CN101880682A - 61-family inscribe cellulases eg gene clone of thermoascus aurantiacus var. levisporus and coding protein thereof - Google Patents
61-family inscribe cellulases eg gene clone of thermoascus aurantiacus var. levisporus and coding protein thereof Download PDFInfo
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- CN101880682A CN101880682A CN2010101867044A CN201010186704A CN101880682A CN 101880682 A CN101880682 A CN 101880682A CN 2010101867044 A CN2010101867044 A CN 2010101867044A CN 201010186704 A CN201010186704 A CN 201010186704A CN 101880682 A CN101880682 A CN 101880682A
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Abstract
The invention provides a 61-family inscribe cellulases eg gene clone of thermoascus aurantiacus var. levisporus and coding protein thereof, which is realized by the following steps: taking thermoascus aurantiacus var. levisporus cDNA as a masterplate, designing degenerate primer according to cellulases N-end sequence of original strain purification and 61-family cellulases conservative locuses, increasing interval segments in the cellulases and adopting a 3' RACE and 5' TAIL-PCR method to obtain the full-length cDNA sequence of an endoglucanase eg gene and a derived amino acid sequence thereof. The clone of the gene plays an important role on researches of catalytic core, action mechanism, space structure and the like for family cellulases. Thermophilic fungus cellulases have thermostability, are applicable to field of converting fiber waste materials and other industrial fields and have economic value and social value.
Description
(1) technical field
The present invention relates to biotechnology, be masterplate specifically with thermophilic ascomycete light spore mutation Thermoascusaurantiacus var.levisporus cDNA, with cellulase N terminal sequence and the conservative site design of 61 family's cellulases degenerate primer by the original strain purifying, amplification cellulase intermediate segment, the back obtains endoglucanase eg full length gene cDNA sequence and deduced amino acid with the method for 3 ' RACE, 5 ' TAIL-PCR.
(2) background technology
Mierocrystalline cellulose is the material the widest, that standing stock are the abundantest that distributes on the earth, is first renewable resources.Cellulosic biological degradation realizes by cellulase.Cellulase is prozyme system, comprise endo-beta-1,4-glucanase (EC 3.2.1.4) (EG), β-1,4-cellobiohydrolase (EC 3.2.1.91) (CBH) and β-1,4-glucuroide (EC 3.2.1.21) is (BGL).These three kinds of enzyme synergies are thoroughly cut β-1, the 4-glycosidic link, and making the Mierocrystalline cellulose eventual degradation is glucose.Wherein endo-beta-1,4-glucanase is the topmost composition of cellulase system, and it at first acts on Mierocrystalline cellulose, the β-1 of hydrocellulose chain noncrystalline domain, and the 4-glycosidic link is opened cellulose chain, and it plays an important role to whole cellulosic degraded.Cellulase belongs to the glycoside hydrolysis enzyme, according to the homology of aminoacid sequence and the similarity of cellulase structure, is divided into different families, can reflect the constructional feature of enzyme, discloses the evolutionary relationship of enzyme, and can infer the mechanism of action of enzyme.So far, 61 family's cellulases do not appear in the newspapers in thermophilic fungus, and the research of catalytic core, mechanism of action, space structure etc. of cloning this family's cellulase of this gene pairs is significant.
Cellulase is widely used in the aspects such as comprehensive utilization of light industry, medicine, environmental protection, food-processing, drink industry, textile industry, renewable resources, be considered to have most application potential zymin it.At present both at home and abroad cellulase is mainly by having a liking for warm fungi aspergillus and wooden mould production, but have that the bacterial classification yield of enzyme is low, vigor is low, problems such as enzyme stability difference and cost height, the production of its cellulase and application are very limited.Because therefore heat-stable cellulase thermally-stabilised under hot conditions have important application prospect and commercial value.
Thermophilic ascomycete light spore mutation Thermoascus aurantiacus var.levisporus is a kind of thermophilic fungus that is distributed widely in the soil.This bacterium is can produce heat-staple cellulase under 50 ℃ of conditions in the substratum of sole carbon source at Microcrystalline Cellulose.
(3) summary of the invention
The present invention obtains inscribe β-1 from thermophilic ascomycete light spore mutation Thermoascus aurantiacus var.levisporus, the full length cDNA sequence of 4-glucanase gene, called after eg, eg gene cDNA total length 1325bp, comprise initiator codon and poly A tract, open reading frame with a 747bp, 249 amino acid of encoding.Have a signal peptide sequence (1-21aa MSFSKIIATAGVLASASLVAG) and a potential N glycosylation site (NNS).
The eg amino acid sequence coded is retrieved in international gene pool, found to belong to glycoside hydrolase the 61st family, wherein 3 motif GNYV-RHE, GAQNYPQC and Y-IPGP tool conservative property.
DNAMAN analyzes its molecular weight and is about 26.5Da, and iso-electric point is about 5.12.The homology of the endoglucanase of the basket bacterium Talaromyces of the endoglucanase EG of Thermoascusaurantiacus var.levisporus and filamentous fungus Ya Xi island stipitatus, Penicillium marneffei bacterium penicillium marneffei is higher, reaches 72%.
(4) embodiment
Embodiment 1: the isolation identification of thermophilic ascomycete light spore mutation Thermoascus aurantiacus var.levisporus
(1) collection of specimens: from compost, gather.
(2) separation and Culture: collect specimen is got 0.5 gram be placed on the dull and stereotyped last 50 ℃ of cultivations of PDA after 3 days, carry out separation and purification.Operation steps is with reference to Cooney and Emerson (1964) document.
(3) identify: with reference to Cooney and Emerson (1964) and LaTouche (1950) document.
Embodiment 2: inscribe-1, the clone of 4-beta-glucanase gene eg
(1) extraction of the total RNA of thermophilic ascomycete light spore mutation Thermoascus aurantiacus var.levisporus: with reference to the explanation of Trizol test kit.
(2) cDNA article one chain is synthetic: TaKaRa RNA PCR kit (AMV) the Ver3.0 test kit specification sheets according to Takara company carries out: get the total RNA of 1～2 μ g, add RNase Free ddH
2O to 9.5 μ L at 75 ℃ of sex change 5min, cools off 5min with the RNA sample immediately in ice bath, once centrifugal a little then, various compositions below adding successively in ice bath: 10mmol/L dNTP Mixture 2 μ L, 10 * RTBuffer (Mg
2+) 2 μ L, 25mmol/L MgCl
24 μ L, Oligo d (T)-Adaptor Primer 1 μ L, RNaseInhibiter 0.5 μ L, AMV Reverse Transcriptase 1 μ L (Final Volume 20 μ L), after the reaction solution mixing, room temperature is transferred 10min, and 42 ℃ of incubation 60min boil 5min again with the deactivation ThermoScript II then.Add the ddH that 180 μ L DEPC handle
2O is diluted to 200 μ L, and mixing is centrifugal a little, is stored in-20 ℃, and is standby.
(3) separation of intermediate segment: according to thermophilic ascomycete light spore mutation Thermoascus aurantiacusvar.levisporus purifying cellulose enzyme N end sequencing result and 61 families cellulase homology conserved sequence design degenerate primer.Upstream primer is: 5 '-GTCCARAAYATYGTYATYGATGG-3 ', downstream primer is: 5 '-GTTGANGCAYTGDGGRTAGTT-3 '
(4) 3 ' with the separating of 5 ' sequence: carry out according to TaKaRa RNA PCR kit (AMV) Ver3.0 operation instruction.Proteinase gene 3 '-RACE the primer is 61-3.2,61-3.3, Oligo dT-Adaptor primer and M13Primer M4, and its sequence is:
61-3.2：5’CGCCTGGTCTACTACGGCAACTGAT-3’
61-3.3：5’-AGACAATCTGATAGCAGCCAACAACA-3’
M13?Primer?M4：5’-GTTTTCCCAGTCACGAC-3’
Oligo dT-Adaptor primer: comprise dT zone and M13 Primer M4 sequence.
Proteinase gene 5 ' terminal sequence clone, the method for use Tail-PCR is a masterplate with the genomic dna,
The primer is 3 outside special nested primer P5-1, P5-2, P5-3, and the public degenerate primer AD in upstream
1, AD
2, AD
3, AD
4, AD
5, its sequence is:
61-5.1：5’-ATCCAGTACCGTCCACAAATCCAAGA-3’
61-5.2：5’-CAGTTGCCGTAGTAGACCAGGCGATGA-3’
61-5.3：5’-GCTGTTGCCGCTTATGTGCGTCTTG-3’
AD
1：5’-NTCGASTWTSGWGTT-3’
AD
2：5’-NGTCGASWGANAWGAA-3’
AD
3：5’-WGTGNAGWANCANAGA-3’
AD
4：5’-TGWGNAGSANCASAGA-3’
AD
5：5’-AGWGNAGWANCAWAGG-3’
(5) gene clone: get 0.5 μ l PCR product and be connected with the pMD18-T carrier, operation steps is carried out according to TAKARA company product description.Connect product transformed into escherichia coli DH5 α bacterial strain then, scribble grow overnight on the LB flat board that contains acillin (100 μ g/mL) of X-gal and IPTG on the surface.The picking white colony, overnight incubation in the LB liquid nutrient medium.
(6) extraction of plasmid DNA: alkaline process extracts plasmid DNA.
(7) sequencing: dna double deoxidation method is measured nucleotide sequence, and company limited carries out at the Shanghai biotechnology.Aligning primer is the M13 promoter primer.Thermophilic ascomycete light spore mutation eg gene cDNA total length 1325bp comprises initiator codon and poly A tract, has the open reading frame of a 747bp, 249 amino acid of encoding.This aminoacid sequence is retrieved in international gene pool, found to belong to glycoside hydrolase the 61st family, wherein 3 motif GNYV-RHE, GAQNYPQC and Y-IPGP tool conservative property.This sequence is as follows:
(A) information of SEQ ID NO 1
(a) sequence signature:
*Length: 1325 base pairs;
*Type: nucleic acid;
*Chain: two strands;
*Topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: thermophilic ascomycete light spore mutation (Thermoascus aurantiacus var.levisporus)
(f) sequence description:
1 CTCGGACACC?GTTTCTTGAA?GTTGCTTTAT?ATTATTAGCT?TTGGTCCATC?GCGATCGAAT
61 TGGCACATCC?TATTTGAAAG?ATGTCCTTTT?CCAAGATAAT?TGCTACTGCC?GGCGTTCTTG
121 CCTCTGCTTC?TCTAGTGGCT?GGCCATGGCT?TCGTTCAGAA?CATCGTGATT?GATGGTAAAA
181 AGTATGTCAT?TGCAAGACGC?AACCAGTATC?CATACATGTC?CAATCCTCCA?GAGGTCATCG
241 CCTGGTCTAC?TACGGCAACT?GATCTTGGAT?TTGTGGACGG?TACTGGATAC?CAAACCCCAG
301 ATATCATCTG?CCATAGGGGC?GCCAAGCCTG?GAGCCCTGAC?TGCTCCAGTC?TCTCCAGGAG
361 GAACTGTTGA?GCTTCAATGG?ACTCCATGGC?CTGATTCTCA?CCATGGCCCA?GTTATCAACT
421 ACCTTGCTCC?GTGCAATGGT?GATTGTTCCA?CTGTGGATAA?GACCCAATTA?GAATTCTTCA
481 AAATTGCCGA?GAGCGGTCTC?ATCAATGATG?ACAATCCTCC?TGGGATCTGG?GCTTCAGACA
541 ATCTGATAGC?AGCCAACAAC?AGCTGGACTG?TCACCATTCC?AACCACAATT?GCACCTGGAA
601 ACTATGTTCT?GAGGCATGAG?ATTATTGCTC?TTCACTCAGC?TCAGAACCAG?GATGGTGCCC
661 AGAACTATCC?CCAGTGCATC?AATCTGCAGG?TCACTGGAGG?TGGTTCTGAT?AACCCTGCTG
721 GAACTCTTGG?AACGGCACTC?TACCACGATA?CCGATCCTGG?AACTCTGATC?AACATCTATC
781 AGAAACTTTC?CAGCTATATC?ATCCCTGGTC?CTCCTCTGTA?TACTGGTTAG?AGGAACGTCC
841 GGCGGTCAGA?ATTGGTGCTG?GTATTGTGAG?AAGGGTCAGC?GGTAGAGGTC?TCGTTCGGAG
901 GGTTGCAAAA?ACATGACGCT?AGGTGGTTTT?GTGCGATTCT?TCCCATCTCG?CCTCTTCCAC
961 TTCTGCCAAA?TCCGGTTCTG?CGATTGCTAT?TTCGCCGTGA?CCGGCTGTTA?CTTTCGAACC
1021?TGTCTCTACC?AGGAACAACA?CCATTTCGTT?CTCCATTCTG?TGGTCCCCCA?GGGCCTCTAC
1081?TAGTCTACCG?GGAATTTCTC?AACAGCGTCC?GTGAATTCCA?CGAGGCTAAA?TGAAAGCAAC
1141?CCTGTGGCTC?CTCAGGTTTG?AACGAGGGAG?GGGACATATC?TTTTTGGAAA?CACATGTGTG
1201?ACCTCCAACT?TCTACGTTCT?TTTCTACCTT?GATTGTTTGT?ATCAAGTACT?TTGACTTGAC
1261?TGTCTTAGTG?TGGATACAAA?TCGTTACATC?AATCCATGTA?TTCTCATTGC?GACAAAAAAA
1321?AAAAA
(B) information of SEQ ID NO 2
(a) sequence signature:
*Length: 249 amino acid;
*Type: amino acid;
*Chain: strand;
*Topological framework: linearity
(b) molecule type: protein
(c) sequence description:
1 MSFSKIIATA?GVLASASLVA?GHGFVQNIVI?DGKKYVIARR?NQYPYMSNPP?EVIAWSTTAT
61 DLGFVDGTGY?QTPDIICHRG?AKPGALTAPV?SPGGTVELQW?TPWPDSHHGP?VINYLAPCNG
121?DCSTVDKTQL?EFFKIAESGL?INDDNPPGIW?ASDNLIAANN?SWTVTIPTTI?APGNYVLRHE
181?IIALHSAQNQ?DGAQNYPQCI?NLQVTGGGSD?NPAGTLGTAL?YHDTDPGTLI?NIYQKLSSYI
241?IPGPPLYTG
Embodiment 3: the preservation of thermophilic ascomycete light spore mutation
The depositary institution of thermophilic ascomycete light spore mutation: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); Address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on May 10th, 2010; Strain number is: CGMCC No.3804; Strain classification called after: thermophilic ascomycete light spore mutation Thermoascus aurantiacus var.levisporus.
Sequence table
＜110〉Shandong Agricultural University
＜120〉thermophilic ascomycete light spore mutation 61 families endo cellulase eg gene clone and proteins encoded thereof
<140>
<141>
<160>2
<170>pantent?In?3.1
<210>1
<211>1325
<212>DNA
＜213〉thermophilic ascomycete light spore mutation (Thermoascus aurantiacus var.levisporus)
<220>
<221>CDS
<222>(81)-(827)
<223>
<400>1
ctcggacacc?gtttcttgaa?gttgctttat?attattagct?ttggtccatc?gcgatcgaat 60
tggcacatcc?tatttgaaag?atg?tcc?ttt?tcc?aag?ata?att?gct?act?gcc?ggc 113
Met?Ser?Phe?Ser?Lys?Ile?Ile?Ala?Thr?Ala?Gly
1 5 10
gtt?ctt?gcc?tct?gct?tct?cta?gtg?gct?ggc?cat?ggc?ttc?gtt?cag?aac 161
Val?Leu?Ala?Ser?Ala?Ser?Leu?Val?Ala?Gly?His?Gly?Phe?Val?Gln?Asn
15 20 25
atc?gtg?att?gat?ggt?aaa?aag?tat?gtc?att?gca?aga?cgc?aac?cag?tat 209
Ile?Val?Ile?Asp?Gly?Lys?Lys?Tyr?Val?Ile?Ala?Arg?Arg?Asn?Gln?Tyr
30 35 40
cca?tac?atg?tcc?aat?cct?cca?gag?gtc?atc?gcc?tgg?tct?act?acg?gca 257
Pro?Tyr?Met?Ser?Asn?Pro?Pro?Glu Val?Ile?Ala?Trp?Ser?Thr?Thr?Ala
45 50 55
act?gat?ctt?gga?ttt?gtg?gac?ggt?act?gga?tac?caa?acc?cca?gat?atc 305
Thr?Asp?Leu?Gly?Phe?Val?Asp?Gly?Thr?Gly?Tyr?Gln?Thr?Pro?Asp?Ile
60 65 70 75
atc?tgc?cat?agg?ggc?gcc?aag?cct?gga?gcc?ctg?act?gct?cca?gtc?tct 353
Ile?Cys?His?Arg?Gly?Ala?Lys?Pro?Gly?Ala?Leu?Thr?Ala?Pro?Val?Ser
80 85 90
cca?gga?gga?act?gtt?gag?ctt?caa?tgg?act?cca?tgg?cct?gat?tct?cac 401
Pro?Gly?Gly?Thr?Val?Glu?Leu?Gln?Trp?Thr?Pro?Trp?Pro?Asp?Ser?His
95 100 105
cat?ggc?cca?gtt?atc?aac?tac?ctt?gct?ccg?tgc?aat?ggt?gat?tgt?tcc 449
His?Gly?Pro?Val?Ile?Asn?Tyr?Leu?Ala?Pro?Cys?Asn?Gly?Asp?Cys?Ser
110 115 120
act?gtg?gat?aag?acc?caa?tta?gaa?ttc?ttc?aaa?att?gcc?gag?agc?ggt 497
Thr?Val?Asp?Lys?Thr?Gln?Leu?Glu?Phe?Phe?Lys?Ile?Ala?Glu?Ser?Gly
125 130 135
ctc?atc?aat?gat?gac?aat?cct?cct?ggg?atc?tgg?gct?tca?gac?aat?ctg 545
Leu?Ile?Asn?Asp?Asp?Asn?Pro?Pro?Gly?Ile?Trp?Ala?Ser?Asp?Asn?Leu
140 145 150 155
ata?gca?gcc?aac?aac?agc?tgg?act?gtc?acc?att?cca?acc?aca?att?gca 593
Ile?Ala?Ala?Asn?Asn?Ser?Trp?Thr?Val?Thr?Ile?Pro?Thr?Thr?Ile?Ala
160 165 170
cct?gga?aac?tat?gtt?ctg?agg?cat?gag?att?att?gct?ctt?cac?tca?gct 641
Pro?Gly?Asn?Tyr?Val?Leu?Arg?His?Glu?Ile?Ile?Ala?Leu?His?Ser?Ala
175 180 185
cag?aac?cag?gat?ggt?gcc?cag?aac?tat?ccc?cag?tgc?atc?aat?ctg?cag 689
Gln?Asn?Gln?Asp?Gly?Ala?Gln?Asn?Tyr?Pro?Gln?Cys?Ile?Asn?Leu?Gln
190 195 200
gtc?act?gga?ggt?ggt?tct?gat?aac?cct?gct?gga?act?ctt?gga?acg?gca 737
Val?Thr?Gly?Gly?Gly?Ser?Asp?Asn?Pro?Ala?Gly?Thr?Leu?Gly?Thr?Ala
205 210 215
ctc?tac?cac?gat?acc?gat?cct?gga?act?ctg?atc?aac?atc?tat?cag?aaa 785
Leu?Tyr?His?Asp?Thr?Asp?Pro?Gly?Thr?Leu?Ile?Asn?Ile?Tyr?Gln?Lys
220 225 230 235
ctt?tcc?agc?tat?atc?atc?cct?ggt?cct?cct?ctg?tat?act?ggt?tagagga 834
Leu?Ser?Ser?Tyr?Ile?Ile?Pro?Gly?Pro?Pro?Leu?Tyr?Thr?Gly
240 245
acgtccggcg?gtcagaattg?gtgctggtat?tgtgagaagg?gtcagcggta?gaggtctcgt 894
tcggagggtt?gcaaaaacat?gacgctaggt?ggttttgtgc?gattcttccc?atctcgcctc 954
ttccacttct?gccaaatccg?gttctgcgat?tgctatttcg?ccgtgaccgg?ctgttacttt 1014
cgaacctgtc?tctaccagga?acaacaccat?ttcgttctcc?attctgtggt?cccccagggc 1074
ctctactagt?ctaccgggaa?tttctcaaca?gcgtccgtga?attccacgag?gctaaatgaa 1134
agcaaccctg?tggctcctca?ggtttgaacg?agggagggga?catatctttt?tggaaacaca 1194
tgtgtgacct?ccaacttcta?cgttcttttc?taccttgatt?gtttgtatca?agtactttga 1254
cttgactgtc?ttagtgtgga?tacaaatcgt?tacatcaatc?catgtattct?cattgcgaca 1314
aaaaaaaaaa?a 1325
<210>2
<211>249
<212>PRT
＜213〉thermophilic ascomycete light spore mutation (Thermoascus aurantiacus var.levisporus)
<400>2
MET?Ser?Phe?Ser?Lys?Ile?Ile?Ala?Thr?Ala?Gly?Val?Leu?Ala?Ser?Ala
1 5 10 15
Ser?Leu?Val?Ala?Gly?His?Gly?Phe?Val?Gln?Asn?Ile?Val?Ile?Asp?Gly
20 25 30
Lys?Lys?Tyr?Val?Ile?Ala?Arg?Arg?Asn?Gln?Tyr?Pro?Tyr?MET?Ser?Asn
35 40 45
Pro?Pro?Glu?Val?Ile?Ala?Trp?Ser?Thr?Thr?Ala?Thr?Asp?Leu?Gly?Phe
50 55 60
Val?Asp?Gly?Thr?Gly?Tyr?Gln?Thr?Pro?Asp?Ile?Ile?Cys?His?Arg?Gly
65 70 75 80
Ala?Lys?Pro?Gly?Ala?Leu?Thr?Ala?Pro?Val?Ser?Pro?Gly?Gly?Thr?Val
85 90 95
Glu?Leu?Gln?Trp?Thr?Pro?Trp?Pro?Asp?Ser?His?His?Gly?Pro?Val?Ile
100 105 110
Asn?Tyr?Leu?Ala?Pro?Cys?Asn?Gly?Asp?Cys?Ser?Thr?Val?Asp?Lys?Thr
115 120 125
Gln?Leu?Glu?Phe?Phe?Lys?Ile?Ala?Glu?Ser?Gly?Leu?Ile?Asn?Asp?Asp
130 135 140
Asn?Pro?Pro?Gly?Ile?Trp?Ala?Ser?Asp?Asn?Leu?Ile?Ala?Ala?Asn?Asn
145 150 155 160
Ser?Trp?Thr?Val?Thr?Ile?Pro?Thr?Thr?Ile?Ala?Pro?Gly?Asn?Tyr?Val
165 170 175
Leu?Arg?His?Glu?Ile?Ile?Ala?Leu?His?Ser?Ala?Gln?Asn?Gln?Asp?Gly
180 185 190
Ala?Gln?Asn?Tyr?Pro?Gln?Cys?Ile?Asn?Leu?Gln?Val?Thr?Gly?Gly?Gly
195 200 205
Ser?Asp?Asn?Pro?Ala?Gly?Thr?Leu?Gly?Thr?Ala?Leu?Tyr?His?Asp?Thr
210 215 220
Asp?Pro?Gly?Thr?Leu?Ile?Asn?Ile?Tyr?Gln?Lys?Leu?Ser?Ser?Tyr?Ile
225 230 235 240
Ile?Pro?Gly?Pro?Pro?Leu?Tyr?Thr?Gly
245
Claims (2)
1. the thermophilic ascomycete light spore mutation Thermoascus aurantiacus var.levisporus 61 endo cellulase eg of family genes is characterized in that: be the base sequence shown in the SEQ ID NO.1.
2. the thermophilic ascomycete light spore mutation Thermoascus aurantiacus var.levisporus 61 endo cellulase eg of family gene coded proteins is characterized in that: be the aminoacid sequence shown in the SEQ ID NO.2.
Priority Applications (1)
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|---|---|---|---|
| CN2010101867044A CN101880682A (en) | 2010-05-31 | 2010-05-31 | 61-family inscribe cellulases eg gene clone of thermoascus aurantiacus var. levisporus and coding protein thereof |
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|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232949A (en) * | 2012-12-20 | 2013-08-07 | 山东农业大学 | Pichia pastoris engineered strain expressing 61-family glycoside hydrolase gene |
| CN103255072A (en) * | 2013-05-28 | 2013-08-21 | 山东农业大学 | Pichia pastoris yeast engineering bacterium expressing thermal stability cellulase gene fusant 61F-EG1 |
-
2010
- 2010-05-31 CN CN2010101867044A patent/CN101880682A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232949A (en) * | 2012-12-20 | 2013-08-07 | 山东农业大学 | Pichia pastoris engineered strain expressing 61-family glycoside hydrolase gene |
| CN103255072A (en) * | 2013-05-28 | 2013-08-21 | 山东农业大学 | Pichia pastoris yeast engineering bacterium expressing thermal stability cellulase gene fusant 61F-EG1 |
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Application publication date: 20101110 |