Media and cell lysates were investigated for pantothenate activity using standard assay methods.

(R)-Pantolactonase activity was mainly found in the lysates of all tested A. niger strains. Only very little activity was found in the medium.

Example 7 Trypsin Digestion of Enriched (R)-Pantolactonase from a Lipase Preparation The amino acid sequence of the resulting small peptide was determined

The enriched formulation of Example 5 was analyzed on a 2D gel. Two major spots, 33 and 40 kDa, pi of 4.7 and 5.6, respectively, were digested with trypsin. The resulting peptides were separated by HPLC (equipment: Hewlett Packard 1090, column: Vydac C18 (0.21×25 cm), absorption at 210 nm, flow rate: 0.20 ml/min, buffer A: 0.075% TFA, buffer B: 80% acetonitrile 0.065% TFA in , gradient: 2% B (0-10 min), 2-75% B (10-120 min)). The selected peptides were sequenced by Edman degradation method, point C [pI 5.6, 39kDa (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), point C [pI 4.7, 33kDa (Poll A/B) ]: N-terminal (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 represents an unidentified amino acid, and the amino acid in parentheses cannot be clearly identified.

A database search using all short peptide sequences as targets yielded no reliable information to help determine which of the two major spots represented the lactonase. Therefore, we used a set of anion exchange chromatography to further enrich the lactonase preparation of Example 5. Protein solutions (in 20 mM Tris/HCl, pH 7.5, 2.5 mM MgCl ₂ ) were separated on a prepacked MonoQ column HR 16/10 (Amersham Pharmacia Biotech Europe GmbH, Dübendorf, Switzerland) with a gradient of 0 to 350 mM NaCl. The activity of all fragments was assayed. Active fragments were analyzed by isoelectric focusing and SDS-PAGE as described by the manufacturer (Invitrogen, Carlsbad, CA, USA). Comparison of fragment activity data from SDS-PAGE gels and IEF suggested that the lactonase is a 38 to 40 kDa protein with a pi around pH 5.6. This data fits very well with point PollC. Therefore, the amino acid sequence of this spot was used to design PCR primers and oligonucleotides for Southern blot experiments. The most active fragments were collected and analyzed on a 2D gel, which showed that PollC was more clearly enriched relative to PollA/B than the previous 2D gel.

Embodiment 8 is designed to be used for the degenerate oligonucleotide of Southern blotting experiment and be used for from A.niger PCR primers for cloning (R)-pantolactonase

Using 12 different phytase sequences from different Aspergillus species, the codon usage of Aspergillus was obtained by the CODONFRENCY program of GCG package version 10.2, which was used to reduce possible differences due to the degeneracy of the genetic code The number of DNA sequence combinations. Using Aspergillus codon usage as the determining factor, every possible combination was considered at the 3'-end of the primers, but some rare codons were ignored at the 5'-end of the primers. In the case of hybridization experiments with oligonucleotides, the 3'- and 5'-ends were treated in the same way. Oligonucleotides with "S (sense)" or "AS (anti-sense)" in their names are intended for PCR. Oligonucleotides without S or AS in the name are for hybridization experiments.

The oligonucleotides used to clone (R)-pantolactonase from A. niger are Fxn42 (SEQ ID NO: 31), Fxn42S (SEQ ID NO: 32), Fxn42AS (SEQ ID NO: 33), Fx57 ( 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).

Embodiment 9 is from A.niger ATCC9142, A.niger NRRL3135, A.niger ssp. Genes isolated from awamori (ATCC38854) or A. niger MacRae (ATCC46951) DNA

Use 1×10 ⁶ spores/ml to inoculate 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)]. The cultures were grown overnight at 30°C with vigorous shaking (200 rpm). Mycelia were collected by filtration with Whatman filter paper, washed with PBS (isotonic phosphate buffered saline), and frozen in liquid nitrogen. In an ice-cold mortar, grind the mycelium to a fine powder. 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 inversions. The solution was cooled to 50 °C. Proteinase K was added (final concentration 100 μg/ml). The solution was incubated at 50°C for 1 hour. In addition, 100 mg/ml of proteinase K was added, and the culture was continued for 3 hours. Add 1/3 volume of phenol/chloroform/isoamyl alcohol (50/49/1). After thorough and gentle mixing, the emulsion was centrifuged (12000g, 20 minutes, 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. Add 0.7 volumes of isopropanol to the aqueous phase and mix the solution gently for 15 min. Precipitated DNA was collected by centrifugation (10000 g, 30 min, 4°C). To the remaining supernatant was added 1/10 volume of 3M KCl, pH 5.2, and incubated for 15 minutes with gentle shaking. After another centrifugation step, the two pellets were washed twice with 70% ethanol, air dried and dissolved in 0.5 ml of water. Finally, the DNA is treated with RNase to remove residual RNA.

Example 10 Cloning of (R)-pantothenate lactonase from A. niger ATCC9142

Genomic DNA (gDNA) of A. niger ATCC9142 was prepared as described in Example 6. The Robocycler Infinity from Stratagene (Stratagene, La Jolla, CA, USA) was used as PCR machine, and the TAQ polymerase kit from Roche Molecular Biochemicals (Penzberg, Germany) was used to carry out PCR:

1μl gDNA (1/10 dilution) Step 1: 5 minutes -95°C

1 μl Nucleotide Mixture Step 2: 30 sec -95°C

5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec -50°C

1 μl Primer S (100pM/μl) Step 4: 1 minute -72°C

1μl Primer AS (100pM/μl)

1 μl TAQ polymerase from Roche Molecular Biochemicals

40 μl _H2O

Steps 2 to 4 are repeated 35 times.

Primer combination:

serial number upstream primer downstream primer PCR product [bp] 1 Fxn42S Fxn57AS 300, 310 2 Fxn42S Fx73AS - 3 Fxn42S Fx74AS - 4 Fxn57S Fxn42AS 80 5 Fxn57S Fx73AS 580 6 Fxn57S Fx74AS 510

7 Fx73S Fxn42AS - 8 Fx73S Fxn57AS 300, 315, 320 9 Fx73S Fx74AS 10 Fx74S Fxn42AS 180 11 Fx74S Fxn57AS 340 12 Fx74S Fx73AS 840 13 Fxn42S Fx57AS, Fx73AS, Fx74AS - 14 Fxn57S Fxn42AS, Fx73AS, Fx74AS- 580 15 Fx73S Fxn42AS, Fxn57AS, Fx74AS - 16 Fx74S Fxn42AS, Fxn57AS, Fx73AS 350

All primers are checked for self-priming. Fxn57AS was the only primer that produced a significant amount of self-anchored product, with a self-anchored product of approximately 300 to 320 bp. With a hybridization temperature of 55°C, PCR 5, 6, 8, 10 and 11 still produced one or more products. Using 30-second amplification and 30 cycles at 60°C, PCRs 5, 6, 8, 10, and 11 still produced one or more PCR products corresponding to the bands produced at the lower hybridization temperature.

The 840 bp product of PCR reaction 12 was isolated from the agarose gel with the QIAEX II gel extraction kit from Qiagen (Qiagen, Hilden, Germany) and dissolved in 40 μl _H2O .

1μl 840bp fragment (1/10 dilution) Step 1: 5 minutes -95°C

1 μl nucleotide mixture Step 2: 30 sec -95°C

5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec -55°C

1μl Primer S (10pMol/μl) Step 4: 30 seconds -72°C

1μl Primer AS (10pMol/μl)

1 μl TAQ polymerase from Roche Molecular Biochemicals

40 μl _H2O

Repeat steps 2 to 4 30 times.

Primer combinations for reactions 2, 5, 10, 11 and 12 were used. Except reaction 2, all other reactions produced PCR products. This means that the sequences of primers 42S/AS, 57S/AS, 73S/AS and 74S/AS are part of a 840 bp fragment. The high proximity of these four pairs of primers strongly indicates that the 840bp fragment is part of the lactonase gene of interest. This hypothesis was supported by sequencing. The amino acid sequence of peptide Fx57 (SEQ ID NO: 90) was confirmed in a fragment of 840 bp, whereas the sequence of peptide Fxn42 (SEQ ID NO: 45) was only partially confirmed.

The corresponding DNA fragment of A. niger MacRae can also be isolated. In the case of A. awamori, only the products of primers Fxn57S and Fx73AS could be isolated by this method. The above sequence of Aspergillus has about 90% identity with the sequence of A. niger ATCC9142 at the DNA level.

To isolate the 5'- and 3'-ends of the lactonase gene, the following method was used:

(a) Isolation of the 3'-end:

1 μl gDNA of A. niger ATCC9142 Step 1: 5 minutes -95°C

1μl Nucleotide Mixture Step 2: 30 sec -95°C

5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec -55°C

1μl primer Oal2AS or Oal3S (10pMol/μl) Step 4: 30 seconds -72°C

1 μl TAQ polymerase from Roche Molecular Biochemicals

41 μl _H2O

Repeat steps 2 to 4 30 times.

The PCR mix was extracted twice with equal volumes of phenol/chloroform and once with chloroform alone. The DNA was then precipitated by adding 1/10 volume of 3M potassium acetate pH 5.2 and 2 volumes of ethanol. After 15 minutes of centrifugation (14000 rpm in Eppendorf plate centrifuge tubes), the pellet was washed with 70% ethanol, air-dried and finally dissolved in 20 μl sterile water.

Second PCR:

4 μl of DNA from the first PCR Step 1: 5 min -95°C

1 μl nucleotide mixture Step 2: 30 sec -95°C

5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec -58°C

1 μl primer Oal4S or Oal1AS (10pMol/μl) Step 4: 1 minute -72°C

1 μl High Fidelity Mix from Roche Molecular Biochemicals

38 μl _H2O

Steps 2 to 4 are repeated 35 times.

Using the Oal4S primer, a 900 bp DNA molecule was generated. Sequencing revealed the 3'-end of its encoding gene.

(b) Isolation of the 5'-end:

Using DIG-labeled oligonucleotides Fxn42, Fxn57, Fx73 and Fx74 as probes, the genomic DNA of A. niger ATCC9142 was hybridized using the DIG system (Roche Molecular Biochemicals) with X-ray film chemiluminescence detection. Hybridization and probing were performed according to the manufacturer's recommendations.

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. Two wash steps with 2x SSC, 0.1% SDS at room temperature were followed by two more wash steps with 0.5x SSC, 0.1% SDS at 50°C. With CSPD (Disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1 ^3,7 ]decan}-4-yl)-1 -phenyl phospate) was used as a substrate to detect alkaline phosphatase, and then the X-ray film was exposed to a filter (filter) for 1 hour. No usable signal on film.

Therefore, randomly anchored DIG-labeled PCR products from reactions 5 and 11 (see Example 11) were used as probes. Hybridization was performed in the same manner, except that the washing conditions were slightly changed to 2 x 5 minutes at room temperature and 2 x 15 minutes at 55°C. The following bands were shown on the film:

Restriction enzyme 340bp probe [kb] 580bp probe [kb] BamHI 10 10/2.8 EcoRI 4.5 4.5/5.2 Hind III 4.2/6.5 3.0/4.2/6.5 PstI 4.4 2.8/4.4 SacI twenty three 23/3.9 XhoI 6 6/20

In order to clone the entire gene or at least its missing 5'-end, according to the hybridization experiment, the chromosomal DNA of A. niger ATCC9142 was digested with HindIII to produce a fragment containing the lactonase gene of about 4 kb. The experimental method is as follows:

20 μl chromosomal DNA

20 μl 10x buffer

5μl HindIII (10U/μl)

175 μl _H2O

Incubate at 37°C for 16 hours.

The digested DNA was purified with a PCR purification kit from Qiagen (Qiagen, Hilden, Germany) and eluted in 100 μl. The following ligation reactions were performed overnight at 16°C. Dilute the purified DNA to 2ml with water. Ligase and ligase buffer were added as recommended by the manufacturer (Roche Molecular Biochemicals). The ligated solution was used as a template for a different PCR using internal primers for the partially isolated lactonase gene:

5 μl ligation reaction solution Step 1: 5 minutes -95°C

1μl Nucleotide Mixture Step 2: 30 sec -95°C

5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec -55°C

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

1μl anti-sense primer (10pMol/μl)

1 μl High Fidelity Polymerase Mix from Roche Molecular Biochemicals

40 μl _H2O

Steps 2 to 4 are repeated 32 times.

Four primer combinations, Oal3S/Fxn57AS, Oal4S/Fxn57AS, Oal3S/Oal8AS and Oal4S/Oal8AS, can all generate a single 4kb DNA fragment. All primer pairs are directed to the outside of the gene. Therefore, the lactonase gene should be located on a circular DNA molecule as a self-ligating HindIII fragment.

Fragments were isolated from the gel with the QIAEX II gel extraction kit from Qiagen, and sequenced with the ABI 310 Genetic Analyzer according to the dideoxy method recommended by the manufacturer (Applied Biosystems, Foster City, CA, USA). The sequence obtained confirmed that the isolated fragment contained both ends of the (R)-pantolactonase gene from A. niger ATCC9142.

Using the primers Oall0S and Oal12AS, it was also possible to isolate the homologous gene of A. niger ssp.awamori, which contains the exact same amino acid sequence as all the peptides tested. The only difference is only in the sequence of the peptide Fxn42. At this point Fxn42 comes from the sequencing of two peptides. Most likely, when HPLC was performed to separate the digested enzyme fragments, two different peptides eluted simultaneously. Sequencing first revealed only the sequence of the main peptide, but with a strong background. Once the sequencing of the first peptide is complete, all that remains is the sequence of the second peptide, which has four more 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 the oligonucleotides Fxn42S and Fxn42AS based on the peptide Fxn42 failed to give combined results. Based on the amino acid sequence, the protein from strain ATCC9142 has a molecular weight of 36573 Da with an estimated absorbance at 280 nm of 1.83 (1 mg/ml protein solution), while the molecular weight of the amino acid sequence from A. niger ssp. awamori is calculated to be 36547 Da for 1 mg /ml protein solution, the estimated _E280nm is 1.831 (Pace et al., 1995).

Embodiment 11 is cloned from the (R)-pantothenate lactonase gene of A.niger ATCC 9142 and Express

The sequences obtained by all three methods described in Example 10 were compared and assembled to generate a contiguous 2443 bp sequence covering the entire lactonase gene. To determine the start codon, stop codon and possible introns of the gene, the available amino acid sequences of peptides obtained from purified genes from commercial preparations were compared with the two programs TESTCODE and The results obtained with CODONPREFERENCE are used in conjunction. While both programs determined the stop codon and an intron in the same way, the start codon was not evident, specifically because no other variants of the peptide Fx74 (SEQ ID NO:84) were available. The N-terminus of the peptide has been sequenced. A comparison of all possible translations to homologous amino acid sequences found during database searches was also not helpful, since the N-terminal part of the protein is completely heterologous. Therefore, two methionine residues found upstream of the position of peptide Fx74 but still in the same reading frame were selected and used to construct different 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 does not really favor Met over other amino acids like the start codon, since the distance between this region and the start codon often varies considerably in fungi. In addition, possible polyadenylation sites were identified downstream of the gene.

For the 72 bp long intron of the gene from A. niger ATCC9142, the DNA segment from 976 to 981 nucleotides most likely represents the 5'-splice site, from 1029 to 1035 or from 1016 The segment to 1022 nucleotides is most likely to represent a deduced internal conservative sequence (consensus sequence), and the DNA segment from 1045 to 1047 nucleotides is most likely to represent a 3'-cleavage site (SEQ ID NO: 7 ). The corresponding sequences of the A. niger MacRae gene are located at bp 32 to 37, 66 to 72, and 80 to 82 in a 51 bp long intron (SEQ ID NO: 79). For the 50bp long intron of the oal gene of A. niger ssp.awamori, the 5'-cleavage site starts from the 203rd nucleotide (GTGCCC), and the internal conserved region is at the 236th nucleotide (AACTAAC), the 3'-cleavage site is at the 250th nucleotide (CAG) (SEQID NO: 9).

intron of oal length 5'-site internal site 3'-site A. niger ATCC 9142 72 GTACCC ACCTAAC TAG A. niger MacRae 51 GTAATC AACTAAC CAG A. niger ssp awamori 50 GTGCCC AACTAAC CAG

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

The following cDNA was produced:

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

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

- oalEShort for expression in S.cerevisiae (with a shorter version of 31 amino acids of the second possible Met at position 32 of SEQ ID NO:95)

- oalS for expression in E. coli (oalS was made because of the misleading sequence of the Fxn42 peptide, which was eventually identified as an artifact)

-oalSEco for expression in S.cerevisiae

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

- oalEhis for expression in S. cerevisiae

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

- oalENhis (SEQ ID NO: 96) for expression in S.cerevisiae (construct oalsec, it contains the signal peptide of the phytase of A.terreus cbs, and this signal peptide is used for the expression in S.cerevisiae and secretion)

Usual procedure:

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

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), Oall0S (SEQ ID NO: 55), Oal10SEco (SEQ ID NO: 56), OalENhis (SEQ ID NO: 57), Oal10SShis (SEQ ID NO: 58), Oal11S (SEQ ID NO: 59), Oal12AS (SEQ ID NO: 60), Oal12ASEco (SEQ ID NO: 61), Oal12AShis (SEQ ID NO: 62), Oal12ASHisEco (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).

Oal11S begins directly in front of the site more upstream of the start codon, while Oal9AS begins several nucleotides downstream of the putative stop codon. In order to assemble the two resulting PCR products without introns in the third PCR, Oal14S and Oal14AS are complementary, containing the sequence from the end of exon I to the beginning of exon II.

1μl gDNA (1/10 dilution) Step 1: 5 minutes -95°C

1 μl Nucleotide Mixture Step 2: 30 sec -95°C

5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec -55°C

1 μl Oal11S (10pMol/μl) Step 4: 1 minute -72°C

1μl Oal14AS (10pMol/μl)

1 μl High Fidelity Polymerase Mixture from Roche Molecular Biochemicals 40 μl _H2O

Steps 2 to 4 are repeated 35 times.

1μl gDNA (1/10 dilution) Step 1: 5 minutes -95°C

1 μl Nucleotide Mixture Step 2: 30 sec -95°C

5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec -55°C

1 μl Oal14S (10pMol/μl) Step 4: 1 minute -72°C

1μl Oal9AS (10pMol/μl)

1 μl High Fidelity Polymerase Mixture from Roche Molecular Biochemicals 40 μl _H2O

Steps 2 to 4 are repeated 35 times.

PCR on gDNA of A. niger ATCC9142 gave a PCR product of the expected size. PCR products were purified by agarose gel electrophoresis. The PCR product was extracted from the gel with the QIAEX II 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 minutes -95°C

1 μl Nucleotide Mixture Step 2: 30 sec -95°C

5 μl PCR standard buffer with Mg ²⁺ Step 3: 30 sec -55°C

1 μl Oal12S (10pMol/μl) Step 4: 1 minute -72°C

1μl Oal10AS (10pMol/μl)

1 μl High Fidelity Polymerase Mixture from Roche Molecular Biochemicals 40 μl _H2O

Repeat steps 2 to 4 30 times.

The final product was purified by agarose gel electrophoresis, extracted from the gel, and cloned into a TA-vector according to the method provided by Invitrogen (carlsbad, CA, USA) for direct cloning of PCR products. All other necessary cloning steps were performed as described by Sambrook et al. (1989). The plasmid containing the correct insert (oal1) detected by sequencing was used as template in all subsequent construction steps.

(a) Constructs oal and oalEco for expression in E. coli and Saccharomyces cerevisiae: PCR was performed on oal1 with primers pQE80EcoS and pQE80BamAS to generate construct Eco-oal-Bam according to the following scheme:

1 μl of plasmid (10 ng) containing oal1 as insert

1 μl nucleotide mix

5μl PCR standard buffer with Mg2 ⁺

1μl pQE80EcoS (10pMol/μl)

1μl pQE80BamAS (10pMol/μl)

1 μl High Fidelity Polymerase Mixture from Roche Molecular Biochemicals 40 μl _H2O

Step 1: 5 minutes -95°C

Step 1: 30 seconds -95°C

Step 1: 30 seconds -55°C

Step 4: 1 minute -72°C

Repeat steps 2 to 4 30 times.

The PCR product was purified by agarose gel electrophoresis, the fragment of interest (QIAEX II, Qiagen) was extracted from the gel, digested with EcoRI and BamHI, purified again by agarose gel electrophoresis, and ligated into Transformed into E.coli Top10 on the vector pQE80 from Qiagen. Plasmids of four clones were isolated and the inserts were sequenced. Plasmids containing the correct constructs were transformed into E. coli M15 or equivalent E. coli strains. All molecular techniques are standard techniques known to the skilled person.

For yeast expression constructs, the primers used were changed to Oal10EcoS and Oal12ASEco, the PCR product was digested separately with EcoRI, and the final product was cloned into pYES2 or used for S.cerevisiae through the EcoRI restriction site on the vector. equivalent expression vector. According to Hinnen et al., Proc.Natl.Acad.Sci.USA 75:1929-1933 (1978) or according to the specification sheet provided simultaneously with bacterial strain, carry out to S.cerevisiae bacterial strain, for example INVScl (Invitrogen, Carlsbad, CA, USA ) conversion.

(b) Construct pQE80oalS for expression in E. coli (with a 31 amino acid shortened version of the second possible Met):

Use the same PCR scheme as above, use the same template, and use pQE80EcoSshort and pQE80BamAS as primers. Treat the reaction solution after the PCR reaction as described in (a).

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

Primers Oal16S and Oal12ASEco were used. Everything else was carried out as described in (a).

(d) Construct pQE80oalhis containing a C-terminal his-tag for expression in E. coli:

PCR was performed using primers pQE80EcoS and pQE80BamhisAS, see (a).

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

PCR was performed using primers Oal10Seco and Oal12AShisEco, see (a).

(f) Construct pQE80oalNhis containing the N-terminal His-tag for expression in E. coli:

PCR was performed using primers pQE80EcoNhisS and pQE80BamAS (see a).

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

PCR was performed using primers OalENhis and Oal12ASEco (see a).

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

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

1.0 μl of plasmid (10 ng) containing the conserved phytase gene as an insert

1.0 μl nucleotide mix

5.0μl PCR standard buffer with Mg2 ⁺

1.0μl CP-a (10pMol/μl)

1.0μl OalsecAS (10pMol/μl)

1.0 μl High Fidelity Polymerase Mix from Roche Molecular Biochemicals

40 μl _H2O

Step 1: 5 minutes -95°C

Step 1: 30 seconds -95°C

Step 1: 30 seconds -55°C

Step 4: 30 seconds -72°C

Repeat steps 2 to 4 30 times.

1.0 μl plasmid containing oal1 gene (10ng)

1.0 μl nucleotide mix

5.0μl PCR standard buffer with Mg2 ⁺

1.0μl OalsecS (10pMol/μl)

1.0μl Oal12ASEco (10pMol/μl)

1.0 μl High Fidelity Polymerase Mix from Roche Molecular Biochemicals

40 μl _H2O

Step 1: 5 minutes -95°C

Step 1: 30 seconds -95°C

Step 1: 30 seconds -55°C

Step 4: 45 seconds -72°C

Repeat steps 2 to 4 30 times.

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

0.5 μl PCR1 product

05 μl PCR2 product

1.0 μl nucleotide mix

5.0μl PCR standard buffer with Mg2 ⁺

1.0μl CP-a (10pMol/μl)

1.0μl Oal12ASEco (10pMol/μl)

1.0 μl High Fidelity Polymerase Mix from Roche Molecular Biochemicals

40 μl _H2O

Step 1: 5 minutes -95°C

Step 1: 30 seconds -95°C

Step 1: 30 seconds -55°C

Step 4: 1 minute -72°C

Repeat steps 2 to 4 30 times.

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

The expression of embodiment 12 oal

(a) In E.coli:

pQE80oal, pQE80oalS, pQE80oalhis and pQE80oalNhis were expressed in E. coli M15 containing pREP4 which contains the repressor of the lac promoter (see expression instructions from Qiagen, Hilden, Germany). The overnight culture was diluted to an OD600nm of 0.1 and grown at 30°C to an _OD600nm of 1.5. The cultures were then induced with 0.5 mM IPTG and incubated at 30°C for 6 hours. Cells were harvested by centrifugation (5000 g, 20 minutes) and frozen at -80°C. They were cleaved with B-PER (Pierce, Rockford, IL, USA) according to the protocol provided by the manufacturer. After another centrifugation step, the supernatant was used for the hydrolysis of (R)-pantolactone.

In transformants containing constructs pQE80oalS and pQE80oalhis, no activity was found in the supernatants or cell lysates, whereas with constructs pQE80oal and pQE80oalNhis approximately 5% of the soluble protein was active (R )-pantothenic acid lactonase.

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

(b) In S. cerevisiae:

The constructs oalEco, oalES, oalEhis, oalENhis and oalsec were expressed in S. cerevisiaeINVScl or similar equivalent strains. 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 at 30°C for 3 to After 4 days of cultivation, the cultured colonies were picked out, transferred to 2ml SD ^ura- liquid medium, and cultured at 30° C. for 3 days under vigorous shaking. The above culture was used as a pre-culture in 25 ml of SD ^ura -medium. After an additional 3 days of cultivation at 30°C with vigorous shaking, 500 ml of YPD medium in 21 flasks was inoculated with the prepared culture [Sherman et al., as described above]. After an additional 3 days of culture under the same conditions, cells were separated from the medium by centrifugation and lysed with Y-PER (Pierce, Rockford, IL).

As in the case of expression in E. coli, only the oalEco and oalENhis constructs resulted in the expression of active (R)-pantothenolactonase, which was found only in cell lysates. 300 U of (R)-pantolactonase can also be produced with 75 ml of YPD culture. The corresponding lysates showed a specific activity of approximately 5 U/mg total protein.

The oal gene from A. awamori and the oal gene from A. niger ATCC46951 can also be constructed and expressed in a similar manner using the sequence data of the above intron position.

Example 13 fixes Oal heterologously expressed in E.coli or S.cerevisiae, and Use it for the hydrolytic resolution of (RS)-pantolactone

(R)-Pantolactonase from A. niger was immobilized for multiple uses in bioreactors. After expression in E.coli or S.cerevisiae, by chemical means such as B-PER (E.coli) or Y-PER (S.cerevisiae) (Pierce, Rockford, IL, USA), or by mechanical means such as ultrasound Or high pressure to disrupt the cells. Lysates were clarified by centrifugation. Preparations were used directly for fixation. Alternatively, use another purification step, such as ammonium sulfate precipitation, prior to fixation to further increase Oal specific activity.

The biocatalyst was immobilized as follows:

(1) Immobilized on Silica Sol-Gel:

According to the literature [Gill and Ballesteros, J.Am.Chem.Soc.120:8587-8598 (1998)], poly(glycerol silicate)-1.0 (PGS-1.0) was prepared and dissolved in half of its mass icy water. A 100 or 200 mg portion of this was mixed thoroughly with 50 or 100 mg of ice-cold biocatalyst stock solution preparation (soluble enzyme from several preparations) in 50 mM phosphate in 2 ml or 5 ml vials, pH 7.0 , swirling the vial gently for 2 minutes to form a thin layer of hydrogel on the vial walls. Place the vial on ice for 20 minutes, then transfer (open) to the refrigerator. The hydrogel was matured at 5°C for 48-72 hours to form a xerogel. Wash the vial with xerogel 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-B at pH 7 by shaking at 100 rpm at 5°C. acid salt, which contains 10mM magnesium acetate.

(2) Fix Oal on silica polyvinyl alcohol sol gel (Sol-Gel Silica-Polyvinyl Alcohol):

The method was similar to (1), except that the DGS solution was mixed with the appropriate amount of polyvinyl alcohol (PVA, 85K in water at 13% w/w from Aldrich) before combining with the lactonase stock solution. The follow-up method is shown in (1).

(3) Oal is fixed on the polyethylenimine activated and supported with glutaraldehyde:

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% acetic acid and butyric acid content, ex Aldrich) was dissolved in 5 mL of dichloromethane and 7 g of Fullers Earth (Fullers Earth, 30-60 mesh, ex Aldrich) was coated with the solution. The wet-coated material was dried in air at room temperature for 0.5 hours until approximately 9 g of coated Fuller's earth was given. It was then resuspended in an ice-cold mixture of glutaraldehyde (8 mL, 50% w/w from Aldrich) and phosphate buffer (50 mL, 50 mM, pH 8) and stirred at 200 rpm for 2 hours. The activated support was washed with water (4×10 mL), then with phosphate buffer (4×10 mL, 20 mM, pH 8), and the liquid was drained. The wet support was mixed with ice-cold lactonase stock solution (50 mM phosphoric acid, pH 8) and the suspension was stirred at 200 rpm for 20-30 hours. The liquid was gently removed from the immobilized enzyme, the wet fixation was washed with phosphate (3 × 5 mL), treated with ethanolamine (20 mL, 20 mM, pH 8), and washed with phosphate (2 × 5 mL, 20 mM , pH 7), and then drain the liquid.

(4) Reaction conditions for hydrolytic resolution of (RS)-pantolactone

Reactions were carried out 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 8.5, containing 20 mM magnesium acetate). The vials were incubated at 40°C, 200 rpm for the necessary period of time, the solution was drained and analyzed by chiral HPLC, the catalyst was washed (2 x 1 or 2 mL) with fresh substrate solution before starting the next cycle. The reaction of samples for analysis was stopped with an equal volume of 500 mM MES buffer, pH 5.5 containing 50 mM EDTA, centrifuged (10000 g, 10 min) and then analyzed by HPLC using a 0.46 x 15 cm CHIRADEX column, eluted with 70:30 water-methanol, 1 mL/min, at 20°C. Initial velocity measurements were taken at 15 minutes and E values were determined at the end of each cycle.

Table 2 summarizes the effect of selected immobilized enzyme preparations on the batch resolution of (RS)-pantolactone. Both sol-gel and covalent immobilization schemes proved to be effective, and even after 24 1-hour batch cycles, the catalyst retained 62–72% of its initial activity and showed: Excess values and enantioselectivities to (R)-pantolactone were 89-98% and 71-88%, respectively. Furthermore, the catalyst behaved very similarly when the batches were run for extended periods of time, showing good long-term operational stability when each batch was run for a day instead of 1 hour.

Table 2: Batch resolution of (RS)-pantolactone by immobilized lactonase preparations

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

The Oal expressed by recombinant E.coli that is fixed in embodiment 14 is carried out continuously to (RS)-pantolactone split app

A sol-gel-PVA immobilized recombinant lactonase biocatalyst (expressed in E. coli) prepared as described in Example 14(2) was used in a packed-bed reactor Sequential resolution of racemic lactones: The immobilized biocatalyst (1.1 g, 0.21 kUg ^-1 , 0.231 kU in total) was dry packed into a 0.46×15 cm Omnifit jacketed glass column with Teflon endpieces . It was attached to a 1 x 10 cm Omnifit jacketed glass column filled with 1-2 mm cellulose beads via two syringe pumps fitted with 50 mL glass/Teflon syringes. These columns were connected to a circulating water bath, which was maintained at 40°C during the resolution. The reactor was conditioned as follows: Tris-acetate buffer (100 mM, pH 7, containing 100 mM magnesium acetate) was added at a rate of ⁵ mL min at room temperature for 30 minutes; followed by 1 :1 volume ratio, total flow rate of 2 mL min ^-1 , adding a combination of racemic lactone solution (1.5M in water) and Tris-acetic acid buffer (2.5M, pH 8.25, containing 50mM magnesium acetate) for 30 minute. The flow rate was then reduced to 1.2 mL min ^-1 , the column was heated to 40°C, and the system was allowed to equilibrate for 15 minutes. The reactor was thus operated for approximately 45 minutes, collecting the eluate on an ice bath, under which conditions a total of 52.7 mL of feed (corresponding to 5.14 g RSPL) were processed. The pH of the eluent was adjusted to 6.5 with sulfuric acid (10% v/v aqueous solution), the solution was extracted with dichloromethane (10×75 mL), the organic layer was dried over anhydrous magnesium sulfate, and the rotary evaporation yielded (S)-Pantolactone enrichment (2.67 g, 98% of theory) consisting of 10% RPL and 90% SPL (by chiral HPLC analysis). The aqueous phase was acidified with sulfuric acid (40% w/w) to pH 1.5, heated to 70 °C for 1 h, cooled on ice, saturated with NaCl, and extracted with dichloromethane (10 x 75 mL ), the organic layer was dried over anhydrous magnesium sulfate and rotary evaporated to yield (R)-pantolactone rich (2.23 g, 92% of theoretical yield) with an enantiomeric purity of 93%. The combined recovery for (R)-/(S)- was 95%.

The results of Examples (13) and (14) clearly show that the recombinant lactonase is an effective biocatalyst for the hydrolytic resolution of racemic pantothenate lactone, the catalyst can also be effectively fixed, and the fixed material can be used for the production of Highly enriched (R)-pantolactone with an enantiomeric excess of 90-98%, both in the case of batch and continuous operations.

Example 15 Cloning and expression of (S)-pantolactone-specific lactonase from B. subtilis

Using the amino acid sequences of lactonases from bovine and equine liver, we found several other sequences showing limited homology to the above sequences. Among them, the yvre gene from B. subtilis (YVRE BACSU, annotated as a member of the SMP30/CGR1 family/hypothetical 33.2kDa protein) was cloned from genomic DNA by PCR using the 5' and 3'- The ends also contained restriction sites (EcoRI and PstI) required for subsequent cloning steps into expression vectors. The PCR conditions were the same as those used to isolate the oal gene from A. niger genomic DNA (see Example 8). The PCR product and vector (PET41a from Stratagene (La Jolla, CA, USA)) were digested with EcoRI and PstI, purified by agarose gel electrophoresis, ligated, and transformed into E.coli Top10 cells (Stratagene, La Jolla, CA). Jolla, CA, USA). Using this strategy, the gene was fused into the reading frame of the GST protein gene from E. coli.

Expression and purification were performed according to the protocol provided by the manufacturer. The activity of the purified protein was determined using a standard assay at 40°C with additional 5 mM _CaCl2 . This enzyme shows very high selectivity towards (S)-pantolactone. This preparation has a specific activity of 1-2 U per mg of enriched fusion protein. The enzyme is only active in the presence of Mg ²⁺ or Ca ²⁺ ions.

sequence listing

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Arg His Gln Gly Ser Leu Tyr Ala Leu Phe Pro Asp His His Val Glu

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Asn Trp Asp Leu Glu Lys Leu Asn Pro Pro Gly Phe Gly Arg Lys Pro

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20 25 30

Glu Tyr His Phe Asn Pro Gln Gln Ser Ser Gln Trp Asp Gly Leu Arg

35 40 45

His His Asn Ala Pro Ala Pro Thr Pro Asp Asp Pro Asp Arg Arg Val

50 55 60

Phe Tyr Gly Gly Thr Thr Ser Thr Glu Ile Leu Asp Pro Ser Ser Ala

65 70 75 80

Arg Ile Gly Ile Ala His Trp Ala Lys Lys Gly Ile Ala Gly Arg Gly

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Val Leu Ile Asp Tyr Ala Ser Tyr Ile Gln Arg Thr Lys Gly Ile Pro

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Val Asn Ala Leu Thr Arg His Thr Val Ser Leu Asp Asp Val Leu Thr

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Ile Ala Lys Glu Cys Asn Ile Thr Phe Gln Pro Gly Asp Ile Leu Phe

130 135 140

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180 185 190

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Glu Trp Asp Leu His His Phe Leu Leu Ala Gly Trp Gly Val Pro Ile

210 215 220

Gly Glu Met Phe Asp Leu Glu Gly Leu Lys Glu Val Cys Glu Arg Leu

225 230 235 240

Gly Arg

<210>7

<211>2443

<212>DNA

<213>Aspergillus niger

<220>

<221> CDS

<222>(774)..(977)

<223>

<220>

<221> CDS

<222>(1050)..(1820)

<223>

<400>7

ccgctatcac tcagcggcag tcgggcgcag acaatgctac cgaggattca gggcacaagg 60

gggatcagtt acgtggggcg tgacgatgta tgggtttcat gaggatgcgt ttacgagtgc 120

gtttggggtg gtggaagagt tgggggcgaa gatcccgttt ccggtggctg atagtcggtt 180

gagagggggt gcagtgaagg aggtgagatg gttggaatat gtgttcagat tggtgttgca 240

gggggtgcag ggcttgattt tgtggctggt gggagaggga aaagggaagc agaaggcgag 300

ctagaacctt ttacacactg cctctcgaat gaacattcag aaattttgga tagacaaggt 360

ttctgtcggt gctattctgg gcgttgcaat acctgtcaca ttgaataacc tccgaggtga 420

ttatccacgc aatcccatat catctgatgt accccttgca taatctgtct ttaccaaatc 480

aggtagaaag taagaccatc cggataggct gtatactacc tcgtgtccac atcggggata 540

ctgaacacga ttgtatatca attcgatgat ggagatacaa ccctaatgaa ccaagcacag 600

ccgtggatct ccccggactt tctccgatgg cttccccacc cgccaatcca gcatcaacta 660

aacaacccct cacgctcttc atcatctctc atttcactcc cacctatcat ctacagcttc 720

acaaacaaca ctactccctc ccaagcacac acctctactg acaagatacc acg atg 776

Met

1

gcc gac tcc ctc ccg aaa acc tac gac gac ctc ccc gac aag cgg cgc 824

Ala Asp Ser Leu Pro Lys Thr Tyr Asp Asp Leu Pro Asp Lys Arg Arg

5 10 15

tac tgg ccc gca acc ccc aac tcc gcc gac gag gga ctg ggg atg ctc 872

Tyr Trp Pro Ala Thr Pro Asn Ser Ala Asp Glu Gly Leu Gly Met Leu

20 25 30

cgt ctc ctc act cca gaa att gtc gcc aac gca gca cgc acc cag atc 920

Arg Leu Leu Thr Pro Glu Ile Val Ala Asn Ala Ala Arg Thr Gln Ile

35 40 45

cag acc ggc gag cgc gta tgc ctg aac tgg gac ctg gag aag ctg aac 968

Gln Thr Gly Glu Arg Val Cys Leu Asn Trp Asp Leu Glu Lys Leu Asn

50 55 60 65

cca cca ggt accctccctt ctacctcagt gacataggtt cacccccaac 1017

Pro Pro Gly

ctcacccatc aacctaactt aaaaacatag gc ttc ggc cgc aaa ccc ttc gcc 1070

Phe Gly Arg Lys Pro Phe Ala

70 75

cac cac gta aaa tgg gtc tcc gaa ggc cac gcc ttc gac gac gaa tac 1118

His His Val Lys Trp Val Ser Glu Gly His Ala Phe Asp Asp Glu Tyr

80 85 90

cac ttc aac ccg caa caa tcc tcc caa tgg gac ggt ctg cga cac cac 1166

His Phe Asn Pro Gln Gln Ser Ser Gln Trp Asp Gly Leu Arg His His

95 100 105

aac gcc ccg gct cca acc ccc gac gac cct aac cgc cgg gtc ttc tac 1214

Asn Ala Pro Ala Pro Thr Pro Asp Asp Pro Asn Arg Arg Val Phe Tyr

110 115 120

ggc ggc acc acc tcc acc gag atc ctc gac ccc tcg tcc acc cga atc 1262

Gly Gly Thr Thr Ser Thr Glu Ile Leu Asp Pro Ser Ser Thr Arg Ile

125 130 135

ggc ata gcc cac tgg gcc aag aaa ggc atc gcc ggc cgc ggc gtc ctc 1310

Gly Ile Ala His Trp Ala Lys Lys Gly Ile Ala Gly Arg Gly Val Leu

140 145 150 155

atc gac tac gcc tcc tac gtg caa cga acc aag ggc gtc aca gta aac 1358

Ile Asp Tyr Ala Ser Tyr Val Gln Arg Thr Lys Gly Val Thr Val Asn

160 165 170

gcc cta acc cgc cac acc gtc tcc ctg gac gac gtc ctc acc atc gcg 1406

Ala Leu Thr Arg His Thr Val Ser Leu Asp Asp Val Leu Thr Ile Ala

175 180 185

aag gaa tgc aac atc acc ttc gaa cca ggc gac att ctc ttc ctg cgc 1454

Lys Glu Cys Asn Ile Thr Phe Glu Pro Gly Asp Ile Leu Phe Leu Arg

190 195 200

gtc ggt cta ccg act aca tgg gat aac atg acc gat gag gag agg gtc 1502

Val Gly Leu Pro Thr Thr Trp Asp Asn Met Thr Asp Glu Glu Arg Val

205 210 215

aag tat agt cag cag gaa acg ccc aac cat gca gga tta gag cag agt 1550

Lys Tyr Ser Gln Gln Glu Thr Pro Asn His Ala Gly Leu Glu Gln Ser

220 225 230 235

gag cgt gtg gtg cgg ttc ctt tgg gat cag cat ttc gcc gct gta gcg 1598

Glu Arg Val Val Arg Phe Leu Trp Asp Gln His Phe Ala Ala Val Ala

240 245 250

ggt gat gcg gtc agc ttt gag gtg tat ccg ccg gta gag aag gag tgg 1646

Gly Asp Ala Val Ser Phe Glu Val Tyr Pro Pro Val Glu Lys Glu Trp

255 260 265

gat tta cat cat ttt ttg ctg gct ggg tgg gga cta ccg att ggg gag 1694

Asp Leu His His Phe Leu Leu Ala Gly Trp Gly Leu Pro Ile Gly Glu

270 275 280

atg ttt gat ctt gag ggg ttg aag gag gtg tgt gag agg ttg gga agg 1742

Met Phe Asp Leu Glu Gly Leu Lys Glu Val Cys Glu Arg Leu Gly Arg

285 290 295

tgg acg ttt ttc gtt agt tct agt ccg ttg aac tgt ggg aga ggt gtg 1790

Trp Thr Phe Phe Val Ser Ser Ser Ser Pro Leu Asn Cys Gly Arg Gly Val

300 305 310 315

tcg agt ccg ccg aat tgt atg gca att ttt taaaatgcag aggtggtgtg 1840

Ser Ser Pro Pro Asn Cys Met Ala Ile Phe

320 325

ggaacggagt tggatggacg atggagtgga tagaacagcg ggagttgatg tattgagctg 1900

gggccctagt catgatttgt ctgaatctgg agactataca tagcaccttc agggcatcaa 1960

gaggcataat atgcagttat acttgtgata ttacattctt tggccacagt tatgactgtc 2020

tcttatattg actttgaagt gtcttctttg agccgagtat acttggatta taggattcca 2080

atgacggtac atcccgaagg tctgtcccag cacttcccta atcgcagtgg taatacctac 2140

tactagccag tatatctaaa gtacaccact cggaactcat tcacatatac gaatggtgac 2200

tatgaagggg taatacattc acaacaagat cagtaaatag cagttgcgag gaatgcaaga 2260

ctagcccttc gtacattcaa tatcagaagt tgggactata aaggccactg cctccctcac 2320

tagcccgtaa agcaatacct cgctgctacc attgccaact gcagggtgtt tccagtaaac 2380

ggagtggctg ttcccaccgt gcagcactct gtacccactc cccttcctgc tgtcattctg 2440

att 2443

<210>8

<211>325

<212>PRT

<213>Aspergillus niger

<400>8

Met Ala Asp Ser Leu Pro Lys Thr Tyr Asp Asp Leu Pro Asp Lys Arg

1 5 10 15

Arg Tyr Trp Pro Ala Thr Pro Asn Ser Ala Asp Glu Gly Leu Gly Met

20 25 30

Leu Arg Leu Leu Thr Pro Glu Ile Val Ala Asn Ala Ala Arg Thr Gln

35 40 45

Ile Gln Thr Gly Glu Arg Val Cys Leu Asn Trp Asp Leu Glu Lys Leu

50 55 60

Asn Pro Pro Gly Phe Gly Arg Lys Pro Phe Ala His His Val Lys Trp

65 70 75 80

Val Ser Glu Gly His Ala Phe Asp Asp Glu Tyr His Phe Asn Pro Gln

85 90 95

Gln Ser Ser Gln Trp Asp Gly Leu Arg His His Asn Ala Pro Ala Pro

100 105 110

Thr Pro Asp Asp Pro Asn Arg Arg Val Phe Tyr Gly Gly Thr Thr Ser

115 120 125

Thr Glu Ile Leu Asp Pro Ser Ser Thr Arg Ile Gly Ile Ala His Trp

130 135 140

Ala Lys Lys Gly Ile Ala Gly Arg Gly Val Leu Ile Asp Tyr Ala Ser

145 150 155 160

Tyr Val Gln Arg Thr Lys Gly Val Thr Val Asn Ala Leu Thr Arg His

165 170 175

Thr Val Ser Leu Asp Asp Val Leu Thr Ile Ala Lys Glu Cys Asn Ile

180 185 190

Thr Phe Glu Pro Gly Asp Ile Leu Phe Leu Arg Val Gly Leu Pro Thr

195 200 205

Thr Trp Asp Asn Met Thr Asp Glu Glu Arg Val Lys Tyr Ser Gln Gln

210 215 220

Glu Thr Pro Asn His Ala Gly Leu Glu Gln Ser Glu Arg Val Val Arg

225 230 235 240

Phe Leu Trp Asp Gln His Phe Ala Ala Val Ala Gly Asp Ala Val Ser

245 250 255

Phe Glu Val Tyr Pro Pro Val Glu Lys Glu Trp Asp Leu His His Phe

260 265 270

Leu Leu Ala Gly Trp Gly Leu Pro Ile Gly Glu Met Phe Asp Leu Glu

275 280 285

Gly Leu Lys Glu Val Cys Glu Arg Leu Gly Arg Trp Thr Phe Phe Val

290 295 300

Ser Ser Ser Pro Leu Asn Cys Gly Arg Gly Val Ser Ser Pro Pro Asn

305 310 315 320

Cys Met Ala Ile Phe

325

<210>9

<211>1025

<212>DNA

<213>Aspergillus awamorii

<220>

<221> CDS

<222>(1)..(204)

<223>

<220>

<221> CDS

<222>(255)..(1025)

<223>

<400>9

atg gcc gac tcc ctc ccg aaa acc tac gac gac ctc ccc gac aag cgg 48

Met Ala Asp Ser Leu Pro Lys Thr Tyr Asp Asp Leu Pro Asp Lys Arg

1 5 10 15

cgc tac tgg ccc gca gcc ccc aac tcc gcc gac gag ggg ctg ggg atg 96

Arg Tyr Trp Pro Ala Ala Pro Asn Ser Ala Asp Glu Gly Leu Gly Met

20 25 30

ctc cgt ctc ctc acc cca gaa gtg gtc gcc aac gca gca cgc acc cag 144

Leu Arg Leu Leu Thr Pro Glu Val Val Ala Asn Ala Ala Arg Thr Gln

35 40 45

atc cag acc ggc gag cgt gta tgc ctc aac tgg gac ctg gag aag cta 192

Ile Gln Thr Gly Glu Arg Val Cys Leu Asn Trp Asp Leu Glu Lys Leu

50 55 60

aac cca cca ggt gcccctcctt ccacccccat cgtctctcac caactaacca 244

Asn Pro Pro Gly

65

aaacacaggt ttc ggc cgc aaa ccc ttc gcc cac cag gtt aaa tgg gtc 293

Phe Gly Arg Lys Pro Phe Ala His Gln Val Lys Trp Val

70 75 80

aac gaa ggc cac gcc ttc gac gac gaa tac cac ttc aat ccc cag caa 341

Asn Glu Gly His Ala Phe Asp Asp Glu Tyr His Phe Asn Pro Gln Gln

85 90 95

tcc tcc caa tgg gac ggc ctc cga cac cac aac gcc ccg gcc cca aca 389

Ser Ser Gln Trp Asp Gly Leu Arg His His Asn Ala Pro Ala Pro Thr

100 105 110

ccc gaa gac cct gag cgc cgc gtc ttc tac ggc ggc acc acc tcc acc 437

Pro Glu Asp Pro Glu Arg Arg Val Phe Tyr Gly Gly Thr Thr Ser Thr

115 120 125

gag atc ctc gac ccc tcg tcc gca cgg atc ggc ata gcc cac tgg gcc 485

Glu Ile Leu Asp Pro Ser Ser Ala Arg Ile Gly Ile Ala His Trp Ala

130 135 140 145

aag aag ggc atc gcc ggc cgc ggc gtc ctc ata gac tac gcc tcc tac 533

Lys Lys Gly Ile Ala Gly Arg Gly Val Leu Ile Asp Tyr Ala Ser Tyr

150 155 160

atc cag cga acc aag ggc atc aca gta aac gcc cta acc cgc cac acc 581

Ile Gln Arg Thr Lys Gly Ile Thr Val Asn Ala Leu Thr Arg His Thr

165 170 175

gtc tcc ctc gac gac gtc ctc acc atc gcg aag gaa tgc aac att acc 629

Val Ser Leu Asp Asp Val Leu Thr Ile Ala Lys Glu Cys Asn Ile Thr

180 185 190

ttc cag cca ggc gac att ctc ttc ctg cgc gtt ggt cta ccg aca acg 677

Phe Gln Pro Gly Asp Ile Leu Phe Leu Arg Val Gly Leu Pro Thr Thr

195 200 205

tgg gat aac atg tcc gat gaa gag aag gtc gagtat agt caa cag caa 725

Trp Asp Asn Met Ser Asp Glu Glu Lys Val Glu Tyr Ser Gln Gln Gln

210 215 220 225

aca ccc cag cat gca ggt tta gag cag agc gag cgt gtg gtg cgg ttc 773

Thr Pro Gln His Ala Gly Leu Glu Gln Ser Glu Arg Val Val Arg Phe

230 235 240

ctc tgg gac cag cat ttc gcg gct gtg gcg ggc gat gcg gtc agc ttt 821

Leu Trp Asp Gln His Phe Ala Ala Val Ala Gly Asp Ala Val Ser Phe

245 250 255

gag gtg tat ccg ccg gta gag aag gag tgg gat tta cac cat ttc ttg 869

Glu Val Tyr Pro Pro Val Glu Lys Glu Trp Asp Leu His His Phe Leu

260 265 270

ctt gct ggg tgg gga gtg ccg att gga gag atg ttt gat ctg gag ggg 917

Leu Ala Gly Trp Gly Val Pro Ile Gly Glu Met Phe Asp Leu Glu Gly

275 280 285

ttg aag gag gtg tgt gag agg ttg ggc agg tgg acg ttt ttt gtt agt 965

Leu Lys Glu Val Cys Glu Arg Leu Gly Arg Trp Thr Phe Phe Val Ser

290 295 300 305

tcg agt ccg ttg aat tgt aag acg ggg gtg tcg agt ccg ccg aat tgt 1013

Ser Ser Pro Leu Asn Cys Lys Thr Gly Val Ser Ser Pro Pro Asn Cys

310 315 320

atg gca att ttt 1025

Met Ala Ile Phe

325

<210>10

<211>325

<212>PRT

<213>Aspergillus awamorii

<400>10

Met Ala Asp Ser Leu Pro Lys Thr Tyr Asp Asp Leu Pro Asp Lys Arg

1 5 10 15

Arg Tyr Trp Pro Ala Ala Pro Asn Ser Ala Asp Glu Gly Leu Gly Met

20 25 30

Leu Arg Leu Leu Thr Pro Glu Val Val Ala Asn Ala Ala Arg Thr Gln

35 40 45

Ile Gln Thr Gly Glu Arg Val Cys Leu Asn Trp Asp Leu Glu Lys Leu

50 55 60

Asn Pro Pro Gly Phe Gly Arg Lys Pro Phe Ala His Gln Val Lys Trp

65 70 75 80

Val Asn Glu Gly His Ala Phe Asp Asp Glu Tyr His Phe Asn Pro Gln

85 90 95

Gln Ser Ser Gln Trp Asp Gly Leu Arg His His Asn Ala Pro Ala Pro

100 105 110

Thr Pro Glu Asp Pro Glu Arg Arg Val Phe Tyr Gly Gly Thr Thr Ser

115 120 125

Thr Glu Ile Leu Asp Pro Ser Ser Ala Arg Ile Gly Ile Ala His Trp

130 135 140

Ala Lys Lys Gly Ile Ala Gly Arg Gly Val Leu Ile Asp Tyr Ala Ser

145 150 155 160

Tyr Ile Gln Arg Thr Lys Gly Ile Thr Val Asn Ala Leu Thr Arg His

165 170 175

Thr Val Ser Leu Asp Asp Val Leu Thr Ile Ala Lys Glu Cys Asn Ile

180 185 190

Thr Phe Gln Pro Gly Asp Ile Leu Phe Leu Arg Val Gly Leu Pro Thr

195 200 205

Thr Trp Asp Asn Met Ser Asp Glu Glu Lys Val Glu Tyr Ser Gln Gln

210 215 220

Gln Thr Pro Gln His Ala Gly Leu Glu Gln Ser Glu Arg Val Val Arg

225 230 235 240

Phe Leu Trp Asp Gln His Phe Ala Ala Val Ala Gly Asp Ala Val Ser

245 250 255

Phe Glu Val Tyr Pro Pro Val Glu Lys Glu Trp Asp Leu His His Phe

260 265 270

Leu Leu Ala Gly Trp Gly Val Pro Ile Gly Glu Met Phe Asp Leu Glu

275 280 285

Gly Leu Lys Glu Val Cys Glu Arg Leu Gly Arg Trp Thr Phe Phe Val

290 295 300

Ser Ser Ser Pro Leu Asn Cys Lys Thr Gly Val Ser Ser Pro pro Asn

305 310 315 320

Cys Met Ala Ile Phe

325

<210>11

<211>11

<212>PRT

<213> Eguus caballus

<400>11

His Gln Gly Ser Leu Val Ala Leu Phe Pro Asp

1 5 10

<210>12

<211>23

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(23)

<223>5'-primer

<400>12

atgtcttcca tcaagattga atg 23

<210>13

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223>3'-primer

<400>13

gtcaaaggaa ttccccccta t 21

<210>14

<211>30

<212>DNA

<213>Bos sp.

<400>14

atgtcttcca ttaagattga gtgtgttttg 30

<210>15

<21l>30

<212>DNA

<213>Oryctolagus cuniculus

<400>15

atgtcttcca ttaagatcga gtgtgttttg 30

<210>16

<211>30

<212>DNA

<213>Homo sapiens

<400>16

atgtcttcca ttaagattga gtgtgttttg 30

<210>17

<211>30

<212>DNA

<213>Rattus norvegicus

<400>17

atgtcttcca tcaagattga atgtgtttta 30

<210>18

<211>30

<212>DNA

<213>Mus sp.

<400>18

atgtcttcca tcaaagttga atgtgtttta 30

<210>19

<211>30

<212>DNA

<213>Gallus sp.

<400>19

atgtcgtccg t taagatcga gtgcgtcggg 30

<210>20

<211>30

<212>DNA

<213>Xenopus sp.

<400>20

atgtcttcca tcaagataga atgtgtagtc 30

<210>21

<211>39

<212>DNA

<213>Bos sp.

<400>21

ataactggcc taggagtcaa aggaattcct ccctatcct 39

<210>22

<211>39

<212>DNA

<213>Oryctolagus cuniculus

<400>22

ataactggcc tgggggtcaa aggaattccc ccctactcc 39

<210>23

<211>39

<212>DNA

<213>Homo sapiens

<400>23

ataactggtc tgggggtcaa aggaattgct ccctactcc 39

<210>24

<211>39

<212>DNA

<213>Rattus norvegicus

<400>24

ataacaggtc ttggggtcaa aggaattgct ccatattcc 39

<210>25

<211>39

<212>DNA

<213>Mus sp.

<400>25

ataacaggtc tcggagtcaa aggaattgcc ccatattcc 39

<210>26

<211>39

<212>DNA

<213>Gallus sp.

<400>26

atcactgggc ttggtgtgaa aggaatcccg ccatatcca 39

<210>27

<211>39

<212>DNA

<213>Xenopus sp.

<400>27

attackggac ttggagtaaa aggaatcgca ccaactgca 39

<210>28

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223>Perfectly matched primers

<400>28

aatacacatt ccatctggga t 21

<210>29

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> forward primer

<400>29

gaactgtgaa gttgcctgtt g 21

<210>30

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer

<400>30

ggaagaggat gtcactcatc c 21

<210>31

<211>26

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(6)..(6)

<223>n means C, G or T

<220>

<221>misc_feature

<222>(9)..(9)

<223>n means C or T

<220>

<221>misc_feature

<222>(12)..(12)

<223>n means C, G or T

<220>

<221>misc_feature

<222>(15)..(15)

<223>n means C or T

<220>

<221>misc_feature

<222>(18)..(18)

<223>n means A or G

<220>

<221>misc_feature

<222>(21)..(21)

<223>n means C or G

<220>

<221>misc_feature

<222>(1)..(26)

<223> oligonucleotide Fxn42

<400>31

aagccnttng cnccancangt naagac 26

<210>32

<211>26

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(15)..(15)

<223>n means C or T

<220>

<221>misc_feature

<222>(1)..(26)

<223> oligonucleotide Fxn42S

<220>

<221>misc_feature

<222>(18)..(18)

<223>n means A or G

<220>

<221>misc_feature

<222>(21)..(21)

<223>n means C, G or T

<220>

<221>misc_feature

<222>(24)..(24)

<223>n means A or G

<400>32

aagccgttcg cccancangt naanac 26

<210>33

<211>26

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(6)..(6)

<223>n means C or G

<220>

<221>misc_feature

<222>(15)..(15)

<223>n means C or G

<220>

<221>misc_feature

<222>(18)..(18)

<223>n means A or G

<220>

<221>misc_feature

<222>(21)..(21)

<223>n means A, C, T or G

<220>

<221>misc_feature

<222>(24)..(24)

<223>n means C or T

<220>

<221>misc_feature

<222>(1)..(26)

<223> oligonucleotide Fxn42AS

<400>33

gtcttnacct ggtgngcnaa nggntt 26

<210>34

<211>24

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(3)..(3)

<223>n means C or T

<220>

<221>misc_feature

<222>(6)..(6)

<223>n means A, C or G

<220>

<221>misc_feature

<222>(9)..(9)

<223>n means C or T

<220>

<221>misc_feature

<222>(12)..(12)

<223>n means C or G

<220>

<221>misc_feature

<222>(15)..(15)

<223>n means C or T

<220>

<221>misc_feature

<222>(21)..(21)

<223>n means C or G

<220>

<221>misc_feature

<222>(1)..(24)

<223> oligonucleotide Fxn57

<400>34

atnggnatng cncantgggc naag 24

<210>35

<211>24

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(9)..(9)

<223>n means C or T

<220>

<221>misc_feature

<222>(12)..(12)

<223>n means C or G

<220>

<221>misc_feature

<222>(15)..(15)

<223>n means C or T

<220>

<221>misc_feature

<222>(21)..(21)

<223>n means C, G or T

<220>

<221>misc_feature

<222>(24)..(24)

<223>n means A or G

<220>

<221>misc_feature

<222>(1)..(24)

<223> oligonucleotide Fxn57S

<400>35

atcggcatng cncantgggc naan 24

<210>36

<211>24

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(13)..(13)

<223>n means A, C or G

<220>

<221>misc_feature

<222>(16)..(16)

<223>n means A or G

<220>

<221>misc_feature

<222>(19)..(19)

<223>n means A, C, T or G

<220>

<221>misc_feature

<222>(22)..(22)

<223>n means A or G

<220>

<221>misc_feature

<222>(1)..(24)

<223> oligonucleotide Fxn57AS

<400>36

cttggcccag tgngcnatnc cnat 24

<210>37

<211>39

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(6)..(6)

<223>n means C, G or T

<220>

<221>misc_feature

<222>(9)..(9)

<223>n means C or T

<220>

<221>misc_feature

<222>(12)..(12)

<223>n means C or T

<220>

<221>misc_feature

<222>(15)..(15)

<223>n means C or G

<220>

<221>misc_feature

<222>(18)..(18)

<223>n means C or G

<220>

<221>misc_feature

<222>(21)..(21)

<223>n means C or G

<220>

<221>misc_feature

<222>(24)..(24)

<223>n means C or G

<220>

<221>misc_feature

<222>(27)..(27)

<223>n means C, G or T

<220>

<221>misc_feature

<222>(30)..(30)

<223>n means C or G

<220>

<221>misc_feature

<222>(33)..(33)

<223>n means C or T

<220>

<221>misc_feature

<222>(1)..(39)

<223> oligonucleotide Fx73

<220>

<221>misc_feature

<222>(36)..(36)

<223>n means C or T

<400>37

tggacnttnt tngtntcntc ntcnccnctn aantgnaag 39

<210>38

<211>39

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(24)..(24)

<223>n means C or G

<220>

<221>misc_feature

<222>(27)..(27)

<223>n means C or G

<220>

<221>misc_feature

<222>(30)..(30)

<223>n means C or G

<220>

<221>misc_feature

<222>(33)..(33)

<223>n means C or T

<220>

<221>misc_feature

<222>(36)..(36)

<223>n means C or T

<220>

<221>misc_feature

<222>(39)..(39)

<223>n means A or G

<220>

<221>misc_feature

<222>(1)..(39)

<223> oligonucleotide Fx73S

<400>38

tggaccttgt tggtgtcctc ctcnccnctn aantgnaan 39

<210>39

<211>39

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(22)..(22)

<223>n means C or G

<220>

<221>misc_feature

<222>(25)..(25)

<223>n means C or G

<220>

<221>misc_feature

<222>(28)..(28)

<223>n means A or C

<220>

<221>misc_feature

<222>(31)..(31)

<223>n means A or C

<220>

<221>misc_feature

<222>(34)..(34)

<223>n means A, C or G

<220>

<221>misc_feature

<222>(1)..(39)

<223> oligonucleotide Fx73AS

<400>39

cttgcagttc agcggggagg anganacnaa naangtcca 39

<210>40

<211>38

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(3)..(3)

<223>n means C or G

<220>

<221>misc_feature

<222>(6)..(6)

<223>n means C or T

<220>

<221>misc_feature

<222>(9)..(9)

<223>n means C or G

<220>

<221>misc_feature

<222>(12)..(12)

<223>n means C or T

<220>

<221>misc_feature

<222>(18)..(18)

<223>n means C or T

<220>

<221>misc_feature

<222>(21)..(21)

<223>n means C or G

<220>

<221>misc_feature

<222>(24)..(24)

<223>n means A or G

<220>

<221>misc_feature

<222>(27)..(27)

<223>n means A or G

<220>

<221>misc_feature

<222>(30)..(30)

<223>n means C or G

<220>

<221>misc_feature

<222>(33)..(33)

<223>n means C or T

<220>

<221>misc_feature

<222>(36)..(36)

<223>n means C or G

<220>

<221>misc_feature

<222>(1)..(38)

<223> oligonucleotide FX74

<400>40

gtntgnctna antggganct nganaanctn aanccncc 38

<210>41

<211>29

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(15)..(15)

<223>n means A or G

<220>

<221>misc_feature

<222>(18)..(18)

<223>n means A or G

<220>

<221>misc_feature

<222>(21)..(21)

<223>n means C or G

<220>

<221>misc_feature

<222>(24)..(24)

<223>n means C or T

<220>

<221>misc_feature

<222>(27)..(27)

<223>n means C, G or T

<220>

<221>misc_feature

<222>(1)..(29)

<223> oligonucleotide Fx74S

<400>41

aactgggacc tgganaanct naanccncc 29

<210>42

<211>27

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(12)..(12)

<223>n means C or T

<220>

<221>misc_feature

<222>(15)..(15)

<223>n means C or T

<220>

<221>misc_feature

<222>(18)..(18)

<223>n means C or G

<220>

<221>misc_feature

<222>(21)..(21)

<223>n means A or G

<220>

<221>misc_feature

<222>(27)..(27)

<223>n means A or G

<220>

<221>misc_feature

<222>(1)..(27)

<223> oligonucleotide Fx74AS

<400>42

ggcgggttca gnttntcnag ntcccan 27

<210>43

<211>8

<212>PRT

<213>Aspergillus sp.

<400>43

Lys Pro Phe Ala His Gln Val Lys

1 5

<210>44

<211>16

<212>PRT

<213>Aspergillus sp.

<400>44

Arg His His Asn Ala Pro Ala Pro Thr Pro Glu Asp Pro Glu Arg Arg

1 5 10 15

<210>45

<211>12

<212>PRT

<213>Aspergillus sp.

<220>

<221>MISC_FEATURE

<222>(10)..(10)

<223>x means?

<400>45

Lys Pro Phe Ala His Gln Val Lys Thr Xaa Glu Asp

1 5 10

<210>46

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer Oal1AS

<400>46

cgaagggtttgcggccgaag c 21

<210>47

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer Oal2AS

<400>47

aaggcgtggc c ttcggagac c 21

<210>48

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer Oal3S

<400>48

gtagcgggtg atgcggtcag c 21

<210>49

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer Oal4S

<400>49

tgtatccgcc ggtagagaag g 21

<210>50

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer Oal5S

<400>50

ccctcgtcca cccgaatcgg c 21

<210>51

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer Oal6AS

<400>51

gcgtagtcgatgaggacgcc g 21

<210>52

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer Oal7AS

<400>52

tgactatact tgaccctctc c 21

<210>53

<211>22

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(22)

<223> Primer Oal8AS

<400>53

ggagacccat tttacgtggtgg 22

<210>54

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer Oal9AS

<400>54

ccgttcccac accacctctg c 21

<210>55

<211>24

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(24)

<223> Primer Oal10S

<400>55

atggccgact ccctcccgaa aacc 24

<210>56

<211>27

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(27)

<223> Primer Oal10SEco

<400>56

atataagaat tcaaatggcc gactccc 27

<210>57

<211>53

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(53)

<223> Primer OalENhis

<400>57

gaattcaaat gagaggatcc catcaccatc accatcacgc cgactccctc ccg 53

<210>58

<211>19

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(19)

<223> Primer Oal10SShis

<400>58

atgagaggat cgcatcacc 19

<210>59

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer Oal11S

<400>59

ctctactgac aagataccac g 21

<210>60

<211>26

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(26)

<223> Primer Oal12AS

<400>60

ttaaaaaatt gccatacaat tcggcg 26

<210>61

<211>38

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(38)

<223> Primer Oal12ASEco

<400>61

tatatagaat tcttaaaaaattgccataca attcggcg 38

<210>62

<211>44

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(44)

<223> Primer Oal12AShis

<400>62

tcaatggtga tggtgatgat gaaaaattgc catacaattc ggcg 44

<210>63

<211>56

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(56)

<223> Primer Oal12ASHisEco

<400>63

tatatagaat tcttaatggt gatggtgatg atgaaaaatt gccatacaat tcggcg 56

<210>64

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer Oal13S

<400>64

ggagatacaa ccctaatgaa c 21

<210>65

<211>21

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(21)

<223> Primer Oal13AS

<400>65

gttcattagg gttgtatctc c 21

<210>66

<211>37

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(37)

<223> Primer Oal14S

<400>66

gagaagctga accccaccagg cttcggccgc aaaccct 37

<210>67

<211>37

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(37)

<223> Primer Oal14AS

<400>67

agggtttgcg gccgaagcct ggtgggttca gcttctc 37

<210>68

<211>37

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(37)

<223> Primer Oal15S

<400>68

cttcgcccac cacgtaaaaa cccccgacga ccctaac 37

<210>69

<211>37

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(37)

<223> Primer Oal15AS

<400>69

gttagggtcg tcgggggttt ttacgtggtg ggcgaag 37

<210>70

<211>32

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(32)

<223> Primer Oal16S

<400>70

gaattcaaat gctccgtctc ctcactccac tc 32

<210>71

<211>30

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(30)

<223> Primer OalsecS

<400>71

ggtcctcgtg gtaatgccga ctccctcccg 30

<210>72

<211>27

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(27)

<223> Primer OalsecAS

<400>72

cgggaggggag tcggcattac cacgagg 27

<210>73

<211>48

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(48)

<223> Primer pQE80EcoS

<400>73

tatatagaat tcattaaaga ggagaaatta actatggccg actccctc 48

<210>74

<211>52

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(52)

<223> Primer pQE80EcoNhisS

<400>74

tatatagaat tcattaaaga ggagaaatta actatgagag gatcgcatca cc 52

<210>75

<211>34

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(34)

<223> Primer pQE80BamAS

<400>75

tatatagtcg acttaaaaaa ttgccataca attc 34

<210>76

<211>56

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(56)

<223> Primer pQE80BamhisAS

<400>76

tatatagtcg actcaatggtgatggtgatg atgaaaaatt gccatacaat tcggcg 56

<210>77

<211>57

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(57)

<223> Primer pQE80EcoSshort

<400>77

tatatagaat tcattaaaga ggagaaatta actatgctcc gtctcctcac tccactc 57

<210>78

<211>26

<212>DNA

<213> Artificial

<220>

<221>misc_feature

<222>(1)..(26)

<223> Primer CP-a

<400>78

tatatgaatt catgggcgtg ttcgtc 26

<210>79

<211>777

<212>DNA

<213>Aspergillus niger

<220>

<221> Intron

<222>(32)..(82)

<223>

<220>

<221> CDS

<222>(83)..(777)

<223>

<220>

<221> CDS

<222>(1)..(31)

<223>

<400>79

aac tgg gac ctg gaa aaa ctc aat cct cca g gtaatcccct ccttccaacc 51

Asn Trp Asp Leu Glu Lys Leu Asn Pro Pro

1 5 10

catcctctat cgccaactaa cctaaacaca g gc ttc ggc cgc aaa ccc ttc 102

Gly Phe Gly Arg Lys Pro Phe

                            

gcc cac cag gtt aaa tgg gtc agc gaa ggc cac gcc ttc gac gac gag 150

Ala His Gln Val Lys Trp Val Ser Glu Gly His Ala Phe Asp Asp Glu

20 25 30

tac cac ttc aac cca cag caa tcc tcc caa tgg gac ggc cta cga cac 198

Tyr His Phe Asn Pro Gln Gln Ser Ser Gln Trp Asp Gly Leu Arg His

35 40 45

cac aat gcc cca gcc cca aca ccc gac gac cct gac cgc cgc gtc ttc 246

His Asn Ala Pro Ala Pro Thr Pro Asp Asp Pro Asp Arg Arg Val Phe

50 55 60 65

tac ggc ggc acc acc tcc acc gag atc ctc gac ccg tcc tca gct cgc 294

Tyr Gly Gly Thr Thr Ser Thr Glu Ile Leu Asp Pro Ser Ser Ala Arg

70 75 80

atc ggc atc gct cac tgg gcc aag aag ggc atc gcc ggc cgt ggc gtc 342

Ile Gly Ile Ala His Trp Ala Lys Lys Gly Ile Ala Gly Arg Gly Val

85 90 95

ctc atc gac tac gcg tcc tac atc cag cga acc aag ggc ata cca gta 390

Leu Ile Asp Tyr Ala Ser Tyr Ile Gln Arg Thr Lys Gly Ile Pro Val

100 105 110

aac gcc cta acc cgg cac acc gtc tcc ctg gac gac gtc ctc acc atc 438

Asn Ala Leu Thr Arg His Thr Val Ser Leu Asp Asp Val Leu Thr Ile

115 120 125

gcg aaa gaa tgc aac atc acc ttc cag cca ggc gac att ctc ttc ttg 486

Ala Lys Glu Cys Asn Ile Thr Phe Gln Pro Gly Asp Ile Leu Phe Leu

130 135 140 145

cgt gtc ggc ctg ccc acc aca tgg gat aac atg tcc gat gac gag aag 534

Arg Val Gly Leu Pro Thr Thr Trp Asp Asn Met Ser Asp Asp Glu Lys

150 155 160

gtc aaa tac agt caa cag gaa atg cct cag cat gca ggg tta gag cag 582

Val Lys Tyr Ser Gln Gln Glu Met Pro Gln His Ala Gly Leu Glu Gln

165 170 175

agt gag cgc gtg gtg cgg ttc ctc tgg gac cag cat ttc gcg gct gtg 630

Ser Glu Arg Val Val Arg Phe Leu Trp Asp Gln His Phe Ala Ala Val

180 185 190

gcg ggt gat gcg gtc agc ttc gag gtg tat ccg cct gta gag aag gag 678

Ala Gly Asp Ala Val Ser Phe Glu Val Tyr Pro Pro Val Glu Lys Glu

195 200 205

tgg gat ttg cat cat ttt ctg ttg gcc ggg tgg gga gtg ccg att ggg 726

Trp Asp Leu His His Phe Leu Leu Ala Gly Trp Gly Val Pro Ile Gly

210 215 220 225

gag atg ttt gat ctg gag ggg ttg aag gag gtt tgt gag agg ttg ggc 774

Glu Met Phe Asp Leu Glu Gly Leu Lys Glu Val Cys Glu Arg Leu Gly

230 235 240

agg 777

Arg

<210>80

<211>242

<212>PRT

<213>Aspergillus niger

<400>80

Asn Trp Asp Leu Glu Lys Leu Asn Pro Pro Gly Phe Gly Arg Lys Pro

1 5 10 15

Phe Ala His Gln Val Lys Trp Val Ser Glu Gly His Ala Phe Asp Asp

20 25 30

Glu Tyr His Phe Asn Pro Gln Gln Ser Ser Gln Trp Asp Gly Leu Arg

35 40 45

His His Asn Ala Pro Ala Pro Thr Pro Asp Asp Pro Asp Arg Arg Val

50 55 60

Phe Tyr Gly Gly Thr Thr Ser Thr Glu Ile Leu Asp Pro Ser Ser Ala

65 70 75 80

Arg Ile Gly Ile Ala His Trp Ala Lys Lys Gly Ile Ala Gly Arg Gly

85 90 95

Val Leu Ile Asp Tyr Ala Ser Tyr Ile Gln Arg Thr Lys Gly Ile Pro

100 105 110

Val Asn Ala Leu Thr Arg His Thr Val Ser Leu Asp Asp Val Leu Thr

115 120 125

Ile Ala Lys Glu Cys Asn Ile Thr Phe Gln Pro Gly Asp Ile Leu Phe

130 135 140

Leu Arg Val Gly Leu Pro Thr Thr Trp Asp Asn Met Ser Asp Asp Glu

145 150 155 160

Lys Val Lys Tyr Ser Gln Gln Glu Met Pro Gln His Ala Gly Leu Glu

165 170 175

Gln Ser Glu Arg Val Val Arg Phe Leu Trp Asp Gln His Phe Ala Ala

180 185 190

Val Ala Gly Asp Ala Val Ser Phe Glu Val Tyr Pro Pro Val Glu Lys

195 200 205

Glu Trp Asp Leu His His Phe Leu Leu Ala Gly Trp Gly Val Pro Ile

210 215 220

Gly Glu Met Phe Asp Leu Glu Gly Leu Lys Glu Val Cys Glu Arg Leu

225 230 235 240

Gly Arg

<210>81

<211>12

<212>PRT

<213>Aspergillus niger

<220>

<221>MISC_FEATURE

<222>(10)..(10)

<223>undefined amino acid

<400>81

Lys Pro Phe Ala His Gln Val Lys Thr Xaa Glu Asp

1 5 10

<210>82

<211>8

<212>PRT

<213>Aspergillus niger

<400>82

Ile Gly Ile Ala His Trp Ala Lys

1 5

<210>83

<211>13

<212>PRT

<213>Aspergillus niger

<400>83

Trp Thr Phe Phe Val Ser Ser Ser Ser Pro Leu Asn Cys Lys

1 5 10

<210>84

<211>15

<212>PRT

<213>Aspergillus niger

<220>

<221>MISC_FEATURE

<222>(5)..(5)

<223>Amino acid cannot be unambiguously determined

<220>

<221>MISC_FEATURE

<222>(14)..(14)

<223> Unidentified amino acid

<400>84

Val Cys Leu Asn Trp Asp Leu Glu Lys Leu Asn Pro Pro Xaa Phe

1 5 10 15

<210>85

<211>15

<212>PRT

<213>Aspergillus niger

<400>85

Glu Cys Asn Ile Thr Phe Gln Pro Gly Asp Ile Leu Phe Leu Arg

1 5 10 15

<210>86

<211>11

<212>PAT

<213>Aspergillus niger

<220>

<221>MISC_FEATURE

<222>(8)..(8)

<223> Unidentified amino acid

<400>86

Ala Glu Thr Thr Thr Asn Pro Xaa Tyr Phe Phe

1 5 10

<210>87

<211>8

<212>PRT

<213>Aspergillus niger

<400>87

Tyr His Leu Leu Gln Ala Glu Asp

1 5

<210>88

<211>10

<212>PRT

<213>Aspergillus niger

<400>88

Leu Glu Ser Leu Val Glu Glu Val Tyr Lys

1 5 10

<210>89

<211>16

<212>PRT

<213>Aspergillus niger

<220>

<221>MISC_FEATURE

<222>(15)..(15)

<223> Unidentified amino acid

<400>89

Leu Phe Glu Asp Val Tyr Ala Gln Lys Pro Glu Thr Ala Pro Xaa Asp

1 5 10 15

<210>90

<211>9

<212>PRT

<213>Aspergillus niger

<220>

<221>MISC_FEATURE

<222>(7)..(7)

<223> Amino acids cannot be unambiguously determined

<400>90

Phe Leu Phe Asn Val Pro Tyr Thr Ser

1 5

<210>91

<211>8

<212>PRT

<213>Aspergillus niger

<220>

<221>MISC_FEATURE

<222>(8)..(8)

<223> Amino acids cannot be unambiguously determined

<400>91

Gly Val Met Gly Gly Leu Gly Gly

1 5

<210>92

<211>1005

<212>DNA

<213>Aspergillus niger

<220>

<221> CDS

<222>(1)..(1002)

<223>

<400>92

atg aga gga tcg cat cac cat cac cat cac gcc gac tcc ctc ccg aaa 48

Met Arg Gly Ser His His His His His His Ala Asp Ser Leu Pro Lys

1 5 10 15

acc tac gac gac ctc ccc gac aag cgg cgc tac tgg ccc gca acc ccc 96

Thr Tyr Asp Asp Leu Pro Asp Lys Arg Arg Tyr Trp Pro Ala Thr Pro

20 25 30

aac tcc gcc gac gag gga ctg ggg atg ctc cgt ctc ctc act cca gaa 144

Asn Ser Ala Asp Glu Gly Leu Gly Met Leu Arg Leu Leu Thr Pro Glu

35 40 45

att gtc gcc aac gca gca cgc acc cag atc cag acc ggc gag cgc gta 192

Ile Val Ala Asn Ala Ala Arg Thr Gln Ile Gln Thr Gly Glu Arg Val

50 55 60

tgc ctg aac tgg gac ctg gag aag ctg aac cca cca ggc ttc ggc cgc 240

Cys Leu Asn Trp Asp Leu Glu Lys Leu Asn Pro Pro Gly Phe Gly Arg

65 70 75 80

aaa ccc ttc gcc cac cac gta aaa tgg gtc tcc gaa ggc cac gcc ttc 288

Lys Pro Phe Ala His His Val Lys Trp Val Ser Glu Gly His Ala Phe

85 90 95

gac gac gaa tac cac ttc aac ccg caa caa tcc tcc caa tgg gac ggt 336

Asp Asp Glu Tyr His Phe Asn Pro Gln Gln Ser Ser Gln Trp Asp Gly

100 105 110

ctg cga cac cac aac gcc ccg gct cca acc ccc gac gac cct aac cgc 384

Leu Arg His His Asn Ala Pro Ala Pro Thr Pro Asp Asp Pro Asn Arg

115 120 125

cgg gtc ttc tac ggc ggc acc acc acc tcc acc gag atc ctc gac ccc tcg 432

Arg Val Phe Tyr Gly Gly Thr Thr Ser Thr Glu Ile Leu Asp Pro Ser

130 135 140

tcc acc cga atc ggc ata gcc cac tgg gcc aag aaa ggc atc gcc ggc 480

Ser Thr Arg Ile Gly Ile Ala His Trp Ala Lys Lys Gly Ile Ala Gly

145 150 155 160

cgc ggc gtc ctc atc gac tac gcc tcc tac gtg caa cga acc aag ggc 528

Arg Gly Val Leu Ile Asp Tyr Ala Ser Tyr Val Gln Arg Thr Lys Gly

165 170 175

gtc aca gta aac gcc cta acc cgc cac acc gtc tcc ctg gac gac gtc 576

Val Thr Val Asn Ala Leu Thr Arg His Thr Val Ser Leu Asp Asp Val

180 185 190

ctc acc atc gcg aag gaa tgc aac atc acc ttc gaa cca ggc gac att 624

Leu Thr Ile Ala Lys Glu Cys Asn Ile Thr Phe Glu Pro Gly Asp Ile

195 200 205

ctc ttc ctg cgc gtc ggt cta ccg act aca tgg gat aac atg acc gat 672

Leu Phe Leu Arg Val Gly Leu Pro Thr Thr Trp Asp Asn Met Thr Asp

210 215 220

gag gag agg gtc aag tat agt cag cag gaa acg ccc aac cat gca gga 720

Glu Glu Arg Val Lys Tyr Ser Gln Gln Glu Thr Pro Asn His Ala Gly

225 230 235 240

tta gag cag agt gag cgt gtg gtg cgg ttc ctt tgg gat cag cat ttc 768

Leu Glu Gln Ser Glu Arg Val Val Arg Phe Leu Trp Asp Gln His Phe

245 250 255

gcc gct gta gcg ggt gat gcg gtc agc ttt gag gtg tat ccg ccg gta 816

Ala Ala Val Ala Gly Asp Ala Val Ser Phe Glu Val Tyr Pro Pro Val

260 265 270

gag aag gag tgg gat tta cat cat ttt ttg ctg gct ggg tgg gga cta 864

Glu Lys Glu Trp Asp Leu His His Phe Leu Leu Ala Gly Trp Gly Leu

275 280 285

ccg att ggg gag atg ttt gat ctt gag ggg ttg aag gag gtg tgt gag 912

Pro Ile Gly Glu Met Phe Asp Leu Glu Gly Leu Lys Glu Val Cys Glu

290 295 300

agg ttg gga agg tgg acg ttt ttc gtt agt tct agt ccg ttg aac tgt 960

Arg Leu Gly Arg Trp Thr Phe Phe Val Ser Ser Ser Pro Leu Asn Cys

305 310 315 320

ggg aga ggt gtg tcg agt ccg ccg aat tgt atg gca att ttt taa 1005

Gly Arg Gly Val Ser Ser Pro Pro Asn Cys Met Ala Ile Phe

325 330

<210>93

<211>334

<212>PRT

<213>Aspergillus niger

<400>93

Met Arg Gly Ser His His His His His His Ala Asp Ser Leu Pro Lys

1 5 10 15

Thr Tyr Asp Asp Leu Pro Asp Lys Arg Arg Tyr Trp Pro Ala Thr Pro

20 25 30

Asn Ser Ala Asp Glu Gly Leu Gly Met Leu Arg Leu Leu Thr Pro Glu

35 40 45

Ile Val Ala Asn Ala Ala Arg Thr Gln Ile Gln Thr Gly Glu Arg Val

50 55 60

Cys Leu Asn Trp Asp Leu Glu Lys Leu Asn Pro Pro Gly Phe Gly Arg

65 70 75 80

Lys Pro Phe Ala His His Val Lys Trp Val Ser Glu Gly His Ala Phe

85 90 95

Asp Asp Glu Tyr His Phe Asn Pro Gln Gln Ser Ser Gln Trp Asp Gly

100 105 110

Leu Arg His His Asn Ala Pro Ala Pro Thr Pro Asp Asp Pro Asn Arg

115 120 125

Arg Val Phe Tyr Gly Gly Thr Thr Ser Thr Glu Ile Leu Asp Pro Ser

130 135 140

Ser Thr Arg Ile Gly Ile Ala His Trp Ala Lys Lys Gly Ile Ala Gly

145 150 155 160

Arg Gly Val Leu Ile Asp Tyr Ala Ser Tyr Val Gln Arg Thr Lys Gly

165 170 175

Val Thr Val Asn Ala Leu Thr Arg His Thr Val Ser Leu Asp Asp Val

180 185 190

Leu Thr Ile Ala Lys Glu Cys Asn Ile Thr Phe Glu Pro Gly Asp Ile

195 200 205

Leu Phe Leu Arg Val Gly Leu Pro Thr Thr Trp Asp Asn Met Thr Asp

210 215 220

Glu Glu Arg Val Lys Tyr Ser Gln Gln Glu Thr Pro Asn His Ala Gly

225 230 235 240

Leu Glu Gln Ser Glu Arg Val Val Arg Phe Leu Trp Asp Gln His Phe

245 250 255

Ala Ala Val Ala Gly Asp Ala Val Ser Phe Glu Val Tyr Pro Pro Val

260 265 270

Glu Lys Glu Trp Asp Leu His His Phe Leu Leu Ala Gly Trp Gly Leu

275 280 285

Pro Ile Gly Glu Met Phe Asp Leu Glu Gly Leu Lys Glu Val Cys Glu

290 295 300

Arg Leu Gly Arg Trp Thr Phe Phe Val Ser Ser Ser Pro Leu Asn Cys

305 310 315 320

Gly Arg Gly Val Ser Ser Pro Pro Asn Cys Met Ala Ile Phe

325 330

<210>94

<211>992

<212>DNA

<213>Aspergillus niger

<220>

<221> CDS

<222>(9)..(983)

<223>

<400>94

gaattcaa atg gcc gac tcc ctc ccg aaa acc tac gac gac ctc ccc gac 50

Met Ala Asp Ser Leu Pro Lys Thr Tyr Asp Asp Leu Pro Asp

1 5 10

aag cgg cgc tac tgg ccc gca acc ccc aac tcc gcc gac gag gga ctg 98

Lys Arg Arg Tyr Trp Pro Ala Thr Pro Asn Ser Ala Asp Glu Gly Leu

15 20 25 30

ggg atg ctc cgt ctc ctc act cca gaa att gtc gcc aac gca gca cgc 146

Gly Met Leu Arg Leu Leu Thr Pro Glu Ile Val Ala Asn Ala Ala Arg

35 40 45

acc cag atc cag acc ggc gag cgc gta tgc ctg aac tgg gac ctg gag 194

Thr Gln Ile Gln Thr Gly Glu Arg Val Cys Leu Asn Trp Asp Leu Glu

50 55 60

aag ctg aac cca cca ggc ttc ggc cgc aaa ccc ttc gcc cac cac gta 242

Lys Leu Asn Pro Pro Gly Phe Gly Arg Lys Pro Phe Ala His His Val

65 70 75

aaa tgg gtc tcc gaa ggc cac gcc ttc gac gac gaa tac cac ttc aac 290

Lys Trp Val Ser Glu Gly His Ala Phe Asp Asp Glu Tyr His Phe Asn

80 85 90

ccg caa caa tcc tcc caa tgg gac ggt ctg cga cac cac aac gcc ccg 338

Pro Gln Gln Ser Ser Gln Trp Asp Gly Leu Arg His His Asn Ala Pro

95 100 105 110

gct cca acc ccc gac gac cct aac cgc cgg gtc ttc tac ggc ggc acc 386

Ala Pro Thr Pro Asp Asp Pro Asn Arg Arg Val Phe Tyr Gly Gly Thr

115 120 125

acc tcc acc gag atc ctc gac ccc tcg tcc acc cga atc ggc ata gcc 434

Thr Ser Thr Glu Ile Leu Asp Pro Ser Ser Thr Arg Ile Gly Ile Ala

130 135 140

cac tgg gcc aag aaa ggc atc gcc ggc cgc ggc gtc ctc atc gac tac 482

His Trp Ala Lys Lys Gly Ile Ala Gly Arg Gly Val Leu Ile Asp Tyr

145 150 155

gcc tcc tac gtg caa cga acc aag ggc gtc aca gta aac gcc cta acc 530

Ala Ser Tyr Val Gln Arg Thr Lys Gly Val Thr Val Asn Ala Leu Thr

160 165 170

cgc cac acc gtc tcc ctg gac gac gtc ctc acc atc gcg aag gaa tgc 578

Arg His Thr Val Ser Leu Asp Asp Val Leu Thr Ile Ala Lys Glu Cys

175 180 185 190

aac atc acc ttc gaa cca ggc gac att ctc ttc ctg cgc gtc ggt cta 626

Asn Ile Thr Phe Glu Pro Gly Asp Ile Leu Phe Leu Arg Val Gly Leu

195 200 205

ccg act aca tgg gat aac atg acc gat gag gag agg gtc aag tat agt 674

Pro Thr Thr Trp Asp Asn Met Thr Asp Glu Glu Arg Val Lys Tyr Ser

210 215 220

cag cag gaa acg ccc aac cat gca gga tta gag cag agt gag cgt gtg 722

Gln Gln Glu Thr Pro Asn His Ala Gly Leu Glu Gln Ser Glu Arg Val

225 230 235

gtg cgg ttc ctt tgg gat cag cat ttc gcc gct gta gcg ggt gat gcg 770

Val Arg Phe Leu Trp Asp Gln His Phe Ala Ala Val Ala Gly Asp Ala

240 245 250

gtc agc ttt gag gtg tat ccg ccg gta gag aag gag tgg gat tta cat 818

Val Ser Phe Glu Val Tyr Pro Pro Val Glu Lys Glu Trp Asp Leu His

255 260 265 270

cat ttt ttg ctg gct ggg tgg gga cta ccg att ggg gag atg ttt gat 866

His Phe Leu Leu Ala Gly Trp Gly Leu Pro Ile Gly Glu Met Phe Asp

275 280 285

ctt gag ggg ttg aag gag gtg tgt gag agg ttg gga agg tgg acg ttt 914

Leu Glu Gly Leu Lys Glu Val Cys Glu Arg Leu Gly Arg Trp Thr Phe

290 295 300

ttc gtt agt tct agt ccg ttg aac tgt ggg aga ggt gtg tcg agt ccg 962

Phe Val Ser Ser Ser Pro Leu Asn Cys Gly Arg Gly Val Ser Ser Pro

305 310 315

ccg aat tgt atg gca att ttt taagaattc 992

Pro Asn Cys Met Ala Ile Phe

320 325

<210>95

<211>325

<212>PRT

<213>Aspergillus niger

<400>95

Met Ala Asp Ser Leu Pro Lys Thr Tyr Asp Asp Leu Pro Asp Lys Arg

1 5 10 15

Arg Tyr Trp Pro Ala Thr Pro Asn Ser Ala Asp Glu Gly Leu Gly Met

20 25 30

Leu Arg Leu Leu Thr Pro Glu Ile Val Ala Asn Ala Ala Arg Thr Gln

35 40 45

Ile Gln Thr Gly Glu Arg Val Cys Leu Asn Trp Asp Leu Glu Lys Leu

50 55 60

Asn Pro Pro Gly Phe Gly Arg Lys Pro Phe Ala His His Val Lys Trp

65 70 75 80

Val Ser Glu Gly His Ala Phe Asp Asp Glu Tyr His Phe Asn Pro Gln

85 90 95

Gln Ser Ser Gln Trp Asp Gly Leu Arg His His Asn Ala Pro Ala Pro

100 105 110

Thr Pro Asp Asp Pro Asn Arg Arg Val Phe Tyr Gly Gly Thr Thr Ser

115 120 125

Thr Glu Ile Leu Asp Pro Ser Ser Thr Arg Ile Gly Ile Ala His Trp

130 135 140

Ala Lys Lys Gly Ile Ala Gly Arg Gly Val Leu Ile Asp Tyr Ala Ser

145 150 155 160

Tyr Val Gln Arg Thr Lys Gly Val Thr Val Asn Ala Leu Thr Arg His

165 170 175

Thr Val Ser Leu Asp Asp Val Leu Thr Ile Ala Lys Glu Cys Asn Ile

180 185 190

Thr Phe Glu Pro Gly Asp Ile Leu Phe Leu Arg Val Gly Leu Pro Thr

195 200 205

Thr Trp Asp Asn Met Thr Asp Glu Glu Arg Val Lys Tyr Ser Gln Gln

210 215 220

Glu Thr Pro Asn His Ala Gly Leu Glu Gln Ser Glu Arg Val Val Arg

225 230 235 240

Phe Leu Trp Asp Gln His Phe Ala Ala Val Ala Gly Asp Ala Val Ser

245 250 255

Phe Glu Val Tyr Pro Pro Val Glu Lys Glu Trp Asp Leu His His Phe

260 265 270

Leu Leu Ala Gly Trp Gly Leu Pro Ile Gly Glu Met Phe Asp Leu Glu

275 280 285

Gly Leu Lys Glu Val Cys Glu Arg Leu Gly Arg Trp Thr Phe Phe Val

290 295 300

Ser Ser Ser Pro Leu Asn Cys Gly Arg Gly Val Ser Ser Pro Pro Asn

305 310 315 320

Cys Met Ala Ile Phe

325

<210>96

<211>1019

<212>DNA

<213>Aspergillus niger

<220>

<221> CDS

<222>(9)..(1010)

<223>

<400>96

gaattcaa atg aga gga tcg cat cac cat cac cat cac gcc gac tcc ctc 50

     Met Arg Gly Ser His His His His His His His Ala Asp Ser Leu

1 5 10

ccg aaa acc tac gac gac ctc ccc gac aag cgg cgc tac tgg ccc gca 98

Pro Lys Thr Tyr Asp Asp Leu Pro Asp Lys Arg Arg Tyr Trp Pro Ala

15 20 25 30

acc ccc aac tcc gcc gac gag gga ctg ggg atg ctc cgt ctc ctc act 146

Thr Pro Asn Ser Ala Asp Glu Gly Leu Gly Met Leu Arg Leu Leu Thr

35 40 45

cca gaa att gtc gcc aac gca gca cgc acc cag atc cag acc ggc gag 194

Pro Glu Ile Val Ala Asn Ala Ala Arg Thr Gln Ile Gln Thr Gly Glu

50 55 60

cgc gta tgc ctg aac tgg gac ctg gag aag ctg aac cca cca ggc ttc 242

Arg Val Cys Leu Asn Trp Asp Leu Glu Lys Leu Asn Pro Pro Gly Phe

65 70 75

ggc cgc aaa ccc ttc gcc cac cac gta aaa tgg gtc tcc gaa ggc cac 290

Gly Arg Lys Pro Phe Ala His His Val Lys Trp Val Ser Glu Gly His

80 85 90

gcc ttc gac gac gaa tac cac ttc aac ccg caa caa tcc tcc caa tgg 338

Ala Phe Asp Asp Glu Tyr His Phe Asn Pro Gln Gln Ser Ser Gln Trp

95 100 105 110

gac ggt ctg cga cac cac aac gcc ccg gct cca acc ccc gac gac cct 386

Asp Gly Leu Arg His His Asn Ala Pro Ala Pro Thr Pro Asp Asp Pro

115 120 125

aac cgc cgg gtc ttc tac ggc ggc acc acc tcc acc gag atc ctc gac 434

Asn Arg Arg Val Phe Tyr Gly Gly Thr Thr Ser Thr Glu Ile Leu Asp

130 135 140

ccc tcg tcc acc cga atc ggc ata gcc cac tgg gcc aag aaa ggc atc 482

Pro Ser Ser Thr Arg Ile Gly Ile Ala His Trp Ala Lys Lys Gly Ile

145 150 155

gcc ggc cgc ggc gtc ctc atc gac tac gcc tcc tac gtg caa cga acc 530

Ala Gly Arg Gly Val Leu Ile Asp Tyr Ala Ser Tyr Val Gln Arg Thr

160 165 170

aag ggc gtc aca gta aac gcc cta acc cgc cac acc gtc tcc ctg gac 578

Lys Gly Val Thr Val Asn Ala Leu Thr Arg His Thr Val Ser Leu Asp

175 180 185 190

gac gtc ctc acc atc gcg aag gaa tgc aac atc acc ttc gaa cca ggc 626

Asp Val Leu Thr Ile Ala Lys Glu Cys Asn Ile Thr Phe Glu Pro Gly

195 200 205

gac att ctc ttc ctg cgc gtc ggt cta ccg act aca tgg gat aac atg 674

Asp Ile Leu Phe Leu Arg Val Gly Leu Pro Thr Thr Trp Asp Asn Met

210 215 220

acc gat gag gag agg gtc aag tat agt cag cag gaa acg ccc aac cat 722

Thr Asp Glu Glu Arg Val Lys Tyr Ser Gln Gln Glu Thr Pro Asn His

225 230 235

gca gga tta gag cag agt gag cgt gtg gtg cgg ttc ctt tgg gat cag 770

Ala Gly Leu Glu Gln Ser Glu Arg Val Val Arg Phe Leu Trp Asp Gln

240 245 250

cat ttc gcc gct gta gcg ggt gat gcg gtc agc ttt gag gtg tat ccg 818

His Phe Ala Ala Val Ala Gly Asp A la Val Ser Phe Glu Val Tyr Pro

255 260 265 270

ccg gta gag aag gag tgg gat tta cat cat ttt ttg ctg gct ggg tgg 866

Pro Val Glu Lys Glu Trp Asp Leu His His Phe Leu Leu Ala Gly Trp

275 280 285

gga cta ccg att ggg gag atg ttt gat ctt gag ggg ttg aag gag gtg 914

Gly Leu Pro Ile Gly Glu Met Phe Asp Leu Glu Gly Leu Lys Glu Val

290 295 300

tgt gag agg ttg gga agg tgg acg ttt ttc gtt agt tct agt ccg ttg 962

Cys Glu Arg Leu Gly Arg Trp Thr Phe Phe Val Ser Ser Ser Ser Pro Leu

305 310 315

aac tgt ggg aga ggt gtg tcg agt ccg ccg aat tgt atg gca att ttt 1010

Asn Cys Gly Arg Gly Val Ser Ser Pro Pro Asn Cys Met Ala Ile Phe

320 325 330

taagaattc 1019

<210>97

<211>334

<212>PRT

<213>Aspergillus niger

<400>97

Met Arg Gly Ser His His His His His His Ala Asp Ser Leu Pro Lys

1 5 10 15

Thr Tyr Asp Asp Leu Pro Asp Lys Arg Arg Tyr Trp Pro Ala Thr Pro

20 25 30

Asn Ser Ala Asp Glu Gly Leu Gly Met Leu Arg Leu Leu Thr Pro Glu

35 40 45

Ile Val Ala Asn Ala Ala Arg Thr Gln Ile Gln Thr Gly Glu Arg Val

50 55 60

Cys Leu Asn Trp Asp Leu Glu Lys Leu Asn Pro Pro Gly Phe Gly Arg

65 70 75 80

Lys Pro Phe Ala His His Val Lys Trp Val Ser Glu Gly His Ala Phe

85 90 95

Asp Asp Glu Tyr His Phe Asn Pro Gln Gln Ser Ser Gln Trp Asp Gly

100 105 110

Leu Arg His His Asn Ala Pro Ala Pro Thr Pro Asp Asp Pro Asn Arg

115 120 125

Arg Val Phe Tyr Gly Gly Thr Thr Ser Thr Glu Ile Leu Asp Pro Ser

130 135 140

Ser Thr Arg Ile Gly Ile Ala His Trp Ala Lys Lys Gly Ile Ala Gly

145 150 155 160

Arg Gly Val Leu Ile Asp Tyr Ala Ser Tyr Val Gln Arg Thr Lys Gly

165 170 175

Val Thr Val Asn Ala Leu Thr Arg His Thr Val Ser Leu Asp Asp Val

180 185 190

Leu Thr Ile Ala Lys Glu Cys Asn Ile Thr Phe Glu Pro Gly Asp Ile

195 200 205

Leu Phe Leu Arg Val Gly Leu Pro Thr Thr Trp Asp Asn Met Thr Asp

210 215 220

Glu Glu Arg Val Lys Tyr Ser Gln Gln Glu Thr Pro Asn His Ala Gly

225 230 235 240

Leu Glu Gln Ser Glu Arg Val Val Arg Phe Leu Trp Asp Gln His Phe

245 250 255

Ala Ala Val Ala Gly Asp Ala Val Ser Phe Glu Val Tyr Pro Pro Val

260 265 270

Glu Lys Glu Trp Asp Leu His His Phe Leu Leu Ala Gly Trp Gly Leu

275 280 285

Pro Ile Gly Glu Met Phe Asp Leu Glu Gly Leu Lys Glu Val Cys Glu

290 295 300

Arg Leu Gly Arg Trp Thr Phe Phe Val Ser Ser Ser Pro Leu Asn Cys

305 310 315 320

Gly Arg Gly Val Ser Ser Pro Pro Asn Cys Met Ala Ile Phe

325 330

<210>98

<211>975

<212>DNA

<213>Aspergillus niger

<220>

<221> CDS

<222>(1)..(975)

<223>

<400>98

atg gcc gac tcc ctc ccg aaa acc tac gac gac ctc ccc gac aag cgg 48

Met Ala Asp Ser Leu Pro Lys Thr Tyr Asp Asp Leu Pro Asp Lys Arg

1 5 10 15

cgc tac tgg ccc gca acc ccc aac tcc gcc gac gag gga ctg ggg atg 96

Arg Tyr Trp Pro Ala Thr Pro Asn Ser A la Asp Glu Gly Leu Gly Met

20 25 30

ctc cgt ctc ctc act cca gaa att gtc gcc aac gca gca cgc acc cag 144

Leu Arg Leu Leu Thr Pro Glu Ile Val Ala Asn Ala Ala Arg Thr Gln

35 40 45

atc cag acc ggc gag cgc gta tgc ctg aac tgg gac ctg gag aag ctg 192

Ile Gln Thr Gly Glu Arg Val Cys Leu Asn Trp Asp Leu Glu Lys Leu

50 55 60

aac cca cca ggc ttc ggc cgc aaa ccc ttc gcc cac cac gta aaa tgg 240

Asn Pro Pro Gly Phe Gly Arg Lys Pro Phe Ala His His Val Lys Trp

65 70 75 80

gtc tcc gaa ggc cac gcc ttc gac gac gaa tac cac ttc aac ccg caa 288

Val Ser Glu Gly His Ala Phe Asp Asp Glu Tyr His Phe Asn Pro Gln

85 90 95

caa tcc tcc caa tgg gac ggt ctg cga cac cac aac gcc ccg gct cca 336

Gln Ser Ser Gln Trp Asp Gly Leu Arg His His Asn Ala Pro Ala Pro

100 105 110

acc ccc gac gac cct aac cgc cgg gtc ttc tac ggc ggc acc acc tcc 384

Thr Pro Asp Asp Pro Asn Arg Arg Val Phe Tyr Gly Gly Thr Thr Ser

115 120 125

acc gag atc ctc gac ccc tcg tcc acc cga atc ggc ata gcc cac tgg 432

Thr Glu Ile Leu Asp Pro Ser Ser Thr Arg Ile Gly Ile Ala His Trp

130 135 140

gcc aag aaa ggc atc gcc ggc cgc ggc gtc ctc atc gac tac gcc tcc 480

Ala Lys Lys Gly Ile Ala Gly Arg Gly Val Leu Ile Asp Tyr Ala Ser

145 150 155 160

tac gtg caa cga acc aag ggc gtc aca gta aac gcc cta acc cgc cac 528

Tyr Val Gln Arg Thr Lys Gly Val Thr Val Asn Ala Leu Thr Arg His

165 170 175

acc gtc tcc ctg gac gac gtc ctc acc atc gcg aag gaa tgc aac atc 576

Thr Val Ser Leu Asp Asp Val Leu Thr Ile Ala Lys Glu Cys Asn Ile

180 185 190

acc ttc gaa cca ggc gac att ctc ttc ctg cgc gtc ggt cta ccg act 624

Thr Phe Glu Pro Gly AspIle Leu Phe Leu Arg Val Gly Leu Pro Thr

195 200 205

aca tgg gat aac atg acc gat gag gag agg gtc aag tat agt cag cag 672

Thr Trp Asp Asn Met Thr Asp Glu Glu Arg Val Lys Tyr Ser Gln Gln

210 215 220

gaa acg ccc aac cat gca gga tta gag cag agt gag cgt gtg gtg cgg 720

Glu Thr Pro Asn His Ala Gly Leu Glu Gln Ser Glu Arg Val Val Arg

225 230 235 240

ttc ctt tgg gat cag cat ttc gcc gct gta gcg ggt gat gcg gtc agc 768

Phe Leu Trp Asp Gln His Phe Ala Ala Val Ala Gly Asp Ala Val Ser

245 250 255

ttt gag gtg tat ccg ccg gta gag aag gag tgg gat tta cat cat ttt 816

Phe Glu Val Tyr Pro Pro Val Glu Lys Glu Trp Asp Leu His His Phe

260 265 270

ttg ctg gct ggg tgg gga cta ccg att ggg gag atg ttt gat ctt gag 864

Leu Leu Ala Gly Trp Gly Leu Pro Ile Gly Glu Met Phe Asp Leu Glu

275 280 285

ggg ttg aag gag gtg tgt gag agg ttg gga agg tgg acg ttt ttc gtt 912

Gly Leu Lys Glu Val Cys Glu Arg Leu Gly Arg Trp Thr Phe Phe Val

290 295 300

agt tct agt ccg ttg aac tgt ggg aga ggt gtg tcg agt ccg ccg aat 960

Ser Ser Ser Pro Leu Asn Cys Gly Arg Gly Val Ser Ser Pro Pro Asn

305 310 315 320

tgt atg gca att ttt 975

Cys Met Ala Ile Phe

325

<210>99

<211>325

<212>PRT

<213>Aspergillus niger

<400>99

Met Ala Asp Ser Leu Pro Lys Thr Tyr Asp Asp Leu Pro Asp Lys Arg

1 5 10 15

Arg Tyr Trp Pro Ala Thr Pro Asn Ser Ala Asp Glu Gly Leu Gly Met

20 25 30

Leu Arg Leu Leu Thr Pro Glu Ile Val Ala Asn Ala Ala Arg Thr Gln

35 40 45

Ile Gln Thr Gly Glu Arg Val Cys Leu Asn Trp Asp Leu Glu Lys Leu

50 55 60

Asn Pro Pro Gly Phe Gly Arg Lys Pro Phe Ala His His Val Lys Trp

65 70 75 80

Val Ser Glu Gly His Ala Phe Asp Asp Glu Tyr His Phe Asn Pro Gln

85 90 95

Gln Ser Ser Gln Trp Asp Gly Leu Arg His His Asn Ala Pro Ala Pro

100 105 110

Thr Pro Asp Asp Pro Asn Arg Arg Val Phe Tyr Gly Gly Thr Thr Ser

115 120 125

Thr Glu Ile Leu Asp Pro Ser Ser Thr Arg Ile Gly Ile Ala His Trp

130 135 140

Ala Lys Lys Gly Ile Ala Gly Arg Gly Val Leu Ile Asp Tyr Ala Ser

145 150 155 160

Tyr Val Gln Arg Thr Lys Gly Val Thr Val Asn Ala Leu Thr Arg His

165 170 175

Thr Val Ser Leu Asp Asp Val Leu Thr Ile Ala Lys Glu Cys Asn Ile

180 185 190

Thr Phe Glu Pro Gly Asp Ile Leu Phe Leu Arg Val Gly Leu Pro Thr

195 200 205

Thr Trp Asp Asn Met Thr Asp Glu Glu Arg Val Lys Tyr Ser Gln Gln

210 215 220

Glu Thr Pro Asn His Ala Gly Leu Glu Gln Ser Glu Arg Val Val Arg

225 230 235 240

Phe Leu Trp Asp Gln His Phe Ala Ala Val Ala Gly Asp Ala Val Ser

245 250 255

Phe Glu Val Tyr Pro Pro Val Glu Lys Glu Trp Asp Leu His His Phe

260 265 270

Leu Leu Ala Gly Trp Gly Leu Pro Ile Gly Glu Met Phe Asp Leu Glu

275 280 285

Gly Leu Lys Glu Val Cys Glu Arg Leu Gly Arg Trp Thr Phe Phe Val

290 295 300

Ser Ser Ser Pro Leu Asn Cys Gly Arg Gly Val Ser Ser Pro Pro Asn

305 310 315 320

Cys Met Ala Ile Phe

325

Claims (9)

1. nucleotide sequence, its coding has the active albumen of selective hydrolysis R-or S-pantolactone, and said nucleotide sequence is selected from the group that following polynucleotide constitute:

(a) polynucleotide shown in SEQ ID NO:5;

(b) polynucleotide of the polypeptide of coding shown in SEQ ID NO:6.

2. nucleotide sequence as claimed in claim 1, it obtains from A.niger MacRae ATCC46951.

3. an expression vector wherein comprises nucleotide sequence according to claim 1 or claim 2.

4. the host cell that transformed with expression vector as claimed in claim 3.

5. a peptide species, it has the pantolactone hydrolase activity, and is described nucleotide sequence coded by claim 1.

6. polypeptide as claimed in claim 5, it is SEQ ID NO:6.

7. like any described polypeptide in claim 5 or 6, it obtains from A.niger MacRae ATCC 46951.

8. be used for R-or S-pantolactone are carried out the purposes of selective hydrolysis like any described polypeptide in the claim 5 to 7.

9. method of producing R-pantothenic acid or its salt or R-panthenol, said method comprise the steps: under like the situation that any described polypeptide exists in the claim 5 to 7, R-or S-pantolactone to be carried out selective hydrolysis.