CN1768141A - Pantolactone hydrolase - Google Patents

Abstract

The present invention relates to the to encode nucleotide sequence of gene of pantolactone hydrolase, and the carrier and the host cell that contain above-mentioned nucleotide sequence, and by above-mentioned nucleotide sequence coded pantolactone hydrolase. The present invention also provides the method for making and use pantolactone hydrolase.

Description

Pantolactone hydrolase

The light that the present invention relates to can be used for pantolactone (pantolactone) enantiomter splits the enzyme of (optical resolution), the gene that relates to the described enzyme of encoding also relates to the purposes of described enzyme in the method for preparing R-pantothenic acid or its salt or R-panthenol (R-panthenol).

Enantiomter is in its physiological effect, i.e. toxicity and pharmacodynamics effect, from the reaction of enzyme with and Perception Features (sensorial characteristics) aspect be different. Known the R-pantolactone is only arranged is intermediate product in the R-pantothenic acid preparation, and R-pantothenic acid is vitamin, all is necessary its growth concerning the human and animal, breeding and the normal physiological function. Pantothenic acid is as the precursor of coacetylase and as the acyl carrier of fatty acid synthetase; relate to and surpass 100 kinds different metabolic approach; comprising the energetic supersession of carbohydrate, protein and lipid, and lipid, neurotransmitter, steroid hormone, porphyrin and hormone is synthetic. Until recently, the method that is widely used in the preparation of R-pantolactone is that the light that R-and the S-pantolactone to chemical synthesis carries out splits. This kind method is very expensive, because it need to use expensive light resolution reagent. In addition, to the recovery of the R-enantiomter of pantolactone also difficult. Therefore, the pantolactone hydrolase that provides the light of the R-that can be used for pantolactone and S-enantiomter to split is that people are needed. Using this kind of enzyme, especially be hydrolyzed S-or the R-form of pantolactone, is the more economic and more maneuverable method of separating two kinds of enantiomters of pantolactone. The example of the salt of R-pantothenic acid comprises the R-calcium pantothenate.

In one embodiment, the invention provides the nucleotide sequence of the gene of coding pantolactone hydrolase, described pantolactone hydrolase obtains from horse liver, A.niger ATCC 9142, A.niger awamori ATCC 38854 or A.niger MacRae ATCC 46951. The public can be from American Type Culture Collection (ATCC), 10801 University Boulvard, and Manassas, VA 20110-2209 USA obtains above-mentioned bacterial strains.

In another embodiment, the invention provides the nucleotide sequence of coding pantolactone hydrolase, described sequence comprises: when pantolactone hydrolase during from the horse liver, and the nucleotide sequence shown in SEQ ID NO:1; When pantolactone hydrolase during from A.niger MacRae ATCC 46951, the nucleotide sequence shown in SEQ ID NO:5; When pantolactone hydrolase during from A.niger ATCC 9142, the sequence shown in SEQ ID NO:7; And when pantolactone hydrolase during from A.niger awamori ATCC 38854, the sequence shown in SEQ ID NO:9.

Described nucleotide sequence can comprise coded sequence and the regulating and controlling sequence of above-mentioned every kind of pantolactone hydrolase.

" regulating and controlling sequence " is defined as at this: instruct the arrangement of the nucleic acid control sequence of the transcribed nucleic acid that is operably connected. The example of this type of expression control sequenc is promoter. " promoter " is included near the necessary nucleotide sequence the transcription initiation site. Promoter also comprises alternatively the far-end enhancer or checks element, and it can be positioned reaching on the thousands of base-pairs after the transcription initiation site. " composing type " promoter is activated promoter all under most of environment and developmental condition. " induction type " promoter is the activated promoter of tool in environment or developmental regulation. Term " is operably connected " and refers to functional connection between expression of nucleic acid control sequence (for example arrangement of promoter or transcription factor binding site point) and the second nucleotide sequence, and wherein said expression control sequenc instructs transcribing corresponding to the nucleic acid of the second sequence.

Can use the fragment of nucleotide sequence, for example, in cross experiment as probe.

Insert nucleotide sequence in host living beings, make it transcribe and translate, in the situation with the manufacturing function polypeptide, the technical staff will recognize, because code degeneracy, and a variety of polynucleotide sequences same polypeptide of all will encoding. These variants also comprise within the scope of the present invention clearly. In addition, the present invention also comprises following sequence clearly, described sequence is identical in fact (according to hereinafter described determining) each other, their codings be wild type peptide mutant polypeptide or remain with the polypeptide (for example being obtained by the conservative replacement to polypeptide amino acid) of polypeptide function. In addition, variant can be the following dominant negative mutant of coding.

If according to hereinafter described carrying out most homogeneous when comparison, two nucleotide sequences or polypeptide nucleotide sequence or amino acid residue separately are same, and these two nucleotide sequences or polypeptide can be considered to " identical ". In the context about two or more pieces nucleic acid or peptide sequence, term " identical " or percentage " homogeny " refer to: carry out most homogeneous in comparison window (comparison window) and relatively reach when comparison, in the situation of observing to measure with a kind of in the following sequence comparison algorithm or by manual comparison and eyes, two or more pieces sequence or subsequence are same, or wherein same amino acid residue or nucleotides has specific percentage. When the percentage of sequence homogeny uses for protein or peptide, will be appreciated that, not identical residue position is normally owing to conservative 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor causes different, wherein amino acid residue is substituted by other and (for example has similar chemical property, electric charge or hydrophobicity) amino acid residue, therefore do not change the functional attributes of molecule. When sequence replaces not simultaneously because of conservative, can because of the conservative essence that replaces, percentage sequence homogeny be raised to proofread and correct. The method of carrying out this adjusting is well known to a person skilled in the art. Typically, this comprises and will conservative replace as part but not completely mispairing is marked, thereby improves percentage sequence homogeny. Therefore, for example, divide for identical amino acid/11, giving in 0 minute the situation of non-conservative replacement, giving the conservative mark that replaces between 0 and 1. According to, for example, Meyers ﹠ Miller, the algorithm of Computer Applic.Biol.Sci.4:11-17 (1988), for example, at program PC/GENE (Intelligenetics, Mountain View, Calif., realize in USA), calculate to conservative replace to minute.

In the context about two nucleic acid or polypeptide, phrase " identical in fact " refers to: when comparison window is carried out the most homogeneous comparison, in the situation of observing to measure with a kind of in the following sequence comparison algorithm or by manual comparison and eyes, have at least 60%, preferred 80%, most preferably sequence or the subsequence of the nucleotides of 90-95% or amino acid residue homogeny. This definition also refer to can with the complementary series of the sequence of sequence hybridization to be measured.

To sequence comparatively speaking, typically, a sequence compares sequence to be measured as canonical sequence with it. When using sequence comparison algorithm, to be measured and canonical sequence all are input in the computer, if necessary, specify the subsequence coordinate, and specified sequence algorithm routine parameter. Can use default program parameter, perhaps specify other parameter. Then sequence comparison algorithm can based on program parameter, be treated the percentage sequence homogeny that is listed as with respect to canonical sequence that checks order and calculate.

" comparison window " used herein comprising: refer to a kind of segment, the quantity of its continuous position is selected from by 20 to 600, common about 50 to about 200, be more typically about 100 to about 150 groups that consist of, wherein, sequence can compare with the canonical sequence of the continuous position with same quantity, and this carries out after two sequences is by optimal alignment. The method that sequence is arranged to compare is well known in the art.

" through the conservative variant of modifying " is used for amino acid and nucleotide sequence. With regard to concrete nucleotide sequence, through the conservative variant of modifying refer to the to encode nucleic acid of identical or essentially identical amino acid sequence, perhaps, when nucleic acid not during encoding amino acid sequence, refer to essentially identical sequence. Because the degeneracy of genetic code, nucleic acid codon identical on the several functions any given protein of encoding is arranged. For example, codon GCA, GCC, GCG and GCU this seed amino acid of alanine of all encoding. Therefore, on each position that alanine is determined by codon, codon can be changed into any one in the above-mentioned corresponding codon, and can not change the polypeptide of encoding out. The variation of this type of nucleic acid is " silent variant ", and it is a kind of in conservative variation of modifying. The nucleotide sequence of every coded polypeptide is yet described every kind of possible silent variant of this nucleic acid herein. The technical staff will recognize that each codon in the nucleic acid (except the AUG, unique codon of its methionine of normally encoding) all can be modified, to produce identical molecule on the function. Therefore, every kind of silent variant of the nucleic acid of coded polypeptide all lies in every sequence that was described.

For amino acid sequence, the technical staff will recognize, indivedual replacements, disappearance or increase to nucleic acid, or in the sequence that is encoded, change single amino acids or little percentage (namely to what peptide, polypeptide or protein sequence carried out, less than 20%, for example 15%, 10%, 5%, 4%, 3%, 2% or 1%) amino acid whose replacement is " through the conservative variant of modifying ", and change wherein is that amino acid is replaced by chemically similar amino acid. Provide amino acid whose conservative replacement table similar on the function to be known in the art.

Each group in following six groups all contains can guard mutually the amino acid that replaces:

Alanine (A), serine (S), threonine (T);

Aspartic acid (D), glutamic acid (E);

Asparagine (N), glutamine (Q);

Arginine (R), lysine (K);

Isoleucine (I), leucine (L), methionine (M), valine (V); And

Phenylalanine (F), tyrosine (Y), tryptophan (W). (see, for example, Creighton, Proteins (1984)).

Article two, the sign that nucleotide sequence or polypeptide are identical in fact is: the antibody that the polypeptide of article one nucleic acid coding can produce with the polypeptide because of antagonism second nucleic acid coding carries out immunological cross-reaction. Therefore, for example, when two peptides only owing to conservative the replacement, have not simultaneously, then typically, wherein a polypeptide is identical in fact with the second polypeptide. Article two, the another kind sign that nucleotide sequence is identical in fact is: these two molecules or its complement can the phase mutual crosses under rigorous condition as mentioned below.

Phrase " with ... specific hybrid " refer to: under rigorous hybridization conditions, when sequence (for example is present in complex mixture, the total DNA of cell or RNA, or library DNA or RNA) in the time, molecule only be combined, is formed two strands with specific nucleotide sequence or hybridizes.

Phrase " rigorous hybridization conditions " refer to probe will be with its target sequence not with the condition of other sequence hybridization, typically, its target sequence is in the complex mixture of nucleotide sequence. Rigorous condition depends on sequence, and is also different in different situations. Long sequence can be under higher temperature specific hybrid. Usually, high rigor condition is selected as: melt temperature (T than the heat of specific sequence under given ionic strength and pH_m) low about 5-10 ℃. Low rigor condition is selected as comparing T usually_mLow about 15-30 ℃. T_mBe: (under given ionic strength, pH and nucleic acid concentration) during balance, (target sequence is excessive when existing, T to be complementary to the temperature of 50% in the probe of target and target sequence hybridization_mThe place, 50% probe is combined during balance). Rigorous condition will be following condition: salinity is lower than the sodium ion of about 1.0M, typically, about Na ion concentration of 0.01 to 1.0M (or other salt), pH 7.0 to 8.3, short probe (for example, 10 to 50 nucleotides) temperature is about at least 30 ℃, to long probe (for example, more than 50 nucleotides), temperature is about at least 60 ℃. Rigorous condition can also be removed stable reagent by adding, and for example formamide obtains. For selective or specific hybrid, positive signal is at least twice of background, preferred 10 times to background hybridization.

If the coded polypeptide of nucleic acid that can not the phase mutual cross under rigorous condition is that identical in fact, so such nucleic acid also remains identical in fact. This can betide, and for example, uses the maximum codon degeneracy degree that is allowed by genetic code to produce in the situation of nucleic acid copy. In this type of situation, typically, nucleic acid is hybridized under the rigorous hybridization conditions of moderate.

In the present invention, use nucleotide sequence disclosed herein, under rigorous condition, carry out the Southern Blot experiment of standard, can identify the genomic DNA (gDNA) or the cDNA that contain nucleic acid of the present invention. Plant disclosed purpose at this point, the rigorous condition that is suitable for this type of hybridization comprises: hybridizing in 40% formamide, 1M NaCl, 1% lauryl sodium sulfate (SDS) buffer solution under 37 ℃, at least in 0.2X SSC, be washed till under about 50 ℃ temperature less once, 20 minutes, temperature was normally at about 55 ℃ to about 60 ℃; Or suitable condition. Positive hybridization is the twice of background at least. Those of ordinary skill is easy to recognize that other hybridization and wash conditions can be used for providing the condition with similar rigor.

About two identical another kind signs of polynucleotides be: the canonical sequence that increases out by a pair of Oligonucleolide primers, under rigorous hybridization conditions, can be used as probe, from cDNA or genomic library, isolating sequence to be measured, or in northern or Southern Blot experiment, identify sequence to be measured.

The present invention also comprises expression vector defined herein. Expression vector comprises in the polynucleotide sequence listed above any one or more copies. Expression vector of the present invention can contain any polynucleotide sequence of this paper definition.

Therefore, in another embodiment, the invention provides a kind of expression vector, described carrier comprises the nucleotide sequence of the gene of the pantolactone hydrolase of encoding, and described pantolactone hydrolase is from horse liver, Bacillus subtilis, A.niger ATCC 9142, A.niger awamori ATCC 38854 or A.niger MacRae ATCC 46951; Perhaps described carrier comprises the nucleotide sequence of the gene of the pantolactone hydrolase of encoding, described pantolactone hydrolase is from the horse liver, shown in SEQ ID NO:2, from Bacillus subtilis, shown in SEQ ID NO:4, from A. niger ATCC 9142, shown in SEQ ID NO:8, from A.niger awamori ATCC 38854, shown in SEQ ID NO:10, and from A.niger MacRae ATCC 46951, part is shown in SEQ ID NO:6.

Alternatively, the polynucleotide sequence in the expression vector can be operably connected with expression control sequenc defined above.

The phrase " expression vector " that uses in this article is reproducible (replicatable) carrier, the dna sequence dna of the polynucleotide sequence that it is listed with coding this paper, and can regulate and control the expression of these dna sequence dnas. In this context, term " reproducible " refers to that in the host cell of the given type of introducing carrier, carrier can be replicated. In the place of interested polynucleotide sequence upstream near these polynucleotides, may provide the sequence of code signal peptide, its existence has been guaranteed to be secreted with the expressed polypeptide that is encoded of the host cell of carrier. Burst can be to be connected with the polynucleotide sequence that is selected is natural, perhaps can originate from another kind.

Carrier can be any carrier that tradition is used for recombinant DNA technique, will depend on usually that to the selection of carrier it is with the host cell that is introduced into. Therefore, carrier can be autonomously replicationg vector, that is, as the carrier that the outer material of chromosome exists, it copies and is independent of chromosome replication; The example of this kind carrier is plasmid, bacteriophage, clay or mini chromosome. In addition, carrier can be following carrier, when it is introduced in the host cell, can be incorporated in the host cell gene group, and copy with the host cell gene group that it is integrated into. Expression vector of the present invention can with any dna sequence dna of the present invention, can be used to express any polypeptide of the present invention.

The present invention also comprises cell or the host cell through cultivating, and wherein contains one or more polynucleotide sequence disclosed herein and/or one or more expression vector disclosed herein. " cell through cultivating " that use in this article comprises, any can under specified criteria, growth, and can express the cell of one or more polypeptide of polynucleotide encoding of the present invention. Preferably, the cell through cultivating is yeast, fungi, bacterium or algae. More preferably, the cell through cultivating is Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Aspergillus niger or Bacillus subtilis.

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

In another embodiment, the invention provides a kind of pantolactone hydrolase, its amino acid sequence comprises: when from the horse liver, the amino acid sequence shown in the SEQ ID NO:2; When from A.niger MacRae ATCC 46951, the amino acid sequence shown in the SEQ ID NO:6; When from A.niger ATCC 9142, the amino acid sequence shown in the SEQ ID NO:8; When from A. niger awamori ATCC 38854, the amino acid sequence shown in the SEQ ID NO:10.

Shown in the SEQ ID NO:2 from the horse liver with show that from the pantolactone hydrolase of Bacillus subtilis the S-pantolactone hydrolase is active shown in the SEQ ID NO:4. Shown in the SEQ ID NO:8 from A.niger ATCC 9142, show that with the pantolactone hydrolase from A.niger MacRae ATCC 46951 of part shown in SEQ ID NO:6 the R-pantolactone hydrolase is active from A. niger awamori ATCC 38854 shown in the SEQ ID NO:10.

In another embodiment, the present invention comprises the fragment of above-mentioned amino acid sequence, and described fragment has the enzymatic activity of whole protein.

" fragment of amino acid sequence " according to the present invention can be defined as lacking one section amino acid whose sequence, and this section amino acid may be necessary concerning protein correctly folds. Yet in case protein is folded, this section amino acid just can be deleted, and can not destroy the function of single protein. These sections may reside in the folded protein, and for example ring perhaps can be N-or the C-end sequence not directly related with the activity of folded protein.

In addition, the present invention includes the derivative of the protein that comprises described R-and S-pantolactone hydrolase activity. Described derivative comprises the enzyme that is fixed, and for example is fixed by including (inclusion), and wherein said enzyme is retained on the film device, for example doughnut, polymer network structure or microcapsule. Also may be by crosslinked or come enzyme is fixed by producing enzyme crystal. The another kind of possible method that enzyme is fixed is that it is attached on the polymer of growing, for example silicon dioxide gel gel or silica graphite sol gel, perhaps by enzyme being attached on the prefabricated carrier, for example EupergitC, Eupergit 250L, PEG, Celite and Amberlite XAD-7. To the technology of the applicable another kind of immobilized enzyme of R-of the present invention and S-pantolactone hydrolase be with its be dispersed in can't be miscible with water organic solvent in. Usually, by nearly all technique for fixing known in the art, can be to having the protein of enzymatic activity, for example enzyme of the present invention is fixed.

In another embodiment, the present invention relates to the purposes with separated pantolactone hydrolase selective hydrolysis R-or S-pantolactone hydrolase, this can be used for the industrial production to the R-enantiomter of pantolactone, and the R-enantiomter of pantolactone is to produce the precursor of R-pantothenic acid and R-panthenol.

Therefore, an object of the present invention is to provide method or the approach that comes R-or S-pantolactone are carried out selective hydrolysis with separated pantolactone hydrolase as herein described.

In another embodiment, the invention provides the method for producing R-pantothenic acid or its salt or R-panthenol, comprising the step of in the presence of pantolactone hydrolase of the present invention, R-or S-pantolactone being carried out selective hydrolysis.

In another embodiment, the invention provides the method for the nucleotide sequence of clone and known array direct neighbor, for example the pantolactone hydrolase of coding novelty or the nucleotide sequence of its part.

Particularly, the present invention includes the new PCR strategy of energy amplify unknown sequence. Described new PCR strategy is included in the synthetic middle same primer that uses of article one and second DNA chain. This primer can with the known area hybridization of the gDNA that will be amplified. This primer shows the homology with known array 100%. Under the help of this primer, carry out the PCR first time, article one strand copy of genomic DNA is provided, arrive unknown nucleotide sequence. Carry out the PCR second time with the part PCR product first time as template, use therein (nested) primer can with the 3 ' direction that is positioned primer used among the PCR for the first time on dna fragmentation hybridization, but still be in logic the known portions of sequence. After this nested primers is hybridized at random with the single stranded DNA that the first time, polymeric enzyme reaction produced, produce the antisense strand of this single stranded DNA, himself is as being used for for the second time template of the same primer of PCR, generation second chain. In the end, originate in known array inside, extend contiguous genomic DNA or extend the dna fragmentation of any other DNA of disappearance partial sequence out manufactured by a kind of primer that can both hybridize with the new segment two ends.

Therefore, in another embodiment, the invention provides the method for the nucleotide sequence of clone and known array direct neighbor, described method comprises pcr amplification, and described amplification comprises:

A) carry out PCR second time with the single stranded DNA of PCR for the first time as template, in described second time PCR with the primer that can hybridize at random with the single stranded DNA of the PCR generation first time; And

B) thus, still use the at random hybridized primer of step in a), produce self as the antisense strand of second chain template.

Following embodiment is described in detail evaluation, sign and the expression of coding pantolactone hydrolase gene. These embodiment only are exemplary, but not want by any way scope of the present invention to be limited.

Embodiment 1 comes purifying (S)-pantolactone hydrolysing activity from commercially available horse liver esterase acetone powder

1g horse liver esterase acetone powder (Fluka Holding AG, Industriestrasse 25, P.O. Box 260, CH-9471 Buchs, Switzerland) is dissolved among the Tris/HCl of 40ml 50mM, and pH 7.5, wherein contain 2M ammonium sulfate, 1mM CaCl₂、1mM MgCl ₂With 10 μ M EDTA (buffer A). Go out undissolved material by centrifugation, afterwards, carry out filtration sterilization with 0.45 μ M filter. Protein solution is loaded among the butyl-agarose post HR26/10 (Amersham Pharmacia Biotech Europe GmbH, D ü bendorf, Switzerland) that crosses with the buffer A balance. Wash this post with the 100ml buffer A. Use afterwards the buffer B (buffer A that does not have ammonium sulfate) of 400ml from 0 to 100% linear gradient. Lactonase is middle by wash-out out in gradient. Collect the segment that shows the pantolactone hydrolysing activity, buffer solution is changed to 20mM Tris/HCl, the 1mM CaCl of pH 8.0₂、1mM MgCl ₂And 10 μ M EDTA. The segment of collecting is loaded to MonoQ-and fills out in advance among (pre-packed) post HR 26/10 (Amersham Pharmacia Biotech Europe GmbH, D ü bendorf, Switzerland), uses the linear gradient of 0 to 350mM NaCl. Protein begins namely by wash-out out this gradient. Again collect active part, be concentrated to 1 ml, loading is to Superdex 200 posts (Amersham Pharmacia Biotech Europe GmbH, D ü bendorf, Switzerland). 50mM Tris/HCl, 1mM CaCl with pH 8.0₂、1mM MgCl ₂And 10 μ M EDTA as elution buffer, lactonase as last peak by wash-out out, it shows the molecular size of about 31kDa, conforms to the size shown in the SDS-PAGE.

By 37 ℃ with enzyme in the 200mM of pH 9.0 Tris/ acetate buffer, 5mM MgCl₂Middle cultivation was measured in 60 minutes, and the discovery maximum specific activity is 13.3U/ (mg protein). Measure the concentration of (S)-and (R)-pantolactone by HPLC.

2 pairs of (S)-pantolactone hydrolases from the horse liver of embodiment carry out preliminary sign

(a) molecular size, structure and isoelectric point:

According to SDA-PAGE, albuminate has the molecular weight of about 35kDa. With the elution time of calibrated solvent resistant column, the molecular weight of native protein is calculated as about 31kDa. This means that lactonase from the horse liver is 30 to 35kDa monomer. Use the 2D gel, isoelectric point is measured as in the scope of pH 5.5.

(b) the terminal and internal amino acid order-checking of N-:

Digest with the protein of trypsase to enrichment after the 2D gel electrophoresis. By mass spectrum the peptide that produces is analyzed. The N-end sequence of peptide No.66 has been shown among the SEQ NO:11.

This sequence shows that with the MS-spectrum this protein belongs to the protein families that is known as Senescence Marker Protein-30 or calmodulin (regucalcins), and it mainly is found in the vertebrate. From the calmodulin sequence of rat, mouse, people, chicken, rabbit, ox, Xenopus laevis, and determined from the sequence of two less homologys of Drosophila melanogaster. Yet, from the also not separated and order-checking of this gene of horse.

(c) metal dependence:

For measuring different divalent metals to the impact of the pantolactone enzymatic activity of horse liver enzyme, with 1.3,2.6,5.2 or the Ca of 10.4mM²⁺、Mg ²⁺Or Mn²⁺Join in the reactant mixture that 200mM Tris/ acetate and 50mM (R, S)-pantolactone by pH 9.0 form. At 37 ℃ reactant mixture is carried out 60 minutes cultivation, come cessation reaction with the methyl alcohol of the isopyknic 2mM of containing EDTA. Measure the minimizing of (S)-pantolactone by HPLC. Calcium makes enzymatic activity reduce, and magnesium and manganese then make enzymatic activity increase.

(d) heat endurance:

Under 30 to 80 ℃ temperature, with purified enzyme at 5.2mM MgCl₂With cultivated 15 minutes in the 200mM Tris/ acetate of pH 9.0. After 30 minutes, measure activity (60 minutes, 37 ℃) with standard test method on ice. Until 60 ℃ of enzymes still are stable. It is at 65 ℃ of beginning inactivations, 70 ℃ of activity of losing above 50%.

          Embodiment 3 clones are from (S)-pantolactone enzyme of horse liver

Use the sequence information from all known mammalian genes (seeing below) to design primer, be used for the PCR that carries out for the cDNA that makes from the horse liver from age " Hafflinger " of Bavaria, described cDNA is by Roche Molecular Biochemicals, Penzberg, German friendship provides. Prepare total RNA from liver organization with QIAGEN RNeasy Maxi Kit (Qiagen, Hilden, Germany).

Homology between horse liver lactonase and other member of calmodulin family is shown in the table 1. Mensuration is that the Application standard parameter is measured by the GAP program in the GCG program package. The numeral of similitude is shown in cornerwise the right in the table, and the numeral of homogeny is shown in the diagonal left side.

Table 1: the amino acid sequence similarity in the calmodulin family and homogeny

	Horse	Ox	Rabbit	The 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
The 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

With Titan One Tube RT-PCT Kit (Roche Molecular Biochemicals, Penzberg, Germany) and covered the homologous primer of initiation codon and terminator codon upstream 50bp sequence, utilize RT-PCT to amplify the fragment (the 1-885 position nucleotides of SEQ ID NO:1) of 885bp. The method that reaction is recommended according to manufacturer is carried out.

The structure that is used for the N-end 5 ' primer (SEQ ID NO:12) of PCR for the first time and C-end 3 '-primer (3 '-CAGTTTCCTTAAGGGGGGATA-5 ') (SEQ ID NO:13) is based on from the N-end sequence of ox (SEQ ID NO:14), rabbit (SEQ ID NO:15), people (SEQ ID NO:16), rat (SEQ ID NO:17), mouse (SEQ ID NO:18), chicken (SEQ ID NO:19) and Xenopus (SEQ ID NO:20) and from the C-end sequence of ox (SEQ ID NO:21), rabbit (SEQ ID NO:22), people (SEQ ID NO:23), rat (SEQ ID NO:24), mouse (SEQ ID NO:25), chicken (SEQ ID NO:26) and Xenopus (SEQ ID NO:27).

This fragment is cloned in the TA cloning vector (Invitrogen, Carlsberg, CA, USA). Order-checking proves that it is from horse liver calmodulin. By being called as the method for 5 '/3 ' RACE, with Roche Molecular Biochemicals, Penzberg, the independent kit that Germany provides is isolated 5 ' of disappearance-end: use the primer (SEQ ID NO:28) of perfect matching from separated horse liver cDNA manufacture order chain DNA. This single stranded DNA of purifying adds the polyC tail, and nested primers, the polyG primer held with article one primer 5 ' carry out another time PCR. The 5 '-terminal dna fragmentation that is representing the disappearance of gene is amplified out. With cloning 3 '-end according to the forward primer of known sequences Design (SEQ ID NO:29) and last the amino acid whose primer of gene (SEQ ID NO:30) that originates in that mainly designs according to ox and rabbit gene. The last time among the PCR, design 5 ' of covering gene initial sum terminator codon-and 3 '-primer with the sequence information that obtains, according to the full-length cDNA of from total RNA, isolating lactonase mentioned above. This cDNA and amino acid sequence are showed among SEQ ID NO:1 and the SEQ ID NO:2. This gene is by 900bp/299 Amino acid profile.

With the obtainable expression vector of a kind of commercial sources, for example from the pQE60 of Qiagen, the gene of the expressed in E. coli, its C-is terminal to be merged with the His label. According to standardization program the protein that adds the His label is expressed and purifying, produce the protein of 33 kDa of demonstration (S)-pantolactone enzymatic activity.

Embodiment 4 comes purifying (R)-pantolactone hydrolysis to live by commercially available A.niger lipase A crude preparation by using The property

In the enzyme product that is obtained by the A.niger fermentation not of the same race that can obtain from commercial channels, identified the activity of specificity hydrolysis (R)-pantolactone. Be the concrete enzyme of purifying, used the Co.Ltd. from Amano Pharmaceuticals, Nagoya, the commercial formulation that is called as Lipase A " Amano " of Japan. According to the sequence data that obtains from two enzymes with the common purifying of lactonase, said preparation freeze-drying and the broken overexpression A. awamori cell from the lipase of A.tubigensis of hanging oneself. This material is dissolved in the 20mM Tris/HCl of pH 7.5, the MgCl of 2.5mM₂In (standard buffer solution). The ethanol of adding 5% is to increase the solubility of material. By centrifugal (30 minutes, 15000rpm, 4 ℃) and the filtration (with the filter of 0.45 μ m) followed, isolate the material that can't enter into solution. First by ultrafiltration the part of having dissolved is carried out the purifying first time in Amicon cell ultrafilter (50kDa burble point), to remove any salt and micromolecular compound, they can upset follow-up anion-exchange chromatography step. Then the protein loading is filled out Q-Ago-Gel post (Amersham Pharmacia Biotech Europe GmbH, D ü bendorf, Switzerland) in advance to HR26/10. Wash post with the 200ml standard buffer solution, then the linear gradient (400m1) by 0 to 350mM NaCl elutes protein. Containing activated segment is collected. Add ammonium sulfate, to final concentration be 1.5M, treated protein solution loading to HR 26/10 butyl-agarose Gel Pre is filled out in the post (Amersham Pharmacia Biotech Europe GmbH, D ü bendorf, Switzerland). The standard buffer solution that contains 1.5M ammonium sulfate with 200ml washs, and isolates protein by 1.5 to the 0M ammonium sulphate gradients that successively decrease (400ml). This moment, activity was eluted at two peaks, and was collected respectively. The volume of twice gleanings is reduced to less than 2ml. Use standard buffer solution, isolate two kinds of concentrates by carrying out gel filtration at Superdex 200 posts. The active fragment of every kind of separator is collected and concentrates. The activity of gleanings II trends towards a little earlier in gleanings I by wash-out out, and this shows that the molecular weight of respective egg white matter may be slightly high.

80U/ml (40 ℃) is the high activity of having measured respectively for gleanings I and gleanings II. Shown in the 2D gel, protein formulation not exclusively is homology, this real specific activity of expression even can be higher. Yet because the lactonase activity corresponding to the protein band of 38kDa in the gel filtration elution curve, also is dominant protein in the final preparation, this band most possibly is lactonase. According to the result of gel filtration, natural lactonase has the molecular weight of 75kDa. This is hinting the homodimer structure of this enzyme.

          Embodiment 5 carries out preliminary sign to (R)-pantolactone hydrolase

(a) structure of native form and molecular weight:

With condition as described in Example 4, at the 72ml place, elute enzyme with Superdex 200 (Amersham Pharmacia Biotech Europe GmbH, D ü bendorf, Switzerland). Infer that from calibration curve this has reflected the molecular weight of 75kDa. SDS-PAGE shows that molecular weight is 38kDa. The homodimer that the combination of above-mentioned data hint is comprised of two 38 identical kDa subunits from (R)-pantolactone enzyme of A.niger.

(b) heat endurance:

In the 20mM of pH 7.5 Tris/HCl, in 0,30,40,50,55,60,70 ℃ the diluted enzymes of 50 μ l (0.1U) are carried out one hour cultivation. After 15 minutes, measure residual activity with standard test method on ice. Until 50 ℃ of enzymes all are stable. Under higher temperature, it begins inactivation.

(c) optimum temperature:

Adopt following condition, in 20,30,40,50,55 and 60 ℃, (the R)-pantolactone enzyme from the intense enrichment of commercial source (seeing embodiment 5) carried out 30 minutes cultivation:

100mM (R, S)-pantolactone

100mM MgCl ₂

200mM Tris/HCl，pH 8.0

0.05U (R)-and the pantolactone enzyme, final volume is 10 μ l.

Under used condition, it is about 50 ℃ from the optimum temperature of (R)-pantolactone enzyme of commercialization A.niger preparation.

(d) confactor dependence and inhibition:

Activity from (R)-pantolactone enzyme of A.niger needs Mg²⁺Or Mn²⁺ Add 1mM EDTA and can suppress its activity fully.

(e) best pH:

When pH changed between 7.0 to 11.0, this enzyme did not demonstrate " normally " pH-activity curve. At 40 ℃, 10 mg Amano Lipase A in the 200mM TEA-acetate buffer (pH 7.0 to 11.0) are carried out 20 minutes cultivation, described buffer solution contains 0.1%Span 80, the 20mM magnesium acetate. Add isopyknic 500mM MES buffer solution and stop the reaction of sample, described pH of buffer is 5.5, contains 50mM EDTA; Sample is carried out centrifugal (10,000g, 10 minutes), then analyze by HPLC, wherein, with 0.46 * 15cm CHIRADEX post, come wash-out with 70: 30 water-methanols, 1ml/ minute, 20 ℃. High activity is to reach in about 10 o'clock at pH. 11.0, activity there is no considerable change from this position to pH. High selectivity reached at pH in about 9.0 o'clock.

Embodiment 6 cultivates A.niger ATCC 9142,9029,26875,62863 and 10864, and right Its (R)-pantolactone enzymatic activity is evaluated and tested

For know that whether the lactonase activity extensively exists, and cultivates five A.niger bacterial strains under the following conditions in Aspergillus belongs to:

Culture medium (with respect to every liter amount): 1.2g NaNO₃、0.5g KH ₂PO ₄、0.2g MgSO ₄×7H ₂The trace metal solution of O, 0.5g yeast extract, 5% glucose and 0.04ml, described solution such as Vishniac and Santer, Bacteriol.Rev.21:195-213 (1957) is described.

With every ml 5 * 10⁶Individual spore is in 30 ℃ of 300ml preculture things that go to inoculate in 11 bottles. After 8 hours, the preculture thing is joined in 1.71 culture mediums in 21 fermentation tanks, fermentation tank is with pH and dissolved oxygen control. By automatic adding 5M NaOH pH is remained on 5.5. Under low ventilation condition (air saturation of 5-10%), cell is carried out 14 hours cultivation. Afterwards dissolved oxygen levels is increased to the 30-50% air saturation. After 5 hours, the results mycelium filters it, spends mineral matter washing twice, is frozen in the liquid nitrogen. With French Press a part is wherein directly carried out cracking. With standard test method culture medium and cell pyrolysis liquid are carried out research for the pantolactone enzymatic activity.

(R)-the pantolactone enzymatic activity mainly is found in the lysate of all tested A.niger bacterial strains. In culture medium, only found very little activity.

7 pairs of (R)-pantolactone enzymes through enrichment from the lipase preparation of embodiment carry out Trypsin Induced The amino acid sequence of the little peptide that produces is measured

The preparation through enrichment to embodiment 5 on the 2D gel is analyzed. With trypsase two main points are digested, it is respectively 33 and 40kDa, 4.7 and 5.6 pI. Peptide (the equipment: Hewlett Packard 1090 that separates generation by HPLC, post: Vydac C18 (0.21 * 25cm), the 210nm place absorbs, flow velocity: 0.20ml/ minute, buffer A: 0.075%TFA, buffer B: the 0.065%TFA in 80% acetonitrile, gradient: 2%B (0-10 minute), 2-75%B (10-120 minute)). Come the peptide of selecting is checked order by the Edman edman degradation Edman, 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 undetermined amino acid, and the amino acid in the round parentheses can't clearly be determined.

Carry out database search with all short peptide sequences as target, do not obtain reliable information and can help to determine that in two main points which representing lactonase. Therefore, we utilize a cover anion-exchange chromatography that the lactonase preparation of embodiment 5 is carried out further enrichment. Filling out in advance on the MonoQ post HR 16/10 (Amersham Pharmacia Biotech Europe GmbH, D ü bendorf, Switzerland), under 0 to the 350mM NaCl gradient, to protein solution (at the 20mM of pH 7.5 Tris/HCl, 2.5mM MgCl₂In) separate. Activity to all segments is measured. According to described isoelectric focusing and the SDS-PAGE of carrying out of manufacturer (Invitrogen, Carlsbad, CA, USA), active part is analyzed. Compare the segment activity data that SDS-PAGE gel and IEF draw, the hint lactonase is 38 to 40kDa protein, and pI is at pH about 5.6. These data are coincide very well with some PollC. Therefore, the amino acid sequence of this point just is used to design the PCR primer and is used for the oligonucleotides that the Southern trace is tested. Collect the activated segment of tool, analyze at the 2D gel again, analyze and show that than 2D gel before, PollC is with respect to more clearly enrichment of PollA/B.

Embodiment 8 is designed for the degenerate oligonucleotide of Southern trace test and is used for from A.niger The PCR primer of clone's (R)-pantolactone enzyme

Use is from 12 of different Aspergillus kinds different phytase sequences, CODONFREQUENCY program by GCG package version 10.2, obtain the codon operating position of Aspergillus, use it for and reduce because the number of the possible different dna sequence dna combinations that genetic code degeneration causes. As deciding factor, primer 3 '-end has all been considered every kind of possible combination with Aspergillus codon operating position, but primer 5 '-end has just been ignored some rare codons. Carrying out with oligonucleotides in the situation of hybrid experiment, in the same way to 3 '-and 5 '-end process. Contain the oligonucleotides of " S (sense) " or " AS (anti-sense) " in the title for PCR. Do not contain the oligonucleotides of S or AS in the title for hybrid experiment.

Being used for from the oligonucleotides of A.niger clone (R)-pantolactone enzyme is Fxn42 (SEQ ID NO:31), Fxn42S (SEQ ID NO:32), Fxn42AS (SEQ ID NO:33), Fx 57 (SEQ ID NO:34), Fxn57S (SEQ ID NO:35), Fxn57AS (SEQ ID NO:36), Fx 73 (SEQ ID NO:37), Fx 73S (SEQ ID NO:38), Fx 73AS (SEQ ID NO:39), Fx 74 (SEQ ID NO:40), Fx 74S (SEQ ID NO:41) and Fx 74AS (SEQ ID NO:42).

Embodiment 9 is from A.niger ATCC 9142, A.niger NRRI3135, A.niger ssp. Isolated genes among awamori (ATCC 38854) or the A.niger MacRae (ATCC 46951) Group DNA

With 1 * 10⁶Spore/ml removes to inoculate YPD culture medium [the Sherman et al. of 300ml, Laboratory course manual for methods in yeast genetics, Cold Spring Harbor Laoratory, Cold Spring Harbor, New York (1986)]. Under fierce vibration (200 rpm), in 30 ℃ culture is carried out incubated overnight. Collect mycelium with Whatman filter paper by filtering, wash with PBS (waiting the phosphatizing acid buffer), freezing in liquid nitrogen. In ice-cold mortar, mycelium is worn into fine powder. This powder Eddy diffusion in 50 mM Tris/HCl, the 1.0%SDS of the pH 8.0 of 1/3 volume, 50mM EDTA, is frequently put upside down under the condition in 65 ℃ and to be cultivated 15 minutes. Solution is cooled to 50 ℃. Add Proteinase K (final concentration is 100 μ g/ml). Solution was cultivated 1 hour at 50 ℃. The Proteinase K that adds in addition 100mg/ml continues to cultivate 3 hours. Phenol/chloroform/the isoamyl alcohol (50/49/1) that adds 1/3 volume. After the thorough and soft mixing, emulsion is carried out centrifugal (12000g, 20 minutes, 4 ℃). Shift out water, again extraction. Another centrifugation step (12000g, 10 minutes, 4 ℃) afterwards, extracts water with chloroform. To the isopropyl alcohol of 0.7 times of volume of aqueous phase adding, soft mixed solution 15 minutes. By centrifugal (10000g, 30 minutes, 4 ℃) collecting precipitation DNA. The 3M KCl that adds the pH 5.2 of 1/10 volume in the residual supernatant cultivated 15 minutes under soft vibration. After the centrifugation step, the ethanol with 70% washes twice to two parts of precipitations again, and air drying is dissolved in the 0.5ml water. At last, process DNA to remove residual RNA with RNase.

Embodiment 10 clones are from (R)-pantolactone enzyme of A.niger ATCC 9142

According to the embodiment 6 described genomic DNAs (gDNA) that prepare A.niger ATCC 9142. Use (the Stratagene from Stratagene, La Jolla, CA, USA) Robocycler Infinity as the PCR instrument, use from the TAQ polymerase kit of Roche Molecular Biochemicals (Penzberg, Germany) and carry out PCR:

-95 ℃ of 1 μ l gDNA (1/10 dilution) step 1:5 minute

1 μ l mixture of ribonucleotides step 2:30 second-95 ℃

5 μ l are with Mg²⁺PCR standard buffer solution step 3:30 second-50 ℃

-72 ℃ of 1 μ l primer S (100pM/ μ l) step 4:1 minute

1 μ l primer AS (100pM/ μ l)

1 μ l is from the TAQ polymerase of Roche Molecular Biochemicals

40μl H ₂O

Step 2 is to 4 repetitions 35 times.

Combination of primers:

Numbering	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 can carry out inspection about self grappling (self-priming) to all primers. Fxn57AS be unique can produce a great deal of self in conjunction with the primer of product, self grappling product is about 300 to 320bp. With 55 ℃ hybridization temperature, PCR 5,6,8,10 and 11 still can produce one or more product. 60 ℃ lower adopts amplification in 30 seconds and 30 circulations, PCR 5,6,8,10 and 11 still can produce the one or more PCR product corresponding with hanging down the band that produces under the hybridization temperature.

Use the product of from Ago-Gel, isolating the 840bp of PCR reaction 12 from the QIAEX II gel extraction agent box of Qiagen (Qiagen, Hilden, Germany), it is dissolved among the 40 μ l H2O.

-95 ℃ of the fragments of 1 μ l 840bp (110 dilution) step 1:5 minute

1 μ l mixture of ribonucleotides step 2:30 second-95 ℃

5 μ l are with Mg²⁺PCR standard buffer solution step 3:30 second-55 ℃

1 μ l primer S (10pMol/ μ l) step 4:30 second-72 ℃

1 μ l primer AS (10pMol/ μ l)

1 μ l is from the TAQ polymerase of Roche Molecular Biochemicals

40μl H ₂O

Step 2 is to 4 repetitions 30 times.

Used and reacted 2,5,10,11 and 12 combination of primers. Except reaction 2, other PCR product that responded produces. The sequence that this means primer 42S/AS, 57S/AS, 73S/AS and 74S/AS is the part of 840bp fragment. The height proximity of these four pairs of primers indicates that strongly the fragment of 840bp is the part of interested lactonase gene. This hypothesis has obtained the support of order-checking. The amino acid sequence of peptide Fx 57 (SEQ ID NO:90) has obtained confirmation in the fragment of 840bp, and the sequence of peptide Fxn 42 (SEQ ID NO:45) has only obtained partly confirming.

Can also isolate the corresponding DNA fragments of A.niger MacRae. About the situation of A.awamori, only can isolate the product of primers F xn57S and Fx73AS by the method. The sequence of above-mentioned Aspergillus has about 90% homogeny with the sequence of A.niger ATCC 9142 on dna level.

For separating 5 ' of lactonase gene-and 3 '-end, use following method:

(a) separate 3 '-end:

-95 ℃ of the gDNA steps of 1 μ l A.niger ATCC 9142 1:5 minute

1 μ l mixture of ribonucleotides step 2:30 second-95 ℃

5 μ l are with Mg²⁺PCR standard buffer solution step 3:30 second-55 ℃

1 μ l primer Oal2AS or Oal3S (10pMol/ μ l) step 4:30 second-72 ℃

1 μ l is from the TAQ polymerase of Roche Molecular Biochemicals

41μl H ₂O

Step 2 is to 4 repetitions 30 times.

With isopyknic phenol/chloroform PCR is mixed and to carry out twice extraction, carry out once separately with chloroform again. Then the 3M potassium acetate of the pH 5.2 by adding 1/10 volume and the ethanol of 2 times of volumes are settled out DNA. (in the board-like centrifuge tube of Eppendorf, 14000rpm) afterwards, wash precipitation with 70% ethanol, air is dry, finally is dissolved in the 20 μ l aqua sterilisas for 15 minutes centrifugal.

PCR for the second time:

4 μ l are from-95 ℃ of DNA steps 1:5 minute of the PCR first time

1 μ l mixture of ribonucleotides step 2:30 second-95 ℃

5 μ l are with Mg²⁺PCR standard buffer solution step 3:30 second-58 ℃

-72 ℃ of 1 μ l primer Oal4S or Oal1AS (10pMol/ μ l) step 4:1 minute

1 μ l is from the High Fidelity Mix 38 μ l H of Roche Molecular Biochemicals₂O

Step 2 is to 4 repetitions 35 times.

Use the Oal4S primer, produced the dna molecular of one section 900bp. Order-checking shows 3 '-end of its encoding gene.

(b) separate 5 '-end:

Oligonucleotides Fxn42, the Fxn57, Fx73 and the Fx74 that cross with the DIG mark are as probe, the DIG system (Roche Molecular Biochemicals) that use is surveyed with the x-ray film chemiluminescence carries out hybrid experiment to the genomic DNA of A.niger ATCC 9142. Hybridization and detection are carried out according to the recommendation of manufacturer.

Come the genomic DNA of 10 μ g is digested with BamHI, EcoRI, HindIII, PstI, SacI and XhoI. With Roche Molecular Biochemicals hybridization solution 42 ℃ of hybridization of spending the night. Room temperature carries out with 2 * SSC, 0.1% SDS then carrying out twice washing step at 50 ℃ with 0.5 * SSC, 0.1% SDS after twice washing step again. 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) as substrate, alkaline phosphatase is surveyed, afterwards x-ray film is exposed to filter (filter) 1 hour. The signal that does not share on the film.

Therefore, use PCR product (seeing embodiment 11) that the DIG mark of the at random grappling of reaction 5 and 11 crosses as probe. Hybridize after the same method, just wash conditions is changed under the room temperature 2 * 5 minutes a little, 55 ℃ lower 2 * 15 minutes. Shown following band on the film:

Restriction Enzyme	The probe of 340bp [kb]	The probe of 580bp [kb]
			BamHI	10	10/2.8
EcoRI	4.5	4.5/5.2
			HindIII	4.2/6.5	3.0/4.2/6.5
PstI	4.4	2.8/4.4
			SacI	23	23/3.9
XhoI	6	6/20

For cloning whole gene or being 5 ' of its disappearance-end at least, according to hybrid experiment, with HindIII the chromosomal DNA of A.niger ATCC 9142 is digested, produce the fragment that contains the lactonase gene of about 4kb, experimental technique is as follows:

20 μ l chromosomal DNAs

20 μ l 10x buffer solutions

5μl HindIII(10U/μl)

175μl H ₂O

Cultivated 16 hours at 37 ℃.

Use the PCR purification kit from Qiagen (Qiagen, Hilden, Germany) that the DNA through digestion is carried out purifying, and wash-out is 100 μ l. Following coupled reaction is spent the night at 16 ℃ and is carried out. Purified DNA is diluted with water to 2ml. Recommend to add ligase and ligase buffer solution according to manufacturer (Roche Molecular Biochemicals). Connect the solution of finishing and be used as template, carry out different PCR, wherein use the inside primer of the lactonase gene that separates through part:

-95 ℃ of 5 μ l coupled reaction solution steps 1:5 minute

1 μ l mixture of ribonucleotides step 2:30 second-95 ℃

5 μ l are with Mg²⁺PCR standard buffer solution step 3:30 second-55 ℃

-72 ℃ of 1 μ l sense primers (10pMol/ μ l) step 4:3 minute

1 μ i anti-sense primer (10pMol/ μ l)

1 μ l is from the High Fidelity polymerase mixed liquor 40 μ l H of Roche Molecular Biochemicals₂O

Step 2 is to 4 repetitions 32 times.

Four kinds of combination of primers, Oal3S/Fxn57AS, Oal4S/Fxn57AS, Oal3S/Oal8AS and Oal4S/Oal8AS can both produce the dna fragmentation of wall scroll 4kb. All primers are to all being oriented to the outside of gene. Therefore, the lactonase gene should be positioned at as certainly connecting on the ring-shaped DNA molecule of HindIII fragment.

Use from the QIAEX II gel extraction agent box of Qiagen and from gel, isolate fragment, according to manufacturer (Applied Biosystems, Foster City, CA, the two deoxidation methods of USA) recommending check order with ABI 310Genetic Analyzer. The sequence that obtains confirms that isolated fragment contains two ends from (R)-pantolactone enzyme gene of A.niger ATCC 9142.

With primer Oal10S and Oal12AS, also may isolate the homologous gene of A.niger ssp.awamori, wherein contain and the duplicate amino acid sequence of all measured peptides. Unique difference only is the sequence of peptide Fxn42. This moment, Fxn42 was from the order-checking to two peptides. Most probable situation is, when carrying out HPLC when separating enzyme fragment through digestion, eluted simultaneously two different peptides. Order-checking has at first only disclosed the sequence of main peptide, but it is with very strong background. After the order-checking of first peptide finished, remaining was exactly the sequence of second peptide, and it has other four amino acid:

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) provides

K-P-F-A-H-Q-V-K-T-x-E-D (SEQ ID NO:45), it is Fxn 42.

Why this explained also that oligonucleotides Fxn42S and Fxn42AS based on peptide Fxn 42 can't provide the result who share. According to amino acid sequence, the molecular weight that has 36573Da from the protein of strains A TCC 9142, the absorptance that the 280nm place estimates is 1.83 (1mg/ml protein solutions), and be calculated as 36547 Da from the molecular weight of the amino acid sequence of A.niger ssp.awamori, for the 1mg/ml protein solution, the E of estimation280nmBe 1.831 (Pace et al., 1995).

11 pairs of (R)-pantolactone enzyme genes from A.niger ATCC 9142 of embodiment clone and Express

The sequence that obtains according to the whole three kinds of methods described in the embodiment 10 is compared, it is assembled, produce the sequence of the 2443bp of a whole lactonase gene of continuous covering. Be initiation codon, terminator codon and the possible introne of measuring this gene, the obtainable amino acid sequence of the peptide that will obtain from the purified genes from commercial formulation, the result who obtains with two program TESTCODE and the CODONPREFERENCE of GCG package version 10.2 unites use. Although two programs have all determined terminator codon and an introne in the same way, and initiation codon is also not obvious, particularly, because the peptide that does not have other obtainable N-end of peptide Fx 74 (SEQ ID NO:84) to be sequenced. There is not help to what all possible translation to homologous amino acid sequence of finding during the data library searching was carried out, because the N-end portion of protein is complete allos more yet. Therefore, in the position upstream of peptide Fx 74 but still two methionine residues in same reading frame, finding be selected, use it for and make up different expression cassettes. The zone of between the 666-742 base-pair of SEQ ID NO:7, having found to be rich in CT. Yet this position of being rich in the zone of CT is not veritably preference Met rather than other amino acid as initiation codon because should the zone in fungi and initiation codon between distance sizable variation is arranged usually. In addition, identified possible poly-putative adenylylation site in the downstream of gene.

For from the long introne of the 72bp of the gene of A.niger ATCC 9142, the DNA section most probable of the from the 976th to 981 nucleotides represents 5 '-shearing site, the section most probable of the from the 1029th to 1035 or from 1016 to 1022 nucleotides represents the inside conserved sequence (consensus sequence) of inferring, and the DNA section most probable of the from the 1045th to 1047 nucleotides represents 3 '-shearing site (SEQ ID NO:7). The corresponding sequence of A.niger MacRae gene is positioned bp 32 to 37,66 to 72 and 80 to 82 in the long introne (SEQ ID NO:79) of 51 bp. The introne long for the 50bp of the oal gene of A.niger ssp.awamori, 5 '-shearing site starts from the 203rd nucleotides (GTGCCC), inner conservative region is at the 236th nucleotides place (AACTAAC), and 3 '-shearing site is (SEQ ID NO:9) at the 250th nucleotides place (CAG).

The introne of oal	Length	5 '-site	Inner 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 of 5 '-shearing site is GTPuNGPy. Neither one has G at the 5th in the site of listing. The conserved sequence that is used for the yeast in the site, inside that lasso trick (lariat) forms is TACTAAC. 3 '-shearing site has the conserved sequence [Unkles of PyAG, Gene organization in industrial filamentous fungi.In Applied molecular genetics of filamentous fungi (Kinghoun, ed.), pp.28-53.Blackie Academic ﹠ Professional, Wester Cleddens Road, Bishopbriggs, Glasgow G642NZ, UK, Glasgow (1992)].

Produced following cDNA:

-for the oal (SEQ ID NO:98) that expresses at E.coli

-for the oalEco (SEQ ID NO:94) that expresses at Saccharomyces cerevisiae

-for the oalEShort that expresses at S.cerevisiae (31 amino acid whose short versions of second possible Met at the 32nd place of usefulness SEQ ID NO:95)

-the oalS (it is because the misleading sequence of Fxn 42 peptides that oalS is made into, and it finally is accredited as artefact) that is used for expressing at E.coli

-for the oalSEco that expresses at S.cerevisiae

-for the oalhis that contains the terminal his-label of C-that expresses at E.coli

-for the oalEhis that expresses at S.cerevisiae

-for the oalNhis (SEQ ID NO:92) that contains the terminal his-label of N-that expresses at E.coli

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

Common operation:

At first, in twice PCR, isolate the hypothesis coded sequence (cDNA) of oal gene. By PCR for the third time two kinds of PCR products are assembled up. Use following primer:

OallAS (SEQ ID NO:46), Oal2AS (SEQ ID NO:47), Oal3S (SEQ ID NO:48), Oal4S (SEQ ID NO:49), Oal5S (SEQ ID NO:50), Oal6AS (SEQ ID NO:51), Oal7AS (SEQ ID NO:52), Oal8AS (SEQ ID NO:53), Oal9AS (SEQ ID NO:54), Oa110S (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), Oa113AS (SEQ ID NO:65), Oal14S (SEQ ID NO:66), Oal14AS (SEQ ID NO:67), Oal15S (SEQ ID NO:68), Oal15AS (SEQ ID NO:69), Oa116S (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 directly starts from the front, site of the more upstream of initiation codon, and Oal9AS starts from some nucleotides place, terminator codon downstream of hypothesis. For two kinds of PCR products that can will obtain without introne in PCR for the third time assemble up, Oal14S and Oal14AS are complementary, and it contains terminal point from extron I to the sequence the starting point of extron II.

-95 ℃ of 1 μ l gDNA (1/10 dilution) step 1:5 minute

1 μ l mixture of ribonucleotides step 2:30 second-95 ℃

5 μ l are with Mg²⁺PCR standard buffer solution step 3:30 second-55 ℃

-72 ℃ of 1 μ l Oal11S (10pMol/ μ l) step 4:1 minute

1μl Oal14AS(10pMol/μl)

1 μ l is from the High Fidelity polymerase mixed liquor of Roche Molecular Biochemicals

40μl H ₂O

Step 2 is to 4 repetitions 35 times.

-95 ℃ of 1 μ l gDNA (1/10 dilution) step 1:5 minute

1 μ l mixture of ribonucleotides step 2:30 second-95 ℃

5 μ l are with Mg²⁺PCR standard buffer solution step 3:30 second-55 ℃

-72 ℃ of 1 μ l Oal14S (10pMol/ μ l) step 4:1 minute

1μl Oal9AS(10pMol/μl)

1 μ l is from the High Fidelity polymerase mixed liquor of Roche Molecular Biochemicals

40μl H ₂O

Step 2 is to 4 repetitions 35 times.

The PCR that the gDNA of A.niger ATCC 9142 is carried out has provided the PCR product with expectation size. By agarose gel electrophoresis the PCR product is carried out purifying. Use the QIAEX II kit from Qiagen (Qiagen, Hilden, Germany) from gel, to extract the PCR product, use it for for the third time PCR:

-95 ℃ of PCR product 1 and 2 each 0.5 μ l steps 1:5 minute

1 μ l mixture of ribonucleotides step 2:30 second-95 ℃

5 μ l are with Mg²⁺PCR standard buffer solution step 3:30 second-55 ℃

-72 ℃ of 1 μ l Oal12S (10pMol/ μ l) step 4:1 minute

1μl Oal10AS(10pMol/μl)

1 μ l is from the High Fidelity polymerase mixed liquor of Roche Molecular Biochemicals

40μl H ₂O

Step 2 is to 4 repetitions 30 times.

By agarose gel electrophoresis end-product is carried out purifying, from gel, extract, and according to the method that Invitrogen (carlsbad, CA, USA) provides it is cloned the carrier into TA-, be used for the PCR product is carried out Direct Cloning. All other essential clone's steps are all according to described the carrying out of Sambrook et al. (1989). The plasmid that contains correct insertion (oal1) that detects by order-checking is used as template in all follow-up construction steps.

(a) be used for the construct oal and the oalEco that express at E.coli and Saccharomyces cerevisiae: according to following proposal, with primer pQE80EcoS and pQE80BamAS oal1 is carried out PCR, with generation construct Eco-oal-Bam:

1 μ l contains the plasmid (10ng) as the oal1 that inserts

1 μ l mixture of ribonucleotides

5 μ l are with Mg²⁺The PCR standard buffer solution

1μl pQE80EcoS(10pMol/μl)

1μl pQE80BamAS(10pMol/μl)

1 μ l is from the High Fidelity polymerase mixed liquor of Roche Molecular Biochemicals

40μl H ₂O

Step 1:5 minute-95 ℃

Step 1:30 second-95 ℃

Step 1:30 second-55 ℃

Step 4:1 minute-72 ℃

Step 2 is to 4 repetitions 30 times.

By agarose gel electrophoresis the PCR product is carried out purifying, from gel, extract the interested fragment of people (QIAEX II, Qiagen), digest with EcoRI and BamHI, again carry out purifying with agarose gel electrophoresis, be connected on the carrier pQE80 from Qiagen, transform E.coli Top10. Isolate four clones' plasmid, insertion is checked order. The plasmid that contains correct construct is converted into E.coli M15 or suitable E.coli bacterial strain. All molecular engineerings all are standard techniques known to the skilled.

For the Yeast expression construct, used primer is changed to Oal10EcoS and Oal12ASEco, with EcoRI the PCR product is digested separately, by the EcoRI restriction site on the carrier, clone end-product into pYES2 or be used for the suitable expression vector of S.cerevisiae. According to Hinnen et al., Proc.Natl.Acad.Sci.USA 75:1929-1933 (1978) or according to the specification that provides simultaneously with bacterial strain carries out the bacterial strain to S.cerevisiae, INVScl (Invitrogen for example, Carlsbad, CA, USA) conversion.

(b) for the construct pQE80oalS (with 31 amino acid whose versions that shorten of second possible Met) that expresses at E.coli:

State the same PCR scheme of content with catching up with, with same template, primer pQE80EcoSshort and pQE80BamAS. Process according to the reactant liquor that described in (a) PCR is reacted after finishing.

(c) for the construct oalS that expresses at S.cerevisiae:

Use primer Oal16S and Oal12ASEco. Other all carries out described in (a).

(d) for the construct pQE80oalhis that contains the terminal his-label of C-that expresses at E.coli:

Carry out PCR with primer pQE80EcoS and pQE80BamhisAS, see (a).

(e) for the construct oalhis that expresses at S.cerevisiae:

Carry out PCR with primer Oal10Seco and Oal12ASHisEco, see (a).

(f) for the construct pQE80oalNhis that contains the terminal His-label of N-that expresses at E.coli:

Carrying out PCR with primer pQE80EcoNhisS and pQE80BamAS (sees a).

(g) for the construct oalENhis that expresses at S.cerevisiae:

Carrying out PCR with primer OalENhis and Oal12ASEco (sees a).

(h) construct oalsec, it contains the signal peptide of the phytase of A.terreus cbs, and this signal peptide is used for the expression and secretion at S.cerevisiae:

With three times independently PCR prepare this construct. At first, by being carried out PCR, conservative phytase gene isolates burst [Lehmann et al., Protein Eng.13:49-57 (2000)].

1.0 μ l contains the plasmid (10ng) as the conservative phytase gene that inserts

1.0 μ l mixture of ribonucleotides

5.0 μ l is with Mg²⁺The PCR standard buffer solution

1.0μl CP-a(10pMol/μl)

1.0μl OalsecAS(10pMol/μl)

1.0 μ l is from the High Fidelity polymerase mixed liquor of Roche Molecular Biochemicals

40μl H ₂O

Step 1:5 minute-95 ℃

Step 1:30 second-95 ℃

Step 1:30 second-55 ℃

Step 4:30 second-72 ℃

Step 2 is to 4 repetitions 30 times.

1.0 μ l contains the plasmid (10ng) of oal1 gene

1.0 μ l mixture of ribonucleotides

5.0 μ l is with Mg²⁺The PCR standard buffer solution

1.0μl OalsecS(10pMol/μl)

1.0μl Oal12ASEco(10pMol/μl)

1.0 μ l is from the High Fidelity polymerase mixed liquor of Roche Molecular Biochemicals

40μl H ₂O

Step 1:5 minute-95 ℃

Step 1:30 second-95 ℃

Step 1:30 second-55 ℃

Step 4:45 second-72 ℃

Step 2 is to 4 repetitions 30 times.

By coming purified pcr product at the enterprising row agarose gel electrophoresis of 1.5% Ago-Gel, from gel, extract PCR product (QIAEX II, Qiagen), use it for for the third time PCR:

0.5 the product of μ l PCR1

0.5 the product of μ l PCR2

1.0 μ l mixture of ribonucleotides

5.0 μ l is with Mg²⁺The PCR standard buffer solution

1.0μl CP-a(10pMol/μl)

1.0μl Oal12ASEco(10pMol/μl)

1.0 μ l is from the High Fidelity polymerase mixed liquor of Roche Molecular Biochemicals

40μl H ₂O

Step 1:5 minute-95 ℃

Step 1:30 second-95 ℃

Step 1:30 second-55 ℃

Step 4:1 minute-72 ℃

Step 2 is to 4 repetitions 30 times.

By agarose gel electrophoresis the PCR product is carried out purifying, from gel, extract the PCR product, digest with EcoRI, again carry out purifying with agarose gel electrophoresis, be connected in the S.cerevisiae expression vector according to above-mentioned.

                        The expression of embodiment 12oal

(a) in E.coli:

Express pQE80oal, pQE80oalS, pQE80oalhis and pQE80oalNhis in E.coli M15, described E.coli M15 contains pREP4, and it contains the repressor (seeing Germany, the expression specification of the Qiagen of Hilden) of lac promoter. The OD that overnight culture is diluted to 600 nm is 0.1, and being cultured to OD600nm at 30 ℃ is 1.5. Then induce culture with 0.5mM IPTG, cultivated 6 hours at 30 ℃ again. By centrifugal (5000g, 20 minutes) harvesting, be frozen in-80 ℃. According to the scheme that manufacturer provides, with B-PER (Pierce, Rockford, IL, USA) they are carried out cracking. Again after the centrifugation step, supernatant is used for hydrolysis to (R)-pantolactone.

In the transformant that contains construct pQE80oalS and pQE80oalhis, all do not find active in supernatant and the cell pyrolysis liquid, and in the situation with construct pQE80oal and pQE80oalNhis, about 5% in the soluble protein is activated (the R)-pantolactone of tool enzyme.

With the method same with the gene that does not have the His-label, express the construct that contains the terminal His-label of N-. Use " QIAexpressionist " the described scheme from Qiagen (Hilden, Germany), the protein that adds the His-label to native form from cell pyrolysis liquid carries out purifying.

(b) in S.cerevisiae:

Construct oalEco, oalES, oalEhis, oalENhis and oalsec express in S.cerevisiae INVScl or similar suitable bacterial strain. Transform and at SDura-culture medium [Sherman et al., Laboratory course manual for methods in yeast genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1986)] go up after the coating, at 30 ℃ flat board is carried out 3 to 4 days cultivation, pick the bacterium colony of turning out, transfer them in the 2ml SDura-fluid nutrient medium, under fierce vibration, cultivated 3 days in 30 ℃. Above-mentioned culture is used as the preculture thing of 25ml SDura-culture medium. Under the fierce vibration in 30 ℃ cultivate again 3 days after, with the culture of preparation [Sherman et al., as mentioned above] the 500ml YPD culture medium in 21 bottles of the inoculations. After cultivating under the same conditions 3 days again, by the centrifugal cell of from culture medium, isolating, carry out cracking with Y-PER (Pierce, Rockford, IL).

As situation about expressing among the E.coli, only there are oalEco and oalENhis construct to cause the expression of active (R)-pantolactone enzyme, it only is found in cell pyrolysis liquid. With 75ml YPD culture, also can produce (R)-pantolactone enzyme of 300U. Corresponding lysate shows the specific activity of about 5 U/mg total proteins.

With the sequence data of above-mentioned introne position, also available similar mode is to making up and express from the oal gene of A.awamori with from the oal gene of A.niger ATCC 46951.

The Oal of 13 pairs of heterogenous expressions in E.coli or S.cerevisiae of embodiment is fixed, and

Use it for the Hydrolysis Resolution to (RS)-pantolactone

For in bioreactor, bringing into play multiple use, (the R)-pantolactone enzyme from A.niger is fixed. After in E.coli or S.cerevisiae, expressing, by chemical means, for example B-PER (E.coli) or Y-PER (S.cerevisiae) (Pierce, Rockford, IL, USA), or by mechanical means for example ultrasonic wave or high pressure carry out fragmentation to cell. Come cleared lysate by centrifugal. Preparation is directly used in fixing. Perhaps, before fixing, use another purification step, for example ammonium sulfate precipitation increases the Oal specific activity further.

Fix by the following method this biocatalyst:

(1) fixing at silicon dioxide gel gel (Silica Sol-Gel):

According to described poly-(the silicic acid glycerine)-1.0 (PGS-1.0) for preparing of document [Gill and Ballesteros, J.Am.Chem.Soc.120:8587-8598 (1998)], it is dissolved in half the icy water of its quality. Get wherein 100 or the part of 200mg, with 50 or the ice-cold living things catalysis developing agent storage liquid formulation (from the soluble enzyme of some preparations) of 100mg mix up hill and dale, be mixed in the 50mM phosphate in 2ml or the 5ml bottle and carry out, pH 7.0, with the soft rotation of bottle 2 minutes, to form the thin layer of hydrogel at the bottle wall. Bottle was placed 20 minutes on ice, then (unlatching) be transferred to refrigerator. Hydrogel is at 5 ℃ of ripe 48-72 hours, to form xerogel (xerogel). By 5 ℃ with 100rpm vibration, with 2 * 1 or 2 * 4ml pH be the bottle that 7 50mM phosphate buffer washs xerogel, then be 2 * 1 or 2 * 4ml pH be 7 50mM TEA-acetate, wherein contain the 10mM magnesium acetate.

(2) Oal is fixed on the silica polyvinyl alcohol collosol and gel (Sol-Gel Silica-Polyvinyl Alcohol):

Similar in method and (1), difference is to store before liquid be combined with lactonase, DGS solution will with the polyvinyl alcohol of appropriate quantity (PVA, from Aldrich, the 85K in the water of 13%w/w) mixing. Follow-up method is shown in (1).

(3) Oal is fixed on the polymine that activates and support with glutaraldehyde:

With polymine (1g, anhydrous, HMW, branched polymer is from Aldrich), CA (0.2g, 36%Ac) and cellulose acetate-butyrate (0.2g, 48% acetic acid and butyric acid content are from Aldrich) be dissolved in the carrene of 5mL, with solution to 7g bleaching earth (Fullers Earth, the 30-60 order is from Aldrich) carry out dressing. Through the material of wet dressing in air under room temperature dry 0.5 hour, until provide about 9g by the bleaching earth of dressing. Then with its Eddy diffusion in the ice-cold mixture of glutaraldehyde (8mL, 50%w/w is from Aldrich) and phosphate buffer (pH 8 for 50mL, 50mM), stirred 2 hours with 200rpm. Wash that (then 4 * 10mL) holders through overactivation wash (pH 8 for 4 * 10 mL, 20mM) with phosphate buffer, then discharge liquid with water. Wet holder and ice-cold lactonase are stored liquid (in the 50mM phosphoric acid, pH 8) mixing, suspension is carried out 20-30 hour stirring with 200rpm. Liquid is softly removed from the enzyme that is fixed, washed with phosphate that (3 * 5mL) wet fixtures are processed with monoethanolamine (pH 8 for 20mL, 20mM), wash (pH 7 for 2 * 5mL, 20mM) with phosphate again, then discharge liquid.

(4) for the reaction condition that (RS)-pantolactone is hydrolyzed and splits

Reaction 2 or the bottle of 4mL in carry out, wherein use 50 or the biocatalyst of 100mg and 1 or the 0.5M racemic lactone (in 0.75M triethylamine-acetate, pH 8.5, contain the magnesium acetate of 20mM) of 2mL. Bottle is cultivated the essential time period under 40 ℃, 200rpm, solution is discharged, and analyze with chirality HPLC, before beginning circulates, wash (2 * 1 or 2mL) catalyst with fresh substrate solution next time. Stop reaction for the sample of analyzing with isopyknic 500mM MES buffer solution, described pH of buffer is 5.5, wherein contain 50 mM EDTA, centrifugal (10000g, 10 minutes) then analyze by HPLC, wherein use the CHIRADEX post of 0.46 * 15cm, carry out wash-out with the water-methanol of 70:30,1mL/ minute, carry out at 20 ℃. In 15 minutes, initial velocity is measured, measured the E value at the end of each circulation.

Summarized the effect in (RS)-pantolactone being carried out split process in batches of the enzyme preparation that is fixed selected in the table 2. Collosol and gel and covalency fixed solution all are proved to be as effectively, even 24 every batch be 1 hour circulation after, catalyst has kept the 62-72% of its initial activity, and demonstrates: excessive (excess) value of enantiomter and the enantio-selectivity of (R)-pantolactone is respectively 89-98% and 71-88%. In addition, when prolonging batch time of carrying out when testing, the catalyst function mode also is very similar, namely carries out in the situation of a day rather than 1 hour when every batch reaction, and it has shown good long-time operation stability.

Table 2: (RS)-pantolactone is split in batches by the lactonase preparation that is fixed

Catalyst	Load (U/g)	Efficient (%)	Active (U/g)	The number of times of residual activity (% is after the 1h circulation) batch reactions circulation
																2	4	6	8	10	12	14	16	18	20	22	24
				Silicon dioxide gel gel (pure silicon dioxide xerogel) fixture
Yeast 26U/mL	78	71	55																	96	91	88	87	83	86	85	80	78	74	71	68
				E.coli   8.2U/mg	820	58	476	92	87	87	84	80	77	73	72	70	72	69	66
E.coli   2.6U/mg	260	48	124																	90	87	81	85	82	80	73	76	70	69	65	62
				Silica-PVA collosol and gel (5: the 1w/w xerogel) fixture

Yeast 26U/mL	78	84	64	93	90	91	85	83	81	76	72	70	-	66	63
																E.coli   8.2U/mg	820	82	671	91	87	83	82	80	77	78	72	70	67	67	66
E.coli   2.6U/mg	260	80	207	92	87	84	80	79	73	75	70	69	65	63
																Bleaching earth-acetic acid/cellulose butyrate-poly-(aziridine)-glutaraldehyde fixture
Yeast 26U/mL	52	34	18	91	88	85	82	82	81
																E.coli   8.2U/mg	164	50	82	95	92	88	85	84	86	81	79	79	76	74	71
E.coli   8.2U/mg	410	23	95	93	90	86	85	83	80	79	75	72
																E.coli   2.6U/mg	130	25	33	97	88	87	87	87	86	83	80	77	78	75	72
Enantio-selectivity: value (E) scope of the value of excessive (%ee) of enantiomter and enantio-selectivity is respectively above 89-98% and 71-88%.

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

The Oal that is expressed by restructuring E.coli that embodiment 14 is fixed carries out (RS)-pantolactone continuously

                               The application that splits

According to prepare described in the embodiment 14 (2) through the fixing restructuring lactonase biocatalyst (expressing among the E.coli) of collosol and gel-PVA, be used in packed bed (packed-bed) reactor, the racemic lactone be split continuously: with biocatalyst (1.1g, the 0.21kU g that is fixed^-1, 0.231kU altogether) and the dried Omnifit that inserts with 0.46 * 15cm of Teflon terminal (endpiece) adds in the cover glass column. It adds the cover glass column with the Omnifit of 1 * 10cm and links to each other, and is filled with the cellulose bead of 1-2mm in the glass column of 1 * 10 cm, and it is to insert by two syringe pumps that 50mL glass/Teflon syringe is housed. These posts all link to each other with circulator bath, are maintained at 40 ℃ during fractionation is carried out. By the following method reactor is regulated: under the room temperature with 5mL min^-1Speed add Tris-acetate buffer (100mM, pH 7, contain the 100mM magnesium acetate), carried out 30 minutes; Then be at room temperature with 1: 1 volume ratio, 2mL min^-1Overall flow rate, add the combination of ester solution (1.5M in the water) and Tris-acetate buffer (2.5M, pH 8.25, contain the 50mM magnesium acetate) in the racemic, carried out 30 minutes. Then flow velocity is reduced to 1.2mL min^-1, pillar is heated to 40 ℃, system was allowed to balance 15 minutes. Therefore reactor has been carried out about 45 minutes operation, collected eluent at ice bath, with this understanding, with the constant conversion ratio of 46-48% the charging thing of 52.7mL (being equivalent to 5.14g RSPL) has altogether been processed. With sulfuric acid (the 10%v/v aqueous solution) pH of eluent is adjusted to 6.5, with carrene solution is extracted (10 * 75mL), organic layer is dry on anhydrous magnesium sulfate, rotary evaporation, (S)-pantolactone that generation is comprised of the SPL of 10% RPL and 90% (analyzed draw by chirality HPLC) is rich in thing (2.67g, in theory 98%). (40% w/w) carries out acidifying to water with sulfuric acid, to pH 1.5, be heated to 70 ℃, 1 hour, again in cooled on ice, saturated with sodium chloride, (10 * 75mL), organic layer is dry on anhydrous magnesium sulfate then to use dichloromethane extraction, rotary evaporation, produce (R)-pantolactone and be rich in thing (2.23g, theoretical yield 92%), its enantiomeric purity is 93%. To (R)-/(S)-the combination rate of recovery be 95%.

The result of embodiment (13) and (14) clearly illustrates that, the restructuring lactonase is effective biocatalyst for Hydrolysis Resolution racemic pantolactone, this catalyst also can effectively be fixed, fixture can be used for producing highly enriched (R)-pantolactone of the excessive 90-98% of reaching of enantiomerism, no matter be all like this in batch operation or the situation of continued operation.

Embodiment 15 gram falls and expresses (S)-pantolactone specificity lactonase from B.subtilis

Use the amino acid sequence from the lactonase of ox and horse liver, we have found that several other shows the sequence that has limited homology with above-mentioned sequence. By PCR with in the middle of their from yvre gene of B. subtilis (YVRE BACSUBe noted as the member's of SMP30/CGR1 family/suppose 33.2kDa albumen) from genomic DNA the clone out, 5 ' and 3 ' of use therein primer-end also contains the follow-up needed restriction site of step (EcoRI and PstI) of cloning in the expression vector. It is identical that PCR condition and genomic DNA from A.niger separate the used condition of oal gene (seeing embodiment 8). Digest PCR product and carrier (from Stratagene (La Jolla with EcoRI and PstI, CA, USA) PET41a), come purifying by agarose gel electrophoresis, connect, and it is transformed into E.coli Top10 cell (Stratagene, La Jolla, CA, USA). Use this kind strategy, this gene is integrated in the reading frame from the GST GFP of E.coli.

Express and purifying according to the scheme that manufacturer provides. With wherein additionally containing 5mM CaCl₂Code test measure the activity of the protein that is purified at 40C. This enzyme has shown very high selective to (S)-pantolactone. Said preparation has the specific activity with respect to every mg enrichment fusion 1-2U. This enzyme is only at Mg²⁺Or Ca²⁺Has activity in the situation that ion exists.

Sequence table

＜110〉DSM IP Assets BV

＜120〉novel enzyme

<130>21717

<160>99

<170>PatentIn version 3.1

<210>1

<211>900

<212>DNA

<213>Equus caballus

<220>

<221>CDS

<222>(1)..(900)

<223>

<400>1

atg tct tcc att aag att gag tgt gtt ctg ccc gag aac tgc cag tgc        48

Met Ser Ser Ile Lys Ile Glu Cys Val Leu Pro Glu Asn Cys Gln Cys

1               5                   10                  15

ggt gag tcg cca gtg tgg gag gaa gcg tcc agc tct ctg ctc ttc gta        96

Gly Glu Ser Pro Val Trp Glu Glu Ala Ser Ser Ser Leu Leu Phe Val

            20                  25                  30

gat att cct gcc aaa aag gtt tgc cgg tgg gat gcg ctc agc aag caa       144

Asp Ile Pro Ala Lys Lys Val Cys Arg Trp Asp Ala Leu Ser Lys Gln

        35                  40                  45

gtg cag cga atg acc gtg gat gcc ccg gtt ggc tca gtg gcc ctc cgc       192

Val Gln Arg Met Thr Val Asp Ala Pro Val Gly Ser Val Ala Leu Arg

    50                  55                  60

cag tcg gga ggc tat gtc gcc aca att gga acg aag ttc tgt gct ttg       240

Gln Ser Gly Gly Tyr Val Ala Thr Ile Gly Thr Lys Phe Cys Ala Leu

65                  70                  75                  80

aac tgg gaa gat cag tca gta gtt gtc ctg gcc gcg gta gag aaa gac       288

Asn Trp Glu Asp Gln Ser Val Val Val Leu Ala Ala Val Glu Lys Asp

                85                  90                  95

aag aaa aac aat cga ttc aat gac ggg aag gtg gat ccc gcg ggg aga       336

Lys Lys Asn Asn Arg Phe Asn Asp Gly Lys Val Asp Pro Ala Gly Arg

            100                 105                 110

tac ttt gct ggc acc atg gct gag gaa aca gcc ccg gca gtt acg gag       384

Tyr Phe Ala Gly Thr Met Ala Glu Glu Thr Ala Pro Ala Val Thr Glu

        115                 120                 125

cgg cac caa ggg tcc ctg tac gcc ctc ttt cct gac cac cac gtg gaa       432

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

    130                 135                 140

aag tac ttt gac cag gtg gac atc tcc aac ggt ttg gat tgg tcc ctg       480

Lys Tyr Phe Asp Gln Val Asp Ile Ser Asn Gly Leu Asp Trp Ser Leu

145                 150                 155                 160

gac cac aaa atc ttc tat tac atc gac agc ctg tcc tac tct gtg gat       528

Asp His Lys Ile Phe Tyr Tyr Ile Asp Ser Leu Ser Tyr Ser Val Asp

                165                 170                 175

gcc ttt gac tat gac ctg ctg aca gga agg atc tct aac cgc aga agt       576

Ala Phe Asp Tyr Asp Leu Leu Thr Gly Arg Ile Ser Asn Arg Arg Ser

            180                 185                 190

gtt tac aag ctg gag aag gaa gaa cat atc cca gat gga atg tgt att       624

Val Tyr Lys Leu Glu Lys Glu Glu His Ile Pro Asp Gly Met Cys Ile

        195                 200                 205

gat act gag ggg aag ctc tgg gtg gcc tgt tac aac gga gga aga gtg       672

Asp Thr Glu Gly Lys Leu Trp Val Ala Cys Tyr Asn Gly Gly Arg Val

    210                 215                 220

att cgt tta gat cct gag aca ggg aaa aga ctc caa act gtg aag ttg       720

Ile Arg Leu Asp Pro Glu Thr Gly Lys Arg Leu Gln Thr Val Lys Leu

225                 230                 235                 240

cct gtt gat aaa acc act tcg tgc tgc ttc gga ggg aag gat tac tct       768

Pro Val Asp Lys Thr Thr Ser Cys Cys Phe Gly Gly Lys Asp Tyr Ser

                245                 250                 255

gaa atg tac gtg acc tgc gcc cgg gct gga ctc gac ccc gag gct ctc       816

Glu Met Tyr Val Thr Cys Ala Arg Ala Gly Leu Asp Pro Glu Ala Leu

            260                 265                 270

tcg cgg cag cct gaa gct ggt ggc att ttc aag ata act ggc ctg ggg       864

Ser Arg Gln Pro Glu Ala Gly Gly Ile Phe Lys Ile Thr Gly Leu Gly

        275                 280                 285

gtc aaa gga att cct ccc tat gcc tac gca gga tga                       900

Val Lys Gly Ile Pro Pro Tyr Ala Tyr Ala Gly

    290                 295

<210>2

<211>299

<212>PRT

<213>Equus caballus

<400>2

Met Ser Ser Ile Lys Ile Glu Cys Val Leu Pro Glu Asn Cys Gln Cys

1               5                   10                  15

Gly Glu Ser Pro Val Trp Glu Glu Ala Ser Ser Ser Leu Leu Phe Val

            20                  25                  30

Asp Ile Pro Ala Lys Lys Val Cys Arg Trp Asp Ala Leu Ser Lys Gln

        35                  40                  45

Val Gln Arg Met Thr Val Asp Ala Pro Val Gly Ser Val Ala Leu Arg

    50                  55                  60

Gln Ser Gly Gly Tyr Val Ala Thr Ile Gly Thr Lys Phe Cys Ala Leu

65                  70                  75                  80

Asn Trp Glu Asp Gln Ser Val Val Val Leu Ala Ala Val Glu Lys Asp

                85                  90                  95

Lys Lys Asn Asn Arg Phe Asn Asp Gly Lys Val Asp Pro Ala Gly Arg

            100                 105                 110

Tyr Phe Ala Gly Thr Met Ala Glu Glu Thr Ala Pro Ala Val Thr Glu

        115                 120                 125

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

    130                 135                 140

Lys Tyr Phe Asp Gln Val Asp Ile Ser Asn Gly Leu Asp Trp Ser Leu

145                 150                 155                 160

Asp His Lys Ile Phe Tyr Tyr Ile Asp Ser Leu Ser Tyr Ser Val Asp

                165                 170                 175

Ala Phe Asp Tyr Asp Leu Leu Thr Gly Arg Ile Ser Asn Arg Arg Ser

            180                 185                 190

Val Tyr Lys Leu Glu Lys Glu Glu His Ile Pro Asp Gly Met Cys Ile

        195                 200                 205

Asp Thr Glu Gly Lys Leu Trp Val Ala Cys Tyr Asn Gly Gly Arg Val

    210                 215                 220

Ile Arg Leu Asp Pro Glu Thr Gly Lys Arg Leu Gln Thr Val Lys Leu

225                 230                 235                 240

Pro Val Asp Lys Thr Thr Ser Cys Cys Phe Gly Gly Lys Asp Tyr Ser

                245                 250                 255

Glu Met Tyr Val Thr Cys Ala Arg Ala Gly Leu Asp Pro Glu Ala Leu

            260                 265                 270

Ser Arg Gln Pro Glu Ala Gly Gly Ile Phe Lys Ile Thr Gly Leu Gly

        275                 280                 285

Val Lys Gly Ile Pro Pro Tyr Ala Tyr Ala Gly

    290                 295

<210>3

<211>879

<212>DNA

<213>Bacillus subtilis

<220>

<221>CDS

<222>(1)..(879)

<223>

<400>3

atg gat gca gta ttg gaa gca gac act cgg gca gtg att ggt gaa ggc        48

Met Asp Ala Val Leu Glu Ala Asp Thr Arg Ala Val Ile Gly Glu Gly

1               5                   10                  15

ccg tta tgg gat gaa gag aac ggc cgc tta tat tgg gtt gat atc ctg        96

Pro Leu Trp Asp Glu Glu Asn Gly Arg Leu Tyr Trp Val Asp Ile Leu

            20                  25                  30

ggg agc gag ctc cac atc ttt gac cct gaa gaa aaa atc aac cga tca       144

Gly Ser Glu Leu His Ile Phe Asp Pro Glu Glu Lys Ile Asn Arg Ser

        35                  40                  45

atc aaa ttc aaa tcc ttt gtg acg gcg ctt gcg aaa tat tca aag gat       192

Ile Lys Phe Lys Ser Phe Val Thr Ala Leu Ala Lys Tyr Ser Lys Asp

    50                  55                  60

gaa ctg att atg acg atg aag gac ggg ttt tac ctg tat cat ctt cgg       240

Glu Leu Ile Met Thr Met Lys Asp Gly Phe Tyr Leu Tyr His Leu Arg

65                  70                  75                  80

gat gac agc ttg gaa aaa att aaa cag ccg aag gac atg cat gag agc       288

Asp Asp Ser Leu Glu Lys Ile Lys Gln Pro Lys Asp Met His Glu Ser

                85                  90                  95

ctg aga ttt aat gat gct aaa tgt gac ccg tac gga agg ctt tgg gcg       336

Leu Arg Phe Asn Asp Ala Lys Cys Asp Pro Tyr Gly Arg Leu Trp Ala

            100                 105                 110

ggg acg acg agc atg gaa ggc gag caa aaa cag gcg tcg ctg tac cgt       384

Gly Thr Thr Ser Met Glu Gly Glu Gln Lys Gln Ala Ser Leu Tyr Arg

        115                 120                 125

ttg aat cta gac ggc agt ctt gtc aaa atc aag gat caa gtc tcc acc       432

Leu Asn Leu Asp Gly Ser Leu Val Lys Ile Lys Asp Gln Val Ser Thr

    130                 135                 140

tca aac ggt ttg gat tgg gac cgc gag cgg aat ttg atg tat tac atc       480

Ser Asn Gly Leu Asp Trp Asp Arg Glu Arg Asn Leu Met Tyr Tyr Ile

145                 150                 155                 160

gac acg ccg acc cag gag att gta cgt tac agc tat gat cct caa agc       528

Asp Thr Pro Thr Gln Glu Ile Val Arg Tyr Ser Tyr Asp Pro Gln Ser

                165                 170                 175

gga gat gtt tcg aat cca gaa cct gtc tat cgt ttt gat cag tca gac       576

Gly Asp Val Ser Asn Pro Glu Pro Val Tyr Arg Phe Asp Gln Ser Asp

            180                 185                 190

gga ttg ccg gac ggc atg aca att gac caa aat ggc atg ctg tgg gtg       624

Gly Leu Pro Asp Gly Met Thr Ile Asp Gln Asn Gly Met Leu Trp Val

        195                 200                 205

gcg ctg ttt ggc ggc agc cgc gtt gtt cac att gac ccg ttt cag aaa       672

Ala Leu Phe Gly Gly Ser Arg Val Val His Ile Asp Pro Phe Gln Lys

    210                 215                 220

aaa gaa atc aat tca atc agc gtg ccg gct aaa tat gtc acg tgc tgc       720

Lys Glu Ile Asn Ser Ile Ser Val Pro Ala Lys Tyr Val Thr Cys Cys

225                 230                 235                 240

gcg ttc ggc ggc aga gac tta aaa acc ctt tac att aca acg gca aca       768

Ala Phe Gly Gly Arg Asp Leu Lys Thr Leu Tyr Ile Thr Thr Ala Thr

                245                 250                 255

gaa caa atg aca gaa aaa gag aga tac gag cag cct cac gct gga ggg       816

Glu Gln Met Thr Glu Lys Glu Arg Tyr Glu Gln Pro His Ala Gly Gly

            260                 265                 270

ttg ttt tca gca caa ctg gaa aca ggc gga tat cag ccg gta cca ttt       864

Leu Phe Ser Ala Gln Leu Glu Thr Gly Gly Tyr Gln Pro Val Pro Phe

        275                 280                 285

gct gga gac gta taa                                                   879

Ala Gly Asp Val

    290

<210>4

<211>292

<212>PRT

<213>Bacillus subtilis

<400>4

Met Asp Ala Val Leu Glu Ala Asp Thr Arg Ala Val Ile Gly Glu Gly

1               5                   10                  15

Pro Leu Trp Asp Glu Glu Asn Gly Arg Leu Tyr Trp Val Asp Ile Leu

            20                  25                  30

Gly Ser Glu Leu His Ile Phe Asp Pro Glu Glu Lys Ile Asn Arg Ser

        35                  40                  45

Ile Lys Phe Lys Ser Phe Val Thr Ala Leu Ala Lys Tyr Ser Lys Asp

    50                  55                  60

Glu Leu Ile Met Thr Met Lys Asp Gly Phe Tyr Leu Tyr His Leu Arg

65                  70                  75                  80

Asp Asp Ser Leu Glu Lys Ile Lys Gln Pro Lys Asp Met His Glu Ser

                85                  90                  95

Leu Arg Phe Asn Asp Ala Lys Cys Asp Pro Tyr Gly Arg Leu Trp Ala

            100                 105                 110

Gly Thr Thr Ser Met Glu Gly Glu Gln Lys Gln Ala Ser Leu Tyr Arg

        115                 120                 125

Leu Asn Leu Asp Gly Ser Leu Val Lys Ile Lys Asp Gln Val Ser Thr

    130                 135                 140

Ser Asn Gly Leu Asp Trp Asp Arg Glu Arg Asn Leu Met Tyr Tyr Ile

145                 150                 155                 160

Asp Thr Pro Thr Gln Glu Ile Val Arg Tyr Ser Tyr Asp Pro Gln Ser

                165                 170                 175

Gly Asp Val Ser Asn Pro Glu Pro Val Tyr Arg Phe Asp Gln Ser Asp

            180                 185                 190

Gly Leu Pro Asp Gly Met Thr Ile Asp Gln Asn Gly Met Leu Trp Val

        195                 200                 205

Ala Leu Phe Gly Gly Ser Arg Val Val His Ile Asp Pro Phe Gln Lys

    210                 215                 220

Lys Glu Ile Asn Ser Ile Ser Val Pro Ala Lys Tyr Val Thr Cys Cys

225                 230                 235                 240

Ala Phe Gly Gly Arg Asp Leu Lys Thr Leu Tyr Ile Thr Thr Ala Thr

                245                 250                 255

Glu Gln Met Thr Glu Lys Glu Arg Tyr Glu Gln Pro His Ala Gly Gly

            260                 265                 270

Leu Phe Ser Ala Gln Leu Glu Thr Gly Gly Tyr Gln Pro Val Pro Phe

        275                 280                 285

Ala Gly Asp Val

    290

<210>5

<211>726

<212>DNA

<213>Aspergillus niger

<220>

<221>CDS

<222>(1)..(726)

<223>

<400>5

aac tgg gac ctg gaa aaa ctc aat cct cca ggc ttc ggc cgc aaa ccc        48

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

1               5                   10                  15

ttc gcc cac cag gtt aaa tgg gtc agc gaa ggc cac gcc ttc gac gac        96

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

            20                  25                  30

gag tac cac ttc aac cca cag caa tcc tcc caa tgg gac ggc cta cga       144

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

        35                  40                  45

cac cac aat gcc cca gcc cca aca ccc gac gac cct gac cgc cgc gtc       192

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

    50                  55                  60

ttc tac ggc ggc acc acc tcc acc gag atc ctc gac ccg tcc tca gct       240

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

65                  70                  75                  80

cgc atc ggc atc gct cac tgg gcc aag aag ggc atc gcc ggc cgt ggc       288

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

                85                  90                  95

gtc ctc atc gac tac gcg tcc tac atc cag cga acc aag ggc ata cca       336

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

            100                 105                 110

gta aac gcc cta acc cgg cac acc gtc tcc ctg gac gac gtc ctc acc       384

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

        115                 120                 125

atc gcg aaa gaa tgc aac atc acc ttc cag cca ggc gac att ctc ttc       432

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

    130                 135                 140

ttg cgt gtc ggc ctg ccc acc aca tgg gat aac atg tcc gat gac gag       480

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

145                 150                 155                 160

aag gtc aaa tac agt caa cag gaa atg cct cag cat gca ggg tta gag       528

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

                165                 170                 175

cag agt gag cgc gtg gtg cgg ttc ctc tgg gac cag cat ttc gcg gct       576

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

            180                 185                 190

gtg gcg ggt gat gcg gtc agc ttc gag gtg tat ccg cct gta gag aag       624

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

        195                 200                 205

gag tgg gat ttg cat cat ttt ctg ttg gcc ggg tgg gga gtg ccg att       672

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

    210                 215                 220

ggg gag atg ttt gat ctg gag ggg ttg aag gag gtt tgt gag agg ttg       720

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

225                 230                 235                 240

ggc agg                                                               726

Gly Arg

<210>6

<211>242

<212>PRT

<213>Aspergillus niger

<400>6

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>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 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 Va1 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 gag tat 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>Equus 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

<211>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 ttaagatcga 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

attactggac ttggagtaaa aggaatcgca ccaactgca                             39

<210>28

<211>21

<212>DNA

＜213〉artificial

<220>

<221>misc_feature

<222>(1)..(21)

＜223〉primer of perfect matching

<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 gtcac tcatc c                                               21

<210>31

<211>26

<212>DNA

＜213〉artificial

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉n represents C, G or T

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(12)..(12)

＜223〉n represents C, G or T

<220>

<221>misc_feature

<222>(15).，(15)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(18)..(18)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(1)..(26)

＜223〉oligonucleotides Fxn42

<400>31

aagccnttng cncancangt naagac                                           26

<210>32

<211>26

<212>DNA

＜213〉artificial

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(1)..(26)

＜223〉oligonucleotides Fxn42S

<220>

<221>misc_feature

<222>(18)..(18)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉n represents C, G or T

<220>

<221>misc_feature

<222>(24)..(24)

＜223〉n represents 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 represents C or G

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(18)..(18)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉n represents A, C, T or G

<220>

<221>misc_feature

<222>(24)..(24)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(1)..(26)

＜223〉oligonucleotides Fxn42AS

<400>33

gtcttnacct ggtgngcnaa nggntt                                           26

<210>34

<211>24

<212>DNA

＜213〉artificial

<220>

<221>misc_feature

<222>(3)..(3)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉n represents A, C or G

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(12)..(12)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(1)..(24)

＜223〉oligonucleotides Fxn57

<400>34

atnggnatng cncantgggc naag                                             24

<210>35

<211>24

<212>DNA

＜213〉people T

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(12)..(12)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉n represents C, G or T

<220>

<221>misc_feature

<222>(24)..(24)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(1)..(24)

＜223〉oligonucleotides Fxn57S

<400>35

atcggcatng cncantgggc naan                                             24

<210>36

<211>24

<212>DNA

＜213〉artificial

<220>

<221>misc_feature

<222>(13)..(13)

＜223〉n represents A, C or G

<220>

<221>misc_feature

<222>(16)..(16)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(19)..(19)

＜223〉n represents A, C, T or G

<220>

<221>misc_feature

<222>(22)..(22)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(1)..(24)

＜223〉oligonucleotides Fxn57AS

<400>36

cttggcccag tgngcnatnc cnat                                             24

<210>37

<211>39

<212>DNA

＜213〉artificial

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉n represents C, G or T

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(12)..(12)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(18)..(18)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(24)..(24)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(27)..(27)

＜223〉n represents C, G or T

<220>

<221>misc_feature

<222>(30)..(30)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(33)..(33)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(1)..(39)

＜223〉oligonucleotides Fx73

<220>

<221>misc_feature

<222>(36)..(36)

＜223〉n represents 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 represents C or G

<220>

<221>misc_feature

<222>(27)..(27)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(30)..(30)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(33)..(33)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(36)..(36)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(39)..(39)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(1)..(39)

＜223〉oligonucleotides 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 represents C or G

<220>

<221>misc_feature

<222>(25)..(25)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(28)..(28)

＜223〉n represents A or C

<220>

<221>misc_feature

<222>(31)..(31)

＜223〉n represents A or C

<220>

<221>misc_feature

<222>(34)..(34)

＜223〉n represents A, C or G

<220>

<221>misc_feature

<222>(1)..(39)

＜223〉oligonucleotides 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 represents C or G

<220>

<221>misc_feature

<222>(6)..(6)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(9)..(9)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(12)..(12)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(18)..(18)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(24)..(24)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(27)..(27)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(30)..(30)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(33)..(33)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(36)..(36)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(1)..(38)

＜223〉oligonucleotides 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 represents A or G

<220>

<221>misc_feature

<222>(18)..(18)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(24)..(24)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(27)..(27)

＜223〉n represents C, G or T

<220>

<221>misc_feature

<222>(1)..(29)

＜223〉oligonucleotides Fx74S

<400>41

aactgggacc tgganaanct naanccncc                                        29

<210>42

<211>27

<212>DNA

＜213〉artificial

<220>

<221>misc_feature

<222>(12)..(12)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(15)..(15)

＜223〉n represents C or T

<220>

<221>misc_feature

<222>(18)..(18)

＜223〉n represents C or G

<220>

<221>misc_feature

<222>(21)..(21)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(27)..(27)

＜223〉n represents A or G

<220>

<221>misc_feature

<222>(1)..(27)

＜223〉oligonucleotides Fx74AS

<400>42

ggcgggt tca gnttntcnag ntcccan                                         27

<210>43

<211>8

<212>PRT

<213>Aspergillus sp.

<400>43

Iys 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)

Does＜223〉x represent?

<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

cgaagggttt gcggccgaag c                                                21

<210>47

<211>21

<212>DNA

＜213〉artificial

<220>

<221>misc_feature

<222>(1)..(21)

＜223〉primer Oal2AS

<400>47

aaggcgtggc cttcggagac 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

gcgtagtcga tgaggacgcc 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 tttacgtggt gg                                               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 tcttaaaaaa ttgccataca 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 acccaccagg 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

cgggagggag 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 actcaatggt gatggtgatg 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

                                                   15

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 Set 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 Als 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_FFATURE

<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 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 can't clearly be determined

<220>

<221>MISC_FEATURE

<222>(14)..(14)

＜223〉undetermined 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>PRT

<213>Aspergillus niger

<220>

<221>MISC_FEATURE

<222>(8)..(8)

＜223〉undetermined 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〉undetermined 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 acid can't clearly be 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 acid can't clearly be 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 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 Als 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 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 Ala 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 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 Ala 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 Asp Ile 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 (10)

1. the nucleotide sequence of the gene of the pantolactone hydrolase of encoding, described pantolactone hydrolase is from horse liver, A.niger ATCC 9142, A.niger awamori ATCC 38854 or A. niger MacRae ATCC 46951.

2. nucleotide sequence as claimed in claim 1 wherein comprises: when from the horse liver, and the nucleotide sequence shown in SEQ ID NO:1; When from A.niger MacRae ATCC 46951, the nucleotide sequence shown in SEQ ID NO:5; When from A.niger ATCC 9142, the sequence shown in SEQ ID NO:7; And when from A.niger awamori ATCC 38854, the sequence shown in SEQ ID NO:9.

3. expression vector, the nucleotide sequence that wherein comprises the gene of the pantolactone hydrolase of encoding, described pantolactone hydrolase is from horse liver, Bacillus subtilis, A.niger ATCC 9142, A.niger awamori ATCC 38854 or A.niger MacRae ATCC 46951.

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

5. pantolactone hydrolase is from horse liver, A.niger ATCC 9142, A.niger awamori ATCC 38854 or A.niger MacRae ATCC 46951.

6. pantolactone hydrolase, its amino acid sequence comprises: when from the horse liver, the amino acid sequence shown in SEQ ID NO:2; When from A.niger MacRae ATCC 46951, the amino acid sequence shown in SEQ ID NO:6; When from A.niger ATCC 9142, the amino acid sequence shown in SEQ ID NO:8; And when from A.niger awamori ATCC 38854, the amino acid sequence shown in SEQ ID NO:10.

7. such as claim 5 or 6 described pantolactone hydrolases, wherein said pantolactone hydrolase is fixed.

8. pantolactone hydrolase as claimed in claim 6 or the pantolactone hydrolase that obtains from Bacillus subtilis shown in SEQ ID NO:4 are in the purposes of R-or S-pantolactone being carried out the selective hydrolysis.

9. method of producing R-pantothenic acid or its salt or R-panthenol, described method comprises the steps: in the situation of pantolactone hydrolase as claimed in claim 6 or the pantolactone hydrolase existence that obtains from Bacillus subtilis shown in SEQ ID NO:4 R-or S-pantolactone to be carried out selective hydrolysis.

10. the method for the nucleotide sequence of a clone and known array direct neighbor, described method comprises pcr amplification, described amplification comprises:

A) carry out PCR second time with the single stranded DNA of PCR for the first time as template, in described second time PCR with the primer that can hybridize at random with the single stranded DNA of the PCR generation first time; And

B) thus, still use the at random hybridized primer of step in a), produce self as the antisense strand of the template of second chain.