CN102827817A - 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, promptly (glucoamylase EC.3.2.1.3), is the important enzyme in the starch hydrolytic process to glucoamylase.Saccharifying enzyme is a kind of circumscribed-type Glycosylase, and main effect is α-1,4 glycosidic link and the α-1 on the carbochains such as non-reducing end hydrolyzed starch, dextrin, glycogen; 6 glycosidic links; Downcut glucose unit one by one, and the glucose generation change of configuration that hydrolysis is got off, end product β-D-glucose obtained.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 be used 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 foodstuffs industry such as glucose, fructose syrup, organic acid, monosodium glutamate.
Saccharifying enzyme is present in animal, plant and the mikrobe widely.Wherein mikrobe is the important source (Pardeep K and Satyanarayana T, 2009, Crit Rev Biotechnol 29:225-255) of saccharifying enzyme.The saccharifying enzyme of using in the industry mainly obtains from mould genus (Aspergillus), Rhizopus filamentous funguss such as (Rhizopus) and yeast belong (Saccharmyces).Along with the widespread use of genetic engineering technique, a lot of glucoamylase genes are by the clone and 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 by catalytic domain (catalytic domain, CD), starch binding domain (starch-binding domain, SBD) and 0-glycosylation link field (the O-glycosylated linker domain) composition that connects CD and SBD.
It is also inequality that the pH of the saccharifying enzyme that different microorganisms produced fits property, thermotolerance, catalysis characteristics etc.Mensuration to saccharifying enzyme character mainly concentrates in the research of optimum temperuture and optimum pH at present, and mostly the saccharifying enzyme of using in the industry is to use its thermostability.The optimal reaction pH value of most of fungi saccharifying enzyme is 4.5-5.0, and is stable under acidic conditions.Optimal reactive temperature is 40-60 ℃ (Norouzian D et al.2006, Biotechnol Adv 24:80-85).At present; It is the report of 70 ℃ saccharifying enzyme that optimum temperuture is also arranged; Derive from Trichoderma Reesi (Fagerstrom R et al.1995 respectively; 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; The mashing process is accomplished as starch-liquefying at short notice; Thereby reduce energy consumption and production cost, will open up more wide prospect in Industrial Application, have important commercial and be worth to saccharifying enzyme.
Summary of the invention
The object of the invention provides a kind of heat-resisting saccharifying enzyme.
A purpose more of the present invention provides the gene of the above-mentioned saccharifying enzyme of above-mentioned coding.
A purpose more of the present invention provides the recombinant vectors that comprises above-mentioned saccharifying enzyme encoding sox.
A purpose more of the present invention provides the recombinant bacterial strain that comprises above-mentioned glucoamylase gene.
A purpose more of the present invention provides a kind of method for preparing heat-resisting saccharifying enzyme.
A purpose more of the present invention provides the application of above-mentioned heat-resisting saccharifying enzyme.
The present invention's technical problem at first to be solved is the deficiency that overcomes prior art, provides a kind of acidproof, heat-stable character good new saccharifying enzyme.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 produced be suitable for 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, its aminoacid sequence is 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 sophisticated saccharifying enzyme GAI such 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 made up 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 the BLAST comparison find that this gene is up to 64.2% with the saccharifying enzyme sequence identity that derives from Aureobasidium pullulans in GenBank.Explain 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 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 is 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 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 contains 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 sox of the present invention is inserted between the suitable restriction enzyme site of expression vector, makes the exercisable and expression regulation sequence of its nucleotide sequence, and the carrier signal peptide sequence is connected.As a most preferred embodiment of the present invention; Being preferably the glucoamylase gene that will remove signal peptide is inserted between the EcoR I and Not I restriction enzyme site on the plasmid pPIC9; Make this nucleotide sequence be positioned at the downstream of AOX1 promotor and regulated and control 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, may further comprise the steps:
1), gets recombinant bacterial strain with above-mentioned recombinant vectors transformed host cell;
2) cultivate recombinant bacterial strain, induce the expression of reorganization glucoamylase gene; And
3) reclaim the also expressed saccharifying enzyme of purifying.
Wherein, Preferred said host cell is pichia spp cell, cerevisiae, Bacillus coli cells or filamentous fungal cells; Preferably the expression of recombinant yeast plasmid is transformed pichia spp cell (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 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.
Description of drawings
The bacterial strain Bispora sp.MEY-1 that uses among the present invention; Be stored in (Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms 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 1: low molecular weight protein Marker; Saccharifying enzyme GAI behind the 2:endoH de-glycosylation; 3: the saccharifying enzyme GAI of purifying; 4: the unpurified saccharifying enzyme GAI of Pichia anomala expression
The ph optimum 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 common micro-organisms center, and its preserving number is: CGMCC No.2500.Yeast expression vector pPIC9 and bacterial strain GS115 are available from Invitrogen company.
2, enzyme and other biochemical reagents: restriction endonuclease is available from TaKaRa company, and ligase enzyme is available from Invitrogen company.Zulkovsky starch is available from Sigma company, and other all is domestic reagent (all can buy from common biochemical reagents company and obtain).
3, substratum:
(1) intestinal bacteria substratum 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.
Explain: the genetic recombination of using learns a skill and is the routine techniques in this area among the present invention.The technology that in following examples, does not describe in detail is all carried out according to related Sections in following laboratory manual or the document or part, comprising: 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 sox gaI
Extract Bispora sp.MEY-1CGMCC2500 genomic dna:
3 days mycelium of liquid culture is put into mortar with the aseptic filter paper filtration, add the 2mL extracting solution, grind 5min; Then lapping liquid is placed the 50mL centrifuge tube; 65 ℃ of water-bath cracking 20min, whenever once at a distance from the 10min mixing, at 4 ℃ of centrifugal 5min of following 10000rpm.Get supernatant extrct foreigh protein removing in phenol/chloroform, get supernatant again and add the equal-volume Virahol, after room temperature leaves standstill 5min, 4 ℃ of centrifugal 10min of following 10000rpm.Abandon supernatant, deposition is with 70% washing with alcohol twice, and vacuum-drying adds an amount of TE and dissolves, place-20 ℃ subsequent use.
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).
With the total DNA of Bispora sp.MEY-1CGMCC2500 is that template utilizes degenerated primer P1 and P2 to carry out pcr amplification.The PCR reaction parameter is: 95 ℃ of 5min; 94 ℃ of 30sec, 55-45 ℃ of 30sec (wherein each circulation back renaturation temperature descends 1 ℃), 72 ℃ of 1min, 10 circulations get into second cycling program then: 94 ℃ of 30sec, 45 ℃ of 30sec, 72 ℃ of 1min are after 25 circulations; 72 ℃ of 10min, agarose electrophoresis detects.Obtain the fragment of about 700bp, reclaim the back and link to each other, send Beijing three rich Bioisystech Co., Ltd to carry out sequencing with the pEASY-T3 carrier.
According to measuring sequence results, in the GenBank of NCBI, utilize BLASTX [http://www.ncbi.nlm.nih.gov/BLAST] to carry out sequence alignment, judge that tentatively this gene fragment is the glucoamylase gene fragment, and carry out this segmental Study on Similarity.This sheet segment length 704bp is up to 80% with the sequence identity of the saccharifying enzyme in Aureobasidium pullulans source.
According to the nucleotide sequence that order-checking obtains, each three TAIL-PCR Auele Specific Primer 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 per two primers does not have strict regulation, the general 25～35nt of primer length, and annealing temperature is at 60～65 ℃.And with they difference called after Gamyusp1, Gamyusp2, Gamyusp3 (upper reaches Auele Specific Primer), Gamydsp1, Gamydsp2, Gamy dsp3 (downstream Auele Specific Primer) sees table 1.
Table 1. saccharifying enzyme GAI TAIL-PCR Auele Specific Primer
Obtain the flanking sequence of known sequence through reverse TAIL-PCR, amplification obtains sending after product reclaims the order-checking of three rich Bioisystech Co., Ltd.The core fragment that degenerated primer is obtained splices with the flanking sequence that obtains through TAIL-PCR and obtains the gaI full-length gene.Show that through sequential analysis this gene DNA total length is the gene fragment of a long 2001bp.
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; The primer of designing proper (GAI F:5 '-ATGCAAGTATCAACTTGCCTATTTGCGCTATG-3 ' then; GAI R:5 '-TTACTGCCAACTATCATTCACCTCAGCAACCCC-3 ') this strand cDNA that increases obtains the cDNA sequence of saccharifying enzyme, and amplification obtains sending after product reclaims 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, and 001bp, cDNA is long by 1,827bp.Contain 3 introns, its sequence is respectively: 1, and 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 the GeneBank is carried out homology relatively, and is the highest with the consistence of the saccharifying enzyme in Aureobasidium pullulans source, is 64.2%.With the consistence of the saccharifying enzyme in Talaromyces emersonii source be 62.3%.
The preparation of embodiment 3 reorganization saccharifying enzyme.
Expression vector pPIC9 is carried out double digestion (EcoRI+NotI); The gene pgI of saccharifying enzyme of will encoding simultaneously removes signal peptide sequence through 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, and the gene fragment that obtains the encoding mature saccharifying enzyme is connected with expression vector pPIC9, obtains to contain the recombinant plasmid pPIC-gaI of glucoamylase gene gaI and transform pichia spp GS115; Coating MD is dull and stereotyped; Treat after 3 days that bacterium colony grows, the toothpick of using the bacterium of going out is put on the MM from growing picking list bacterium colony on the MD plate that transformant is arranged earlier according to numbering; The MD that puts corresponding numbering again cultivated 1～2 day for dull and stereotyped last 30 ℃, grew to bacterium colony.Can be inoculated in the centrifuge tube that 5mL BMGY substratum is housed by the normal growth transformant on the MD flat board; 30 ℃, 260rpm shaking table are cultivated the centrifugal supernatant that goes behind the 48h; Add 1mL in the centrifuge tube again and contain the BMMY substratum of 0.5% methyl alcohol; Behind 30 ℃, 260rpm inducing culture 48h, the centrifuging and taking supernatant is used for enzymic activity and detects, and therefrom filters out the transformant with diastatic activity.Obtain recombinant pichia yeast strain GS115/gaI.Screen 96 of transformants altogether, have 66 of diastatic activity are wherein arranged.
The enzyme high bacterial strain of living is re-seeded into and is equipped with in the 400mL BMGY nutrient solution, behind 30 ℃ of 250rpm shaking culture 48h, centrifugal collection thalline.Resuspended in 200mL BMMY substratum then, 30 ℃ of 250rpm shaking culture.After inducing 48h, whenever measured the vigor of saccharifying enzyme in the supernatant and added methyl alcohol at a distance from 24 hours.SDS-PAGE result (Fig. 1) shows that the reorganization saccharifying enzyme has obtained expression in pichia spp.Inducing the expression amount of reorganization saccharifying enzyme after 96 hours is 46U/mL.
The activation analysis of embodiment 4 reorganization saccharifying enzyme
At pH3.5, under 75 ℃ of conditions, the reaction system of 1mL comprises 100 μ L suitable dilution enzyme liquid, 900 μ L, 1% Zulkovsky starch, and reaction 10min, boiling water boils 3min.To cooled on ice.The amount of the glucose that discharges is measured with the method for P-FAD.1 enzyme unit (U) that lives is defined as the enzyme amount that under given condition PM discharges 1 μ mol glucose.
The property testing of embodiment 5 reorganization saccharifying enzyme GAI
Purified saccharifying enzyme GAI carries out enzymatic reaction to measure its ph optimum under different pH.Used damping fluid is the 0.1mol/L KCl-HCl damping fluid of 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 75 ℃ of pH that measure down fit property result (Fig. 2) and show: the righttest action pH of GAI is 3.5, can keep the enzyme more than 90% to live at pH2.0-4.5.
Enzyme liquid is handled 1h in the damping fluid of different pH values, measure the pH stability of enzymic activity again under 37 ℃ with the research enzyme.The result shows (Fig. 3), and GAI is very stable between 1.5-12.0 in the pH scope.
Carry out enzymatic reaction under the McIlvaine buffer solution system of the 0.1mol/L that is determined at pH3.5 of optimum temperuture and the different temperature (0～90 ℃).The enzyme reaction optimum temperuture is measured result (Fig. 4) and is shown, 75 ℃ of the optimum temperatures of GAI between 60 ℃ and 80 ℃, keep the enzyme more than 50% to live.
The kinetic constant of embodiment 6 reorganization saccharifying enzyme GAI
Zulkovsky starch with different concns (1-5mg/mL) is a substrate; In the McIlvaine buffer solution system of the 0.1mol/L of pH 3.5, measure enzymic activitys, 5 minutes reaction times down for 75 ℃; Calculate its speed of response under 75 ℃, utilize the double-reciprocal plot method to try to achieve its K
mValue and V
MaxThrough measuring, this saccharifying enzyme is the K of substrate with the Zulkovsky starch under 75 ℃
mValue is 2.57mg/ml, maximum reaction velocity V
MaxBe 2110.9 μ mol/min/mg.Be 3750mg/mg than living.
Claims (10)
1. a heat-resisting saccharifying enzyme GAI is characterized in that it has the aminoacid sequence shown in SEQ ID NO.1 or 2.
2. a heat-resisting glucoamylase gene gaI is characterized in that, the described saccharifying enzyme of coding claim 1.
3. glucoamylase gene gaI as claimed in claim 2 is characterized in that, its gene order is shown in SEQ ID NO.5.
4. the expression vector of heat-resisting saccharifying enzyme GAI is characterized in that it includes claim 2 or 3 described glucoamylase gene sequences.
5. expression vector according to claim 4 is characterized in that said expression vector is yeast expression vector, yeast saccharomyces cerevisiae expression vector, filamentous fungus expression vector, coli expression carrier.
6. the recombinant bacterial strain that comprises claim 2 or 3 said glucoamylase genes.
7. recombinant bacterial strain according to claim 6 is characterized in that, said host cell is pichia spp cell, brewing yeast cell, intestinal bacteria, aspergillus or wooden mould cell.
8. a method for preparing heat-resisting saccharifying enzyme GAI is characterized in that, may further comprise the steps:
1), gets recombinant bacterial strain with claim 5 recombinant vectors transformed host cell;
2) cultivate recombinant bacterial strain, induce the expression of reorganization saccharifying enzyme; And
3) reclaim the also expressed saccharifying enzyme GAI of purifying.
9. according to the application of claim 2 or 3 said glucoamylase gene gaI.
10. the application of heat-resisting saccharifying enzyme GAI according to claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667213A (en) * | 2013-12-26 | 2014-03-26 | 中国农业科学院饲料研究所 | Acidic intermediate-temperature saccharifying enzyme GAII as well as gene and application thereof |
| CN107475219A (en) * | 2017-09-29 | 2017-12-15 | 天津科技大学 | Three kinds recombinate carbohydrase and preparation method and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457207A (en) * | 2008-05-28 | 2009-06-17 | 中国农业科学院饲料研究所 | Eosinophil beta-mannanase MAN5A and gene and application thereof |
| CN101748108A (en) * | 2008-12-09 | 2010-06-23 | 中国农业科学院饲料研究所 | Acidophil Beta-glucanase GLU7A and gene and application thereof |
| CN101768578A (en) * | 2008-12-26 | 2010-07-07 | 中国农业科学院饲料研究所 | Eosinophilic lactase BGALA, gene and application thereof |
| CN101818135A (en) * | 2010-01-22 | 2010-09-01 | 中国农业科学院饲料研究所 | Acidophilic alpha-galactosidase AgalB with galactomannan degradation capability and gene and application thereof |
| CN101993863A (en) * | 2009-08-14 | 2011-03-30 | 中国科学院微生物研究所 | Glucamylase as well as encoding gene and application thereof |
-
2011
- 2011-06-15 CN CN2011101597497A patent/CN102827817B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101457207A (en) * | 2008-05-28 | 2009-06-17 | 中国农业科学院饲料研究所 | Eosinophil beta-mannanase MAN5A and gene and application thereof |
| CN101748108A (en) * | 2008-12-09 | 2010-06-23 | 中国农业科学院饲料研究所 | Acidophil Beta-glucanase GLU7A and gene and application thereof |
| CN101768578A (en) * | 2008-12-26 | 2010-07-07 | 中国农业科学院饲料研究所 | Eosinophilic lactase BGALA, gene and application thereof |
| CN101993863A (en) * | 2009-08-14 | 2011-03-30 | 中国科学院微生物研究所 | Glucamylase as well as encoding gene and application thereof |
| CN101818135A (en) * | 2010-01-22 | 2010-09-01 | 中国农业科学院饲料研究所 | Acidophilic alpha-galactosidase AgalB with galactomannan degradation capability and gene and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| HUIYING LUO ET AL: "A novel highly acidic B-mannanase from the acidophilic fungus bispora sp. MEY-1:gene cloning and overexpression in Pichia pastoris", 《APPL MICROBIOL BIOTECHNOL》 * |
| HUIYING LUO ET AL: "A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp. MEY-1", 《EXTREMOPHILES》 * |
| LI HF ET AL: "GI:307590719", 《GENBANK》 * |
| 罗会颖: "极端嗜酸真菌Bispora sp. MEY-1胞外糖苷水解酶类的产酶分析及其相关基因的克隆与表达", 《中国博士学位论文全文数据库基础科学辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667213A (en) * | 2013-12-26 | 2014-03-26 | 中国农业科学院饲料研究所 | Acidic intermediate-temperature saccharifying enzyme GAII as well as gene and application thereof |
| CN103667213B (en) * | 2013-12-26 | 2016-05-04 | 中国农业科学院饲料研究所 | Warm carbohydrase GA II and gene and application in a kind of acidity |
| CN107475219A (en) * | 2017-09-29 | 2017-12-15 | 天津科技大学 | Three kinds recombinate carbohydrase and preparation method and application |
| CN107475219B (en) * | 2017-09-29 | 2020-06-09 | 天津科技大学 | Three recombinant saccharifying enzymes and preparation method and application thereof |
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