CN103667213B

CN103667213B - Warm carbohydrase GA II and gene and application in a kind of acidity

Info

Publication number: CN103667213B
Application number: CN201310739004.7A
Authority: CN
Inventors: 姚斌; 罗会颖; 华慧芳; 石鹏君; 黄火清; 王亚茹; 柏映国; 杨培龙; 孟昆
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Animal Science of CAAS
Priority date: 2013-12-26
Filing date: 2013-12-26
Publication date: 2016-05-04
Anticipated expiration: 2033-12-26
Also published as: CN103667213A

Abstract

The present invention relates to warm carbohydrase and gene and application in a kind of acidity. Concrete, the invention provides one and derive from Talaromyces? emersonii? warm carbohydrase GA II in the new acidity of CBS204.75, does it have as SEQ? ID? amino acid sequence shown in NO.1 or 2, the gene of the above-mentioned carbohydrase of encoding is provided, does it have as SEQ? ID? nucleotide sequence shown in NO.3 or 4, and the recombinant vector that comprises this gene, recombinant bacterial strain and recombinase and application thereof. The pH4.0 of carbohydrase of the present invention, is very stable between 3-6 in pH scope, is typical acid carbohydrase; 50 DEG C of optimum temperatures, can keep more than 50% activity between 37 DEG C-55 DEG C. Be suitable for the food industry such as the production of drinks and fuel alcohol, glucose, fructose syrup, organic acid, monosodium glutamate etc. use in multiple industries.

Description

Warm carbohydrase GA II and gene and application in a kind of acidity

Technical field

The present invention relates to biological technical field, particularly, relate in a kind of acidity warm carbohydrase GA II and gene thereof andApplication.

Background technology

Carbohydrase, i.e. glucoamylase (glucoamylase, E.C.3.2.1.3), is the weight in Starch Hydrolysis processWant enzyme. Carbohydrase is a kind of circumscribed-type glycosidase, and Main Function is from non-reducing end hydrolyzed starch, dextrin, glycogenEtc. α-Isosorbide-5-Nitrae glycosidic bond and α-1 in carbochain, 6 glycosidic bonds, cut glucose unit one by one, and make to be hydrolyzedGlucose generation change of configuration, obtains end-product β-D-Glucose. Carbohydrase also can weakly hydrolyse α-1 in addition, 3 glucosidesKey. Carbohydrase is output maximum, enzyme preparation that range of application is the widest in the world. Not only for drinks and fuel wineThe production of essence, is also widely used in multiple fields of the food industry such as glucose, fructose syrup, organic acid, monosodium glutamate（PolakovicandBryjak,2004,BiochemEngJ18:57–64）。

Carbohydrase is present in animal, plant and microorganism widely. Wherein microorganism is the important sources of carbohydrase(PardeepKandSatyanarayanaT, 2009, CritRevBiotechnol29:225 – 255). In industry, applyCarbohydrase be mainly from filamentous fungi and saccharomyces such as mould genus (Aspergillus), rhizopus (Rhizopus)(Saccharmyces) in, obtain. Along with the extensive use of technique for gene engineering, a lot of glucoamylase genes by gramGrand and carried out heterogenous expression (PardeepKandSatyanarayanaT, 2009, CritRevBiotechnol29:225 – 255). Report carbohydrase belong to more glycoside hydrolase 15 families (HenrissatBetal.1991,BiochemJ). Generally by catalytic domain (catalyticdomain, CD), starch binding domain (starch-bindingdomain,SBD) and the O-glycosylation link field (O-glycosylatedlinkerdomain) that connects CD and SBD form.

The pH adaptive of the carbohydrase that different microorganisms produces, heat resistance, catalysis characteristics etc. are not identical yet. Right at presentThe mensuration of carbohydrase character mainly concentrates in the research of optimum temperature and optimum pH, the carbohydrase of industrial applicationMostly be its heat endurance of application. The optimal reaction pH value of most of fungi carbohydrase is 4.5-5.0, at acid conditionLower stable.

Carbohydrase of the present invention has high enzyme vigor under acid condition, and very steady under acid and medium temperature conditionFixed, be adapted at a lot of industrial application. Further screening Novel saccharification enzyme or clonal expression Novel saccharification enzyme gene,Improve the operative temperature of carbohydrase by site-directed mutagenesis technique, can make starch saccharification process as starch liquefacation shortIn time, complete, thereby reduce energy resource consumption and production cost, will open up more wide to the application of carbohydrase in industryWealthy prospect, has important commercial value.

Summary of the invention

The object of the invention is to provide a kind of carbohydrase.

A further object of the present invention is to provide the gene of the above-mentioned carbohydrase of above-mentioned coding.

A further object of the present invention is to provide the recombinant vector that comprises above-mentioned carbohydrase encoding gene.

A further object of the present invention is to provide the recombinant bacterial strain that comprises above-mentioned glucoamylase gene.

A further object of the present invention is to provide a kind of method of preparing Novel saccharification enzyme.

A further object of the present invention is to provide the application of above-mentioned Novel saccharification enzyme.

The present invention's technical problem first to be solved is to overcome the deficiencies in the prior art, provides a kind of acidproof, heat-resistingThe good new enzyme of character. The present invention from TalaromycesemersoniiCBS204.75 obtain one new acidproof,The carbohydrase that action pH is wide. The carbohydrase that it produces be suitable for the production of drinks and fuel alcohol, glucose,The food industry such as fructose syrup, organic acid, monosodium glutamate etc. use in multiple industries.

The present invention has obtained a kind of heat-resisting carbohydrase GA II from above-mentioned bacterial strains, and its amino acid sequence is as SEQIDNO.Shown in 1:

1MLVKPLLSQVALLAVSLGASAESLVTRASDLNAFIASESDIALRGVLANIGPNGKAVPGA

61DAGIVVASPSKTNPDYWYTWTRDAALTFKYLIDRFVDGDSSLQGLIQDYISAQAKLQTVQ

121NPSGDLSSGAGLAEPKFYINETAFLGQWGRPQRDGPALRATALIAYSQWLIDNGYSDVAL

181SHVWPIASNDLAYVAQYWNQTGYDLWEEVDGSSFFTIAVSHRALVEGAALAKRLNQTVGD

241YSSVASQILCFQQSFWTGSYIDSNINLVHDVNRTGKDANSILASIHVFDPEAACDDSTFQ

301PCSSYALANHKAVVDSFRNLYSVNDGIKKGSAVAIGRYLCTLAAAEQLYDALYQWQKQGS

361LNITSISLDFFRDFDSSVNTGTYPSSSDTYASLTKAIKTYADGFVSIVQKYTPDNGALSE

421QFSKVNGSQTSAIDLTWSYAAFLTAASRRNGTMGPSWGASQANSVPSSWATTTLKGTYKA

481AMVTSWPTGLVSKNSSSSATGTATGSASASTTSKSAANPGAAGAMMASGY GGNQLTFAFL

541SILFLYLIF*

Wherein, 549 amino acid of this zymoprotein total length and a terminator codon, 21 amino acid of N end are signalPeptide sequence " MLVKPLLSQVALLAVSLGASA ".

Therefore, the amino acid sequence of ripe carbohydrase GA II is as SEQIDNO.2:

1ESLVTRASDLNAFIASESDIALRGVLANIGPNGKAVPGADAGIVVASPSKTNPDYWYTWT

61RDAALTFKYLIDRFVDGDSSLQGLIQDYISAQAKLQTVQNPSGDLSSGAGLAEPKFYINE

121TAFLGQWGRPQRDGPALRATALIAYSQWLIDNGYSDVALSHVWPIASNDLAYVAQYWNQT

181GYDLWEEVDGSSFFTIAVSHRALVEGAALAKRLNQTVGDYSSVASQILCFQQSFWTGSYI

241DSNINLVHDVNRTGKDANSILASIHVFDPEAACDDSTFQPCSSYALANHKAVVDSFRNLY

301SVNDGIKKGSAVAIGRYLCTLAAAEQLYDALYQWQKQGSLNITSISLDFFRDFDSSVNTG

361TYPSSSDTYASLTKAIKTYADGFVSIVQKYTPDNGALSEQFSKVNGSQTSAIDLTWSYAA

421FLTAASRRNGTMGPSWGASQANSVPSSWATTTLKGTYKAAMVTSWPTGLVSKNSSSSATG

481TATGSASASTTSKSAANPGAAGAMMASGYGGNQLTFAFLSILFLYLIF*

Maturation protein is made up of 528 amino acid and a terminator codon, and theoretical molecular is 56.4kDa, this enzymeBelong to glycosyl hydrolase the 15th family. The carbohydrase amino acid sequence of deriving is carried out to BLAST in GenBankIt is a kind of new carbohydrase that comparison is found.

The invention provides the gene of the above-mentioned heat-resisting carbohydrase of coding.

This enzyme full length gene 1858bp, sequence is as shown in SEQIDNO.3:

1ATGTTGGTCAAGCCATTGTTGTCTCAGGTCGCCCTGCTGGCGGTCTCCTTGGGTGCCTCG

61GCAGAGTCGTTGGTGACACGAGCTTCCGATCTGAATGCCTTCATTGCGTCAGAATCAGAT

121ATCGCTCTGCGTGGCGTCTTGGCCAATATTGGCCCTAACGGAAAAGCCGTTCCGGGAGCA

181GATGCGGGCATTGTGGTTGCCAGCCCATCCAAGACGAATCCTGACTGTGAGTAGATGTCG

241TAGATCCCACTATTGCCATGCCAGATCTAGGACGCTAATCTAATCGCGCTCTTTCAAGAC

301TGGTACACCTGGACGAGAGATGCTGCCTTGACATTCAAGTACTTGATTGACCGGTTCGTC

361GACGGCGATTCGTCCCTGCAGGGCCTGATCCAGGATTACATTTCCGCCCAAGCAAAGCTG

421CAGACCGTCCAGAACCCATCAGGAGACCTGTCCTCCGGAGCCGGTCTTGCAGAGCCCAAG

481TTTTACATCAACGAGACGGCCTTTCTGGGCCAGTGGGGTCGTCCCCAACGTGATGGGCCG

541GCATTGCGTGCCACGGCTCTGATCGCATATTCCCAATGGCTGATAGACAATGGCTACTCG

601GACGTTGCTCTGTCGCACGTCTGGCCCATTGCGAGCAACGATCTGGCCTATGTTGCCCGG

661TACTGGAACCAGGCCGGCTATGACCTCTGGGAAGAGGTCGACGGTTCGTCGTTCTTCACC

721ATTGCCGTCTCTCACCGGGCCCTCGTCGAGGGAGCTGCGCTGGCGAAGCGCCTCAACCAG

781ACTGTTGGCGACTACAGTTCGGTGGCCTCCCAGATCCTGTGCTTCCAGCAGTCCTTCTGG

841ACAGGGAGCTACATCGACTCGAATATCAATCTGGTGCACGACGTCAACCGGACTGGCAAA

901GACGCCAACTCCATTCTGGCCTCGATCCACGTCTTTGACCCGGAGGCCGCATGTGACGAT

961TCAACATTCCAGCCCTGCTCGTCATACGCGCTGGCGAACCACAAAGCTGTCGTGGACTCG

1021TTCAGAAACCTCTATTCTGTCAACGATGGCATTAAGAAGGGATCCGCAGTGGCAATCGGA

1081CGGTATGCGGAGGATATATACTACGACGGGAATCCATGGTAGGGAGCTTCCTTTGCTGTT

1141TTTCAAACCTTTTTTTTTACCGAATCTACTTTTTATCTACTTCGATGAACCTGGAAACAA

1201CGCTGATCTATGGTCTAGGTACCTGTGCACACTGGCGGCAGCCGAACAGCTCTACGATGC

1261CCTCTATCAGTGGCAGAAACGGGGCTCGCTGAACATCACATCCATCTCTCTGGATTTCTT

1321CCGCGACTTTGACTCCTCCGTCAACACCGGGACGTACCCGTCTTCCTCCGACACCTACGC

1381CTCGCTGACGAAGGCGATCAAGACCTATGCAGACGGCTTCGTCAGCATCGTGCAGAAATA

1441CACGCCAGACAACGGGGCCCTCTCAGAGCAATTCTCCAAAGTCAACGGCAGCCAGACGTC

1501CGCCATCGACCTCACATGGTCCTACGCTGCCTTCTTGACGGCGGCCAGCCGTCGCAACGG

1561CACCATGGGGCCGAGCTGGGGAGCATCACAGGCGAACAGCGTCCCGTCTTCGTGGGCGAC

1621GACCACGCTGAAAGGAACCTACAAAGCTGCGATGGTGACTTCGTGGCCGACCGGGTTGGT

1681GAGCAAGAACAGCAGCAGCAGCGCCACGGGGACAGCCACTGGTTCCGCGTCCGCATCCAC

1741CACGAGTAAAAGCGCCGCTAACCCTGGCGCAGCAGGGGCAATGATGGCTTCTGGCTATGG

1801CGGCAACCAGCTGACATTCGCCTTCCTTTCTATTCTCTTTCTCTATCTGATTTTCTGA

The cDNA sequence that the invention provides the above-mentioned heat-resisting carbohydrase of coding, total length 1686bp, as SEQIDNO.Shown in 4.

1ATGTTGGTCAAGCCATTGTTGTCTCAGGTCGCCCTGCTGGCGGTCTCCTTGGGTGCCTCG

61GCAGAGTCGTTGGTGACACGAGCTTCCGATCTGAATGCCTTCATTGCGTCAGAATCAGAT

121ATCGCTCTGCGTGGCGTCTTGGCCAATATTGGCCCTAACGGAAAAGCCGTTCCGGGAGCA

181GATGCGGGCATTGTGGTTGCCAGCCCATCCAAGACGAATCCTGACTACTGGTACACCTGG

241ACGAGAGATGCTGCCTTGACATTCAAGTACTTGATTGACCGGTTCGTCGACGGCGATTCG

301TCCCTGCAGGGCCTGATCCAGGATTACATTTCCGCCCAAGCAAAGCTGCAGACCGTCCAG

361AACCCATCAGGAGACCTGTCCTCCGGAGCCGGTCTTGCAGAGCCCAAGTTTTACATCAAC

421GAGACGGCCTTTCTGGGCCAGTGGGGTCGTCCCCAACGTGATGGGCCGGCATTGCGTGCC

481ACGGCTCTGATCGCATATTCCCAATGGCTGATAGACAATGGCTACTCGGACGTTGCTCTG

541TCGCACGTCTGGCCCATTGCGAGCAACGATCTGGCCTATGTTGCCCGGTACTGGAACCAG

601GCCGGCTATGACCTCTGGGAAGAGGTCGACGGTTCGTCGTTCTTCACCATTGCCGTCTCT

661CACCGGGCCCTCGTCGAGGGAGCTGCGCTGGCGAAGCGCCTCAACCAGACTGTTGGCGAC

721TACAGTTCGGTGGCCTCCCAGATCCTGTGCTTCCAGCAGTCCTTCTGGACAGGGAGCTAC

781ATCGACTCGAATATCAATCTGGTGCACGACGTCAACCGGACTGGCAAAGACGCCAACTCC

841ATTCTGGCCTCGATCCACGTCTTTGACCCGGAGGCCGCATGTGACGATTCAACATTCCAG

901CCCTGCTCGTCATACGCGCTGGCGAACCACAAAGCTGTCGTGGACTCGTTCAGAAACCTC

961TATTCTGTCAACGATGGCATTAAGAAGGGATCCGCAGTGGCAATCGGACGGTATGCGGAG

1021GATATATACTACGACGGGAATCCATGGTACCTGTGCACACTGGCGGCAGCCGAACAGCTC

1081TACGATGCCCTCTATCAGTGGCAGAAACGGGGCTCGCTGAACATCACATCCATCTCTCTG

1141GATTTCTTCCGCGACTTTGACTCCTCCGTCAACACCGGGACGTACCCGTCTTCCTCCGAC

1201ACCTACGCCTCGCTGACGAAGGCGATCAAGACCTATGCAGACGGCTTCGTCAGCATCGTG

1261CAGAAATACACGCCAGACAACGGGGCCCTCTCAGAGCAATTCTCCAAAGTCAACGGCAGC

1321CAGACGTCCGCCATCGACCTCACATGGTCCTACGCTGCCTTCTTGACGGCGGCCAGCCGT

1381CGCAACGGCACCATGGGGCCGAGCTGGGGAGCATCACAGGCGAACAGCGTCCCGTCTTCG

1441TGGGCGACGACCACGCTGAAAGGAACCTACAAAGCTGCGATGGTGACTTCGTGGCCGACC

1501GGGTTGGTGAGCAAGAACAGCAGCAGCAGCGCCACGGGGACAGCCACTGGTTCCGCGTCC

1561GCATCCACCACGAGTAAAAGCGCCGCTAACCCTGGCGCAGCAGGGGCAATGATGGCTTCT

1621GGCTATGGCGGCAACCAGCTGACATTCGCCTTCCTTTCTATTCTCTTTCTCTATCTGATT

1681TTCTGA

Wherein, the base sequence of signal peptide is: ATGTTGGTCAAGCCATTGTTGTCTCAGGTCGCCCTGCTGGCGGTCTCCTTGGGTGCCTCGGCA

DNA sequence dna and cDNA sequence alignment analysis result show: the structural gene ga II of Novel saccharification enzyme GA IITotal length 1,858bp, contains 2 intrones, and its sequence is respectively: 227 – 298bp and 1,122 – 1,221bp, cDNALong 1,686bp.

CDNA sequence after removal signal peptide sequence is as shown in SEQIDNO.5.

The present invention also provides the recombinant vector that comprises above-mentioned glucoamylase gene ga II. Be preferably pPIC9-ga II.Carbohydrase maturation protein encoding gene of the present invention is inserted between the restriction enzyme site that expression vector is suitable,Make the exercisable and expression regulation sequence of its nucleotide sequence, and carrier signal peptide sequence is connected. As the present inventionA most preferred embodiment, be preferably and be inserted on plasmid pPIC9 removing the glucoamylase gene of signal peptideEcoRI and NotI restriction enzyme site between, make this nucleotide sequence be positioned at the downstream of AOX1 promoterAnd regulated and controled by it, obtain expression of recombinant yeast plasmid pPIC9-ga II.

The present invention also provides the recombinant bacterial strain that comprises above-mentioned glucoamylase gene, is preferably recombinant bacterial strain GS115/ga II.

The present invention also provides a kind of method of preparing Novel saccharification enzyme, comprises the following steps:

1) with claim recombinant vector transformed host cell, obtain recombinant bacterial strain;

2) cultivate recombinant bacterial strain, the expression of induction restructuring glucoamylase gene; And

3) reclaim the also expressed carbohydrase of purifying.

Wherein, preferred described host cell is Pichia pastoris, beer yeast cells, Bacillus coli cells or silkShape fungal cell, preferably transforms Pichia pastoris (Pichicpastoris) GS115 by expression of recombinant yeast plasmid,Obtain recombinant bacterial strain GS115/ga II.

The present invention also provides the application of above-mentioned carbohydrase.

The invention provides a new glucoamylase gene, the carbohydrase of its coding has certain acid resistance, can beThe food industry such as the production of drinks and fuel alcohol, glucose, fructose syrup, organic acid, monosodium glutamate etc. multiple industriesMiddle use.

Brief description of the drawings

Fig. 1 recombinate carbohydrase SDS-PAGE analyze, M: low molecular weight protein Marker; 1: finish red fermentThe unpurified carbohydrase GA II that matrix reaches; Carbohydrase GA II after 2:endoH de-glycosylation.

The optimal pH curve of Fig. 2 carbohydrase GA II.

The pH stability curve of Fig. 3 carbohydrase GA II.

The optimum temperature curve of Fig. 4 carbohydrase GA II effect.

The heat endurance curve of Fig. 5 carbohydrase GA II.

Detailed description of the invention

Experiment condition:

1, bacterial strain and carrier: TalaromycesemersoniiCBS204.75 is purchased from Dutch fungi strain preservation center(CBS). Yeast expression vector pPIC9 and bacterial strain GS115 are purchased from Invitrogen company.

2, enzyme and other biochemical reagents: restriction endonuclease is purchased from TaKaRa company, and ligase is purchased from Invitrogen public affairsDepartment. Maltose is purchased from Sigma company, and other is all that domestic reagent (all can be bought from common biochemical reagents companyTo).

3, culture medium:

(1) Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).

(2) TalaromycesemersoniiCBS204.75 culture medium is potato juice culture medium: 1000mL horseBell potato juice, 10g glucose, 25g agar, pH2.5.

(3) MM solid medium: 1.34%YNB, 0.00004%Biotin, 0.5% methyl alcohol, 1.5% agarose.

(4) MD solid medium: 1.34%YNB, 0.00004%Biotin, 2% glucose, 1.5% agarose.

(5) BMGY culture medium: 1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin,1% glycerine (V/V).

(6) BMMY culture medium: replace glycerine divided by 0.5% methyl alcohol, all the other compositions are all identical with BMGY.

Illustrate: in the present invention, the genetic recombination of using learns a skill and is the routine techniques in this area. In following enforcementThe technology not describing in detail in example, all carries out according to the related Sections in following laboratory manual or document or part,Comprise: the people such as Sambrook, MolecularCloning, ALaboratoryManual (the 3rd edition .2001);Kriegler, GeneTransferandExpression:ALaboratoryManual (1990); And CurrentProtocolsinMolecularBiology (people such as Ausubel compiles, 1994).

The clone of embodiment 1TalaromycesemersoniiCBS204.75 carbohydrase encoding gene ga II

Extract TalaromycesemersoniiCBS204.75 genomic DNA:

The Liquid Culture mycelium of 3 days is filtered and puts into mortar with aseptic filter paper, add 2mL extract, grind5min, is then placed in lapping liquid 50mL centrifuge tube, and 65 DEG C of water-bath cracking 20min, mix every 10minOnce, the centrifugal 5min of 10000rpm at 4 DEG C. Get supernatant extrct foreigh protein removing in phenol/chloroform, then getReset and add into equal-volume isopropyl alcohol, leave standstill after 5min in room temperature, the centrifugal 10min of 10000rpm at 4 DEG C. Abandon supernatant,70% ethanol washed twice for precipitation, vacuum drying, adds appropriate TE dissolving, be placed in-20 DEG C for subsequent use.

Close according to the conservative WGRPQRDG of the 15th family's glucoamylase gene and YDAV (I/L) YQW sequences DesignBecome degenerate primer P1(5'-TGGGGHCGTCCDCARMGNGAYGG-3'), P2(5'-CCACTGRTARAYNGCRTCRTA-3') (wherein: Y=C/T, R=A/G, M=A/C, H=A/C/T, N=A/T/G/C）。

Utilize degenerate primer P1 and P2 to carry out taking the total DNA of TalaromycesemersoniiCBS204.75 as templatePcr amplification. PCR response parameter is: 95 DEG C of 5min; 94 DEG C of 30sec, 55-45 DEG C of 30sec (wherein each followingAfter ring, renaturation temperature declines 1 DEG C), 72 DEG C of 1min, 10 circulations, then enter second cyclic program: 94 DEG C 30Sec, 45 DEG C of 30sec, 72 DEG C of 1min, after 25 circulations; 72 DEG C of 10min, agarose electrophoresis detects. ObtainThe fragment of about 700bp, reclaims rear and pEASY-T₃Carrier is connected, and send Beijing Bioisystech Co., Ltd of farsighted Boxing sectionCarry out sequencing.

According to measuring sequence results, in the GenBank of NCBI, utilize BLASTX[http://www.ncbi.nlm.nih.gov/BLAST] carries out sequence alignment, tentatively judges that this genetic fragment is carbohydrase baseBecause of fragment, and carry out the Study on Similarity of this fragment. This sheet segment length 760bp.

The nucleotide sequence obtaining according to order-checking, each three the TAIL-PCR Auele Specific Primers of design upstream and downstream:Design direction is the zone of ignorance direction that needs amplification, and the Position Design of sp2 is in the inner side of sp1, and sp3 is positioned at sp2Inner side. Distance between every two primers does not have strict regulations, the general 18～26nt of primer length, annealing temperatureAt 55～65 DEG C. And by they difference called after Gamyusp1, Gamyusp2, Gamyusp3 (draw by upstream specificityThing), Gamydsp1, Gamydsp2, Gamydsp3 (downstream Auele Specific Primer) is in table 1.

Table 1. carbohydrase GA II TAIL-PCR Auele Specific Primer

Obtain the flanking sequence of known sequence by reverse TAIL-PCR, amplification obtains after product reclaims sending threeThe order-checking of rich Bioisystech Co., Ltd. The core fragment that degenerate primer is obtained and the flank obtaining through TAIL-PCRSequence is spliced and is obtained ga II full-length gene. Show through sequence analysis, this gene DNA total length be one long by 1,The genetic fragment of 858bp.

The RT-PCR of embodiment 2 glucoamylase genes analyzes

The total RNA that extracts TalaromycesemersoniiCBS204.75, utilizes reverse transcriptase to obtain one of cDNABar chain, then design appropriate primer (GA II F:5'-CGTCCATTCGTTTGAGCACATTCGTTCGTC-3'，GAⅡR：5'-GGCCGGGTCAAAGATATTGATACGTGGCT-3'

) this strand cDNA that increases, the cDNA sequence of acquisition carbohydrase, amplification obtains product and reclaims Hou Songrui Boxing section biologyTechnology Co., Ltd.'s order-checking.

The genomic dna sequence of carbohydrase and cDNA the sequencing results show, the knot of heat-resisting carbohydrase GA IIStructure full length gene 1,858bp, long 1, the 686bp of cDNA. Contain 2 intrones, its sequence is respectively: 227 – 298Bp and 1,122 – 1,221bp. 21 amino acid of N end are signal peptide sequence.

The recombinate preparation of carbohydrase of embodiment 3.

Expression vector pPIC9 is carried out to double digestion (EcoRI+NotI), simultaneously by logical the gene ga II of coding carbohydraseCross PCR and remove signal peptide sequence and add restriction enzyme site EcoRI and NotI, the primer be (GamyFcom-s:ATGGAATTCGAGTCGTTGGTGACACGAGC；GamyRcom：GATGCGGCCGCTCAGAAAATCAGATAGAGAAAGAGAATAGAAAGG), product warp(EcoRI+NotI) double digestion, the genetic fragment that obtains encoding mature carbohydrase is connected with expression vector pPIC9,The recombinant plasmid pPIC-ga II that acquisition contains glucoamylase gene ga II also transforms Pichia pastoris GS115, coating MDFlat board, treats after 3 days that bacterium colony grows, and with sterilized toothpick picking list bacterium colony from the long MD plate that has transformant, pressesFirst put MM according to numbering upper, more dull and more stereotyped upper 30 DEG C of the MD that puts corresponding numbering cultivates 1～2 day, grow to bacterium colony.On MD flat board, can be inoculated in the centrifuge tube that 5mLBMGY culture medium is housed by normal growth transformant 30 DEG C, 260The centrifugal supernatant that goes after rpm shaking table cultivation 48h, the BMMY training that adds again 1mL to contain 0.5% methyl alcohol in centrifuge tubeSupport base, cultivate after 48h at 30 DEG C, 260rpm induction, centrifuging and taking supernatant detects for enzymatic activity, therefrom filters outThere is the transformant of diastatic activity. Obtain recombinant pichia yeast strain GS115/ga II. Screen altogether transformant 48Individual, wherein there are have 19 of diastatic activity.

Expression vector with same method member containing signal peptide sequence, and transform Pichia anomala expression.

High bacterial strain alive enzyme is re-seeded into and is equipped with in 400mLBMGY nutrient solution, 30 DEG C of 250rpm vibration trainingsSupport after 48h centrifugal collection thalline. Then resuspended in 200mLBMMY culture medium, 30 DEG C of 250rpm vibrationsCultivate. After induction 48h, measured the vigor of carbohydrase in supernatant and added methyl alcohol every 24 hours. SDS-PAGEResult (Fig. 1) shows, restructuring carbohydrase has obtained expression in Pichia pastoris. Induce the saccharification of recombinating after 60 hoursThe expression of enzyme is 10U/mL.

The recombinate activity analysis of carbohydrase of embodiment 4

At pH4.0, under 50 DEG C of conditions, the reaction system of 140 μ L comprises the dilution enzyme liquid that 5 μ L are suitable, 65 μ LbufferWith 70 μ L2% maltose, reaction 10min, boiling water boiling 3min. To cooled on ice. The amount of the glucose discharging is usedThe method of glucose oxidase is measured. 1 Ge Meihuo unit (U) is defined as and per minutely under given condition discharges 1The enzyme amount that μ mol glucose is required.

The recombinate property testing of carbohydrase GA II of embodiment 5

Purified carbohydrase GA II is carried out enzymatic reaction to measure its optimal pH under different pH. Buffering usedLiquid is the 0.1mol/LKCl-HCl buffer solution of pH0.5 – 2.2, the McIlvaine buffering of the 0.1mol/L of pH2.5～3.0Liquid, 0.1mol/L acetic acid-sodium acetate buffer of pH3.5 – 6.0, the 0.1mol/LMcIlvaine buffer solution of pH7.0 – 8.0,The 0.1Mglycine-NaOH buffer solution of pH9.0 – 12.0. The carbohydrase GA II of purifying is in the buffer system of different pH,The pH adaptive result (Fig. 2) of measuring at 50 DEG C shows: the suitableeest action pH of GA II is 4.0, can protect at pH3.5-4.5The enzyme of holding more than 80% is lived.

Enzyme liquid is processed to 1h in the buffer solution of different pH values at 37 DEG C, then measure the pH of enzymatic activity with studying enzymeStability. Result shows (Fig. 3), and GA II is all very stable between 3.0-6.0 in pH scope.

The acetate salt buffer liquid system of the 0.1mol/L that is determined at pH4.0 of optimum temperature and different temperatureUnder (20～70 DEG C), carry out enzymatic reaction. Enzyme reaction optimum temperature measurement result (Fig. 4) shows, GA II the suitableeest50 DEG C of operative temperatures, between 37 DEG C and 55 DEG C, keep more than 50% enzyme to live.

Measure carbohydrase and under 50 DEG C and 40 DEG C of conditions, be incubated respectively different time and measure enzyme activity, drafting enzymeHeat endurance curve. At 40 DEG C, GA II heat endurance is fine, after processing 60min, still can retain nearly 70% enzymeLive. (Fig. 5).

The recombinate kinetic constant of carbohydrase GA II of embodiment 6

With the maltose of variable concentrations (0.4-3mmol/L) be substrate, slow at the acetate of the 0.1mol/L of pH4.0Rush in liquid system, measure enzymatic activity at 50 DEG C, 4 minutes reaction time, calculate its reaction speed at 50 DEG C,Utilize double-reciprocal plot method to try to achieve its K_mValue and V_max. After measured, this carbohydrase at 50 DEG C taking maltose as substrateK_mValue is 0.45mg/ml, maximum reaction velocity V_maxBe 769 μ mol/min/mg. Specific activity is 784U/mg.

Claims

1. a carbohydrase GA II, is characterized in that, its amino acid sequence by nucleotide sequence as SEQIDNO.3,4 or 5Shown gene code.

2. glucoamylase gene ga II, is characterized in that, its gene order is as shown in SEQIDNO.3,4 or 5.

3. comprise the recombinant expression carrier of glucoamylase gene ga II described in claim 2.

4. comprise the recombinant expression carrier pPIC9-ga II of glucoamylase gene ga II described in claim 2, wherein, by sequence asGlucoamylase gene ga II shown in SEQIDNO.5 is inserted into EcoRI and the NotI restriction enzyme site on plasmid pPIC9Between, make this nucleotide sequence be positioned at the downstream of AOX1 promoter and regulated and controled by it, obtain recombinant expression plasmid pPIC9-ga II.

5. comprise the recombinant bacterial strain of glucoamylase gene ga II described in claim 2.

6. comprise the recombinant bacterial strain GS115/ga II of glucoamylase gene ga II described in claim 2, wherein by described in claim 4Recombinant expression carrier pPIC9-ga II transform Pichia pastoris GS115, obtain recombinant bacterial strain GS115/ga II.

7. a method of preparing carbohydrase GA II, is characterized in that, comprises the following steps:

1) with the recombinant vector transformed host cell of claim 4, obtain recombinant bacterial strain;

2) cultivate recombinant bacterial strain, the expression of induction restructuring carbohydrase;

3) reclaim the also expressed carbohydrase GA II of purifying.

8. the application of glucoamylase gene ga II in suitability for industrialized production carbohydrase according to claim 2.