CN103602604B

CN103602604B - Pichia pastoris engineered strain expressing 9-family glycoside hydrolase gene CtAA9d

Info

Publication number: CN103602604B
Application number: CN201310422852.5A
Authority: CN
Inventors: 李多川; 韩超
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2013-09-17
Filing date: 2013-09-17
Publication date: 2015-03-04
Anticipated expiration: 2033-09-17
Also published as: CN103602604A

Abstract

The invention relates to a Pichia pastoris engineered strain GS-CT-9D expressing Chaetomium thermophilum thermally stable 9-family glycoside hydrolase gene CtAA9d. Through an RT-PCR method, the glycoside hydrolase gene CtAA9d is obtained from the Chaetomium thermophilum, and is cloned and inserted into a Pichia pastoris integration expression vector pPIC9K. Then, the obtained glycoside hydrolase gene CtAA9d expression vector pPIC9K/CtAA9d is introduced into Pichia pastoris GS115, and yeast engineered strain GS-CT-9D expressing the glycoside hydrolase gene CtAA9d is obtained by screening. The CtAA9D expressed by the engineered strain has s obvious promotion effects upon endo-cellulase activity. The cellulose degradation activity of mixed enzyme is increased by 1.6 times than that of an original endonuclease N24. Under Mn<2+> conditions, the cellulase activity of the CtAA9D and the cellulase activity the CtAA9D+N24 are respectively increased by 15 times and 2.5 times. The CtAA9D has good thermal stability and capacity for improving the cellulase activity, and can be widely applied in fiber waste and other industrial fields. The engineered strain has important economic and social values.

Description

Express the pichia pastoris engineered strain of 9 family glycoside hydrolase gene C tAA9d

(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-9D of chaetomium thermophilum Chaetomium thermophilum9 family glycoside hydrolase gene C tAA9d.

(2) background technology

Glycoside hydrolase (English: Glycoside hydrolases, also known as Glycosylase) is that one is hydrolyzed glycosidic linkage (glycosidic bond) specially, and produces the ferment of two less glycan molecules, is also one of common ferment of occurring in nature.This proteinoid is also applied to some extent in the industry of the mankind, such as, be used for the biomass energy such as Mierocrystalline cellulose and hemicellulose to be degraded into available small molecules.Glycoside hydrolase has multiple family.

Chaetomium thermophilum Chaetomium thermophilum is a kind of thermophilic fungus be distributed widely in soil.This bacterium is can produce heat-staple cellulase and 9 family's glycoside hydrolases under 50 DEG C of conditions in the substratum of sole carbon source at Microcrystalline Cellulose.

The 9 family glycoside hydrolase CtAA9D that chaetomium thermophilum produces have faint cellulase activity, but have obvious promoter action to the enzymic activity of the endo cellulase N24 that this bacterial strain produces, and can make its active raising 1.6 times.At different metal ion as Mn ²⁺effect under, the activity of CtAA9D and CtAA9D+N24 can improve 15 times and 2.5 times respectively.

(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 CtAA9d, CtAA9d gene DNA total length 870bp, comprise signal peptide sequence, initiator codon and terminator codon, intronless, to encode 224 amino acid, there is signal peptide sequence (1-22aa MKLSLASLLTAALSVQGHAIFQ).The aminoacid sequence that CtAA9d encodes is retrieved in international gene pool, finds to belong to glycoside hydrolase the 9th family.

Build recombinant expression plasmid carrier pPIC9K/CtAA9d, electroporation is utilized to carry out electroporated Pichia pastoris GS115, MD and MM substratum screens, through PCR evaluation and screening positive transformant, G418 screens multiple copied transformant, then carry out methanol induction expression, obtain pichia pastoris engineered strain GS-CT-9D.This project inoculation is in containing in BMGY substratum, and after 6d cultivated by 28 DEG C of 200rpm/min shaking tables, the expression amount of glycoside hydrolase is 2.51mg/ml, molecular weight is 25KDa, enzyme activity is 0.0862U/ml, CtAA9D is stable at 65 DEG C, still has the enzymic activity of more than 95% after process 60min; The activity of 72.8% is still kept after 70 DEG C of process 30min; The activity of 20.4% is kept after 80 DEG C of process 30min; 90 DEG C of process 30min activity almost completely lose.

The enzymic activity of 9 family glycoside hydrolase CtAA9D to chaetomium thermophilum Chaetomium thermophilum endo cellulase N24 has obvious promoter action, and mixed enzyme improves 1.1 times than the ability of the degraded cellulose of protoenzyme N24.Different metal ion pair CtAA9D and CtAA9D+N24 mixed enzyme activity have promotion or restraining effect, wherein at Mn ²⁺under condition, the cellulase activity of CtAA9D and CtAA9D+N24 can improve 33 times and 2.5 times respectively.

(4) accompanying drawing illustrates:

The SDS-PAGE of Figure 19 family glycoside hydrolase CtAA9D analyzes

Swimming lane 1: low molecular weight protein (LMWP) standard

Swimming lane 2: 9 family glycoside hydrolase CtAA9D of purifying

The optimum temperuture of Fig. 2 A:9 family glycoside hydrolase CtAA9D

The thermal stability determination of B:9 family glycoside hydrolase CtAA9D

Fig. 39 family glycoside hydrolase CtAA9D is to the promoter action of endo cellulase N24 activity

Fig. 4 metal ions M n ²⁺on the impact of CtAA9D 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 0.5 gram and be placed on the dull and stereotyped upper 50 DEG C 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 tAA9d

(1) extraction of chaetomium thermophilum Chaetomium thermophilum total serum IgE: with reference to the explanation of Trizol test kit.

(2) synthesis of cDNA Article 1 chain: carry out according to TaKaRa RNA PCR kit (AMV) the Ver3.0 test kit specification sheets of Takara company: get 1 ~ 2 μ g total serum IgE, add RNase Free ddH ₂o to 9.5 μ L, by RNA sample at 75 DEG C of sex change 5min, cools 5min immediately in ice bath, then once centrifugal a little, adds following various composition in ice bath successively: 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, ambient temperatare 10min, then 42 DEG C of incubation 60min, then boil 5min with deactivation ThermoScript II.Add the ddH of 180 μ L DEPC process ₂o, is diluted to 200 μ L, mixing, centrifugal a little, is stored in-20 DEG C, for subsequent use.

(3) PCR reaction (25 μ L): cDNA2 μ L, 10 × Buffer2.5 μ L, 10mmol/L dNTP2 μ L, 2,5mm,ol/,LMg,Cl2 2 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 DEG C of 5min denaturations; 94 DEG C of 40s, 57 DEG C of 40s, 72 DEG C of 1min, totally 32 circulations, 72 DEG C extend 10min, 4 DEG C of preservations.

Upstream primer: 5 '-ATGAAACTTTCCTTGGCGTCAC-3 '

Downstream primer: 5 '-TTAGCACGTGATGGGAGCCG-3 '

(4) gene clone: get 0.5 μ l PCR primer and be connected with pMD18-T carrier, operation steps is carried out according to TAKARA Products specification sheets.Then connect product conversion e.colistraindh5α, the LB grow on plates scribbling acillin (100 μ g/mL) on surface spends the night.Picking white colony, overnight incubation in LB liquid nutrient medium.

(5) extraction of plasmid DNA: alkalinity extraction plasmid DNA.`

(6) sequencing: DNA double deoxidation method measures nucleotide sequence, and biotechnology company limited carries out in Shanghai.Aligning primer is M13 promoter primer.Chaetomium thermophilum CtAA9d gene cDNA total length 869bp, comprises initiator codon and terminator codon, intronless, 175 amino acid of encoding.This aminoacid sequence is retrieved in international gene pool, finds to belong to glycoside hydrolase the 9th family.This sequence is as follows:

(A) information of SEQ ID NO 1

(a) sequence signature: * length: 870 base pairs; * type: nucleic acid; * chain: double-strand; * topological framework: linear

(b) molecule type: DNA

C () is supposed: no

(d) antisense: no

E () is originated at first: chaetomium thermophilum Chaetomium thermophilum

(f) sequence description:

(B) information of SEQ ID NO 2

(a) sequence signature: * length: 224 amino acid; * type: amino acid; * chain: strand; * topological framework: linear

(b) molecule type: protein

(c) sequence description:

Embodiment 3: the structure of expression vector

(1) express primer according to the nucleotide sequence design of isolated CtAA9d gene, introduce Ecor I and Not I restriction enzyme site respectively at 5 ' end of primer, downstream primer introduces 6 histidine sequences, as purification tag: upstream primer: 5 '-CCG gAATTCcACCACCACCACCACCACATCTTTCAGAA AGTC-3 ' (Ecor I); Downstream primer: 5 '-TT gCGGCCGCtTAGCACGTG ATGGGAGCCG-3 ' (Not I) (histidine sequences)

(2) extraction of chaetomium thermophilum total serum IgE: utilize Trizol reagent to extract.

(3) reverse transcription synthesis cDNA Article 1 chain: get 2 μ g total serum IgE, add 5 × reaction buffer 4 μ L, 10mM dNTP2 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 DEG C of reaction 60min, then 85 DEG C of 10min termination reactions, are diluted to 200 μ L.

(4) PCR reaction (25 μ L): cDNA2 μ L, 10 × Buffer2.5 μ L, 10mmol/L dNTP2 μ L, 25mmol/LMgCl ₂2 μ L, each 2 μ L of upstream and downstream primer, Taq archaeal dna polymerase 0.5 μ L (5U/ μ L), ddH ₂o12 μ L.Reaction conditions is 94 DEG C of 5min denaturations; 94 DEG C of 40s, 57 DEG C of 40s, 72 DEG C of 1min, totally 32 circulations, 72 DEG C extend 10min, 4 DEG C of preservations.

(5) gene clone: get 2 μ L PCR primer and be connected with pMD18-T carrier, obtains recombinant plasmid pMD18T/CtAA9d operation steps and is undertaken by TAKARA Products specification sheets.Then connect product conversion bacillus coli DH 5 alpha, spend the night at the LB grow on plates scribbling AMP.Picking white colony, overnight incubation in LB liquid nutrient medium.

(6) extraction of plasmid DNA: alkalinity extraction plasmid DNA.

(7) carry out double digestion with Ecor I and Not I pair of recombinant plasmid pMD18-T/CtAA9d product, use Ecor I and Not I double digestion expression plasmid of yeast pPIC9K simultaneously, DNA glue reclaims test kit and reclaims purifying 9a gene and carrier pPIC9K segment.Then carry out Ligation in vitro, obtain recombinant plasmid pPIC9K/CtAA9d, recombinant plasmid transformed e. coli jm109, picking individual colonies on the LB transformation plate containing Amp, extraction plasmid DNA, carries out PCR and enzyme cuts qualification, and 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 tAA9d

(1) linearizing of recombinant expression plasmid: by recombinant expression plasmid pPIC9K/CtAA9d restriction enzyme Sac I linearizing.

(2) transform: electroporated yeast strain Pichia pastoris GS115 competent yeast cells, method for transformation is see Invitrogen company pichia spp operational manual.

(3) screen: with the corresponding dibbling of sterilizing toothpick picking transformant on MM and MD flat board, cultivate 2-4d for 30 DEG C, picking all well-grown transformant on MD/MM flat board is positive transformant.Picking positive transformant respectively dibbling screens 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 Activity determination

(1) be inoculated in by positive transformant containing in 25mL BMGY substratum, 24h(OD cultivated by 30 DEG C of 250rpm/min shaking tables ₆₀₀reach 2-6), collected by centrifugation thalline, is resuspended in the BMMY substratum of proper volume, to OD by cell ₆₀₀value is that 1.0,30 DEG C of 250rpm/min continue to cultivate, and it is 0.5% that every 24h supplements methyl alcohol to final concentration, and every 24h sampling, the centrifugal 5min of room temperature 10,000g, gets supernatant and carry out SDS-PAGE analysis.

(2) Enzyme assay: enzyme assay adopts DNS method, reaction system and reaction conditions are the enzyme liquid of 100 μ L, add 0.5% Microcrystalline Cellulose of 200 μ L, acetate buffer solution (0.4mol/L, pH5.0) the 60 DEG C reaction 30min of 100 μ L, add 400 μ L DNS reagent, boil 10min, under 540nm wavelength, measure absorbance value.The enzyme liquid of control group inactivation contrasts.Be defined as 100% with the activity of fermentoid liquid, measure the relative activity of glycoside hydrolase CtAA9D.In triplicate, average.Determining the protein quantity adopts Bradford method.

(3) to recombinate the optimal reactive temperature of glycoside hydrolase CtAA9D, pH and thermostability: the restructuring glycoside hydrolase of abduction delivering through the HisTrap FF crude metal-ligand affinity chromatography column purification of GE company with the recombinant protein of histidine mark, obtain the purifying protein that electrophoresis is homogeneous, by its character of restructuring glycoside hydrolysis enzymatic determination of purifying.Measure respectively under differing temps and pH condition restructuring glycoside hydrolase to cellulosic enzyme activity, the highest enzyme activity is defined as 100%, calculates the relative activity of glycoside hydrolase under condition of different temperatures respectively; Restructuring glycoside hydrolase is incubated 30min at different temperature, detects residual enzyme vigor.In triplicate, average.

Embodiment 6:9 family glycoside hydrolase is on the impact of the promoter action of endo cellulase N24 activity and its activity of different metal ion pair

(1) glycoside hydrolase is to the promoter action of endo cellulase N24 activity

Adopt DNS method, measure CtAA9D, N24 and the endo cellulase activity of mixed enzyme under N24 optimum reaction conditions respectively, mixed enzyme is the mixing of CtAA9D and N24 equal-volume, reaction system is: CtAA9D 50 μ L, N2450 μ L, the 0.5%CMC-Na of 200 μ L, acetate buffer solution (0.4mol/L, pH5.0) the 50 DEG C reaction 30min of 100 μ L, add 400 μ L DNS reagent, boil 10min, under 540nm wavelength, measure absorbance value.Be defined as 100% with the enzymic activity do not added in the reaction system of glycoside hydrolase CtAA9D, calculate the relative activity of glycoside hydrolase CtAA9D, endo cellulase N24 and mixed enzyme respectively.In triplicate, average.

(2) metal ions M n ²⁺on the impact of glycoside hydrolase CtAA9D cellulase activity

Different metal ions is added in reaction system, its final concentration is made to be 1mmol/L, its endo cellulase is measured active under glycoside hydrolase CtAA9D optimum reaction conditions, the enzymic activity do not added in metal ion reaction system is defined as 100%, the relative activity of glycoside hydrolase CtAA9D under calculating different metal ionic conditions.In triplicate, average.

(3) metal ions M n ²⁺on the impact of mixed enzyme activity

Different metal ions is added in reaction system, its final concentration is made to be 1mmol/L, the endo cellulase measuring mixed enzyme under endo cellulase N24 optimum reaction conditions is active, the enzymic activity do not added in metal ion reaction system is defined as 100%, the relative activity of mixed enzyme under calculating different metal ionic conditions.In triplicate, average.

Embodiment 7: the preservation expressing the pichia pastoris engineered strain GS-CT9D of CtAA9d gene

Express the depositary institution of the pichia pastoris engineered strain GS-CT9D of CtAA9d 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.7945; The Classification And Nomenclature of pichia pastoris engineered strain is: pichia pastoris Pichia pastoris.

Claims

1. one kind express chaetomium thermophilum ( chaetomium thermophilum) Yeast engineering bacterium strain of 9 family glycoside hydrolase gene C tAA9d, it is characterized in that this bacterium is a kind of pichia spp, thermally-stabilised 9 family glycoside hydrolase gene C tAA9d are obtained from chaetomium thermophilum by RT-PCR method, by this gene clone to pichia spp secreted expression carrier pPIC9K, obtain recombinant expression pPIC9K/CtAA9d, transform Pichia pastoris GS115, therefrom filter out the pichia pastoris engineered strain GS-CT-9D expressing thermally-stabilised glycoside hydrolase gene C tAA9d, this pichia pastoris engineered strain is expressed the activity of glycoside hydrolase CtAA9D to endo cellulase produced and is had obvious promoter action, the nucleotide sequence of described chaetomium thermophilum 9 family glycoside hydrolase gene C tAA9d is as shown in SEQ ID NO.1.