CN103484390A - Pichia pastoris engineering strains expressing nine-family glucoside hydrolase gene CtAA9b - Google Patents
Pichia pastoris engineering strains expressing nine-family glucoside hydrolase gene CtAA9b Download PDFInfo
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Abstract
The invention provides pichia pastoris GS-CT-9B expressing Chaetomium thermophilum thermal stability nine-family glucoside hydrolase gene CtAA9b. The glucoside hydrolase gene CtAA9b is obtained from the Chaetomium thermophilum in an RT-PCR method, cloned and inserted into pichia pastoris integration expression vectors pPIC9K, then the obtained glucoside hydrolase gene CtAA9b expression vectors pPIC9K/CtAA9b are led in pichia pastoris GS115, and one kind of pichia pastoris GS-CT-9B expressing the glucoside hydrolase gene CtAA9b is screened from the pichia pastoris GS115. The glucoside hydrolase gene CtAA9b expressed by the GS-CT-9B has obvious prompting effects on activity of enco-cellulase, and activity of mixed enzyme is improved by 1.7 times that of original incision enzyme N24. Under the condition of Mn2+, the activity of the cellulose CtAA9B and cellulose CtAA9B+N24 are improved by 15 times and 2.54 times. The CtAA9B has good thermal stability and the capacity of improving activity of the cellulose, and the pichia pastoris GS-CT-9B can be widely used for the fields of fiber waste and other industries, and has large economic value and society value.
Description
(1) technical field
The present invention relates to biotechnology, specifically a kind of expression is specifically with the pichia pastoris engineered strain Pichia pastoris GS-CT-9B of the glycoside hydrolase gene C tAA9b of chaetomium thermophilum Chaetomium thermophilum9 family.
(2) background technology
Glycoside hydrolase (English: Glycoside hydrolases claims again Glycosylase) is a kind of special hydrolysis glycosidic linkage (glycosidic bond), and produces the ferment of two less glycan molecules, is also one of common ferment of occurring in nature.This proteinoid is also application to some extent on the mankind's industry, such as being used for, the biomass energies such as Mierocrystalline cellulose and hemicellulose is degraded into to available small molecules.Glycoside hydrolase has a plurality of families.
Chaetomium thermophilum Chaetomium thermophilum is a kind of thermophilic fungus be distributed widely in soil.Can produce heat-staple cellulase and 9 family's glycoside hydrolases under 50 ℃ of conditions in the substratum that this bacterium is sole carbon source at Microcrystalline Cellulose.
The 9 glycoside hydrolase CtAA9B of family that chaetomium thermophilum produces have faint cellulase activity, but the enzymic activity of the endo cellulase N24 that this bacterial strain is produced has obvious promoter action, can make 1.7 times of its active raisings.At the different metal ion as Mn
2+effect under, the activity of CtAA9B and CtAA9B+N24 can improve respectively 15 times and 2.54 times.
(3) summary of the invention
The present invention obtains inscribe β-1 from chaetomium thermophilum Chaetomium thermophilum, the cDNA sequence total length of 4-glucanase gene, called after CtAA9b, CtAA9b gene DNA total length 1041bp, comprise signal peptide sequence, initiator codon and terminator codon, 322 amino acid of encoding, have signal peptide sequence (1-24aa MVKAKKSAFLATVAGASLVAAHGY).The aminoacid sequence of CtAA9b coding is retrieved in international gene pool, found to belong to glycoside hydrolase the 9th family.
Build recombinant expression plasmid carrier pPIC9K/CtAA9b, utilize electroporation to shock by electricity and transform Pichia pastoris GS115, on MD and MM substratum, screen, through PCR evaluation and screening positive transformant, G418 screening multiple copied transformant, then carry out the methanol induction expression, obtain pichia pastoris engineered strain GS-CT-9B.This project inoculation is in containing the BMGY substratum, and after 28 ℃ of 200rpm/min shaking tables are cultivated 6d, the expression amount of glycoside hydrolase is 3.16mg/ml, molecular weight is 55KDa, enzyme activity is 0.0365U/ml, and CtAA9b is stable under 65 ℃, after processing 60min, still has the enzymic activity more than 95%; Still keep 64% activity after 70 ℃ of processing 30min; Keep 13.5% activity after 80 ℃ of processing 30min; Processing the 30min activity for 90 ℃ almost completely loses.
The 9 glycoside hydrolase CtAA9B of family have obvious promoter action to the enzymic activity of chaetomium thermophilum Chaetomium thermophilum endo cellulase N24, and mixed enzyme improves 1.7 times than the ability of the degraded cellulose of protoenzyme N24.Different metal ion pair CtAA9B and CtAA9B+N24 mixed enzyme activity have promotion or restraining effect, wherein at Mn
2+under condition, the cellulase activity of CtAA9B and CtAA9B+N24 can improve respectively 33 times and 2.54 times.
(4) accompanying drawing explanation:
The SDS-PAGE of the glycoside hydrolase CtAA9B of Figure 19 family analyzes
Swimming lane 1: low molecular weight protein (LMWP) standard
Swimming lane 2: the 9 glycoside hydrolase CtAA9B of family of purifying
The optimum temperuture of the glycoside hydrolase CtAA9B of Fig. 2 A:9 family
The thermal stability determination of the glycoside hydrolase CtAA9B of B:9 family
The promoter action of the glycoside hydrolase CtAA9B of Figure 39 family to endo cellulase N24 activity
Fig. 4 metal ions M n
2+impact on CtAA9B cellulase and mixed enzyme activity
(5) embodiment
Embodiment 1: the isolation identification of chaetomium thermophilum Chaetomium thermophilum
(1) collection of specimens: gather from compost.
(2) separation and Culture: collect specimen is got to 0.5 gram and be placed on the dull and stereotyped upper 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 glycoside hydrolase gene C tAA9b
(1) extraction of the total RNA of chaetomium thermophilum Chaetomium thermophilum: 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, by the RNA sample, at 75 ℃ of sex change 5min, cooling 5min in ice bath, then once centrifugal a little immediately, adds successively following various composition in ice bath: 10mmol/L dNTP Mixture2 μ L, 10 * RT Buffer (Mg
2+) 2 μ L, 25mmol/L MgCl
24 μ L, Oligo d (T)-Adaptor Primer1 μ L, RNase Inhibiter0.5 μ L, AMV Reverse Transcriptase1 μ L(Final Volume20 μ L), after reaction solution is mixed, room temperature is transferred 10min, 42 ℃ of incubation 60min then, then boil 5min with the deactivation ThermoScript II.The ddH that adds 180 μ L DEPC to process
2o, be diluted to 200 μ L, mixes, centrifugal a little, is stored in-20 ℃, standby.
(3) PCR reaction (25 μ L): cDNA2 μ L, 10 * Buffer2.5 μ L, 10mmol/L dNTP2 μ L, 25mmol/LMgCl22 μ L, each 2 μ L of upstream and downstream primer, Taq archaeal dna polymerase 0.5 μ L (5U/ μ L), ddH2O12 μ L.Reaction conditions is 94 ℃ of 5min denaturations; 94 ℃ of 40s, 57 ℃ of 40s, 72 ℃ of 1min, totally 32 circulations, 72 ℃ are extended 10min, 4 ℃ of preservations.
Upstream primer: 5 '-ATGGTCAAGGCTAAGAAGTC-3 '
Downstream primer: 5 '-TTAGACGCACTGGTGGTACC-3 '
(4) 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.Then connect product and transform e.colistraindh5α, scribble grow overnight on the LB flat board of acillin (100 μ g/mL) on surface.The picking white colony, overnight incubation in the LB liquid nutrient medium.
(5) extraction of plasmid DNA: alkaline process extracts plasmid DNA.
(6) sequencing: DNA double deoxidation method is measured nucleotide sequence, and in Shanghai, biotechnology company limited carries out.Aligning primer is the M13 promoter primer.Chaetomium thermophilum CtAA9b gene cDNA total length 1041bp, comprise initiator codon and terminator codon, intronless, 322 amino acid of encoding.This aminoacid sequence is retrieved in international gene pool, found to belong to glycoside hydrolase the 9th family.This sequence is as follows:
(A) information of SEQ ID NO1
(a) sequence signature: * length: 1041 base pairs; * type: nucleic acid; * chain: two strands; * topological framework: linearity
(b) molecule type: DNA
(c) suppose: no
(d) antisense: no
(e) initial source: chaetomium thermophilum Chaetomium thermophilum
(f) sequence description:
1 ATGGTCAAGG CTAAGAAGTC TGCTTTTCTC GCCACCGTCG CTGGTGCCAG CCTCGTTGCT
61 GCCCACGGCT ATGTCAACGG AATCGTCGTC AACGGCGTCT ACTACAGGAA CTACAACCCC
121 TCGGTCGACT GGTACCGTGG CAACAACCAG GAGACTCTGA TCGGCTGGAG GGCTGAGAAC
181 ACGGACAACG GCTTCGTCGA GCCCAACAAG TTCAACACTG CTGACATCAT CTGCCACCGC
241 CAGGCCGTCA ATGCCAAGGG CTATGCCACC GTCAAGGCCG GCGACAAGAT TAATATCAAG
301 TGGGACCCAA TCTGGCCTGA GAGCCACGTC GGTCCCGTCA TCGACTACCT TGCCGACTGC
361 AACGGCGACT GCTCTACAGT CTCCAAGAAC TCCCTTCGGT TCTTCAAGAT CGACGGTGCC
421 GGCTACGACA AGGCCAAGGG CAAGTGGGCT GCCGATGTCC TCCGTGAAAA TGGCAACAGC
481 TGGATGGTCC AGATCCCGGC TGACCTGAAG CCCGGTCACT ACGTCCTTCG CCATGAGATC
541 ATCGCCCTCC ACGGTGCTGC CAACCCCAAT GGCGCTCAGG CCTATCCTCA GTGCATCAAC
601 ATCAAGGTTG AGGGCAGCGG CAGCAACTCG CCTTCCGGCG TTGCCGGCAC CTCTCTCTAT
661 ACCGCCAACG ACCCGGGCAT CCTCTTCAAC CCTTGGGTCA GCAACATCGA CTACCCGGTT
721 CCGGGCCCTG CTCTGATCCC CGGTGCTGTC AGCTCCATCC AGCAGAGCAC CTCGGCCGCT
781 ACCAGAACCG CCTCAGCCAC CCCTTACGCG GGCGGCGCTG TTCCAACCAC CACTCAGGGC
841 GGCAGCCAGC CCACTACCAC GGCTCTCCCG ACCACTACCC TCGTCACCAC CACTGCTGTC
901 CCGATCACCA CTGCTCCTCC GGCTGGCCCT ACTCAGAGCA AGTGGGGACA GTGCGGTGGC
961 AGCGGCTACA CCGGCCCGAC TCTCTGCGCT CCTGGCTCGA ACTGCCAGGT CATCAACCCC
1021 TGGTACCACC AGTGCGTCTA A
(B) information of SEQ ID NO2
(a) sequence signature: * length: 322 amino acid; * type: amino acid; * chain: strand; * topological framework: linearity
(b) molecule type: protein
(c) sequence description:
1 VNGIVVNGVY YRNYNPSVDW YRGNNQETLI GWRAENTDNG FVEPNKFNTA DIICHRQAVN
61 AKGYATVKAG DKINIKWDPI WPESHVGPVI DYLADCNGDC STVSKNSLRF FKIDGAGYDK
121 AKGKWAADVL RENGNSWMVQ IPADLKPGHY VLRHEIIALH GAANPNGAQA YPQCINIKVE
181 GSGSNSPSGV AGTSLYTAND PGILFNPWVS NIDYPVPGPA LIPGAVSSIQ QSTSAATRTA
241 SATPYAGGAV PTTTQGGSQP TTTALPTTTL VTTTAVPITT APPAGPTQSK WGQCGGSGYT
301 GPTLCAPGSN CQVINPWYHQ CV
Embodiment 3: the structure of expression vector
(1) according to the nucleotide sequence of isolated CtAA9b gene, primer is expressed in design, at 5 ' end of primer, introduces respectively Ecor I and Not I restriction enzyme site, and downstream primer is introduced 6 Histidine sequences, as purification tag: upstream primer: 5 '-CCG
gAATTCcACCACCACCACCACCACGTCAACGGAATCGTCG-3 '
(EcorI); Downstream primer: 5 '-TT
gCGGCCGCtTAGACGCACTGGTGGTACC-3 '
(Not I) (Histidine sequence)
(2) extraction of the total RNA of chaetomium thermophilum: utilize Trizol reagent to extract.
(3) the synthetic cDNA article one chain of reverse transcription: get the total RNA of 2 μ g, add 5 * reaction buffer, 4 μ L, 10mM dNTP 2 μ L, ribonuclease inhibitor (40-200u/ μ L) 0.5 μ L, primer oligodT (1 μ g/ μ L) 1 μ L, ThermoScript II (10u/ μ L) 2 μ L, 42 ℃ of reaction 60min, then 85 ℃ of 10min termination reactions, be diluted to 200 μ L.
(4) PCR reaction (25 μ L): cDNA2 μ L, 10 * Buffer, 2.5 μ L, 10mmol/L dNTP2 μ L, 25mmol/LMgCl
22 μ L, each 2 μ L of upstream and downstream primer, TaqDNA polysaccharase 0.5 μ L (5U/ μ L), ddH
2o 12 μ L.Reaction conditions is 94 ℃ of 5min denaturations; 94 ℃ of 40s, 57 ℃ of 40s, 72 ℃ of 1m totally 32 circulations, 72 ℃ are extended 10min, 4 ℃ of preservations.
(5) gene clone: get 2 μ L PCR products and be connected with the pMD18-T carrier, obtain recombinant plasmid pMD18T/CtAA9b operation steps and undertaken by TAKARA company product description.Then connect product and transform bacillus coli DH 5 alpha, scribbling grow overnight on the LB flat board of AMP.The picking white colony, overnight incubation in the LB liquid nutrient medium.
(6) extraction of plasmid DNA: alkaline process extracts plasmid DNA.
(7) by Ecor I and Not I, recombinant plasmid pMD18-T/CtAA9b product is carried out to double digestion, use EcorI and Not I double digestion expression plasmid of yeast pPIC9K simultaneously, DNA glue reclaims test kit and reclaims purifying CtAA9b gene and carrier pPIC9K segment.Then carry out external connection, obtain recombinant plasmid pPIC9K/CtAA9b, the recombinant plasmid transformed e. coli jm109, transform on flat board and select single bacterium colony at the LB containing Amp, 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. expresses structure and the screening of the Yeast engineering bacterium strain of glycoside hydrolase gene C tAA9b
(1) linearizing of recombinant expression plasmid: by restriction enzyme Sac I linearizing for recombinant expression plasmid pPIC9K/CtAA9b.
(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 sterilizing toothpick picking transformant for 30 ℃, picking equal positive transformant of well-grown transformant on the MD/MM flat board.Picking positive transformant dibbling respectively screens the multiple copied transformant on the YPD flat board of 250 μ g/mL, 300 μ g/mL, 400 μ g/mL, 500 μ g/mL, 600 μ g/mLG418.
Embodiment 5: the abduction delivering of pichia spp Pichia pastoris GS115 and active the detection
(1) positive transformant is inoculated in containing in 25mL BMGY substratum, 30 ℃ of 250rpm/min shaking tables are cultivated 24h(OD
600reach 2-6), centrifugal collection thalline, be resuspended in cell in the BMMY substratum of proper volume, to OD
600value is that 1.0,30 ℃ of 250rpm/min continue to cultivate, and it is 0.5% that every 24h supplements methyl alcohol to final concentration, every 24h sampling, and room temperature 10, the centrifugal 5min of 000g, get supernatant and carry out the SDS-PAGE analysis.
(2) enzymic activity detects: enzyme assay adopts the DNS method, the enzyme liquid that reaction system and reaction conditions are 100 μ L, 0.5% Microcrystalline Cellulose that adds 200 μ L, 60 ℃ of reaction 30min of the acetate buffer solution of 100 μ L (0.4mol/L, pH5.0), add 400 μ L DNS reagent, boil 10min, under the 540nm wavelength, measure absorbance value.Control group is done contrast with the enzyme liquid of inactivation.Activity with fermentoid liquid is defined as 100%, measures the relative activity of glycoside hydrolase CtAA9B.Triplicate, average.Determining the protein quantity adopts the Bradford method.
(3) optimal reactive temperature, pH and the thermostability of restructuring glycoside hydrolase CtAA9B: the HisTrap FF crude metal-ligand affinity chromatography column purification of the restructuring glycoside hydrolase process GE company of abduction delivering is with the recombinant protein of histidine mark, obtain the purifying protein of electrophoresis homogeneous, by its character of restructuring glycoside hydrolysis enzymatic determination of purifying.Under differing temps and pH condition, measure respectively the restructuring glycoside hydrolase to cellulosic enzyme activity, the highest enzyme activity is defined as to 100%, calculate respectively the relative activity of glycoside hydrolase under condition of different temperatures; The restructuring glycoside hydrolase is incubated to 30min at different temperature, detects the residual enzyme vigor.Triplicate, average.
The impact of embodiment 6:9 family glycoside hydrolase on promoter action and its activity of different metal ion pair of endo cellulase N24 activity
(1) promoter action of glycoside hydrolase to endo cellulase N24 activity
Adopt the DNS method, measure respectively CtAA9B, N24 and the mixed enzyme endo cellulase activity under the N24 optimum reaction conditions, mixed enzyme is that CtAA9B and N24 equal-volume mix, reaction system is: 9B50 μ L, N2450 μ L, the 0.5%CMC-Na of 200 μ L, 50 ℃ of reaction 30min of the acetate buffer solution of 100 μ L (0.4mol/L, pH5.0), add 400 μ L DNS reagent, boil 10min, under the 540nm wavelength, measure absorbance value.Be defined as 100% with the enzymic activity in the reaction system that does not add glucosides lytic enzyme CtAA9B, calculate respectively the relative activity of glycoside hydrolase CtAA9B, endo cellulase N24 and mixed enzyme.Triplicate, average.
(2) metal ions M n
2+impact on glycoside hydrolase CtAA9B cellulase activity
Add different metal ions in reaction system, making its final concentration is 1mmol/L, measure its endo cellulase activity under glycoside hydrolase CtAA9B optimum reaction conditions, the enzymic activity do not added in the metal ion reaction system is defined as 100%, calculates the relative activity of glycoside hydrolase CtAA9B under different metal ion condition.Triplicate, average.
(3) metal ions M n
2+impact on the mixed enzyme activity
Add different metal ions in reaction system, making its final concentration is 1mmol/L, measure the endo cellulase activity of mixed enzyme under endo cellulase N24 optimum reaction conditions, the enzymic activity do not added in the metal ion reaction system is defined as 100%, calculates the relative activity of mixed enzyme under different metal ion condition.Triplicate, average.
Embodiment 7: express the preservation of the pichia pastoris engineered strain GS-CT-9B of CtAA9b gene
Express the depositary institution of the pichia pastoris engineered strain GS-CT-9B of CtAA9b gene: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC); Address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on July 19th, 2013; Yeast engineering bacterium strain is numbered: CGMCC No.7943; The Classification And Nomenclature of pichia pastoris engineered strain is: pichia pastoris Pichia pastoris.
Claims (1)
1. a Yeast engineering bacterium strain of expressing the glycoside hydrolase gene C tAA9b of chaetomium thermophilum Chaetomium thermophilum9 family, it is characterized in that this bacterium is a kind of pichia spp, obtain the thermally-stabilised 9 glycoside hydrolase gene C tAA9b of family by the RT-PCR method from chaetomium thermophilum Chaetomium thermophilum, this gene clone is arrived to pichia spp secreted expression carrier pPIC9K, obtain recombinant expression pPIC9K/CtAA9b, transform Pichia pastoris GS115, therefrom filter out the pichia pastoris engineered strain GS-CT-9B that expresses thermally-stabilised glycoside hydrolase gene C tAA9b, this glycoside hydrolase CtAA9B has obvious promoter action to the activity of endo cellulase.
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Citations (3)
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| CN1353751A (en) * | 1999-05-28 | 2002-06-12 | 诺沃奇梅兹有限公司 | NOvel endo-beta-1,4-glucanases |
| CN101307295A (en) * | 2008-06-05 | 2008-11-19 | 山东农业大学 | Pichia yeast engineering strain for expressing chaetomium thermophilum gene cbh3 |
| FI122937B (en) * | 2009-12-30 | 2012-09-14 | Roal Oy | Method for treating cellulosic material and CBH II / Cel6A enzymes useful herein |
-
2013
- 2013-09-17 CN CN201310423132.0A patent/CN103484390A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1353751A (en) * | 1999-05-28 | 2002-06-12 | 诺沃奇梅兹有限公司 | NOvel endo-beta-1,4-glucanases |
| CN101307295A (en) * | 2008-06-05 | 2008-11-19 | 山东农业大学 | Pichia yeast engineering strain for expressing chaetomium thermophilum gene cbh3 |
| FI122937B (en) * | 2009-12-30 | 2012-09-14 | Roal Oy | Method for treating cellulosic material and CBH II / Cel6A enzymes useful herein |
Non-Patent Citations (1)
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| 路梅 等: "嗜热毛壳菌内切β-葡聚糖酶的分离纯化及特性", 《微生物学报》 * |
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Application publication date: 20140101 |