CN1119418C

CN1119418C - D-amino acid oxidase of red teliosporeae

Info

Publication number: CN1119418C
Application number: CN98117470A
Authority: CN
Inventors: 廖国祯; 李易展; 李韫慧; 陈丽玲; 朱文深
Original assignee: FOODSTUFF INDUSTRIAL AND DEVELOPMENT INST
Current assignee: FOODSTUFF INDUSTRIAL AND DEVELOPMENT INST
Priority date: 1998-07-31
Filing date: 1998-07-31
Publication date: 2003-08-27
Anticipated expiration: 2018-07-31
Also published as: CN1243877A

Abstract

The present invention relates to D-amino acid oxidase in red teliosporeae and a base group encoding the oxidase.

Description

D-amino acid oxidase of red teliosporeae

Background technology

D-amino-acid oxidase (" DAO ") is a kind of flavoprotein, and known this albumen can be converted into α-ketone group hexanedioyl cephalosporonic acid (" α-ketone group hexanedioyl 7-ACA "), hydrogen peroxide and ammonia to cephalosporin (" Ceph C ").With hydroperoxidation the time, α-ketone group hexanedioyl 7-ACA can further be transformed into Glularyl-7-amino cephalosporonic acid (" GL-7-ACA ").Because GL-7-ACA is the antibiotic initial substance of cephem, thereby DAO has huge industrial value.

Remove alpha-ketoacid product (Tosa etc., agricultural biochemistry (Agric Biol Chem) [1974] 38,1529-1536), DAO has been used for the (Tu etc. that take apart of amino acid whose racemic mixture, separation science (Separation Sci) [1972] 7,403-408) and amino acid whose assay determination (Guilbault etc., analytical chemistry journal (Anal Chim Actal) [1971] 56,285-290).DAO can find in different microorganisms, insect and animal.

Summary of the invention

The present invention relates to a kind of isolating nucleic acid, the DAO of this nucleic acid encoding red teliosporeae, for example, the red winter spore of two obovate, the red winter spore of ball, R.kratochvilovae, the red winter spore of class yeast, the red winter spore of Portugal, R.paludignenum, R.dacryoidum, R.babjevae and R.fluviale.Term " D-amino acid oxidase of red teliosporeae " refers to any natural or artificial variant of red teliosporeae DAO.For example, this nucleic acid can have sequence SEQ ID NO:1 or SEQ ID NO:2 (the two all shows hereinafter), have under stringent condition the sequence with the sequence hybridization of SEQ ID NO:1 or 2, perhaps have the proteinic sequence of coding SEQ ID NO:3.

The carrier and the transformed host cells that comprise this nucleic acid are included in the scope of the present invention." carrier " refers to comprise any nucleic acid molecule or the virus of regulatory element or reporter gene, and this regulatory element or reporter gene are used for but are not limited to expressing at protokaryon or eukaryotic cell or organism." conversion " host cell " refer to by Protocols in Molecular Biology therein (or in its ancestors) introduced the host cell of nucleic acid that coding comes from the DAO of red teliosporeae.After introducing cell, this nucleic acid can exist outside karyomit(e) or be incorporated in the host genome.

The invention still further relates to the isolating DAO of red teliosporeae, replace and different protein by a conserved amino acid at least as SEQ ID NO:3 (showing below) or with SEQ ID NO:3.

Term " nucleic acid " comprises RNA and DNA (comprising cDNA, genomic dna, and synthetic, DNA for example chemosynthesis or that modify).This nucleic acid can be two strands or strand.Wherein single-chain nucleic acid can be sense strand or antisense strand.Term " isolating nucleic acid " is meant the nucleic acid that the non-natural sequence can be arranged at its flank, as the nucleic acid of plasmid or virus.Therefore, this nucleic acid can not comprise, can comprise some or all 5 ' non-coding (for example promotor) sequences, the tight adjacency of this sequence and encoding sequence yet.Therefore; this term comprises; recombinant DNA for example; this recombinant DNA is incorporated into carrier (plasmid or the virus that comprise self-replicating) or is different from the protokaryon or eukaryotic gene group DNA of red teliosporeae; perhaps as the independent molecule that is independent of other sequence (as, handle cDNA or the genomic DNA fragment that produces by PCR or restriction endonuclease) exist.This term also comprises DNA or the RNA as the reorganization of the part of the heterozygous genes of the additional peptide sequence of coding.In addition, the nucleic acid fragment that this term is intended to comprise and non-natural exists and can not find in native state with the segment form.

" hybridize under stringent condition " means and is being construed to strict and by (molecular clonings such as Sambrook, laboratory manual (Molecular Cloning, A Laboratory Manual), Sambrook, J., Fritsch, E.F. and Maniatis, T. press of second edition [1989] cold spring harbor laboratory) under the less salt and hot conditions that (Cold Spring HarborLaboratory Press) proposes, nucleic acid forms stable with the nucleic acid of SEQ ID NO:1 or SEQ ID NO:2 in solution or on solid support, sequence-specific, non-covalent key.For example, the reference nucleic acid that resembles SEQ ID NO:1 or SEQ ID NO:2 and so on can be immobilized on the nitrocellulose, and 0.2 * SSC (1.75g/1NaCl, 0.88g/l Na are arranged under 68 ℃ ₃Citrate2H ₂O; PH7.0) and 0.1% (w/v) when sodium laurylsulfonate exists, any other nucleic acid specificity and non-covalent be attached to immobilized with reference to just being considered to hybridize under stringent condition on the nucleic acid.

" isolating D-amino-acid oxidase " is meant DAO purifying or enrichment from its natural surroundings (for example, by the DAO-enrichment extract by the preparation of DAO expression vector transformed host cells)." the conservative replacement " is meant the replacement in following certain group: Xie Ansuan, Isoleucine and leucine; Aspartic acid and L-glutamic acid; L-asparagine and desire glutamine; Serine and Threonine; Methionin and arginine; And phenylalanine and tyrosine.

The separation of red teliosporeae DAO gene can produce the proteinic mutation form of DAO with identify making, this protein variant form can have favourable activity (as the Km of the reduction that changes for substrate) or bigger protein stability.

Other characteristic of the present invention or advantage will be from the detailed descriptions of following accompanying drawing and several embodiments, and display in additional claims.

The present invention relates to red teliosporeae DAO, and for example two red winter spores of obovate of the nucleic acid that relates to coding red teliosporeae DAO, said red teliosporeae, the red winter spore of ball, R.kratochvilovae, the red winter spore of class yeast, the red winter spore of Portugal, R.paludignenum, R.dacryoidum, R.babjevae and R.fluviale.

Within limit of consideration of the present invention recombinant nucleic acid or virus arranged, they make can produce DAO protein in transformant or transgenosis organism, perhaps generation specificity or non-specific sudden change easily in the DAO reading frame.These recombinant nucleic acids or virus may further comprise any in the upstream sequence of various DAO encoding sequences, as strong constitutive promoter; Sequence in the DAO encoding sequence, as comprise can be so that the intron of the cis element of high level expression; Perhaps comprise any in the downstream sequence of various DAO encoding sequences, as effective polyadenylation signal.The present invention further comprises any cell that contains or produce this nucleic acid or virus, and comprises any DAO protein that is produced by this cell.

Though further do not elaborate, people know, based on separating of above-mentioned open and following DAO protein and the DAO gene that derives from the red winter spore of class yeast, those skilled in the art can utilize the present invention to the full extent.Following two embodiment only are used for setting forth those skilled in the art and how separate DAO gene and protein from red teliosporeae, and go up restriction rest part of the present disclosure never in any form.Any publication this paper reference in the lump of in the disclosure, being quoted.

Embodiment

Embodiment 1

Present embodiment explanation purifying and identify from red teliosporeae to the activated D-amino-acid oxidase of cephalosporin.

The red winter spore of class yeast (No.10788) that obtains from American type culture collection is used as the proteinic source of separation DAO.

The following purifying that carries out of protein: added the D-L-Ala in the cell-free extract of the red winter spore of class yeast (1), and final concentration is 0.3M, with mixture heating up to 70 ℃ 4 minutes.(2) under 13000 * g centrifugal 40 minutes with disgorging.(3) with 1.5 liters of buffer A (50mM potassium phosphate buffer, pH8.0,2mM EDTA, 5mM beta-mercaptoethanol, 1mM PMSF and 2.5g/ml pepstatin A) dialysis supernatant liquor.(4) protein soln of dialysing is added to DEAE-Sephacel post and (on 15 * 2.4cm), carries out wash-out with buffer A.Collection has the active component of DAO, and concentrates in Amicon pond (YM30).(5) (on 15 * 2.0cm), this post has replenished buffer B (the same with buffer A, as still the not have pepstatin A) balance of 20% glycerine with 150ml enriched material to be added to phenyl-Sephacel CL-4B post.Replenished the DAO of the buffer B elution of bound of 40% glycerine.Collection has the active component of DAO, and dialyses with the buffer B that contains 10% glycerine.(6) dialysate is concentrated into 2ml, and carries out chromatography on fast protein liquid chromatography (FPLC) Mono QHR 5/5 post, this chromatography column carries out balance with the buffer B that 30ml has added 10% glycerine in advance.At once wash-out DAO after the free column volume.Collection comprises the component of enzymic activity and is concentrated into 1ml.(7) concentrated solution is added on FPLC gel-filtration Superose 12HR 10/30 post and carries out wash-out with the buffer B that contains 10% glycerine and 0.25M NaCl.Collect active ingredient, be stored under-20 ℃.

Utilize the final chromatography of Superose 12HR 10/30 post to make from protein example gel infiltration and effectively remove a small amount of impurity.

From the female red winter spore of about 100g (weight in wet base) fermentoid, obtain 0.88mg purifying DAO, productive rate is 21%, proteinic specific activity is 60U/mg (Lee etc., biotechnology communication (BiotechnolLett) [1994] 16,467-472 have provided definition of DAO units activity and condition determination).The preparation that obtains under natural PAGE is single band.Enzyme is insensitive to Periodic acid/Schiff's reagent dyeing, and this shows that it is not a glycoprotein.

On FPLC Superase 12 posts by gel-filtration be 72kDa with reference to the proteinic typical curve molecular weight that estimates natural enzyme of comparing.SDS/PAGE shows that molecular weight is single band of 37kDa.Its result shows that the natural form of spore DAO of red winter of class yeast is made up of the subunit of two identical sizes.Preceding 22 residues of NH2-terminal amino acid sequence are defined as Met His Ser Gln Lys Arg Val ValVal Leu Gly Ser Gly Val Ile Gly Leu Ser Ser Ala Leu Ile (SEQ ID NO:10).

The absorption spectrum that derives from the DAO of the red winter spore of class yeast shows two distorted peaks, and respectively at 370nm and 455nm place, this is typical for flavine chromophore.E280/E445 ratio (it is often in order to estimate the parameter of flavoprotein purity) is 8.1.Efficient liquid chromatography (HPLC) analysis shows that the flavine that discharges from the protein formulation that boils has identical retention time with standard flavin adenine dinucleotide FAD.The concentration of FAD is defined as 6.34g/ml, and this FAD extracts from the protein soln of 0.297mg/ml.Analyze based on this result and SDS/PAGE, obtain ratio and be approximately 1.73 moles of FAD/ mole protein, this shows all a FAD in each subunit.

Under differing temps, measure the enzymic activity of DAO.This protein shows maximum activity in the time of 35 ℃.This enzyme all has activity (be higher than maximum activity 70%) in 30-50 ℃ temperature range.This enzymic activity is reduced to 30% of maximum activity suddenly in the time of 60 ℃.This enzyme is instability when the temperature that is higher than 35 ℃.When 40 ℃ and 50 ℃, carry out respectively only keeping 20% and 10% of maximum activity after the incubation 10 minutes.

The active optimal pH of DAO is 9.0.This enzyme has activity in pH scope 7.5-10.0.DAO is stable in pH scope 7.0-8.0, determines this moment to surpass 80% residual activity.Yet, be higher than 8.5 at pH and lose considerable activity, this enzyme 70% inactivation during pH11.0.

The relative reactivity of measurement DAO of purifying on various D-amino acid substrate.This purifying enzyme all has activity on the D-of all tests amino acid.Its best substrate is the D-tryptophane, is the D-methionine(Met) secondly, D-phenylalanine, D-L-Ala and D-leucine.What this enzyme showed less activity (be less than maximum activity 20%) has D-Threonine, D-L-glutamic acid, D-aspartic acid and D-Methionin.Determining on L-amino acid does not have activity, comprises the L-L-Ala, L-proline(Pro), L-phenylalanine and L-methionine(Met).The D-tryptophane, the D-methionine(Met), the apparent Km of D-L-Ala and D-Serine is respectively 0.18mM, 0.22mM, 0.68mM and 3.4mM.At para-amino benzoic acid, difference 72%, 49% and 21% ground suppressed the activity of this enzyme under phenylformic acid and nicotinic acid existed.These aromatic acids all are defined as the competitive inhibitor of spore DAO of red winter of class yeast.In the aromatic acid of being tested, para-amino benzoic acid has minimum Ki (0.3mM).

The DAO protein that will have the purifying of sequence SEQ ID NO:3 (showing below) is used for transforming Ceph C.Compare with enzymic activity on the D-L-Ala, this enzyme has 95% activity on Ceph C.This enzyme is 0.65mM to the apparent Km of Ceph C.Productive rate is 91%, and further hatches and do not increase productive rate.This reaction does not need external source H ₂O ₂

Embodiment 2

The present embodiment explanation is from the separation and the expression of the daao gene of red teliosporeae.

As the DAO protein of embodiment 1 described method purifying from the red winter spore of class yeast.Substitute last twice column chromatography (MonoQ HR 5/5 and Supersoe 10HR) with sodium laurylsulfonate (SDS)/polyacrylamide gel electrophoresis (PAGE).From the SDS/PAGE gel, be purified into the DAO of about 300 picomole, go out peptide sequence by Stanford Univ USA's protein and nucleic acid experimental determination.Determined three kinds of internal peptide sequences (YCQYLARELQ[SEQ ID NO:11], IAGGIDDQAAEPIR[SEQID NO:12] and RCTMDSSDP[SEQ ID NO:13]).In order to separate DAO gene, serve as the synthetic four kinds of full degeneracy oligonucleotide primers in basis with this peptide sequence from the red winter spore of class yeast.They be primer 15 '-AARTAYTGYCARTAYC-3 ') (SEQ ID NO:6), primer 25 '-ATNGAYGAYCAYGCNGC-3 ' (SEQ ID NO:7), primer 35 '-GCNGCYTGRTCRTCNAT-3 ' (SEQ ID NO:8), and primer 45-ATGGAYAGYAGYGAYCC-3 ' (SEQ ID NO:9), wherein N represents G, A, T or C; Y represents T or C; And R represents A or G.

When primer 1 uses separately, there is not product to produce as the PCR reaction of template with the chromosomal DNA of the red winter spore of class yeast.Only comprise primer 2, synthesizing several non-specific products in 3 or 4 the PCR reaction.PCR reaction with primer 2 and 4 also obtains several non-specific products.Utilize primer 1 and at 3 o'clock except that non-specific product, its typical consequence is the specific PCR product that also obtains being about 210bp.When during as template, in the PCR reaction, can heavily increasing with primer 1 and 3 with this fragment.

Measure this segmental nucleotide sequence.It comprises 214 following Nucleotide: 5 '-AAGTACTGCCAGTACCTTGCAAGAGAGCTGCAGAAGCTCGGCGCGACGTTTGAGAG ACGGACCGTTACGTCGCTTGAGCAGGCGTTCGACGGTGCGGATTTGGTGGTCAACG CTACGGGACTTGgtatgtcccgaactgcccctctctacctgcaattttgctgattg atatgctcgcagGCGCCAAGTCGATTGCGGGCATCGACGACCAAGC-3 ' (SEQID NO:15)

(SEQ ID NO:15) (lowercase is represented intron sequences, based on following cDNA.)

YCQYLARELQ (SEQ ID NO:11) and IAGIDDQA (SEQ ID NO:14), they are the part by the aminoacid sequence of inferring of said 214bp fragment coding, and are identical with the part of determined DAO protein sequence.The result shows that this 214bpDNA fragment is the part of coding class red winter of yeast spore DAO gene.So, the probe of this fragment as separation DAO gene.

Identify the hybridized fragment of about 3.3kb with the Southern blot hybridization of the above-mentioned 214bp dna fragmentation of digoxigenin mark.By being inserted into, the HindIII-digested genomic dna fragment in 3.0 to the 3.5kb scopes makes up minigene group DNA library among the pBluescript SK+.Screen this library with identical probe.From about 200 clones of screening, obtain 15 positive colonies that in the radioautograph signal, have two kinds of other intensity of level.From various, select three clones at random and analyze the segmental restriction zymogram of insertion.Its result shows that these clones comprise two kinds of different DNA and insert fragment, and this causes having two kinds of other intensity of level in signal.Select one type clone to be used for further research, this research is based on their more high strength on signal and the hybridization collection of illustrative plates that inserts segment and 214bp probe.Find that these clones include genomic DNA fragment, this genomic DNA fragment comprises the DAO gene order.The genome sequence of DAO gene coding region is shown below:

5′-ATGCACTCTCAGAAGCGCGTCGTTGTCCTCGGATCAGGCGgtgcgtcttttcc

ctctcctccccacacccgacagtcctcgacgaggtgtaggacggcgagcaaagctgc

cgagggcgatctgggctgactgagcgctcgagtgtacagTTATCGGTCTGAGCAGCG

CCCTCATCCTCGCTCGGAAGGGCTACAGCGTGCATATTCTCGCGCGCGACTTGCCGG

AGGACGTCTCGAGCCAGACTTTCGCTTCACCATGGGCTgtgcgtcgtctcactgtag

ttggaggatgtcagcgagagctgatcaatctcgtcatccccgcagGGCGCGAATTGG

ACGCCTTTCATGACGCTTACAGACGGTCCTCGACAAGCAAAATGGGAAGAATCGACT

TTgtgcgtctccttctacctcattcttggcctcgagctgacgagtgtatgatacaca

gCAAGAAGTGGGTCGAGTTGGTCCCGACGGGCCATGCCATGTGGCTCAAGGGGACGA

GGCGGTTCGCGCAGAACGAAGACGGCTTGCTCGGGCACTGGTACAAGGACATCACGC

CAAATgtgcgcccacattcactcttcccttcgcatgtctccgtttactgacccgccc

tctttcgccgtgcgcagTACCGCCCCCTCCCATCTTCCGAATGTCCACCTGGCGCTA

TCGGCGTAACCTACGACACCCICTCCGTCCACGCACCAAAGTACTGCCAGTACCTTG

CAAGAGAGCTGCAGAAGCTCGGCGCGACGTTTGAGAGACGGACCGTTACGTCGCTTG

AGCAGGCGTTCGACGGTGCGGATTTGGTGGTCAACGCTACGGGACTTGgtatgtCcc

gaactgcccctctctacctgcaattttgctgattgatatgctcgcagGCGCCAAGTC

GATTGCGGGCATCGACGACCAAGCCGCCGAGCCAATCCGCGGCCAAACCGTCCTCGT

CAAGTCCCCATGCAAGCGATGCACGATGGACTCGTCCGACCCCGCTTCTCCCGCCTA

CATCATTCCCCGACCAGGTGGCGAAGTCATCTGCGGCGGGACGTACGGCGTGGGAGA

CTGGGACTTGTCTGTCAACCCAGAGACGGTCCAGCGGATCCTCAAGCACTGCTTGCG

CCTCGACCCGACCATCTCGAGCGACGGAACGATCGAAGGCATCGAGGTCCTCCGCCA

CAACGTCGGCTTGCGACCTGCACGACGAGGCGGACCCCGCGTCGAGGCAGAACGGAT

CGTCCTGCCTCTCGACCGGACAAAGTCGCCCCTCTCGCTCGGCAGGGGCAGCGCACG

AGCCGCGAAGGAGAAGGAGGTCACGCTTGTGCATGCGTATGGCTTCTCGAGTGCGGG

ATACCAGCAGAGTTGGGGCGCGGCGGAGGATGTCGCGCAGCTCGTCGACGAGGCGTT

CCAGCGGTACCACGGCGCGGCGCGGGAGTCGAAGTTG-3′(SEQ?ID?NO：1)

(lowercase is represented intron sequences, based on following cDNA).

Carry out 5 of cDNA end ' the separate cDNA end of DAO gene with 3 ' rapid amplifying (RACE) .Scheme according to manufacturers is used to carry out RACE from the RACE of Gibco BRL test kit.In 5 ' RACE and 3 ' RACE; Amplify the dna fragmentation of about 550bp and 720bp respectively.For the definite kernel nucleotide sequence; with these fragment clonings in the pGEM-T carrier.Its result shows that two cDNA fragments have the overlap of 124bp.The nucleotide sequence that makes up two bar segment produces the opening code-reading frame ( ORF ) of the encoding amino acid sequence of expecting, this encoding amino acid sequence comprises and three above-mentioned zones that internal peptide has complete identity property.DAOcDNA：5′-ATGCACTCTCAGAAGCGCGTCGTTGTCCTCGGATCAGGCGTTATCGGTCTGAGCAGCGCCCTCATCCTCGCTCGGAAGGGCTACAGCGTGCATATTCTCGCGCGCGACTTGCCGGAGGACGTCTCGAGCCAGACTTTCGCTTCACCATGGGCTGGCGCGAATTGGACGCCTTTCATGACGCTTACAGACGGTCCTCGACAAGCAAAATGGGAAGAATCGACTTTCAAGAAGTGGGTCGAGTTGGTCCCGACGGGCCATGCCATGTGGCTCAAGGGGACGAGGCGGTTCGCGCAGAACGAAGACGGCTTGCTCGGGCACTGGTACAAGGACATCACGCCAAATTACCGCCCCCTCCCATCTTCCGAATGTCCACCTGGCGCTATCGGCGTAACCTACGACACCCTCTCCGTCCACGCACCAAAGTACTGCCAGTACCTTGCAAGAGAGCTGCAGAAGCTCGGCGCGACGTTTGAGAGACGGACCGTTACGTCGCTTGAGCAGGCGTTCGACGGTGCGGATTTGGTGGTCAACGCTACGGGACTTGGCGCCAAGTCGATTGCGGGCATCGACGACCAAGCCGCCGAGCCAATCCGCGGCCAAACCGTCCTCGTCAAGTCCCCATGCAAGCGATGCACGATGGACTCGTCCGACCCCGCTTCTCCCGCCTACATCATTCCCCGACCAGGTGGCGAAGTCATCTGCGGCGGGACGTACGGCGTGGGAGACTGGGACTTGTCTGTCAACCCAGAGACGGTCCAGCGGATCCTCAAGCACTGCTTGCGCCTCGACCCGACCATCTCGAGCGACGGAACGATCGAAGGCATCGAGGTCCTCCGCCACAACGTCGGCTTGCGACCTGCACGACGAGGCGGACCCCGCGTCGAGGCAGAACGGATCGTCCTGCCTCTCGACCGGACAAAGTCGCCCCTCTCGCTCGGCAGGGGCAGCGCACGAGCCGCGAAGGAGAAGGAGGTCACGCTTGTGCATGCGTATGGCTTCTCGAGTGCGGGATACCAGCAGAGTTGGGGCGCGGCGGAGGATGTCGCGCAGCTCGTCGACGAGGCGTTCCAGCGGTACCACGGCGCGGCGCGGGAGTCGAAGTTG-3′ ( SEQ ID NO：2 )

ORF length is 1104 Nucleotide, and its coding molecule amount is 40079Da, 368 amino acid whose protein.This protein sequence (from the N-end to the C-end) is shown below:

M H S Q K R V V V L G S G V I G L S S

A L I L A R K G Y S V H I L A R D L P

E D V S S Q T F A S P W A G A N W T P

F M T L T D G P R Q A K W E E S T F K

K W V E L V P T G H A M W L K G T R R

F A Q N E D G L L G H W Y K D I T P N

Y R P L P S S E C P P G A I G V T Y D

T L S V H A P K Y C Q Y L A R E L Q K

L G A T F E R R T V T S L E Q A F D G

A D L V V N A T G L G A K S I A G I D

D Q A A E P I R G Q T V L V K S P C K

R C T M D S S D P A S P A Y I I P R P

G G E V I C G G T Y G V G D W D L S V

N P E T V Q R I L K H C L R L D P T I

S S D G T I E G I E V L R H N V G L R

P A R R G G P R V E A E R I V L P L D

R T K S P L S L G R G S A R A A K E K

E V T L V H A Y G F S S A G Y Q Q S W

G A A E D V A Q L V D E A F Q R Y H G

A A R E S K L (SEQ?ID?NO：3)

Relatively the nucleotide sequence between cDNA and the genomic dna shows that the DAO gene comprises 6 exons and 5 introns (referring to top SEQ ID NO:1).The intron of DAO gene is less relatively, and its size is in the 56bp-109bp scope.Two ends of 5 introns all comprise the consensus sequence GT that exon-intron connects ... AG.

Closely adjacent with initiator codon 5 ' end and be positioned at the nucleotide sequence and the consensus sequence CCACC around the translation initiation site of inferring of DAO gene ATGC (SEQ ID NO:16) consistent (Kozak, nucleic acids research (Nuc Acids Res) [1984] 12,857-872), always G or A of the nucleosides of position-3 (the A residue of initiator codon be decided to be+1) wherein.5 of initiator codon ' end is as follows:

5′-AAGCTTCGGCACGAGCATGAGTGTGAATGATGGTCCAAGGAGGACAGCGCAGAG

TCAACAGGAGGGCACATGGAGGCAGAGCGTGGGGCGGAGGAGGCAGATGGGGAGTCG

CGCTGGGGGACGAGGGGGTGTCGCTCGACTAACAGCTCTCTATCGCTCTTGCTGCTG

CTTGTACTACTCGAACGACGCC-3′(SEQ?ID?NO：4)

Consistent with fungal gene, do not find tangible TATA box at 5 ' flanking region of spore DAO gene of red winter of class yeast.

In the closely adjacent sequence of 3 of ORF ', there is not the consensus sequence of polyadenylation signal.Yet the sequence TGTATTGC (SEQ IDNO:18) that is positioned at upstream, poly (A) addition site 11 to 18 residues is similar with the known sequence YGTGTTYY (SEQ ID NO:17) (wherein Y represents pyrimidine) that relates in correct 3 ' terminal formation of mammals mRNA.ORF3 ' the end of sequence is expressed as follows:

5′-GGCGGGATTTGTGGCTGTATTGCGGGCATCTACAAGACCAGCTTCATCTCGGAC

GACAACACGAGAGCGGCGAGTCTTCGTACCGTCTGACCTTCGCAATCCGCCGAGTCC

TTGCCCGTTGCCCTGCTTGCTCCTTTCGTATCTCCTGTGACTCGGAACGTCGCTCTT

CGCCTCTGTCACTTGCCAGGCCGTCCCTTCAAACTGTCGCCGCCCTCCTCCTCCCAA

TCTCCTCCTCACGCGACTTGCCACTACTCTCACTTCTCCTGCTTACCAAGGCTTACA

CTACGCAACACTAAGCGCGGCATCAGGTTCCGTTGCGCGCCTCGTCACGACCCGACT

TTTTTTCGCCCGTTCGCTCGCCTCGCTCCGTTGCCGAGCGAAGAACTTCGCCTGCCT

TCGAATCTCTCGCCTTGCTCGTCTCGTCCTGCTCCGTTCCACCACAGATAGACTCAC

AGCAACACACTCACAATGGTCAAGGTGCGCACCCACTCCCGCCAGTCCAACTCGCGC

GACGAGAGACCACCGTCTCGTTGACATCACTGACCTCGTCGCTCGCCACCCACCCTC

CCGCTCGTCCATGCACACAGGCAGCACCACGATCAGACGAGCAGCAGCACTACCACG

CCACAGTCATGGGCGGACTCAAGGGCGGCGCGATGGGGTTGGCGGCGGGTGGAGCGG

GTGCGGTTGCGCTGCAGAGGGCGAACGTACAGGCGTTCACGAGGTTGACGCTGCCTC

TCAAGGCGTTTGCTGTCACGTACGTCCCGCGATCCCTTACTATGCGACTCCCTCGGT

GAATTC-3′(SEQ?ID?NO：5)

Utilize pET23a vector construction expression plasmid pDAO-23 (carrying the cDNA of spore DAO of red winter of class yeast), and this plasmid is incorporated in the e. coli bl21 [DE3].Isopropylthio-(IPTG)-induce transformant to show the DAO activity in cell pyrolysis liquid, this shows that active DAO is at expression in escherichia coli.The SDS/PAGE of cell pyrolysis liquid analyzes the protein band (the estimation size of DAO subunit) that demonstrates a tangible 37kDa, and this district's band does not exist from the lysate of pET23a conversion host with the similarity method preparation.This albumen was fully induced 30 minutes with IPTG.Utilize nickel-column chromatography that DAO is purified near homogeneity.One step can obtain to be about the protein of 4mg from the 250ml culture of Escherichia coli.Protein purification shows 1, and the 211U/mg specific activity increases by 7 times than crude extract, increases by 20 times than the spore DAO protein of red winter of class yeast of purifying.This activity is active similar with the enzyme for preparing by dialysis in the presence of FAD, and this shows that reorganization DAO may comprise the FAD as prothetic group.

Also prepared the coli strain (the DH5 α that pDA023-transforms) of expressing DAO, and be deposited in American type culture collection, its preserving number is No.98485.

Other embodiment

For the purpose of understanding, though in conjunction with having described the present invention, the description of front is intended to explanation, rather than limits the scope of the invention, and scope of the present invention is limited by the scope of additional claims.Others (advantage and improvement) are within the scope of the invention.

For example, DAO protein of the present invention includes but not limited to recombinant protein, natural protein and synthetic protein and preceding albumen or former albumen.Reorganization DAO protein comprises DAO that contains aminoacid deletion adjacency or non-adjacent or the DAO that comprises signal peptide, and this signal peptide allows the various compartments of transporte to cells, as periplasmic space, endoplasmic reticulum, plastosome or extracellular space.

Sequence table (1) general information

(i) applicant: Lao, Gwo-Jen

Lee，Yi-Jang

Lee，Yun-Huey

Chen，Li-Lin

Chu，Wen-Shen

(ii) denomination of invention: D-amino acid oxidase of red teliosporeae

(iii) sequence number: 18

(iv) address:

(A) addressee: Fish ﹠amp; Richardson, P.C.

(B) street: 225 Franklin Street

(C) city: Boston (Boston)

(D) state: MA

(E) country: US

(F)ZIP：02110-2804

(v) computer-reader form:

(R) media type: floppy disk

(R) computer: IBM compatible

(C) operating system: Windows 95

(D) software: Fast SEQ for Windows Version 2.0

(the vi) data of current application:

(A) application number:

(R) applying date: on July 30th, 1997

(C) classification number:

(vii) in the data of first to file:

(A) application number:

(B) applying date:

(viii) lawyer/proxy's information:

(A) title: Tsao, Y.Rocky

(B) registration number: 34,053

(C) certificate number: 06497/006001

(ix) telecommunication information:

(A) phone: 617-542-5070

(B) fax: 617-542-8906

(C) fax: the information of 200154 (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 1458 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: genomic dna

( xi ) ：SEQ ID NO：1：ATGCACTCTC AGAAGCGCGT CGTTGTCCTC GGATCAGGCG GTGCGTCTTT TCCCTCTCCT 60CCCCACACCC GACAGTCCTC GACGAGGTGT AGGACGGCGA GCAAAGCTGC CGAGGGCGAT 120CTGGGCTGAC TGAGCGCTCG AGTGTACAGT TATCGGTCTG AGCAGCGCCC TCATCCTCGC 180TCGGAAGGGC TACAGCGTGC ATATTCTCGC GCGCGACTTG CCGGAGGACG TCTCGAGCCA 240GACTTTCGCT TCACCATGGG CTGTGCGTCG TCTCACTGTA GTTGGAGGAT GTCAGCGAGA 300GCTGATCAAT CTCGTCATCC CCGCAGGGCG CGAATTGGAC GCCTTTCATG ACGCTTACAG 360ACGGTCCTCG ACAAGCAAAA TGGGAAGAAT CGACTTTGTG CGTCTCCTTC TACCTCATTC 420TTGGCCTCGA GCTGACGAGT GTATGATACA CAGCAAGAAG TGGGTCGAGT TGGTCCCGAC 480GGGCCATGCC ATGTGGCTCA AGGGGACGAG GCGGTTCGCG CAGAACGAAG ACGGCTTGCT 540CGGGCACTGG TACAAGGACA TCACGCCAAA TGTGCGCCCA CATTCACTCT TCCCTTCGCA 600TGTCTCCGTT TACTGACCCG CCCTCTTTCG CCGTGCGCAG TACCGCCCCC TCCCATCTTC 660CGAATGTCCA CCTGGCGCTA TCGGCGTAAC CTACGACACC CTCTCCGTCC ACGCACCAAA 720GTACTGCCAG TACCTTGCAA GAGAGCTGCA GAAGCTCGGC GCGACGTTTG AGAGACGGAC 780CGTTACGTCG CTTGAGCAGG CGTTCGACGG TGCGGATTTG GTGGTCAACG CTACGGGACT 840TGGTATGTCC CGAACTGCCC CTCTCTACCT GCAATTTTGC TGATTGATAT GCTCGCAGGC 900GCCAAGTCGA TTGCGGGCAT CGACGACCAA GCCGCCGAGC CAATCCGCGG CCAAACCGTC 960CTCGTCAAGT CCCCATGCAA GCGATGCACG ATGGACTCGT CCGACCCCGC TTCTCCCGCC 1020TACATCATTC CCCGACCAGG TGGCGAAGTC ATCTGCGGCG GGACGTACGG CGTGGGAGAC 1080TGGGACTTGT CTGTCAACCC AGAGACGGTC CAGCGGATCC TCAAGCACTG CTTGCGCCTC 1140GACCCGACCA TCTCGAGCGA CGGAACGATC GAAGGCATCG AGGTCCTCCG CCACAACGTC 1200GGCTTGCGAC CTGCACGACG AGGCGGACCC CGCGTCGAGG CAGAACGGAT CGTCCTGCCT 1260CTCGACCGGA CAAAGTCGCC CCTCTCGCTC GGCAGGGGCA GCGCACGAGC CGCGAAGGAG 1320AAGGAGGTCA CGCTTGTGCA TGCGTATGGC TTCTCGAGTG CGGGATACCA GCAGAGTTGG 1380GGCGCGGCGG AGGATGTCGC GCAGCTCGTC GACGAGGCGT TCCAGCGGTA CCACGGCGCG 1440GCGCGGGAGT CGAACTTG 1458 ( 2 ) SEQ ID NO：2：

(i) sequence signature:

(A) length: 1104 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: cDNA

(ix) feature

(A) title/keyword: encoding sequence

(B) position: 1 ... 1104

(xi) sequence description: SEQ ID NO:2:ATG CAC TCT CAG AAG CGC GTC GTT GTC CTC GGA TCA GGC GTT ATC GGT 48Met His Ser Gln Lys Arg Val Val Val Leu Gly Ser Gly Val Ile Gly 15 10 15CTG AGC AGC GCC CTC ATC CTC GCT CGG AAG GGC TAC AGC GTG CAT ATT 96Leu Ser Ser Ala Leu Ile Leu Ala Arg Lys Gly Tyr Ser Val His Ile

20 25 30CTC?GCG?CGC?GAC?TTG?CCG?GAG?GAC?GTC?TCG?AGC?CAG?ACT?TTC?GCT?TCA 144Leu?Ala?Arg?Asp?Leu?Pro?Glu?Asp?Val?Ser?Ser?Gln?Thr?Phe?Ala?Ser

35 40 45CCA?TGG?GCT?GGC?GCG?AAT?TGG?ACG?CCT?TTC?ATG?ACG?CTT?ACA?GAC?GGT 192Pro?Trp?Ala?Gly?Ala?Asn?Trp?Thr?Pro?Phe?Met?Thr?Leu?Thr?Asp?Gly

50 55 60CCT?CGA?CAA?GCA?AAA?TGG?GAA?GAA?TCG?ACT?TTC?AAG?AAG?TGG?GTC?GAG 240Pro?Arg?Gln?Ala?Lys?Trp?Glu?Glu?Ser?Thr?Phe?Lys?Lys?Trp?Val?Glu?65 70 75 80TTG?GTC?CCG?ACG?GGC?CAT?GCC?ATG?TGG?CTC?AAG?GGG?ACG?AGG?CGG?TTC 288Leu?Val?Pro?Thr?Gly?His?Ala?Met?Trp?Leu?Lys?Gly?Thr?Arg?Arg?Phe

85 90 95GCG?CAG?AAC?GAA?GAC?GGC?TTG?CTC?GGG?CAC?TGG?TAC?AAG?GAC?ATC?ACG 336Ala?Gln?Asn?Glu?Asp?Gly?Leu?Leu?Gly?His?Trp?Tyr?Lys?Asp?Ile?Thr

100 105 110CCA?AAT?TAC?CGC?CCC?CTC?CCA?TCT?TCC?GAA?TGT?CCA?CCT?GGC?GCT?ATC 384Pro?Asn?Tyr?Arg?Pro?Leu?Pro?Ser?Ser?Glu?Cys?Pro?Pro?Gly?Ala?Ile

115 120 125GGC?GTA?ACC?TAC?GAC?ACC?CTC?TCC?GTC?CAC?GCA?CCA?AAG?TAC?TGC?CAG 432Gly?Val?Thr?Tyr?Asp?Thr?Leu?Ser?Val?His?Ala?Pro?Lys?Tyr?Cys?Gln

130 135 140TAC?CTT?GCA?AGA?GAG?CTG?CAG?AAG?CTC?GGC?GCG?ACG?TTT?GAG?AGA?CGG 480Tyr?Leu?Ala?Arg?Glu?Leu?Gln?Lys?Leu?Gly?Ala?Thr?Phe?Glu?Arg?Arg145 150 155 160ACC?GTT?ACG?TCG?CTT?GAG?CAG?GCG?TTC?GAC?GGT?GCG?GAT?TTG?GTG?GTC 528Thr?Val?Thr?Ser?Leu?Glu?Gln?Ala?Phe?Asp?Gly?Ala?Asp?Leu?Val?Val

165 170 175AAC?GCT?ACG?GGA?CTT?GGC?GCC?AAG?TCG?ATT?GCG?GGC?ATC?GAC?GAC?CAA 576Asn?Ala?Thr?Gly?Leu?Gly?Ala?Lys?Ser?Ile?Ala?Gly?Ile?Asp?Asp?Gln

180 185 190GCC?GCC?GAG?CCA?ATC?CGC?GGC?CAA?ACC?GTC?CTC?GTC?AAG?TCC?CCA?TGC 624Ala?Ala?Glu?Pro?Ile?Arg?Gly?Gln?Thr?Val?Leu?Val?Lys?Ser?Pro?Cys

195 200 205AAG?CGA?TGC?ACG?ATG?GAC?TCG?TCC?GAC?CCC?GCT?TCT?CCC?GCC?TAC?ATC 672Lys?Arg?Cys?Thr?Met?Asp?Ser?Ser?Asp?Pro?Ala?Ser?Pro?Ala?Tyr?Ile

210 215 220ATT?CCC?CGA?CCA?GGT?GGC?GAA?GTC?ATC?TGC?GGC?GGG?ACG?TAC?GGC?GTG 720Ile?Pro?Arg?Pro?Gly?Gly?Glu?Val?Ile?Cys?Gly?Gly?Thr?Tyr?Gly?Val225 230 235 240GGA?GAC?TGG?GAC?TTG?TCT?GTC?AAC?CCA?GAG?ACG?GTC?CAG?CGG?ATC?CTC 768Gly?Asp?Trp?Asp?Leu?Ser?Val?Asn?Pro?Glu?Thr?Val?Gln?Arg?Ile?Leu

245 250 255AAG?CAC?TGC?TTG?CGC?CTC?GAC?CCG?ACC?ATC?TCG?AGC?GAC?GGA?ACG?ATC 816Lys?His?Cys?Leu?Arg?Leu?Asp?Pro?Thr?Ile?Ser?Ser?Asp?Gly?Thr?Ile

260 265 270GAA?GGC?ATC?GAG?GTC?CTC?CGC?CAC?AAC?GTC?GGC?TTG?CGA?CCT?GCA?CGA 864Glu?Gly?Ile?Glu?Val?Leu?Arg?His?Asn?Val?Gly?Leu?Arg?Pro?Ala?Arg

275 280 285CGA?GGC?GGA?CCC?CGC?GTC?GAG?GCA?GAA?CGG?ATC?GTC?CTG?CCT?CTC?GAC 912Arg?Gly?Gly?Pro?Arg?Val?Glu?Ala?Glu?Arg?Ile?Val?Leu?Pro?Leu?Asp290 295 300CGG?ACA?AAG?TCG?CCC?CTC?TCG?CTC?GGC?AGG?GGC?AGC?GCA?CGA?GCC?GCG 960Arg?Thr?Lys?Ser?Pro?Leu?Ser?Leu?Gly?Arg?Gly?Ser?Ala?Arg?Ala?Ala305 310 315 320AAG?GAG?AAG?GAG?GTC?ACG?CTT?GTG?CAT?GCG?TAT?GGC?TTC?TCG?AGT?GCG 1008Lys?Glu?Lys?Glu?Val?Thr?Leu?Val?His?Ala?Tyr?Gly?Phe?Ser?Ser?Ala

325 330 335GGA?TAC?CAG?CAG?AGT?TGG?GGC?GCG?GCG?GAG?GAT?GTC?GCG?CAG?CTC?GTC 1056Gly?Tyr?Gln?Gln?Ser?Trp?Gly?Ala?Ala?Glu?Asp?Val?Ala?Gln?Leu?Val

340 345 350GAC?GAG?GCG?TTC?CAG?CGG?TAC?CAC?GGC?GCG?GCG?CGG?GAG?TCG?AAG?TTG 1104Asp?Glu?Ala?Phe?Gln?Arg?Tyr?His?Gly?Ala?Ala?Arg?Glu?Ser?Lys?Leu

The information of 355 360 365 (2) SEQ ID NO:3:

(i) sequence signature:

(A) length: 368 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: protein

(ii) clip types: inside

(xi) sequence description: SEQ ID NO:3:Met His Ser Gln Lys Arg Val Val Val Leu Gly Ser Gly Val Ile Gly 15 10 15Leu Ser Ser Ala Leu Ile Leu Ala Arg Lys Gly Tyr Ser Val His Ile

20 25 30Leu?Ala?Arg?Asp?Leu?Pro?Glu?Asp?Val?Ser?Ser?Gln?Thr?Phe?Ala?Ser

35 40 45Pro?Trp?Ala?Gly?Ala?Asn?Trp?Thr?Pro?Phe?Met?Thr?Leu?Thr?Asp?Gly

50 55 60Pro?Arg?Gln?Ala?Lys?Trp?Glu?Glu?Ser?Thr?Phe?Lys?Lys?Trp?Val?Glu?65 70 75 80Leu?Val?Pro?Thr?Gly?His?Ala?Met?Trp?Leu?Lys?Gly?Thr?Arg?Arg?Phe

85 90 95Ala?Gln?Asn?Glu?Asp?Gly?Leu?Leu?Gly?His?Trp?Tyr?Lys?Asp?Ile?Thr

100 105 110Pro?Asn?Tyr?Arg?Pro?Leu?Pro?Ser?Ser?Glu?Cys?Pro?Pro?Gly?Ala?Ile

115 120 125Gly?Val?Thr?Tyr?Asp?Thr?Leu?Ser?Val?His?Ala?Pro?Lys?Tyr?Cys?Gln

130 135 140Tyr?Leu?Ala?Arg?Glu?Leu?Gln?Lys?Leu?Gly?Ala?Thr?Phe?Glu?Arg?Arg145 150 155 160Thr?Val?Thr?Ser?Leu?Glu?Gln?Ala?Phe?Asp?Gly?Ala?Asp?Leu?Val?Val

165 170 175Asn?Ala?Thr?Gly?Leu?Gly?Ala?Lys?Ser?Ile?Ala?Gly?Ile?Asp?Asp?Gln

180 185 190Ala?Ala?Glu?Pro?Ile?Arg?Gly?Gln?Thr?Val?Leu?Val?Lys?Ser?Pro?Cys

195 200 205Lys?Arg?Cys?Thr?Met?Asp?Ser?Ser?Asp?Pro?Ala?Ser?Pro?Ala?Tyr?Ile

210 215 220Ile?Pro?Arg?Pro?Gly?Gly?Glu?Val?Ile?Cys?Gly?Gly?Thr?Tyr?Gly?Val225 230 235 240Gly?Asp?Trp?Asp?Leu?Ser?Val?Asn?Pro?Glu?Thr?Val?Gln?Arg?Ile?Leu

245 250 255Lys?His?Cys?Leu?Arg?Leu?Asp?Pro?Thr?Ile?Ser?Ser?Asp?Gly?Thr?Ile

260 265 270Glu?Gly?Ile?Glu?Val?Leu?Arg?His?Asn?Val?Gly?Leu?Arg?Pro?Ala?Arg

275 280 285Arg?Gly?Gly?Pro?Arg?Val?Glu?Ala?Glu?Arg?Ile?Val?Leu?Pro?Leu?Asp

290 295 300Arg?Thr?Lys?Ser?Pro?Leu?Ser?Leu?Gly?Arg?Gly?Ser?Ala?Arg?Ala?Ala305 310 315 320Lys?Glu?Lys?Glu?Val?Thr?Leu?Val?His?Ala?Tyr?Gly?Phe?Ser?Ser?Ala

325 330 335Gly?Tyr?Gln?Gln?Ser?Trp?Gly?Ala?Ala?Glu?Asp?Val?Ala?Gln?Leu?Val

340 345 350Asp?Glu?Ala?Phe?Gln?Arg?Tyr?His?Gly?Ala?Ala?Arg?Glu?Ser?Lys?Leu

The information of 355 360 365 (2) SEQ ID NO:4:

(i) sequence signature:

(A) length: 190 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: genomic dna

(xi) sequence description: SEQ ID NO:4:AAGCTTCGGC ACGAGCATGA GTGTGAATGA TGGTCCAAGG AGGACAGCGC AGAGTCAACA 60GGAGGGCACA TGGAGGCAGA GCGTGGGGCG GAGGAGGCAG ATGGGGAGTC GCGCTGGGGG 120ACGAGGGGGT GTCGCTCGAC TAACAGCTCT CTATCGCTCT TGCTGCTGCT TGTACTACTC 180GAACGACGCC 190, (2) information of SEQ ID NO:5:

(i) sequence signature:

(A) length: 801 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: genomic dna

( xi ) ：SEQ ID NO：5：GGCGGGATTT GTGGCTGTAT TGCGGGCATC TACAAGACCA GCTTCATCTC GGACGACAAC 60ACGAGAGCGG CGAGTCTTCG TACCGTCTGA CCTTCGCAAT CCGCCGAGTC CTTGCCCGTT 120GCCCTGCTTG CTCCTTTCGT ATCTCCTGTG ACTCGGAACG TCGCTCTTCG CCTCTGTCAC 180TTGCCAGGCC GTCCCTTCAA ACTGTCGCCG CCCTCCTCCT CCCAATCTCC TCCTCACGCG 240ACTTGCCACT ACTCTCACTT CTCCTGCTTA CCAAGGCTTA CACTACGCAA CACTAAGCGC 300GGCATCAGGT TCCGTTGCGC GCCTCGTCAC GACCCGACTT TTTTTCGCCC GTTCGCTCGC 360CTCGCTCCGT TGCCGAGCGA AGAACTTCGC CTGCCTTCGA ATCTCTCGCC TTGCTCGTCT 420CGTCCTGCTC CGTTCCACCA CAGATAGACT CACAGCAACA CACTCACAAT GGTCAAGGTG 480CGCACCCACT CCCGCCAGTC CAACTCGCGC GACGAGAGAC CACCGTCTCG TTGACATCAC 540TGACCTCGTC GCTCGCCACC CACCCTCCCG CTCGTCCATG CACACAGGCA GCACCACGAT 600CAGACGAGCA GCAGCACTAC CACGCCACAG TCATGGGCGG ACTCAAGGGC GGCGCGATGG 660GGTTGGCGGC GGGTGGAGCG GGTGCGGTTG CGCTGCAGAG GGCGAACGTA CAGGCGTTCA 720CGAGGTTGAC GCTGCCTCTC AAGGCGTTTG CTGTCACGTA CGTCCCGCGA TCCCTTACTA 780TGCGACTCCC TCGGTGAATT C 801 ( 2 ) SEQ ID NO：6：

(i) sequence signature:

(A) length: 16 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: degenerate oligonucleotide

(xi) sequence description: the information of SEQ ID NO:6:AARTAYTGYC ARTAYC 16 (2) SEQ ID NO:7:

(i) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: degenerate oligonucleotide

(xi) sequence description: the information of SEQ ID NO:7:ATNGAYGAYC AYGCNGC 17 (2) SEQ ID NO:8:

(i) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: degenerate oligonucleotide

(xi) sequence description: the information of SEQ ID NO:8:GCNGCYTGRT CRTCNAT 17 (2) SEQ ID NO:9:

(i) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: degenerate oligonucleotide

(xi) sequence description: the information of SEQ ID NO:9:ATGGAYAGYA GYGAYCC 17 (2) SEQ ID NO:10:

(i) sequence signature:

(A) length: 22 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: peptide

(xi) sequence description: SEQ ID NO:10:Met His Ser Gln Lys Arg Val Val Val Leu Gly Ser Gy Val Ile Gly 15 10 15Leu Ser Ser Ala Leu Ile

The information of 20 (2) SEQ ID NO:11:

(i) sequence signature:

(A) length: 10 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: peptide

(xi) sequence description: the information of SEQ ID NO:11:Tyr Cys Gln Tyr Leu Ala Arg Glu Leu Gln1 5 10 (2) SEQ ID NO:12:

(i) sequence signature:

(A) length: 14 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: peptide

(xi) sequence description: the information of SEQ ID NO:12:Ile Ala Gly Gly Ile Asp Asp Gln Ala Ala Glu Pro Ile Arg1 5 10 (2) SEQ ID NO:13:

(i) sequence signature:

(A) length: 9 amino acid

(B) type: amino acid

(ii) molecule type: peptide

(xi) sequence description: the information of SEQ ID NO:13:Arg Cys Thr Met Asp Ser Ser Asp Pro1 5 (2) SEQ ID NO:14:

(i) sequence signature:

(A) length: 8 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: peptide

(xi) sequence description: the information of SEQ ID NO:14:Ile Ala Gly Ile Asp Asp Gln Ala1 5 (2) SEQ ID NO:15:

(i) sequence signature:

(A) length: 214 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: genomic dna

(xi) sequence description: SEQ ID NO:15:AAGTACTGCC AGTACCTTGC AAGAGAGCTG CAGAAGCTCG GCGCGACGTT TGAGAGACGG 60ACCGTTACGT CGCTTGAGCA GGCGTTCGAC GGTGCGGATT TGGTGGTCAA CGCTACGGGA 120CTTGGTATGT CCCGAACTGC CCCTCTCTAC CTGCAATTTT GCTGATTGAT ATGCTCGCAG 180GCGCCAAGTC GATTGCGGGC ATCGACGACC AAGC 214, (2) information of SEQ ID NO:16:

(i) sequence signature:

(A) length: 10 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: Kozak sequence

(xi) sequence description: the information of SEQ ID NO:16:CCACCATGGC 10 (2) SEQ ID NO:17:

(i) sequence signature:

(A) length: 8 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: consensus sequence

(xi) sequence description: the information of SEQ ID NO:17:YGTGTTYY 8 (2) SEQ ID NO:18:

(i) sequence signature:

(A) length: 8 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: genomic dna

(xi) sequence description: SEQ ID NO:18:TGTATTGC 8

Sequence table (1) general information

(i) applicant: foodstuffs industry research and development institute (Food Industry Research andDevelopment Institute)

(ii) denomination of invention: D-amino acid oxidase of red teliosporeae

(iii) sequence number: 18

(iv) address:

(A) addressee: Fish ﹠amp; Richardson P.C.

(B) street: 225 Franklin Street

(C) city: Boston (Boston)

(D) state: MA

(E) country: USA

(F)ZIP：02110-2804

(v) computer-reader form:

(R) media type: floppy disk

(R) computer: IBM compatible

(C) operating system: Windows95

(D) software: Fast SEQ for Windows Version 2.0b

(the vi) data of current application:

(A) application number: 08/903,624

(R) applying date: on July 31st, 1997

(vii) in the data of first to file:

(A) application number:

(B) applying date:

(viii) lawyer/proxy's information:

(A) title: Tsao, Y.Rocky

(B) registration number: 34,053

(C) certificate number: 06497/006CA1

(ix) telecommunication information:

(A) phone: 617-542-5070

(B) fax: 617-542-8906

(C) fax: the information of 200154 (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 1458 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: genomic dna

( xi ) ：SEQ ID NO：1：ATGCACTCTC AGAAGCGCGT CGTTGTCCTC GGATCAGGCG GTGCGTCTTT TCCCTCTCCT 60CCCCACACCC GACAGTCCTC GACGAGGTGT AGGACGGCGA GCAAAGCTGC CGAGGGCGAT 120CTGGGCTGAC TGAGCGCTCG AGTGTACAGT TATCGGTCTG AGCAGCGCCC TCATCCTCGC 180TCGGAAGGGC TACAGCGTGC ATATTCTCGC GCGCGACTTG CCGGAGGACG TCTCGAGCCA 240GACTTTCGCT TCACCATGGG CTGTGCGTCG TCTCACTGTA GTTGGAGGAT GTCAGCGAGA 300GCTGATCAAT CTCGTCATCC CCGCAGGGCG CGAATTGGAC GCCTTTCATG ACGCTTACAG 360ACGGTCCTCG ACAAGCAAAA TGGGAAGAAT CGACTTTGTG CGTCTCCTTC TACCTCATTC 420TTGGCCTCGA GCTGACGAGT GTATGATACA CAGCAAGAAG TGGGTCGAGT TGGTCCCGAC 480GGGCCATGCC ATGTGGCTCA AGGGGACGAG GCGGTTCGCG CAGAACGAAG ACGGCTTGCT 540CGGGCACTGG TACAAGGACA TCACGCCAAA TGTGCGCCCA CATTCACTCT TCCCTTCGCA 600TGTCTCCGTT TACTGACCCG CCCTCTTTCG CCGTGCGCAG TACCGCCCCC TCCCATCTTC 660CGAATGTCCA CCTGGCGCTA TCGGCGTAAC CTACGACACC CTCTCCGTCC ACGCACCAAA 720GTACTGCCAG TACCTTGCAA GAGAGCTGCA GAAGCTCGGC GCGACGTTTG AGAGACGGAC 780CGTTACGTCG CTTGAGCAGG CGTTCGACGG TGCGGATTTG GTGGTCAACG CTACGGGACT 840TGGTATGTCC CGAACTGCCC CTCTCTACCT GCAATTTTGC TGATTGATAT GCTCGCAGGC 900GCCAAGTCGA TTGCGGGCAT CGACGACCAA GCCGCCGAGC CAATCCGCGG CCAAACCGTC 960CTCGTCAAGT CCCCATGCAA GCGATGCACG ATGGACTCGT CCGACCCCGC TTCTCCCGCC 1020TACATCATTC CCCGACCAGG TGGCGAAGTC ATCTGCGGCG GGACGTACGG CGTGGGAGAC 1080TGGGACTTGT CTGTCAACCC AGAGACGGTC CAGCGGATCC TCAAGCACTG CTTGCGCCTC 1140GACCCGACCA TCTCGAGCGA CGGAACGATC GAAGGCATCG AGGTCCTCCG CCACAACGTC 1200GGCTTGCGAC CTGCACGACG AGGCGGACCC CGCGTCGAGG CAGAACGGAT CGTCCTGCCT 1260CTCGACCGGA CAAAGTCGCC CCTCTCGCTC GGCAGGGGCA GCGCACGAGC CGCGAAGGAG 1320AAGGAGGTCA CGCTTGTGCA TGCGTATGGC TTCTCGAGTG CGGGATACCA GCAGAGTTGG 1380GGCGCGGCGG AGGATGTCGC GCAGCTCGTC GACGAGGCGT TCCAGCGGTA CCACGGCGCG 1440GCGCGGGAGT CGAAGTTG 1458 ( 2 ) SEQ ID NO：2：

(i) sequence signature:

(A) length: 1104 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: cDNA

(ix) feature

(A) title/keyword: encoding sequence

(B) position: 1 ... 1104

275 280 285CGA?GGC?GGA?CCC?CGC?GTC?GAG?GCA?GAA?CGG?ATC?GTC?CTG?CCT?CTC?GAC 912Arg?Gly?Gly?Pro?Arg?Val?Glu?Ala?Glu?Arg?Ile?Val?Leu?Pro?Leu?Asp

290 295 300CGG?ACA?AAG?TCG?CCC?CTC?TCG?CTC?GGC?AGG?GGC?AGC?GCA?CGA?GCC?GCG 960Arg?Thr?Lys?Ser?Pro?Leu?Ser?Leu?Gly?Arg?Gly?Ser?Ala?Arg?Ala?Ala305 310 315 320AAG?GAG?AAG?GAG?GTC?ACG?CTT?GTG?CAT?GCG?TAT?GGC?TTC?TCG?AGT?GCG 1008Lys?Glu?Lys?Glu?Val?Thr?Leu?Val?His?Ala?Tyr?Gly?Phe?Ser?Ser?Ala

The information of 355 360 365 (2) SEQ ID NO:3:

(i) sequence signature:

(A) length: 368 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: protein

(ii) clip types: inner (xi) sequence description: SEQ ID NO:3:Met His Ser Gln Lys Arg Val Val Val Leu Gly Ser Gly Val Ile Gly 15 10 15Leu Ser Ser Ala Leu Ile Leu Ala Arg Lys Gly Tyr Ser Val His Ile

20 25 30Leu?Ala?Arg?Asp?Leu?Pro?Glu?Asp?Val?Ser?Ser?Gln?Thr?Phe?Ala?Ser

35 40 45Pro?Trp?Ala?Gly?Ala?Asn?Trp?Thr?Pro?Phe?Met?Thr?Leu?Thr?Asp?Gly

85 90 95Ala?Gln?Asn?Glu?Asp?Gly?Leu?Leu?Gly?His?Trp?Tyr?Lys?Asp?Ile?Thr

100 105 110Pro?Asn?Tyr?Arg?Pro?Leu?Pro?Ser?Ser?Glu?Cys?Pro?Pro?Gly?Ala?Ile

115 120 125Gly?Val?Thr?Tyr?Asp?Thr?Leu?Ser?Val?His?Ala?Pro?Lys?Tyr?Cys?Gln

165 170 175Asn?Ala?Thr?Gly?Leu?Gly?Ala?Lys?Ser?Ile?Ala?Gly?Ile?Asp?Asp?Gln

180 185 190Ala?Ala?Glu?Pro?Ile?Arg?Gly?Gln?Thr?Val?Leu?Val?Lys?Ser?Pro?Cys

195 200 205Lys?Arg?Cys?Thr?Met?Asp?Ser?Ser?Asp?Pro?Ala?Ser?Pro?Ala?Tyr?Ile

245 250 255Lys?His?Cys?Leu?Arg?Leu?Asp?Pro?Thr?Ile?Ser?Ser?Asp?Gly?Thr?Ile

260 265 270Glu?Gly?Ile?Glu?Val?Leu?Arg?His?Asn?Val?Gly?Leu?Arg?Pro?Ala?Arg

275 280 285Arg?Gly?Gly?Pro?Arg?Val?Glu?Ala?Glu?Arg?Ile?Val?Leu?Pro?Leu?Asp

325 330 335Gly?Tyr?Gln?Gln?Ser?Trp?Gly?Ala?Ala?Glu?Asp?Val?Ala?Gln?Leu?Val

340 345 350Asp?Glu?Ala?Phe?Gln?Arg?Tyr?His?Gly?Ala?Ala?Arg?Glu?Ser?Lys?Leu

The information of 355 360 365 (2) SEQ ID NO:4:

(i) sequence signature:

(A) length: 190 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: genomic DNA, (xi) sequence description: SEQ ID NO:4:AAGCTTCGGC ACGAGCATGA GTGTGAATGA TGGTCCAAGG AGGACAGCGC AGAGTCAACA 60GGAGGGCACA TGGAGGCAGA GCGTGGGGCG GAGGAGGCAG ATGGGGAGTC GCGCTGGGGG 120ACGAGGGGGT GTCGCTCGAC TAACAGCTCT CTATCGCTCT TGCTGCTGCT TGTACTACTC 180GAACGACGCC 190, (2) information of SEQ ID NO:5:

(i) sequence signature:

(A) length: 801 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: genomic dna

(i) sequence signature:

(A) length: 16 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: other

(i) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: degenerate oligonucleotide

(i) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: degenerate oligonucleotide

(i) sequence signature:

(A) length: 17 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: line style

(ii) molecule type: degenerate oligonucleotide

(i) sequence signature:

(A) length: 22 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: peptide

The information of 20 (2) SEQ ID NO:11:

(i) sequence signature:

(A) length: 10 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: peptide

(i) sequence signature:

(A) length: 14 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: peptide

(i) sequence signature:

(A) length: 9 amino acid

(B) type: amino acid

(ii) molecule type: peptide

(i) sequence signature:

(A) length: 8 amino acid

(B) type: amino acid

(D) topological framework: line style

(ii) molecule type: peptide

(i) sequence signature:

(A) length: 214 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: genomic dna

(xi) preface is to describing: SEQ ID NO:15:AAGTACTGCC AGTACCTTGC AAGAGAGCTG CAGAAGCTCG GCGCGACGTT TGAGAGACGG 60ACCGTTACGT CGCTTGAGCA GGCGTTCGAC GGTGCGGATT TGGTGGTCAA CGCTACGGGA 120CTTGGTATGT CCCGAACTGC CCCTCTCTAC CTGCAATTTT GCTGATTGAT ATGCTCGCAG 180GCGCCAAGTC CATTGCGGGC ATCGACGACC AAGC 214, (2) information of SEQ ID NO:16:

(i) sequence signature:

(A) length: 10 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: Kozak sequence

(i) sequence signature:

(A) length: 8 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: consensus sequence

(i) sequence signature:

(A) length: 8 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: line style

(ii) molecule type: genomic dna

(xi) sequence description: SEQ ID NO:18:TGTATTGC 8

Claims

1. isolating nucleic acid, it contains SEQ ID NO:1 or SEQ ID NO:2.

2. carrier, it contains the nucleic acid of claim 1.

3. transformed host cells, it comprises the nucleic acid of claim 1.

4. isolating nucleic acid, it is made up of SEQ ID NO:1 or SEQ ID NO:2.

5. carrier, it contains the nucleic acid of claim 4.

6. transformed host cells, it comprises the nucleic acid of claim 4.

7. isolating D-amino-acid oxidase, wherein said D-amino-acid oxidase is SEQ ID NO:3.

8. isolating spore D-amino-acid oxidase of red winter of class yeast.

9. the D-amino-acid oxidase of claim 8, wherein said D-amino-acid oxidase is SEQ ID NO:3.