CN102827817B - Thermotolerant glucoamylase GAI, its gene and application thereof - Google Patents
Thermotolerant glucoamylase GAI, its gene and application thereof Download PDFInfo
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Abstract
The invention relates to a novel thermotolerant glucoamylase and its application. Specifically, the invention provides novel thermotolerant glucoamylase GAI derived from Bispora sp.MEY-1CGMCC 2500. The glucoamylase GAI has an amino acid sequence as shown in SEQ ID NO.1 or 2. The invention also provides a gene for encoding the above glucoamylase, a recombinant vector, a recombinant bacterial strain and recombinase containing the gene, and an application thereof, wherein the gene has a nucleotide sequence as shown in SEQID NO.3 or 4. The glucoamylase provided by the invention has the following properties: the most appropriate operative pH of GAI is 3.5, and more than 90% of enzyme activity can be maintained when pH is 2.0-4.5; the glucoamylase is stable when pH is 1.5-12.0; the most appropriate operative temperature is 75 DEG C; and GAI has good thermal stability at the temperature of 70-75 DEG C.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, relate to a kind of heat-resisting saccharifying enzyme GAI and gene and application.
Background technology
Saccharifying enzyme, glucoamylase (glucoamylase, EC.3.2.1.3), be the important enzyme in the Starch Hydrolysis process.Saccharifying enzyme is a kind of circumscribed-type Glycosylase, and Main Function is α the carbochains such as non-reducing end hydrolyzed starch, dextrin, glycogen-Isosorbide-5-Nitrae glycosidic link and α-1,6 glycosidic links, cut glucose unit one by one, and make the glucose generation change of configuration be hydrolyzed, obtain end product β-D-Glucose.Saccharifying enzyme also can weakly hydrolyse α-1,3 glycosidic link in addition.Saccharifying enzyme is the zymin that turnout is maximum in the world, range of application is the widest.Not only, for the production of drinks and fuel alcohol, also be widely used in a plurality of fields (Polakovic and Bryjak, 2004, Biochem Eng J18:57-64) of the foodstuffs industry such as glucose, fructose syrup, organic acid, monosodium glutamate.
Saccharifying enzyme is present in animal, plant and microorganism widely.Wherein microorganism is the important sources (Pardeep K and Satyanarayana T, 2009, Crit Rev Biotechnol 29:225-255) of saccharifying enzyme.The saccharifying enzyme of applying in industry mainly obtains from the filamentous fungus such as mould genus (Aspergillus), Rhizopus (Rhizopus) and yeast belong (Saccharmyces).Along with the widespread use of genetic engineering technique, a lot of glucoamylase genes have been cloned and have carried out heterogenous expression (Pardeep K and Satyanarayana T, 2009, Crit Rev Biotechnol 29:225-255).The saccharifying enzyme of report belongs to glycoside hydrolase 15 families (Henrissat B et al.1991, Biochem J) more.Generally the 0-glycosylation link field (O-glycosylated linker domain) by catalytic domain (catalytic domain, CD), starch binding domain (starch-binding domain, SBD) and connection CD and SBD forms.
The pH adaptive of the saccharifying enzyme that different microorganisms produces, thermotolerance, catalysis characteristics etc. are not identical yet.At present the mensuration of saccharifying enzyme character mainly concentrated in the research of optimum temperuture and optimum pH, mostly the saccharifying enzyme of industrial application is its thermostability of application.The optimal reaction pH value of most of fungi saccharifying enzyme is 4.5-5.0, stable under acidic conditions.Optimal reactive temperature is 40-60 ℃ (Norouzian D et al.2006, Biotechnol Adv 24:80-85).At present, the report that the saccharifying enzyme that optimum temperuture is 70 ℃ is also arranged, derive from respectively Trichoderma Reesi (Fagerstrom R et al.1995, Biotechnol Appl Biochem 21:223-231), Talaromyces emersonii (Nielsen BR.2002, Protein Expression and Purification 26:1-8), Scytalidium thermophilium (Aquino ACMM et al.2001, Folia Microbiol 46:11-16) etc.
Screen the heat-resisting glucoamylase gene of heat-resisting saccharifying enzyme or clonal expression, improve the operative temperature of saccharifying enzyme, can make the mashing process complete at short notice as starch liquefacation, thereby reduce energy consumption and production cost, to the application in industry open up more wide prospect to saccharifying enzyme, there is important commercial value.
Summary of the invention
The object of the invention is to provide a kind of heat-resisting saccharifying enzyme.
A further object of the present invention is to provide the gene of the above-mentioned saccharifying enzyme of above-mentioned coding.
A further object of the present invention is to provide the recombinant vectors that comprises above-mentioned saccharifying enzyme 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 for preparing heat-resisting saccharifying enzyme.
A further object of the present invention is to provide the application of above-mentioned heat-resisting saccharifying enzyme.
The present invention's technical problem at first to be solved is to overcome the deficiencies in the prior art, and a kind of acidproof, heat-resisting good new saccharifying enzyme of character is provided.The inventor obtains new heat-resisting, acidproof a, saccharifying enzyme that action pH is wide from Bispora sp.MEY-1CGMCC 2500.The saccharifying enzyme that it produces be suitable for the foodstuffs industry such as the production of drinks and fuel alcohol, glucose, fructose syrup, organic acid, monosodium glutamate etc. use in a plurality of industries.
The present invention has obtained a kind of heat-resisting saccharifying enzyme GAI from above-mentioned bacterial strains, and its aminoacid sequence is as shown in SEQ ID NO.1:
1 MQVSTCLFAL CSAVLAAPRQ ENLLRRATGS LTSWLAAESP IALQGVLNNI
51 GPNGVDAPGA DSGMVVASPD QVNPDYFYSW TRDSALTFKC LTDQFLAYGT
101SGLEAQIRDY VNAQAAVQTI ENPSGGLCTG GLGEPKFYLN QTAFFGPWGR
151PQRDGPALRA TALIAYARYL ISQGESSSVS SIIWPIVQND LSYVTQYWND
201TTFDLWEEID SSSFWTTAVQ YRALIEGNTL AGQIGESCSN CVSQAPNVLC
251FLQSYWNGEY IVANTGGGRS GKDSNTILAS IHVFDPDASC DDNTFQPCSD
301KMLANHKVLT DSFRSIYSIN SGISEGSGVA VGRYPEDTYQ GGNPWYLCTM
351AAAELLYDAV YQWKRIGSLN VTSTSLAFFQ AIYPSAAVGT YSSSSSTFDS
401IIDAVMTYGD SYMSIAEKYT PSNGSLSEQF SKTTGSPLSA YDLTWSFAAF
451LTAYNARAAA MPASWGAASA SLPSSCSGSS ASGPCSTATA TFTSGTLTPT
501CSAQPSTTVI FNELVTTTYG TNIYLTGDNS ELGNWSTGSE AVPCSASAYT
551STNPLWYCVA TLPAGTDIQY KFIQVASDGT VTWESGSNRE YTVPSNCAGV
601AEVNDSWQ*
Wherein, 608 amino acid of this zymoprotein total length and a terminator codon, N holds 16 signal peptide sequences " MQVSTCLFALCSAVLA " that amino acid is its prediction.
Therefore, the aminoacid sequence of ripe saccharifying enzyme GAI is as SEQ ID NO.2:
1 APRQENLLRR ATGSLTSWLA AESPIALQGV LNNIGPNGVD APGADSGMVV
51 ASPDQVNPDY FYSWTRDSAL TFKCLTDQFL AYGTSGLEAQ IRDYVNAQAA
101VQTIENPSGG LCTGGLGEPK FYLNQTAFFG PWGRPQRDGP ALRATALIAY
151ARYLISQGES SSVSSIIWPI VQNDLSYVTQ YWNDTTFDLW EEIDSSSFWT
201TAVQYRALIE GNTLAGQIGE SCSNCVSQAP NVICFIQSYW NGEYIVANTG
251GGRSGKDSNT ILASIHVFDP DASCDDNTFQ PCSDKMLANH KVLTDSFRSI
301YSINS6ISEG SGVAVGRYPE DTYQGGNPWY LCTMAAAELL YDAVYQWKRI
351GSLNVTSTSL AFFQAIYPSA AVGTYSSSSS TFDSIIDAVM TYGDSYMSIA
401EKYTPSNGSL SEQFSKTTGS PLSAYDLTWS FAAFLTAYNA RAAAMPASWG
451AASASLPSSC SGSSASGPCS TATATFTSGT LTPTCSAQPS TTVIFNELVT
501TTYGTNIYLT GDNSELGNWS TGSEAVPCSA SAYTSTNPLW YCVATLPAGT
551DIQYKFIQVA SDGTVTWESG SNREYTVPSN CAGVAEVNDS WQ*
Maturation protein is comprised of 592 amino acid and a terminator codon, and theoretical molecular is 63.3kDa, and this enzyme belongs to glycosyl hydrolase the 15th family.The saccharifying enzyme aminoacid sequence of deriving is carried out to the BLAST comparison in GenBank and find, this gene is up to 64.2% with the saccharifying enzyme sequence identity that derives from Aureobasidium pullulans.Illustrate that GAI is a kind of new saccharifying enzyme.
The invention provides the gene of the above-mentioned heat-resisting saccharifying enzyme of coding.
This enzyme full length gene 2001bp, sequence is as shown in SEQ ID NO.3:
1 ATGCAAGTAT CAACTTGCCT ATTTGCGCTA TGCAGCGCTG TTCTTGCTGC TCCAAGGCAG
61 GAAAATCTGT TACGGCGTGC TACGGGAAGC CTCACCTCCT GGCTGGCTGC TGAGAGTCCG
121 ATCGCACTCC AAGGTGTCCT CAACAACATT GGACCGAATG GGGTTGATGC TCCGGGCGCT
181 GATTCCGGAA TGGTCGTAGC CTCTCCAGAT CAGGTGAACC CAGACTACTT TTACAGTTGG
241 ACTCGAGATT CTGCATTGAC GTTCAAATGT CTTACTGACC AGTTCCTGGC ATACGGGACT
301 TCGGGGTTGG AGGCTCAGAT ACGGGATTAT GTCAATGCCC AGGCTGCGGT GCAGACAATC
361 GAAAACCCTT CAGGAGGGTT GTGCACGGGT GGGTTGGGAG AGCCAAAGTT CTACCTCAAC
421 CAAACCGCCT TCTTTGGGCC CTGGGGACGG CCCCAACGAG ATGGCCCCGC CCTTCGTGCT
481 ACAGCGCTCA TTGCCTATGC ACGGTACTTG ATCTCTCAGG GGGAAAGCTC GTCGGTTTCT
541 TCCATCATCT GGCCCATTGT GCAAAACGAT CTTTCCTACG TGACGCAGTA TTGGAATGAT
601 ACAACCTTTG ATCTTTGGGA AGAAATAGAC AGCTCGTCCT TTTGGACGAC AGCCGTACAG
661 TACCGGGCTC TCATTGAGGG AAACACTCTC GCCGGTCAGA TCGGAGAGAG CTGCAGCAAC
721 TGTGTCTCGC AAGCCCCTAA TGTCTTGTGC TTCCTTCAGT CGTACTGGAA TGGAGAATAT
781 ATCGTCGCAA ATACTGGCGG GGGCAGAAGT GGCAAAGACT CTAACACAAT CCTCGCCAGC
841 ATTCATGTCT TCGACCCCGA TGCTTCCTGC GATGACAATA CCTTCCAACC CTGCTCAGAC
901 AAGATGCTTG CCAATCACAA GGTGCTGACC GACTCGTTTC GATCCATCTA TTCTATCAAT
961 AGCGGAATCT CGGAGGGCAG CGGCGTTGCC GTTGGCCGTT ACCCTGAGGA CACATATCAA
1021GGTGGAAATC CGTGGTATGC CCCCAACGAA CCCTTACTAC CTGTGTGCAT AGTCTGGATG
1081TGTCACGCAG GTACTTGTGC ACCATGGCCG CCGCCGAGTT ATTATACGAT GCAGTCTATC
1141AGTGGAAAAG GATTGGCTCG CTGAATGTGA CGTCCACCTC TCTCGCTTTC TTCCAAGCCA
1201TTTATCCATC TGCAGCTGTT GGAACGTACT CTTCATCTTC GAGTACTTTC GACTCCATTA
1261TTGATGCTGT TATGACGTAC GGGGATAGCT ATATGTCGAT CGCCGTATGT TAGAGCTGTG
1321CCATCAAAAG TGTTACTCCT GACTAACTTG ATTTGGCCCT CCAGGAAAAG TACACACCTT
1381CCAATGGCTC TTTATCTGAG CAATTTTCAA AGACGACGGG ATCACCTCTG TCGGCTTATG
1441ACCTGACATG GTCGTTCGCT GCTTTTCTGA CTGCTTATAA TGCTCGAGCA GCCGCCATGC
1501CAGCATCTTG GGGCGCTGCA TCAGTAAGGG AGAAGCAAAA AATAACTAAA GAGTAACCAC
1561CGTCAGCTGA CATTGTTATA GGCATCACTG CCATCATCAT GTTCGGGAAG CTCGGCATCC
1621GGACCATGTT CCACGGCAAC GGCCACCTTC ACATCTGGCA CTTTAACCCC TACTTGCTCA
1681GCCCAACCAT CCACAACTGT AATTTTTAAC GAGCTCGTAA CGACTACATA TGGCACGAAT
1741ATCTATCTTA CTGGCGACAA TTCTGAGCTG GGGAACTGGA GCACTGGCTC TGAAGCCGTT
1801CCATGTTCTG CGTCGGCATA CACATCCACC AATCCGCTCT GGTATTGTGT TGCCACGTTA
1861CCCGCTGGGA CTGACATCCA ATACAAATTC ATACAAGTGG CTTCAGATGG TACAGTCACT
1921TGGGAGAGCG GGTCAAACAG GGAATACACC GTCCCTTCTA ACTGTGCAGG GGTTGCTGAG
1981GTGAATGATA GTTGGCAGTA A
The invention provides the cDNA sequence of the above-mentioned heat-resisting saccharifying enzyme of coding, total length 1827bp, as shown in SEQ ID NO.4.
1 ATGCAAGTAT CAACTTGCCT ATTTGCGCTA TGCAGCGCTG TTCTTGCTGC TCCAAGGCAG
61 GAAAATCTGT TACGGCGTGC TACGGGAAGC CTCACCTCCT GGCTGGCTGC TGAGAGTCCG
121 ATCGCACTCC AAGGTGTCCT CAACAACATT GGACCGAATG GGGTTGATGC TCCGGGCGCT
181 GATTCCGGAA TGGTCGTAGC CTCTCCAGAT CAGGTGAACC CAGACTACTT TTACAGTTGG
241 ACTCGAGATT CTGCATTGAC GTTCAAATGT CTTACTGACC AGTTCCTGGC ATACGGGACT
301 TCGGGGTTGG AGGCTCAGAT ACGGGATTAT GTCAATGCCC AGGCTGCGGT GCAGACAATC
361 GAAAACCCTT CAGGAGGGTT GTGCACGGGT GGGTTGGGAG AGCCAAAGTT CTACCTCAAC
421 CAAACCGCCT TCTTTGGGCC CTGGGGACGG CCCCAACGAG ATGGCCCCGC CCTTCGTGCT
481 ACAGCGCTCA TTGCCTATGC ACGGTACTTG ATCTCTCAGG GGGAAAGCTC GTCGGTTTCT
541 TCCATCATCT GGCCCATTGT GCAAAACGAT CTTTCCTACG TGACGCAGTA TTGGAATGAT
601 ACAACCTTTG ATCTTTGGGA AGAAATAGAC AGCTCGTCCT TTTGGACGAC AGCCGTACAG
661 TACCGGGCTC TCATTGAGGG AAACACTCTC GCCGGTCAGA TCGGAGAGAG CTGCAGCAAC
721 TGTGTCTCGC AAGCCCCTAA TGTCTTGTGC TTCCTTCAGT CGTACTGGAA TGGAGAATAT
781 ATCGTCGCAA ATACTGGCGG GGGCAGAAGT GGCAAAGACT CTAACACAAT CCTCGCCAGC
841 ATTCATGTCT TCGACCCCGA TGCTTCCTGC GATGACAATA CCTTCCAACC CTGCTCAGAC
901 AAGATGCTTG CCAATCACAA GGTGCTGACC GACTCGTTTC GATCCATCTA TTCTATCAAT
961 AGCGGAATCT CGGAGGGCAG CGGCGTTGCC GTTGGCCGTT ACCCTGAGGA CACATATCAA
1021GGTGGAAATC CGTGGTACTT GTGCACCATG GCCGCCGCCG AGTTATTATA CGATGCAGTC
1081TATCAGTGGA AAAGGATTGG CTCGCTGAAT GTGACGTCCA CCTCTCTCGC TTTCTTCCAA
1141GCCATTTATC CATCTGCAGC TGTTGGAACG TACTCTTCAT CTTCGAGTAC TTTCGACTCC
1201ATTATTGATG CTGTTATGAC GTACGGGGAT AGCTATATGT CGATCGCCGA AAAGTACACA
1261CCTTCCAATG GCTCTTTATC TGAGCAATTT TCAAAGACGA CGGGATCACC TCTGTCGGCT
1321TATGACCTGA CATGGTCGTT CGCTGCTTTT CTGACTGCTT ATAATGCTCG AGCAGCCGCC
1381ATGCCAGCAT CTTGGGGCGC TGCATCAGCA TCACTGCCAT CATCATGTTC GGGAAGCTCG
1441GCATCCGGAC CATGTTCCAC GGCAACGGCC ACCTTCACAT CTGGCACTTT AACCCCTACT
1501TGCTCAGCCC AACCATCCAC AACTGTAATT TTTAACGAGC TCGTAACGAC TACATATGGC
1561ACGAATATCT ATCTTACTGG CGACAATTCT GAGCTGGGGA ACTGGAGCAC TGGCTCTGAA
1621GCCGTTCCAT GTTCTGCGTC GGCATACACA TCCACCAATC CGCTCTGGTA TTGTGTTGCC
1681ACGTTACCCG CTGGGACTGA CATCCAATAC AAATTCATAC AAGTGGCTTC AGATGGTACA
1741GTCACTTGGG AGAGCGGGTC AAACAGGGAA TACACCGTCC CTTCTAACTG TGCAGGGGTT
1801GCTGAGGTGA ATGATAGTTG GCAGTAA
Wherein, the base sequence of signal peptide is: ATGCAAGTAT CAACTTGCCT ATTTGCGCTA TGCAGCGCTGTTCTTGCT
Therefore, the gene order of encoding mature saccharifying enzyme is as shown in SEQ ID NO.5
gctccaaggc aggaaaatct gttacggcgt gctacgggaa gcctcacctc ctggctggct 60
gctgagagtc cgatcgcact ccaaggtgtc ctcaacaaca ttggaccgaa tggggttgat 120
gctccgggcg ctgattccgg aatggtcgta gcctctccag atcaggtgaa cccagactac 180
ttttacagtt ggactcgaga ttctgcattg acgttcaaat gtcttactga ccagttcctg 240
gcatacggga cttcggggtt ggaggctcag atacgggatt atgtcaatgc ccaggctgcg 300
gtgcagacaa tcgaaaaccc ttcaggaggg ttgtgcacgg gtgggttggg agagccaaag 360
ttctacctca accaaaccgc cttctttggg ccctggggac ggccccaacg agatggcccc 420
gcccttcgtg ctacagcgct cattgcctat gcacggtact tgatctctca gggggaaagc 480
tcgtcggttt cttccatcat ctggcccatt gtgcaaaacg atctttccta cgtgacgcag 540
tattggaatg atacaacctt tgatctttgg gaagaaatag acagctcgtc cttttggacg 600
acagccgtac agtaccgggc tctcattgag ggaaacactc tcgccggtca gatcggagag 660
agctgcagca actgtgtctc gcaagcccct aatgtcttgt gcttccttca gtcgtactgg 720
aatggagaat atatcgtcgc aaatactggc gggggcagaa gtggcaaaga ctctaacaca 780
atcctcgcca gcattcatgt cttcgacccc gatgcttcct gcgatgacaa taccttccaa 840
ccctgctcag acaagatgct tgccaatcac aaggtgctga ccgactcgtt tcgatccatc 900
tattctatca atagcggaat ctcggagggc agcggcgttg ccgttggccg ttaccctgag 960
gacacatatc aaggtggaaa tccgtggtac ttgtgcacca tggccgccgc cgagttatta 1020
tacgatgcag tctatcagtg gaaaaggatt ggctcgctga atgtgacgtc cacctctctc 1080
gctttcttcc aagccattta tccatctgca gctgttggaa cgtactcttc atcttcgagt 1140
actttcgact ccattattga tgctgttatg acgtacgggg atagctatat gtcgatcgcc 1200
gaaaagtaca caccttccaa tggctcttta tctgagcaat tttcaaagac gacgggatca 1260
cctctgtcgg cttatgacct gacatggtcg ttcgctgctt ttctgactgc ttataatgct 1320
cgagcagccg ccatgccagc atcttggggc gctgcatcag catcactgcc atcatcatgt 1380
tcgggaagct cggcatccgg accatgttcc acggcaacgg ccaccttcac atctggcact 1440
ttaaccccta cttgctcagc ccaaccatcc acaactgtaa tttttaacga gctcgtaacg 1500
actacatatg gcacgaatat ctatcttact ggcgacaatt ctgagctggg gaactggagc 1560
actggctctg aagccgttcc atgttctgcg tcggcataca catccaccaa tccgctctgg 1620
tattgtgttg ccacgttacc cgctgggact gacatccaat acaaattcat acaagtggct 1680
tcagatggta cagtcacttg ggagagcggg tcaaacaggg aatacaccgt cccttctaac 1740
tgtgcagggg ttgctgaggt gaatgatagt tggcagtaa 1779
DNA sequence dna and cDNA sequence alignment analytical results show: the structure gene gaI total length 2 of heat-resisting saccharifying enzyme GAI, and 001bp, contain 3 introns, and its sequence is respectively: 1,038-1,093bp, 1,306-1,365bp and 1,525-1,582bp, the long 1827bp of cDNA.
The present invention also provides the recombinant vectors that comprises above-mentioned glucoamylase gene gaI.Be preferably pPIC9-gaI.Saccharifying enzyme maturation protein encoding gene of the present invention is inserted between the restriction enzyme site that expression vector is suitable, makes the exercisable and expression regulation sequence of its nucleotide sequence, and the carrier signal peptide sequence is connected.As the most preferred embodiment of the present invention, be preferably the glucoamylase gene of removing signal peptide is inserted between the EcoR I and Not I restriction enzyme site on plasmid pPIC9, make this nucleotide sequence be positioned at the downstream of AOX1 promotor and regulated and controled by it, obtain expression of recombinant yeast plasmid pPIC9-gaI.
The present invention also provides the recombinant bacterial strain that comprises above-mentioned glucoamylase gene, is preferably recombinant bacterial strain GS115/gaI.
The present invention also provides a kind of method for preparing heat-resisting saccharifying enzyme, comprises the following steps:
1) with above-mentioned recombinant vectors transformed host cell, obtain recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induce the expression of restructuring glucoamylase gene; And
3) reclaim the also expressed saccharifying enzyme of purifying.
Wherein, preferred described host cell is Pichia pastoris, cerevisiae, Bacillus coli cells or filamentous fungal cells, preferably, by expression of recombinant yeast Plasmid Transformation Pichia pastoris (Pichic pastoris) GS115, obtain recombinant bacterial strain GS115/gaI.
The present invention also provides the application of above-mentioned saccharifying enzyme.
The invention provides a new glucoamylase gene, the saccharifying enzyme of its coding has excellent heat resistance, the action pH scope is wide, acid resistance, can the foodstuffs industry such as the production of drinks and fuel alcohol, glucose, fructose syrup, organic acid, monosodium glutamate etc. use in a plurality of industries.
The accompanying drawing explanation
The bacterial strain Bispora sp.MEY-1 used in the present invention, be stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 19th, 2008, Institute of Microorganism, Academia Sinica, 100101), its preserving number is: CGMCC No.2500.
The recombinate SDS-PAGE of saccharifying enzyme of Fig. 1 analyzes, and 1: low molecular weight protein Marker; Saccharifying enzyme GAI after the 2:endoH de-glycosylation; 3: the saccharifying enzyme GAI of purifying; 4: the unpurified saccharifying enzyme GAI of Pichia anomala expression
The optimal pH curve of Fig. 2 saccharifying enzyme GAI.
The pH beta stability line of Fig. 3 saccharifying enzyme GAI.
The optimum temperuture curve of Fig. 4 saccharifying enzyme GAI effect.
The thermostability curve of Fig. 5 saccharifying enzyme GAI.
Embodiment
Experiment condition:
1, bacterial strain and carrier: Bispora sp.MEY-1 is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and its preserving number is: CGMCC No.2500.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 enzyme is purchased from Invitrogen company.Zulkovsky starch is purchased from Sigma company, and other is all domestic reagent (all can buy and obtain from common biochemical reagents company).
3, substratum:
(1) Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
(2) Bispora sp.MEY-1 substratum is the potato juice substratum: 1000mL 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 substratum: 1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% glycerine (V/V).
(6) BMMY substratum: 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.The technology do not described in detail in following examples, all carry out according to the related Sections in following laboratory manual or document or part, comprising: the people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition .2001); Kriegler, Gene Transfer and Expression:A Laboratory Manual (1990); With Current Protocols in Molecular Biology (people such as Ausubel compiles, 1994).
The clone of embodiment 1Bispora sp.MEY-1CGMCC2500 saccharifying enzyme encoding gene gaI
Extract Bispora sp.MEY-1CGMCC2500 genomic dna:
The liquid culture mycelium of 3 days is filtered and puts into mortar with aseptic filter paper, add the 2mL extracting solution, grind 5min, then lapping liquid is placed in to the 50mL centrifuge tube, 65 ℃ of water-bath cracking 20min, mix once every 10min, at 4 ℃ of centrifugal 5min of lower 10000rpm.Get supernatant extrct foreigh protein removing in phenol/chloroform, then get supernatant and add the equal-volume Virahol, after the standing 5min of room temperature, 4 ℃ of centrifugal 10min of lower 10000rpm.Abandon supernatant, 70% washing with alcohol twice for precipitation, vacuum-drying, add appropriate TE dissolving, be placed in-20 ℃ standby.
Synthesized degenerated primer P1 (5 '-TGGGGHCGTCCDCARMGNGAYGG-3 ') according to the conservative WGRPQRDG of the 15th family's glucoamylase gene and YDAV (I/L) YQW sequences Design, P2 (5 '-CCACTGRTARAYNGCRTCRTA-3 ') (wherein: Y=C/T, R=A/G, M=A/C, H=A/C/T, N=A/T/G/C).
The total DNA of Bispora sp.MEY-1CGMCC2500 of take utilizes degenerated primer P1 and P2 as template and carries out pcr amplification.The PCR reaction parameter is: 95 ℃ of 5min; 94 ℃ of 30sec, 55-45 ℃ of 30sec (wherein after each circulation, the renaturation temperature descends 1 ℃), 72 ℃ of 1min, then 10 circulations enter second cycling program: 94 ℃ of 30sec, 45 ℃ of 30sec, 72 ℃ of 1min, after 25 circulations; 72 ℃ of 10min, agarose electrophoresis detects.Obtain the fragment of about 700bp, be connected with the pEASY-T3 carrier after recovery, send Beijing San Bo Bioisystech Co., Ltd to carry out sequencing.
According to measuring sequence results, utilize BLASTX[http in the GenBank of NCBI: //www.ncbi.nlm.nih.gov/BLAST] carry out sequence alignment, tentatively judge that this gene fragment is the glucoamylase gene fragment, and carry out the Study on Similarity of this fragment.This sheet segment length 704bp, be up to 80% with the sequence identity of the saccharifying enzyme in Aureobasidium pullulans source.
The nucleotide sequence obtained according to order-checking, each three TAIL-PCR Auele Specific Primers of design upstream and downstream: design direction is for needing the zone of ignorance direction of amplification, and the Position Design of sp2 is in the inboard of sp1, and sp3 is positioned at the inboard of sp2.Distance between every two primers does not have strict regulation, the general 25～35nt of primer length, and annealing temperature is at 60～65 ℃.And by they difference called after Gamyusp1, Gamyusp2, Gamyusp3 (upstream Auele Specific Primer), Gamydsp1, Gamydsp2, Gamy dsp3 (downstream Auele Specific Primer) is in Table 1.
Table 1. saccharifying enzyme GAI TAIL-PCR Auele Specific Primer
Obtain the flanking sequence of known sequence by reverse TAIL-PCR, amplification obtains after product reclaims sending the order-checking of three rich Bioisystech Co., Ltd.The core fragment that degenerated primer is obtained splices and obtains the gaI full-length gene with the flanking sequence obtained through TAIL-PCR.Show the gene fragment that this gene DNA total length is a long 2001bp through sequential analysis.
The RT-PCR of embodiment 2 glucoamylase genes analyzes
Extract total RNA of Bispora sp.MEY-1CGMCC2500, utilize ThermoScript II to obtain the chain of cDNA, then design appropriate primer (GAI F:5 '-ATGCAAGTATCAACTTGCCTATTTGCGCTATG-3 ', GAI R:5 '-TTACTGCCAACTATCATTCACCTCAGCAACCCC-3 ') this strand cDNA that increases, obtain the cDNA sequence of saccharifying enzyme, amplification obtains after product reclaims sending the order-checking of three rich Bioisystech Co., Ltd.
The genomic dna sequence of saccharifying enzyme and cDNA the sequencing results show, the structure gene total length 2 of heat-resisting saccharifying enzyme GAI, 001bp, long 1, the 827bp of cDNA.Contain 3 introns, its sequence is respectively: 1,038-1,093bp, 1,306-1,365bp, and 1,525-1,582bp.N holds 16 signal peptide sequences that amino acid is its prediction.Saccharifying enzyme sequence on gaI cDNA sequence encoding albumen and GeneBank is carried out homology relatively, and the consistence of the saccharifying enzyme of originating with Aureobasidium pullulans is the highest, is 64.2%.With the consistence of the saccharifying enzyme in Talaromyces emersonii source be 62.3%.
The preparation of embodiment 3 restructuring saccharifying enzyme.
Expression vector pPIC9 is carried out to double digestion (EcoRI+NotI), the gene pgI of saccharifying enzyme of simultaneously encoding removes signal peptide sequence by PCR and adds restriction enzyme site EcoRI and NotI, and the primer is (Gamy F com-s:GCTCCAAGGCAGGAAAATCTGTTACGGCG; Gamy R com:TTACTGCCAACTATCATTCACCTCAGCAACCCC), product is through (EcoRI+NotI) double digestion, the gene fragment that obtains the encoding mature saccharifying enzyme is connected with expression vector pPIC9, the recombinant plasmid pPIC-gaI that acquisition contains glucoamylase gene gaI also transforms Pichia pastoris GS115, coating MD flat board, treat after 3 days that bacterium colony grows, with sterilized toothpick picking list bacterium colony from growing the MD plate that transformant is arranged, according to numbering, first put on MM, dull and stereotyped upper 30 ℃ of the MD that puts again corresponding numbering cultivates 1～2 day, grows to bacterium colony.On the MD flat board, can be inoculated in the centrifuge tube that 5mL BMGY substratum is housed by the normal growth transformant, 30 ℃, 260rpm shaking table are cultivated the centrifugal supernatant that goes after 48h, the BMMY substratum that adds again 1mL to contain 0.5% methyl alcohol in centrifuge tube, after 30 ℃, 260rpm inducing culture 48h, the centrifuging and taking supernatant detects for enzymic activity, therefrom filters out the transformant with diastatic activity.Obtain recombinant pichia yeast strain GS115/gaI.Screen altogether 96 of transformants, have 66 of diastatic activity are wherein arranged.
Enzyme high bacterial strain alive is re-seeded into and is equipped with in 400mL BMGY nutrient solution, after 30 ℃ of 250rpm shaking culture 48h, centrifugal collection thalline.Then resuspended in 200mL BMMY substratum, 30 ℃ of 250rpm shaking culture.After inducing 48h, every 24 hours, measure the vigor of saccharifying enzyme in supernatant and add methyl alcohol.SDS-PAGE result (Fig. 1) shows, the restructuring saccharifying enzyme has obtained expression in pichia spp.Inducing the expression amount of restructuring saccharifying enzyme after 96 hours is 46U/mL.
The activation analysis of embodiment 4 restructuring saccharifying enzyme
At pH3.5, under 75 ℃ of conditions, the reaction system of 1mL comprises the dilution enzyme liquid that 100 μ L are suitable, 900 μ L 1% Zulkovsky starches, reaction 10min, boiling water boiling 3min.To cooled on ice.The amount of the glucose discharged is measured by the method for glucose oxidase.1 enzyme unit (U) that lives is defined as the enzyme amount that under given condition per minute discharges 1 μ mol glucose.
The property testing of embodiment 5 restructuring saccharifying enzyme GAI
Purified saccharifying enzyme GAI carries out enzymatic reaction to measure its optimal pH under different pH.The 0.1mol/L KCl-HCl damping fluid that damping fluid used is pH 0.5-2.2, the McIlvaine damping fluid of the 0.1mol/L of pH 2.0～8.0, the 0.1mol/L Tris-HCl damping fluid of pH 8.0-9.0, the 0.1M glycine-NaOH damping fluid of pH 9.0-12.0.The saccharifying enzyme GAI of purifying is in the buffer system of different pH, and the pH adaptive result (Fig. 2) of measuring under 75 ℃ shows: the suitableeest action pH of GAI is 3.5, at pH2.0-4.5, can keep the enzyme work 90% or more.
Enzyme liquid is processed to 1h in the damping fluid of different pH values under 37 ℃, then measure the pH stability of enzymic activity with studying enzyme.Result shows (Fig. 3), and GAI is very stable between 1.5-12.0 in the pH scope.
Carry out enzymatic reaction at the McIlvaine buffer solution system of the 0.1mol/L that is determined at pH3.5 of optimum temperuture and different temperature (0～90 ℃).Enzyme reaction optimum temperuture measurement result (Fig. 4) shows, 75 ℃ of the optimum temperatures of GAI, between 60 ℃ and 80 ℃, keep the enzyme more than 50% to live.
Measure saccharifying enzyme is incubated respectively different time and measures enzyme activity, the thermostability curve of drafting enzyme under 70 ℃ and 75 ℃ of conditions.Under 70 ℃, the GAI thermostability is fine, still can retain nearly 100% enzyme work after processing 60min.Process 30min under 75 ℃, retain 50% enzyme work, after processing 60min, still retain 42% enzyme (Fig. 5) alive.
The kinetic constant of embodiment 6 restructuring saccharifying enzyme GAI
With the Zulkovsky starch of different concns (1-5mg/mL), it is substrate, in the McIlvaine buffer solution system of the 0.1mol/L of pH 3.5, measure enzymic activity, 5 minutes reaction times under 75 ℃, calculate its speed of response under 75 ℃, utilize the double-reciprocal plot method to try to achieve its K
mValue and V
max.After measured, this saccharifying enzyme be take the K that Zulkovsky starch is substrate under 75 ℃
mValue is 2.57mg/ml, maximum reaction velocity V
maxBe 2110.9 μ mol/min/mg.Specific activity is 3750mg/mg.
Claims (10)
1. a heat-resisting saccharifying enzyme GAI, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.1 or 2.
2. a heat-resisting glucoamylase gene gaI, is characterized in that, the saccharifying enzyme claimed in claim 1 of encoding.
3. glucoamylase gene gaI as claimed in claim 2, is characterized in that, its gene order is as shown in SEQ ID NO.5.
4. the expression vector of heat-resisting saccharifying enzyme GAI, is characterized in that it includes the described glucoamylase gene gaI of claim 2 or 3.
5. expression vector according to claim 4, is characterized in that, described expression vector is yeast expression vector, saccharomyces cerevisiae expression, filamentous fungus expression vector or coli expression carrier.
6. the recombinant bacterial strain that comprises the described glucoamylase gene gaI of claim 2 or 3.
7. recombinant bacterial strain according to claim 6, is characterized in that, described recombinant bacterial strain is that pichia spp, yeast saccharomyces cerevisiae, intestinal bacteria, aspergillus or wood are mould.
8. a method for preparing heat-resisting saccharifying enzyme GAI, is characterized in that, comprises the following steps:
1) with the described expression vector transformed host cell of claim 5, obtain recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induce the expression of restructuring saccharifying enzyme; And
3) reclaim the also expressed saccharifying enzyme GAI of purifying.
9. the application in producing saccharifying enzyme according to the described glucoamylase gene gaI of claim 2 or 3.
10. heat-resisting saccharifying enzyme GAI according to claim 1 is for amylatic application.
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