CN101962621A - Pichia pastoris engineering strains expressing chit-taa of Thermoascus aurantiacus var. aurantiacus - Google Patents
Pichia pastoris engineering strains expressing chit-taa of Thermoascus aurantiacus var. aurantiacus Download PDFInfo
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Abstract
The invention relates to a Pichia pastoris engineering bacteria GS-CHIT-TAA-176 expressing thermostable chit-taa of Thermoascus aurantiacus var. aurantiacus. The chit-taa is obtained from the Thermoascus aurantiacus var. aurantiacus through methods, i.e. RT-PCR (Reverse Transcription-Polymerase Chain Reaction), RACE, and the like to establish an expression vector pPIC9K/chit-taa; the expression vector pPIC9K/chit-taa is led into Pichia pastoris GS115, and the Pichia pastoris engineering bacteria GS-GHIT-TAA-176 expressing chitinase is screened from the Pichia pastoris GS115. The Pichia pastoris engineering bacteria GS-CHIT-TAA-176 can achieve the enzymatic activity of the chitinase by 28.96 U/mg, and the chitinase is stable at 50 DEG C and still has the activity of 70.3 percent at 60 DEG C for 60 minutes; and in addition, the invention has high heat stability, economic value and social value and is used for transforming chitin wastes in environments and other industrial fields.
Description
(1) technical field
The present invention relates to biotechnology, specifically a kind of pichia pastoris engineered strain Pichia pastoris GS-CHIT-TAA-176 that expresses the thermally-stabilised chitinase gene chit-taa of the former mutation Thermoascuaurantiacus of thermophilic ascomycete var.aurantiacus.
(2) background technology
Chitin (chitin) is to pass through β-1 by N-acetyl-β-D-glucosamine, and the linear polymer that the 4-glycosidic link couples together, structure and cellulose family are one of the abundantest natural high moleculer eompounds seemingly.Chitinase can produce chitin oligo saccharide and N-acetylglucosamine by the hydrolysis chitin, and extensively is present in the natural biology, has the important physical function, plays an important role in the biological control of plant pest.
According to the mode of action and enzymolysis product, chitinase is divided into: endochitinase (endochitinase), N-acetyl-β-glucosaminidase and chitobiose enzyme (chitbiosidase).Endochitinase produces the chitin oligosaccharide, as Glu (NAG) from arbitrary position cutting of chitin chain
2, Glu (NAG)
3Deng; N-acetyl β-glucosaminidase claims excision enzyme (exochitinase) again, and this enzyme downcuts N-acetyl-β-glucose chitin monose successively from the non-reduced end of chitin oligosaccharides or chitin chain; The chitobiose enzyme then is single-minded chitin monose that chitobiose is degraded to.These three kinds of enzymes can be analysed with ply of paper and be distinguished, and the SDS-PAGE technology then can effectively be identified.Each enzyme all has a series of isozymes in addition.
Chitinase is widely used in the aspects such as comprehensive utilization of light industry, medicine, environmental protection, food-processing, renewable resources, and being considered to has one of zymin of application potential most.At present both at home and abroad chitin quality enzyme is mainly by the mould production of normal temperature wood, 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 chitinase and application are very limited.Because therefore thermally-stabilised chitinase thermally-stabilised under hot conditions have important application prospect and commercial value.
The former mutation of thermophilic ascomycete (Thermoascus aurantiacus var aurantiacus) can produce heat-staple chitinase under 50 ℃ of conditions in the substratum that with the tobacco brown spot pathogen is sole carbon source; but make it to be used for large-scale industrial production, also have some open questions still.Relatively harsher as the former mutation culture condition of thermophilic ascomycete, thermophilic fermentation needs specific installation, and product enzyme efficient is low, causes cost to increase, and has therefore limited its application.The effective way that addresses this problem is the applied molecular biology means, to efficiently express in the thermally-stabilised chitinase gene importing of the former mutation of the thermophilic ascomycete normal temperature yeast, utilize yeast growth fast, be easy to characteristics such as cultivation, make thermally-stabilised chitinase gene normal temperature and in the short period of time fast, great expression, expectation reaches the purpose that cuts down the consumption of energy and increase economic efficiency.
(3) summary of the invention
The present invention is that material obtains a kind of chitinase gene with the former mutation of thermophilic ascomycete (Thermoascus aurantiacus var aurantiacus), called after chit-taa, accession number in Genbank is GU338053, full-length cDNA is 1385bp, comprise an open reading frame that constitutes by 1188 Nucleotide, 396 amino acid of encoding.This aminoacid sequence is retrieved in international gene pool, found that this chitinase belongs to glycoside hydrolase 18 families, comprises two region S-X-G-G and D-X-D-X-E high conservative.
Make up recombinant expression plasmid carrier pPIC9K/chit-taa, utilize electroporation to shock by electricity and transform Pichiapastoris GS115, on MD and MM substratum, screen, through PCR evaluation and screening positive transformant, G418 screening multiple copied transformant, carry out methanol induction then and express, obtain pichia pastoris engineered strain GS-CHIT-TAA-176.This project inoculation is in containing the BMGY substratum, after 28 ℃ of 200rpm/min shaking tables are cultivated 6d, chitinase work reaches 28.96U/mg, it is 43.9kDa that SDS-PAGE detects this proteic molecular weight, enzyme is at 60 ℃ of insulation 60min, still have 67.7% activity, have certain thermostability, important economic value and social value are arranged.
(4) description of drawings
Fig. 1 chitinase gene chit-taa PCR product electrophoretogram
Swimming lane 1:Marker-DL2000
Swimming lane 2:cDNA (ORF)
The SDS-PAGE of Fig. 2 chitinase CHIT-TAA analyzes
Swimming lane M: low molecular weight protein (LMWP) Marker
Swimming lane 1-7: Yeast engineering bacteria Pichia pastoris GS-CHIT-TAA-176 induces 1-7 days expression
The purifying of Fig. 3 recombinant chitinase CHIT-TAA
Swimming lane 1: low molecular weight protein (LMWP) standard
Swimming lane 2: the recombinant chitinase CHIT-TAA of purifying
(5) embodiment
Embodiment 1: the isolation identification of the former mutation of thermophilic ascomycete (T.aurantiacus var aurantiacus)
(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: the clone of chitinase gene chit-taa
(1) extraction of the total RNA of the former mutation of thermophilic ascomycete: 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 the homology conserved sequence design degenerate primer of chitinase.Upstream primer is: 5 '-TAYTTYGTCAAYTGGGCMAT-3 ', downstream primer is: 5 '-CRGCRTARTCRTARGCCAT-3 '
(4) 3 ' with the separating of 5 ' sequence: chitinase gene 5 ' terminal sequence clone, use the method for Tail-PCR, obtain 3 outside special nested primers of fragment design according to middle front, and by nested primer and the public degenerate primer AD in upstream
1-5Amplification obtains.
P5-41：5’-GTTTCTCGGCTGGCAGGTCTTGAG-3’
P5-103：5’-GGTCAAGTATCTTCGCCGTTGTC-3’
P5-290：5’-CCCGTCCAGCTTCAGTACTC-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’
Chitinase gene 3 ' terminal sequence clone, according to TaKaRa RNAPCR kit (AMV) Ver3.0 (TaKaRa) and 3 ' RACE Kit, 2nd Genneration (Roche Applied Science) operation instruction is carried out.The upstream primer sequence of 3 ' RACE is:
5’-GTCTTGATGGGCTGGAT-3’
5’-GACACGGACAAGCACTATC-3’
Downstream primer is M13M4:GTTTTCCCAGTCACGAC.
(5) gene clone: get 0.5 μ l PCR recovery 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, and aligning primer is the M13 promoter primer.The cDNA of the former mutation chit-taa of thermophilic ascomycete total length is 1385bp.Open reading frame partly is 1188bp, and 396 the amino acid whose one section sequences of encoding are retrieved this aminoacid sequence in international gene pool, finds to belong to glycoside hydrolase the 18th family, wherein 2 motif S-X-G-G and D-X-D-X-E tool conservative property.This sequence is as follows:
(A) information of SEQ ID NO 1
(a) sequence signature:
*Length: 1385 base pairs;
*Type: nucleic acid;
*Chain: two strands;
*Topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: the former mutation of thermophilic ascomycete (Thermoascus aurantiacus var.aurantiacus)
(f) sequence description:
1 CTGCAAAAGA?GATCATCTGG?GATGAAATCT?GTGGTTTATT?TTGTAAATTG?GGCAATCTAC
61 GGCCGCAACC?ACCACCCTCA?AGACCTGCCA?GCCGAGAAAC?TTACTCACGT?TCTCTACGCC
121?TTTGCCAATG?TCCGTCCGGA?CAACGGCGAA?GTATACTTGA?CCGACCCATG?GTCCGACACG
181 GACAAGCACT?ATCCCAGCGA?TTCATGGAAC?GACGTCGGCA?CCAATGTCTA?CGGGTGCATC
241 AAGCAGCTGT?TCCTGCTCAA?GAAGCGCAAC?AGGAATCTGA?AAGTGCTCCT?GTCAATTGGT
301 GGCTGGACGT?ACTCATCAAA?TTTTGCCCAA?CCGGCGAGTA?CTGAAGCTGG?ACGGGCCAGG
361 TTCGCGGAGA?CTGCTGTGCA?GTTGCTTCTT?GATCTGGGTC?TTGATGGGCT?GGATGTTGAT
421 TGGGAATACC?CCAAGGACGA?CAACGAGGCT?CAGAACTTCG?TTCTGCTCCT?TCAGAAATGT
481 CGCGAGACCC?TCGACAGAGT?CGGCGGTCCA?AACCGCAGAT?TCCTTCTCAC?CATTGCCTGT
541 CCAGCTGGAC?CCCAGAACTT?CACCAAACTC?CGCCTTCGCG?AAATGACCCC?CTACCTCGAC
601 TTCTACAACC?TCATGGGCTA?TGACTATGCC?GGTAGCTGGG?ACACCATAGC?CGGCCACCAG
661 ACCAACCTCT?ACCCATCATC?CTCCAACCCA?TCCAGCACCC?CCTTCTCCAC?CGTTGCGGCC
721 CTGGACTACT?ACATCAACGT?GGGCGGTGTC?CCGCACTCGA?AGATGATCCT?GGGCATGCCG
781 CTGTACGGAC?GCGCATTCAC?CAACACCGAT?GGCCCCGGCA?CACCGTTCTC?CGGGACCGGA
841 GAAGGTAGCT?GGGAGCAAGG?TGTCTGGGAC?TACAAGGCTC?TTCCGCGACC?GGGAGCCACT
901 GAGTACTTAG?ACCCGGATGC?GGGTGCGTCG?TGGTGCTATG?ACGGGGCCTC?CCGGACGATG
961 GTCTCGTACG?ACACTGTGCC?GATGGCGGAG?AGGAAGGTGG?AGTTTATCAG?GCAGCGCGGG
1021?CTGGGTGGTG?CTATGTGGTG?GGAGAGCAGC?GGTGATAAAG?GTGGAAAGAC?TGCCAACAAG
1081?GCTGATGGGA?GTCTTATTGG?GGCATTCGTG?GATGGGGTTG?GAGGAACTGG?GGCGCTGGAG
1141?CAGGTACCCA?ACGTTCTGGA?GTTCCCGGAG?AGCAAATACG?ATAACCTGCG?GGCTGGTTTC
1201?CCGGGGGAGT?AGACGGATGG?ATGAGAACGA?GAATGTGCAG?GCCTCGTCAA?GAGGGATGAG
1261?TCGTCCTCTT?CGATTTTTGA?CACATGAGAA?TGTTACCTAC?AGCAGCAGAT?GTACACTATG
1321?ACGGCGGCAA?GTGCCTTTAT?TTTGTTCTTT?ATCCAGCAGC?TACTACCGCT?AAAAAAAAAA
1381?AAAAA
(B) information of SEQ ID NO 2
(a) sequence signature: * length: 396 amino acid; * type: amino acid; * chain: strand; * topological framework: linearity
(b) molecule type: protein
(c) sequence description:
1 MKSVVYFVNW?AIYGRNHHPQ?DLPAEKLTHV?LYAFANVRPD?NGEVYLTDPW?SDTDKHYPSD
61 SWNDVGTNVY?GCIKQLFLLK?KRNRNLKVLL?SIGGWTYSSN?FAQPASTEAG?RARFAETAVQ
121?LLLDLGLDGL?DVDWEYPKDD?NEAQNFVLLL?QKCRETLDRV?GGPNRRFLLT?IACPAGPQNF
181?TKLRLREMTP?YLDFYNLMGY?DYAGSWDTIA?GHQTNLYPSS?SNPSSTPFST?VAALDYYINV
241?GGVPHSKMIL?GMPLYGRAFT?NTDGPGTPFS?GTGEGSWEQG?VWDYKALPRP?GATEYLDPDA
301?GASWCYDGAS?RTMVSYDTVP?MAERKVEFIR?QRGLGGAMWW?ESSGDKGGKT?ANKADGSLIG
361?AFVDGVGGTG?ALEQVPNVLE?FPESKYDNLR?AGFPGE
Embodiment 3: the structure of expression vector
(1) express primer according to the nucleotide sequence design of isolated chit-taa gene, introduce EcoR I and Not I restriction enzyme site respectively at 5 ' end of primer:
Upstream primer: 5 '-CCGAATTCTCTGGGATGAAATCTGTGGT-3 '
Downstream primer: 5 '-GGGCGGCCGCCTACTCCCCCGGGAAAC-3 '
(2) extraction of the total RNA of the former mutation of thermophilic ascomycete: utilize Trizol reagent to extract.
(3) the synthetic cDNA article one chain of reverse transcription: TaKaRa RNA PCR kit (AMV) the Ver3.0 test kit specification sheets according to TaKaRa company carries out, and is synthetic cDNA first chain of primer with Oligo dT-Adaptor primer.Reaction conditions is: 42 ℃ of incubation 60min, boil 5min again with the deactivation ThermoScript II.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.
(4) PCR reaction (25 μ L): cDNA 2 μ L, 10 * Buffer, 2.5 μ L, 10mmol/L dNTP 2 μ L, 25mmol/L MgCl
22 μ L, each 1 μ L of upstream and downstream primer, Taq archaeal dna polymerase 0.5 μ L (5U/ μ L), ddH
2O 14 μ L.Reaction conditions is 94 ℃ of pre-sex change of 3min; 94 ℃ of 1min, 57 ℃ of 1min, 72 ℃ of 3min, totally 30 circulations, 72 ℃ are extended 10min, 10 ℃ of preservations.
(5) gene clone: get 0.5 μ L PCR product and be connected with the pMD18-T carrier, obtain recombinant plasmid pMD18-T/chit-taa, operation steps is undertaken by TaKaRa company product description.Connect product transformed into escherichia coli DH5 α then, scribbling grow overnight on the LB flat board of X-gal and IPTG.The picking white colony, overnight incubation in the LB liquid nutrient medium.
(6) plasmid DNA is extracted: alkaline process extracts plasmid DNA.
(7) with EcoR I and Not I recombinant plasmid pMD18-T/chit-taa product is carried out double digestion, use EcoR I and Not I double digestion expression plasmid of yeast pPIC9K simultaneously, DNA glue reclaims test kit and reclaims purifying chit-taa gene and carrier pPIC9K segment.Carry out external connection then, obtain recombinant plasmid pPIC9K/chit-taa, the recombinant plasmid transformed e. coli jm109, transform on the flat board at the LB that contains Amp and to select single bacterium colony, extract plasmid DNA, carry out PCR and enzyme and cut evaluation, order-checking confirms that the reading frame of recombinant plasmid is correct.
Embodiment 4: express the structure and the screening of the Yeast engineering bacterium strain of chitinase gene chit-taa
(1) linearizing of recombinant expression plasmid: with recombinant expression plasmid pPIC9K/chit-taa restriction enzyme Sac I linearizing.
(2) transform: electric shock transformed yeast bacterial strain Pichia pastoris GS115 yeast competent cell, method for transformation is referring to Invitrogen company pichia spp operational manual.
(3) screening: on MM and MD flat board, cultivate 2-4d with the corresponding dibbling of sterilization toothpick picking transformant for 30 ℃, picking is the positive transformant of all well-grown transformant on the MD/MM flat board.The picking positive transformant is distinguished dibbling in 1.00mg/mL, 2.00mg/mL, 3.00mg/mL, screening multiple copied transformant on the YPD flat board of 4.00mg/mL G418.
Embodiment 5: the abduction delivering of pichia spp Pichiapastoris GS115 and active the detection
(1) positive transformant is inoculated in contains in the 25mL BMGY substratum, 28 ℃ of 200rpm/min shaking tables are cultivated 24h (OD
600Reach 2-6), centrifugal collection thalline is resuspended in cell in the BMMY substratum of proper volume, to OD
600Value is that 1.0,28 ℃ of 200rpm/min continue to cultivate, and it is 1% that every 12h replenishes methyl alcohol to final concentration, every 24h sampling, and room temperature 10, the centrifugal 5min of 000g gets supernatant and carries out the SDS-PAGE analysis.
(2) enzymic activity detects: the DNS method is adopted in enzyme assay, and determining the protein quantity adopts the Bradford method.
(3) optimal reactive temperature of recombinant chitinase CHIT-TAA, pH and thermostability: the recombinant chitinase of abduction delivering is through the DEAE-Sepharose anion exchange chromatography, obtain the purifying protein of electrophoresis homogeneous, measure its character with the recombinant chitinase of purifying.Under differing temps and pH condition, measure the enzyme activity of recombinant chitinase respectively, the highest enzyme activity is defined as 100%, calculate the relative activity of chitinase under the condition of different temperatures respectively; Chitinase is incubated the different time under different temperature (10min, 20min, 30min, 40min, 50min, 60min) after, detect the residual enzyme vigor.
Embodiment 6: express the preservation of the pichia pastoris engineered strain GS-CHIT-TAA-176 of chit-taa gene
Express the depositary institution of the pichia pastoris engineered strain GS-CHIT-TAA-176 of chit-taa gene: 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; Yeast engineering bacterium strain is numbered: CGMCC No.3820; The classification called after of pichia pastoris engineered strain: pichia pastoris Pichia pastoris.
Sequence table
＜110〉Shandong Agricultural University
＜120〉pichia pastoris engineered strain of the former mutation chit-taa of expression thermophilic ascomycete gene
<140>
<141>
<160>2
<170>pantent?In?3.1
<210>1
<211>1369
<212>DNA
＜213〉the former mutation of thermophilic ascomycete (Thermoascus aurantiacus var.aurantiacus)
<220>
<221>CDS
<222>(22)-(1209)
<223>
<400>1
ctgcaaaagag?atcatctggg?atg?aaa?tct?gtg?gtt?tat?ttt?gta?aat?tgg 51
Met?Lys?Ser?Val?Val?Tyr?Phe?Val?Asn?Trp
1 5 10
gca?atc?tac?ggc?cgc?aac?cac?cac?cct?caa?gac?ctg?cca?gcc?gag?aaa 99
Ala?Ile?Tyr?Gly?Arg?Asn?His?His?Pro?Gln?Asp?Leu?Pro?Ala?Glu?Lys
15 20 25
ctt?act?cac?gtt?ctc?tac?gcc?ttt?gcc?aat?gtc?cgt?ccg?gac?aac?ggc 147
Leu?Thr?His?Val?Leu?Tyr?Ala?Phe?Ala?Asn?Val?Arg?Pro?Asp?Asn?Gly
30 35 40
gaa?gta?tac?ttg?acc?gac?cca?tgg?tcc?gac?acg?gac?aag?cac?tat?ccc 195
Glu?Val?Tyr?Leu?Thr?Asp?Pro?Trp?Ser?Asp?Thr?Asp?Lys?His?Tyr?Pro
45 50 55
agc?gat?tca?tgg?aac?gac?gtc?ggc?acc?aat?gtc?tac?ggg?tgc?atc?aag 243
Ser?Asp?Ser?Trp?Asn?Asp?Val?Gly?Thr?Asn?Val?Tyr?Gly?Cys?Ile?Lys
60 65 70
cag?ctg?ttc?ctg?ctc?aag?aag?cgc?aac?agg?aat?ctg?aaa?gtg?ctc?ctg 291
Gln?Leu?Phe?Leu?Leu?Lys?Lys?Arg?Asn?Arg?Asn?Leu?Lys?Val?Leu?Leu
75 80 85 90
tca?att?ggt?ggc?tgg?acg?tac?tca?tca?aat?ttt?gcc?caa?ccg?gcg?agt 339
Ser?Ile?Gly?Gly?Trp?Thr?Tyr?Ser?Ser?Asn?Phe?Ala?Gln?Pro?Ala?Ser
95 100 105
act?gaa?gct?gga?cgg?gcc?agg?ttc?gcg?gag?act?gct?gtg?cag?ttg?ctt 387
Thr?Glu?Ala?Gly?Arg?Ala?Arg?Phe?Ala?Glu?Thr?Ala?Val?Gln?Leu?Leu
110 115 120
ctt?gat?ctg?ggt?ctt?gat?ggg?ctg?gat?gtt?gat?tgg?gaa?tac?ccc?aag 435
Leu?Asp?Leu?Gly?Leu?Asp?Gly?Leu?Asp?Val?Asp?Trp?Glu?Tyr?Pro?Lys
125 130 135
gac?gac?aac?gag?gct?cag?aac?ttc?gtt?ctg?ctc?ctt?cag?aaa?tgt?cgc 483
Asp?Asp?Asn?Glu?Ala?Gln?Asn?Phe?Val?Leu?Leu?Leu?Gln?Lys?Cys?Arg
140 145 150
gag?acc?ctc?gac?aga?gtc?ggc?ggt?cca?aac?cgc?agattc?ctt?ctc?acc 531
Glu?Thr?Leu?Asp?Arg?Val?Gly?Gly?Pro?Asn?Arg?Arg?Phe?Leu?Leu?Thr
155 160 165 170
att?gcc?tgt?cca?gct?gga?ccc?cag?aac?ttc?acc?aaa?ctc?cgc?ctt?cgc 579
Ile?Ala?Cys?Pro?Ala?Gly?Pro?Gln?Asn?Phe?Thr?Lys?Leu?Arg?Leu?Arg
175 180 185
gaa?atg?acc?ccc?tac?ctc?gac?ttc?tac?aac?ctc?atg?ggc?tat?gac?tat 627
Glu?Met?Thr?Pro?Tyr?Leu?Asp?Phe?Tyr?Asn?Leu?Met?Gly?Tyr?Asp?Tyr
190 195 200
gcc?ggt?agc?tgg?gac?acc?ata?gcc?ggc?cac?cag?acc?aac?ctc?tac?cca 675
Ala?Gly?Ser?Trp?Asp?Thr?Ile?Ala?Gly?His?Gln?Thr?Asn?Leu?Tyr?Pro
205 210 215
tca?tcc?tcc?aac?cca?tcc?agc?acc?ccc?ttc?tcc?acc?gtt?gcg?gcc?ctg 723
Ser?Ser?Ser?Asn?Pro?Ser?Ser?Thr?Pro?Phe?Ser?Thr?Val?Ala?Ala?Leu
220 225 230
gac?tac?tac?atc?aac?gtg?ggc?ggt?gtc?ccg?cac?tcg?aag?atg?atc?ctg 771
Asp?Tyr?Tyr?Ile?Asn?Val?Gly?Gly?Val?Pro?His?Ser?Lys?Met?Ile?Leu
235 240 245 250
ggc?atg?ccg?ctg?tac?gga?cgc?gca?ttc?acc?aac?acc?gat?ggc?ccc?ggc 819
Gly?Met?Pro?Leu?Tyr?Gly?Arg?Ala?Phe?Thr?Asn?Thr?Asp?Gly?Pro?Gly
255 260 265
aca?ccg?ttc?tcc?ggg?acc?gga?gaa?ggt?agc?tgg?gag?caa?ggt?gtc?tgg 867
Thr?Pro?Phe?Ser?Gly?Thr?Gly?Glu?Gly?Ser?Trp?Glu?Gln?Gly?Val?Trp
270 275 280
gac?tac?aag?gct?ctt?ccg?cga?ccg?gga?gcc?act?gag?tac?tta?gac?ccg 915
Asp?Tyr?Lys?Ala?Leu?Pro?Arg?Pro?Gly?Ala?Thr?Glu?Tyr?Leu?Asp?Pro
285 290 295
gat?gcg?ggt?gcg?tcg?tgg?tgc?tat?gac?ggg?gcc?tcc?cgg?acg?atg?gtc 963
Asp?Ala?Gly?Ala?Ser?Trp?Cys?Tyr?Asp?Gly?Ala?Ser?Arg?Thr?Met?Val
300 305 310
tcg?tac?gac?act?gtg?ccg?atg?gcg?gag?agg?aag?gtg?gag?ttt?atc?agg 1011
Ser?Tyr?Asp?Thr?Val?Pro?Met?Ala?Glu?Arg?Lys?Val?Glu?Phe?Ile?Arg
315 320 325 330
cag?cgc?ggg?ctg?ggt?ggt?gct?atg?tgg?tgg?gag?agc?agc?ggt?gat?aaa 1059
Gln?Arg?Gly?Leu?Gly?Gly?Ala?Met?Trp?Trp?Glu?Ser?Ser?Gly?Asp?Lys
335 340 345
ggt?gga?aag?act?gcc?aac?aag?gct?gat?ggg?agt?ctt?att?ggg?gca?ttc 1107
Gly?Gly?Lys?Thr?Ala?Asn?Lys?Ala?Asp?Gly?Ser?Leu?Ile?Gly?Ala?Phe
350 355 360
gtg?gat?ggg?gtt?gga?gga?act?ggg?gcg?ctg?gag?cag?gta?ccc?aac?gtt 1155
Val?Asp?Gly?Val?Gly?Gly?Thr?Gly?Ala?Leu?Glu?Gln?Val?Pro?Asn?Val
365 370 375
ctg?gag?ttc?ccg?gag?agc?aaa?tac?gat?aac?ctg?cgg?gct?ggt?ttc?ccg 1203
Leu?Glu?Phe?Pro?Glu?Ser?Lys?Tyr?Asp?Asn?Leu?Arg?Ala?Gly?Phe?Pro
380 385 390
ggg?gag?tagacggatg?gatgagaacg?agaatgtgca?ggcctcgtca?agagggatga 1259
Gly?Glu
395
gtcgtcctct?tcgatttttg?acacatgaga?atgttaccta?cagcagcaga?tgtacactat 1319
gacggcggca?agtgccttta?ttttgttctt?tatccagcag?ctactaccgc?taaaaaaaaa 1379
aaaaaa 1385
<210>2
<211>396
<212>PRT
＜213〉the former mutation of thermophilic ascomycete (Thermoascus aurantiacus var.aurantiacus)
<400>2
MET?Lys?Ser?Val?Val?Tyr?Phe?Val?Asn?Trp?Ala?Ile?Tyr?Gly?Arg?Asn
1 5 10 15
His?His?Pro?Gln?Asp?Leu?Pro?Ala?Glu?Lys?Leu?Thr?His?Val?Leu?Tyr
20 25 30
Ala?Phe?Ala?Asn?Val?Arg?Pro?Asp?Asn?Gly?Glu?Val?Tyr?Leu?Thr?Asp
35 40 45
Pro?Trp?Ser?Asp?Thr?Asp?Lys?His?Tyr?Pro?Ser?Asp?Ser?Trp?Asn?Asp
50 55 60
Val?Gly?Thr?Asn?Val?Tyr?Gly?Cys?Ile?Lys?Gln?Leu?Phe?Leu?Leu?Lys
65 70 75 80
Lys?Arg?Asn?Arg?Asn?Leu?Lys?Val?Leu?Leu?Ser?Ile?Gly?Gly?Trp?Thr
85 90 95
Tyr?Ser?Ser?Asn?Phe?Ala?Gln?Pro?Ala?Ser?Thr?Glu?Ala?Gly?Arg?Ala
100 105 110
Arg?Phe?Ala?Glu?Thr?Ala?Val?Gln?Leu?Leu?Leu?Asp?Leu?Gly?Leu?Asp
115 120 125
Gly?Leu?Asp?Val?Asp?Trp?Glu?Tyr?Pro?Lys?Asp?Asp?Asn?Glu?Ala?Gln
130 135 140
Asn?Phe?Val?Leu?Leu?Leu?Gln?Lys?Cys?Arg?Glu?Thr?Leu?Asp?Arg?Val
145 150 155 160
Gly?Gly?Pro?Asn?Arg?Arg?Phe?Leu?Leu?Thr?Ile?Ala?Cys?Pro?Ala?Gly
165 170 175
Pro?Gln?Asn?Phe?Thr?Lys?Leu?Arg?Leu?Arg?Glu?MET?Thr?Pro?Tyr?Leu
180 185 190
Asp?Phe?Tyr?Asn?Leu?MET?Gly?Tyr?Asp?Tyr?Ala?Gly?Ser?Trp?Asp?Thr
195 200 205
Ile?Ala?Gly?His?Gln?Thr?Asn?Leu?Tyr?Pro?Ser?Ser?Ser?Asn?Pro?Ser
210 215 220
Ser?Thr?Pro?Phe?Ser?Thr?Val?Ala?Ala?Leu?Asp?Tyr?Tyr?Ile?Asn?Val
225 230 235 240
Gly?Gly?Val?Pro?His?Ser?Lys?MET?Ile?Leu?Gly?MET?Pro?Leu?Tyr?Gly
245 250 255
Arg?Ala?Phe?Thr?Asn?Thr?Asp?Gly?Pro?Gly?Thr?Pro?Phe?Ser?Gly?Thr
260 265 270
Gly?Glu?Gly?Ser?Trp?Glu?Gln?Gly?Val?Trp?Asp?Tyr?Lys?Ala?Leu?Pro
275 280 285
Arg?Pro?Gly?Ala?Thr?Glu?Tyr?Leu?Asp?Pro?Asp?Ala?Gly?Ala?Ser?Trp
290 295 300
Cys?Tyr?Asp?Gly?Ala?Ser?Arg?Thr?MET?Val?Ser?Tyr?Asp?Thr?Val?Pro
305 310 315 320
MET?Ala?Glu?Arg?Lys?Val?Glu?Phe?Ile?Arg?Gln?Arg?Gly?Leu?Gly?Gly
325 330 335
Ala?MET?Trp?Trp?Glu?Ser?Ser?Gly?Asp?Lys?Gly?Gly?Lys?Thr?Ala?Asn
340 345 350
Lys?Ala?Asp?Gly?Ser?Leu?Ile?Gly?Ala?Phe?Val?Asp?Gly?Val?Gly?Gly
355 360 365
Thr?Gly?Ala?Leu?Glu?Gln?Val?Pro?Asn?Val?Leu?Glu?Phe?Pro?Glu?Ser
370 375 380
Lys?Tyr?Asp?Asn?Leu?Arg?Ala?Gly?Phe?Pro?Gly?Glu
385 390 395
Claims (1)
1. Yeast engineering bacterium strain of expressing the former mutation of thermophilic ascomycete (Thermoascus aurantiacus var.aurantiacus) chitinase gene chit-taa, it is characterized in that this bacterium is a kind of pichia spp, obtain thermally-stabilised chitinase gene chit-taa by methods such as RT-PCR and RACE from the former mutation of thermophilic ascomycete (Thermoascus aurantiacus var.aurantiacus), pichia spp secreted expression carrier pPIC9K is arrived in this gene clone, obtain recombinant expression pPIC9K/chit-taa, transform pichia spp GS115, therefrom filter out the pichia pastoris engineered strain GS-CHIT-TAA-176 that expresses thermally-stabilised chitinase gene chit-taa.
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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 |
| CN111763665A (en) * | 2019-04-01 | 2020-10-13 | 江苏师范大学 | Preparation method of high-activity sweet potato chitinase and application of high-activity sweet potato chitinase in preparation of plant pathogenic fungus antibacterial agent |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307295A (en) * | 2008-06-05 | 2008-11-19 | 山东农业大学 | Pichia yeast engineering strain for expressing chaetomium thermophilum gene cbh3 |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101307295A (en) * | 2008-06-05 | 2008-11-19 | 山东农业大学 | Pichia yeast engineering strain for expressing chaetomium thermophilum gene cbh3 |
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| Title |
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| 《Biosci.Biotechnol.Biochem.》 20071231 Shijin E et al. Purification,characterization,and molecular cloning of a thermostable superoxide dismutase from Thermoascus aurantiacus 第1090-1093页 1 第71卷, 第4期 2 * |
| 《中国优秀硕士学位论文全文数据库 农业科技辑》 20090315 于恺 嗜热子囊菌光孢变种几丁质酶基因的克隆和表达 摘要，正文第28-46页，第64-71页，第76-84页 1 , 2 * |
| 《中国优秀硕士学位论文全文数据库 基础科技辑》 20080115 赵春青 中国嗜热真菌分类研究 A006-71 1 , 2 * |
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 |
| CN111763665A (en) * | 2019-04-01 | 2020-10-13 | 江苏师范大学 | Preparation method of high-activity sweet potato chitinase and application of high-activity sweet potato chitinase in preparation of plant pathogenic fungus antibacterial agent |
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