CN1757710A - Saccharomyce engineering strain for expressing cbh1 gene and its construction method - Google Patents

Abstract

A genetically engineered yeast Pichia pastoris GS-H is prepared through screening the thermophilic Chaetomium CT2 by PCR method to obtain cellobiohydrolase II gene, cloning it, inserting it in the expression carrier of Pichia yeast, introducing it to Pichia yeast, and screening said genetically engineered yeast. It can generate a great deal of active cbhl protein to convert the rejected fiber material.

Description

Express the Yeast engineering bacterium strain and the construction process thereof of cbh1 gene

(1) technical field:

The present invention relates to biotechnology, specifically a kind of pichia pastoris engineered strain (Pichia pastoris GS-D) and construction process of expressing the cbh1 gene.

(2) technical background:

Mierocrystalline cellulose is a kind of renewable resources of cheapness, is the main component of higher plant cell wall, and its content reaches 35%～55% of plant dry weight, extensively is present in nature.Cellulose hydrolysis is become glucose, can produce Organic Chemicals and fuel such as ethanol, acetone, butanols by fermentation again, also can produce feed, food and medicine etc.Cellulose materials is to solve the human food problem that faces, the most promising resource of energy problem and environmental problem.Research, exploitation cellulose resource have profound significance.Cellulase can cut off cellulosic molecular chain, make it be converted into glucose, except can further be converted into albumen for fermentation, fat is for people to use and do the animal and fowl fodder, also can be used as the carbon source of producing alcohol, butanols, citric acid, L-glutamic acid, glycerine etc., this is for improving the grain utilization ratio, save a large amount of grains, have crucial meaning.The application of cellulase has entered industrial circles such as food, feed, weaving, chemical industry, papermaking, medicine, oil production at present.

At present, the principal element that the restriction cellulase is used on agro-industry has: (1) production cycle is long, the product cost height, and (2) poor heat stability, shelf-life is short.(3) thermophilic fermentation needs special equipment, and these will increase production cost and energy consumption.Therefore the research and development of thermostability cellulase has important commercial value.The current research that should be devoted to the thermophilic enzyme of thermophilic microorganism generation.The Zimadzhunt L 340 that utilizes thermophile bacteria to produce has many advantages as biological catalyst: the cost of (1) zymin reduces, and stability improves, and can at room temperature separate and purify and packed and transported, and can keep active muchly; (2) accelerated kinetic reaction; (3) cooling system to reaction requires to reduce, thereby has reduced energy consumption, both can reduce cost, and can reduce the process of cooling pollution on the environment again; (4) under the condition of Zimadzhunt L 340 catalyzed reaction (above 60 ℃) seldom have living contaminants, thereby have reduced the pollution of opsonigenous substances to product, can improve degree of purity of production, simplify its purification process; (5) biological process at high temperature carries out increasing the solvability and the utilizability of indissoluble material such as starch based, cellulose family, many aromatics and lipid etc., reduces the viscosity of organic compound and is beneficial to the diffusion and the mixing of material.

Chaetomium thermophilum CT2 (Chaetomium thermophilum CT2) is a kind of thermophilic fungus that is distributed widely in the soil, is the very high a kind of fungi of growth ceiling temperature in the eukaryote, might become the optimum strain that produces thermophilic enzyme in the eukaryote.This bacterium is can produce cellobiohydrolase (CBH) under 50 ℃ of conditions in the substratum of sole carbon source at Microcrystalline Cellulose.But make it to be used for large-scale industrial production, also have some open questions still.Relatively harsher as the thermophile bacteria culture condition, the large scale fermentation production of Zimadzhunt L 340 needs specific installation, and product enzyme efficient is low, thereby causes product 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 warm type microorganism yeast in the importing of heat-stable cellulase gene, utilize yeast growth fast, be easy to characteristics such as cultivation, make cellulose enzyme gene normal temperature and in the short period of time fast, overexpression, expectation reaches the purpose that cuts down the consumption of energy and increase economic efficiency.

(3) summary of the invention:

The object of the present invention is to provide a kind of Yeast engineering bacterium strain and construction process thereof of expressing cellobiohydrolase I gene.This bacterium cost is low, and large scale fermentation is convenient for production, is easy to realize industrialization.Another object of the present invention provides the method for preparing Pichi strain, and this method is easy and simple to handle, good reproducibility.

Cbh1 gene involved in the present invention is from Chaetomium thermophile bacteria strain CT2, the cellobiohydrolase I thermostability height of its coding.Chaetomium thermophilum CT2 uses conventional separation method separation by this laboratory and obtains from soil, the cbh1 gene is obtained by this laboratory clone, and the Genbank accession number is AY861347.Nucleotide sequence of a kind of chaetomium thermophilum CT2 cellobiohydrolase I gene and preparation method thereof is provided in one embodiment of the invention, this method is to be template with the total RNA of chaetomium thermophilum, use the specific oligonucleotide primer, obtain cellobiohydrolase I gene cDNA through the reverse transcription PCR method.Gene sequencing result shows that cellobiohydrolase I maturation protein is made of 512 amino acid of encoding 1536 Nucleotide.

A kind of preparation of chaetomium thermophilum Chaetomium thermophilumCT2 cellobiohydrolase I gene is provided in one embodiment of the present of invention and has been cloned into colibacillary method, this method comprises: amplimer is synthetic, the extraction of the total RNA of chaetomium thermophilum, the reverse transcription of mRNA, the pcr amplification of goal gene, the pulsating recovery of goal gene, the goal gene segment is cloned into carrier, the mensuration of chaetomium thermophilum cellobiohydrolase I gene order.

One of technical essential of the present invention is the structure and the abduction delivering of expressing the pichia pastoris engineered strain of cellobiohydrolase I gene.Pichia yeast expression system is a kind of eukaryotic expression system efficiently that development in recent years is got up, and it has the following advantages: the expression amount height, and good stability, active high, be convenient to suitability for industrialized production.

A kind of structure of engineering strain is provided in one embodiment of the invention, and bacterial strain is pichia spp Pichiapastoris GS-H.Express the method for cellobiohydrolase I gene in Pichia yeast, this method is to express cellobiohydrolase I gene with the pPIC9K carrier in pichia spp, comprising: the pcr amplification of (1) cellobiohydrolase I gene; (2) the PCR product cloning is to the T carrier; (3) structure of shuttle expression plasmid; (4) structure of the Yeast engineering bacterium strain of expression cellobiohydrolase I gene.

Two of technical essential of the present invention is abduction delivering and determinations of activity of engineered protein.Gene A OX1 and AOX2 that two coding alcohol oxidases are arranged in pichia spp, can utilize methyl alcohol to grow fast as sole carbon source, in the Pichia yeast engineering, foreign gene is cloned into the control of AOX1 promotor down, realizes efficiently expressing of foreign protein with methyl alcohol as inductor.

A kind of method of pichia pastoris engineered strain abduction delivering is provided in an embodiment of the present invention, the substratum that can be used for the pichia pastoris engineered strain abduction delivering includes but are not limited to following substratum: MM MD BMGYBMMY, various culture medium preparation methods are the method that present technique field personnel are familiar with very much, can obtain detailed information by the reference pertinent literature.

The biologic activity of a kind of cellobiohydrolase I is provided in one embodiment of the invention, by determination of activity, the activity unit of engineering strain fermentation of the present invention surpasses 21U/mL, and expression amount reaches 1.4g/L, has realized foreign gene efficiently expressing in pichia spp.

(4) description of drawings:

The pcr amplification result (1536bp) of Fig. 1 cellobiohydrolase I gene maturation protein encoding gene

The enzyme of Fig. 2 recombinant plasmid is cut qualification result (EcoR I and Not I double digestion)

The enzyme of Fig. 3 recombinant expression vector is cut qualification result

Swimming lane 1 be nucleic acid high molecular electrophoresis marker λ-Hind III digest DNA marker (23130bp, 9416bp, 6557bp, 4361bp, 2322bp, 2027bp, 564bp, 125bp)

Swimming lane 2 is recombinant plasmid pPIC9K/cbh1

Swimming lane 3 is cut result (9.3Kb+1538bp) for pPIC9K/cbh1EcoR I and Not I enzyme

Swimming lane 4 is cut result (8.5Kb+2348bp) for pPIC9K/cbh1EcoR I and Xbal I enzyme

Swimming lane 5 is cut result (10.8Kb) for pPIC9K/cbh1Xbal I enzyme

Swimming lane 6 is pPIC9K/cbh1 special primer C1, C2 amplification (1538bp)

Swimming lane 7 be DL2000 DNA marker (2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp)

Swimming lane 8 be DL15000Marker (15000bp, 10000bp, 7500bp, 5000bp, 2500bp, 1000bp, 250bp)

Fig. 4 G418 screens multiple copied transformant (left figure G418 content is 2.0mg/mL, and right figure G418 content is 3.0mg/mL)

Fig. 5 recombinant protein SDS-PAGE detects

Swimming lane 1 be albumen lower molecular weight Marker (94kDa, 67kDa, 43kDa, 31kDa, 20kDa)

Swimming lane 2 is a recombinant gene expression albumen

Swimming lane 3 transforms the Pichia yeast engineering tunning for empty carrier

(5) embodiment

The invention will be further described below in conjunction with drawings and Examples, substratum among all embodiment and molecular biology working method are familiar with by those skilled in the art, can obtain detailed information with reference to " molecular clonings " such as Sambrook (laboratory manual, 1989) etc.

The pcr amplification and the clone of embodiment 1. chaetomium thermophilum cellobiohydrolase I genes

(1) amplimer is synthetic

Chaetomium thermophilum cellobiohydrolase I cDNA sequences Design Oligonucleolide primers amplification according to the laboratory clone.Long 26 Nucleotide of upstream primer, long 27 Nucleotide of downstream primer, primer are given birth to the biological company limited of worker by Shanghai and are synthesized, through the PAGE purifying.Sequence following (introduced EcoRI and Not I restriction enzyme site in the upstream and downstream primer respectively, the place of underlining is a restriction enzyme site):

Forward: 5 '---CC GAATTCCAGCAGGCTTGCTCCCTC-----3 '

Reverse: 5 '----GG GCGGCCGCTTACAGGCACTGGGCTG--3 '

(2) extraction of total RNA

From Chinese isolation identification one strain thermophilic fungus Chaetomium thermophilum CT2, extract total RNA according to the TRIZOL method: collect and induce the mycelia of generation, aseptic water washing, drying, liquid nitrogen flash freezer grinds.Every 0.2g adds 1mL TRIZOL, leaves standstill 5 minutes, adds the 0.2mL trichloromethane, thermal agitation 15s, 12000g, 4 ℃, centrifugal 15min.Get supernatant, add that precipitation agent is centrifugal must to be precipitated, 75% ethanol is washed precipitation twice, drying.With resolution of precipitate in the distilled water that 50ul DEPC handles.-80 ℃ of preservations, stand-by.

(3) the synthetic cDNA article one chain of reverse transcription: the Reverse TranscriptionReaction test kit specification sheets according to Promega company carries out, with synthetic cDNA first chain of Oligo (dT) 20 primers.The concrete operations step is as follows:

Following reagent is added one in the PCR reaction tubes of DEPC immersion and sterilising treatment: 25mM MgCl ₂12ul; Damping fluid 6ul; 10m M d NTP 6ul; Recombinant Rnasin RibonucleaseInhibitor 2ul; AMV ThermoScript II 4ul; Oligo (dT) 20 6ul; The total RNA 15ul of Chaetomium thermophile; Sterilization distilled water 9ul.Reaction conditions is: 42 ℃ 1 hour; 95 ℃ 5 minutes; 3 ℃ 5 minutes.

(4) pcr amplification of goal gene

Get 8ul reverse transcription reaction primer, carry out the PCR reaction according to the PCR reaction system parameter that provides in the Reverse Transcription Reaction test kit.Reactive component is:

10X damping fluid 2.5ul

25mM?MgCl ₂ 1.5ul

10m?M?d?NTP 2ul

Upstream primer 1ul

Downstream primer 1ul

Reverse transcription product 5ul

RT-PCR enzyme mixture 2ul

Sterilization distilled water 10ul

Its reaction parameter is:

Pre-sex change: 94 ℃, 4min

Sex change: 94 ℃, 1min

Renaturation: 55 ℃, 1min

Extend: 72 ℃, 1min30s

Circulation: 30

Stop extending: 72 ℃ of 10min

The PCR product detects by 1% agarose electrophoresis, and electrophoresis result as shown in Figure 1.

(5) the segmental recovery of purpose

Reclaim the test kit specification sheets according to Shanghai China Shun PCR of bio-engineering corporation product and reclaim the goal gene segment, its process is as follows: get 30ul PCR reaction product and add 1.4mL PB liquid (test kit provides), mixture moved in the adsorption column centrifugal 15 seconds, abandoned waste liquid; In adsorption column, add 400ul PB liquid, leave standstill 1 minute after, centrifugal 15 seconds, abandon waste liquid; Add 500ul W1 liquid (test kit provides) again, centrifugal 15 seconds, abandon waste liquid, repeat once; In adsorption column, add 300ul T1 liquid (test kit provides), leave standstill 1 minute after, centrifugal 30 seconds, collect centrifugate, be stored in-20 ℃.

(6) target gene fragment is cloned into carrier

Target gene fragment is cloned into the pMD18-T carrier, and this carrier is available from Dalian Bao Bio-Engineering Company.Reactive component is as follows: PCR product 5ul; PMD18-T carrier 1ul; Ligation solution 4ul; Cumulative volume is 10ul, and 16 ℃ of connections are spent the night.

The preparation of competent cell:

The single DH5 α of picking bacterium colony, being inoculated in 3mL does not contain in the LB substratum of penbritin, 37 ℃ of overnight incubation, get above-mentioned bacterium liquid next day is inoculated in the 50ml LB nutrient solution in proportion at 1: 100,37 ℃ of vibrations 3 hours treat that bacterium liquid OD value is at 0.6 o'clock, centrifugal 8 minutes of 4 ℃ of 5000rpm, abandon supernatant, precipitation 0.1MCaCL ₂Suspend, 5000rpm is centrifugal 8 minutes again, abandons supernatant, and precipitation is with an amount of 0.1M CaCL ₂Resuspended, in the rearmounted ice bath of packing 6 hours standby.

Connect the conversion of product:

Get above-mentioned connection product 5ul and added 100ul competent cell ice bath 60 minutes, 42 ℃ of heat-shockeds 90 seconds, put ice bath again 2 minutes, the LB substratum 0.9ml that adds no penbritin, cultivated 1 hour for 37 ℃, getting 200ul bacterium liquid adds to be coated with behind 40ul 20mg/mL5-bromo-4 chloro-, 3 indoles (D-galactoside (X-gal)) and 4ul 0.1M isopropylthiogalactoside (IPTG) mixing and contains penbritin LB plate, cultivated 16 hours for 37 ℃, picking white colony rapid extraction plasmid carries out enzyme and cuts evaluation, enzyme is cut product through 1% agarose electrophoresis detected result as shown in Figure 2, its positive colony called after pMDT/cbh1.

(7) sequencing of chaetomium thermophilum cellobiohydrolase I maturation protein encoding gene

Extract the plasmid DNA dideoxy method and measure nucleotide sequence, sequencing is finished by Shanghai biotechnology company limited, and aligning primer is the M13 promoter primer.Chaetomium thermophilum cellobiohydrolase I gene nucleotide series measurement result: long 1539 Nucleotide of chaetomium thermophilum maturation protein coding gene sequence, the content of adenosine (A), cytidine(C (C), guanosine-(G) and thymidine (T) is respectively: A-318, C-539, G-395, T-287.512 amino acid of encoding.This protein maturation albumen terminator codon is positioned at the 1537th of gene, and terminator codon is TAA.

5 ' end to 3 ' terminal sequence is:

1 CAGCAGGCTT?GCTCCCTCAC?CACTGAGACC?CACCCCAGAC?TCACTTGGAA?GCGCTGCACC

61 TCTGGCGGCA?ACTGCTCGAC?CGTGAACGGC?GCCGTCACCA?TCGATGCCAA?CTGGCGCTGG

121 ACTCACACTG?TTTCCGGCTC?GACCAACTGC?TACACCGGCA?ACGAGTGGGA?TACCTCCATC

181 TGCTCTGATG?GCAAGAGCTG?CGCCCAGACC?TGCTGCGTCG?ACGGCGCTGA?CTACTCTTCG

241 ACCTATGGTA?TCACCACCAG?CGGTGACTCC?CTGAACCTCA?AGTTCGTCAC?CAAGCACCAG

301 CACGGCACCA?ATGTCGGCTC?TCGTGTCTAC?CTGATGGAGA?ACGACACCAA?GTACCAGATG

361 TTCGAGCTCC?TCGGCAACGA?GTTCACCTTC?GATGTCGATG?TCTCTAACCT?GGGCTGCGGT

421 CTCAACGGCG?CCCTCTACTT?CGTCTCCATG?GACGCTGATG?GTGGTATGAG?CAAGTACTCT

481 GGCAACAAGG?CTGGCGCCAA?GTACGGTACC?GGCTACTGCG?ATGCTCAGTG?CCCGCGCGAC

541 CTTAAGTTCA?TCAACGGCGA?GGCCAACATT?GAGAACTGGA?CCCCTTCGAC?CAATGATGCC

601 AACGCCGGTT?TCGGCCGCTA?TGGCAGCTGC?TGCTCTGAGA?TGGATATCTG?GGATGCCAAC

661 AACATGGCTA?CTGCCTTCAC?TCCTCACCCT?TGCACCATTA?TCGGCCAGAG?CCGCTGCGAG

721 GGCAACAGCT?GCGGTGGCAC?CTACAGCTCT?GAGCGCTATG?CTGGTGTTTG?CGATCCTGAT

781 GGCTGCGACT?TCAACGCCTA?CCGCCAGGGC?GACAAGACCT?TCTACGGCAA?GGGCATGACC

841 GTCGACACCA?CCAAGAAGAT?GACCGTCGTC?ACCCAGTTCC?ACAAGAACTC?GGCTGGCGTC

901 CTCAGCGAGA?TCAAGCGCTT?CTACGTTCAG?GACGGCAAGA?TCATTGCCAA?CGCCGAGTCC

961 AAGATCCCCG?GCAACCCCGG?CAACTCCATC?ACCCAGGAGT?GGTGCGATGC?CCAGAAGGTC

1021 GCCTTCGGTG?ACATCGATGA?CTTCAACCGC?AAGGGCGGTA?TGGCTCAGAT?GAGCAAGGCC

1081 CTCGAGGGCC?CTATGGTCCT?GGTCATGTCC?GTCTGGGATG?ACCACTACGC?CAACATGCTC

1141 TGGCTCGACT?CGACCTACCC?CATTGACAAG?GCCGGCACCC?CCGGCGCCGA?GCGCGGTGCT

1201 TGCCCGACCA?CCTCCGGTGT?CCCTGCCGAG?ATTGAGGCCC?AGGTCCCCAA?CAGCAACGTT

1261 ATCTTCTCCA?ACATCCGCTT?CGGCCCCATC?GGCTCGACCG?TCCCTGGCCT?CGACGGCAGC

1321 ACCCCCAGCA?ACCCGACCGC?CACCGTTGCT?CCTCCCACTT?CTACCACCAC?CAGCGTGAGA

1381 AGCAGCACTA?CTCAGATTTC?CACCCCGACT?AGCCAGCCCG?GCGGCTGCAC?CACCCAGAAG

1441 TGGGGCCAGT?GCGGTGGTAT?CGGCTACACC?GGCTGCACTA?ACTGCGTTGC?TGGCACTACC

1501 TGCACTGAGC?TCAACCCCTG?GTACAGCCAG?TGCCTGTAA

Chaetomium thermophilum cellobiose I gene mature peptide aminoacid sequence:

1 QQACSLTTET?HPRLTWKRCT?SGGNCSTVNG?AVTIDANWRW?THTVSGSTNC?YTGNEWDTSI

61 CSDGKSCAQT?CCVDGADYSS?TYGITTSGDS?LNLKFVTKHQ?HGTNVGSRVY?LMENDTKYQM

121 FELLGNEFTF?DVDVSNLGCG?LNGALYFVSM?DADGGMSKYS?GNKAGAKYGT?GYCDAQCPRD

181 LKFINGEANI?ENWTPSTNDA?NAGFGRYGSC?CSEMDIWDAN?NMATAFTpHP?CTIIGQSRCE

241 GNSCGGTYSS?ERYAGVCDPD?GCDFNAYRQG?DKTFYGKGMT?VDTTKKMTVV?TQFHKNSAGV

301 LSEIKRFYVQ?DGKIIANAES?KIPGNPGNSI?TQEWCDAQKV?AFGDIDDFNR?KGCMAQMSKA

361 LEGPMVLVMS?VWDDHYANML?WLDSTYPIDK?AGTPGAERGA?CPTTSGVPAE?IFAQVPNSNV

421 IFSNIRFGPI?GSTVPGLDGS?TPSNPTATVA?PPTSTTTSVR?SSTTQISTPT?SQPGGCTTQK

481 WGQCGGIGYT?GCTNCVAGTT?CTELNPWYSQ?CL

Embodiment 2. expresses the structure of the pichia pastoris gene engineering bacterial strain of cellobiohydrolase I gene

(1) structure of shuttle expression plasmid:

Extract recombinant vectors, with EcoR I and Not I double digestion to obtain purpose fragment cbh1; Same enzyme is cut the pPIC9K empty carrier and make it dephosphorylation under the CIAP effect.The CBH I that obtains behind linearizing pPIC9K empty carrier behind the dephosphorylation and the double digestion is spent the night under under the effect of T4DNA ligase enzyme 4 ℃,, this recombinant vectors is changed in the e. coli jm109 increase to obtain recombinant expression vector pPIC9K-CBH I.The single bacterium colony rapid extraction plasmid of picking white carries out enzyme and cuts evaluation (Fig. 3).

(2) structure of the Yeast engineering bacterium strain of expression cellobiohydrolase I gene:

A. the linearizing of shuttle expression plasmid:

Extract recombinant plasmid pPIC9K/CBH I, be cut into linearity with Bpu1102 I enzyme, same enzyme is cut empty plasmid pPIC9K in contrast.

B. transform pichia spp Gs115

Process is as follows:

(a) .YPD substratum incubated overnight yeast Pichia pastoris Gs115 is seeded in the fresh substratum, is cultured to OD ₆₀₀Value is 1.5,

(b) the centrifugal collection thalline of .1500rpm/min successively uses 500, the aseptic washing thalline of 250mL precooling, 4 ℃ of centrifugal supernatant liquors that go of following 3000rpm/min,

(c). the 1mol/L sorbyl alcohol with the 20mL precooling suspends, and get 80 μ L and add the linearizing recombinant plasmid of 5-10ng (Bpu1102 I cuts), ice bath 5min, electric shock instrument electric shock, shock parameters is 1.5kV, 25 μ F.

(d). electric shock finishes the 1mol/L sorbyl alcohol after the back adds the 1mL precooling rapidly, gets 200 μ L coated plate on solid MD substratum, and 30 ℃ of cultivations occur until single transformant.

(3) screening positive transformant:

On screening culture medium MM and MD flat board, cultivate 2d with the corresponding dibbling of sterilization toothpick picking transformant for 30 ℃, growth on the MD normal and on MM the poor growth or the positive transformant of transformant of not growing.The positive transformant that screening obtains is identified with the carrier primer amplification.

(4) G418 screening multiple copied transformant: with positive transformant respectively dibbling to containing 0.25mg/mL, 0.5mg/mL, 0.75mg/mL, 1.0mg/mL, 1.5mg/mL, 2.0mg/mL, 2.5mg/mL, 3.0mg/mL on the YPD flat board of G418, on the flat board that contains 3.0mg/mL G418, screen 8 multiple copied transformants (Fig. 4)

Embodiment 3. genetic engineering bacterium cellobiohydrolase I induction expression of protein

(1) selects a single bacterium colony, place the 250mL that 25mL BMGY substratum is housed to shake bottle, be cultured to 0D600=2-6 (approximately 16-18h) in 28-30 ℃/250-300rpm;

(2) the centrifugal 5min of 1500-3000g under the room temperature collects thalline, with the resuspended thalline of BMMY, makes (about 200mL) about OD600=1.0;

(3) the bacterium liquid with step 2 gained places 1L to shake bottle, seals with double gauze or cheese cloth, is positioned over continued growth on the shaking table of 28-30 ℃/250-300rpm;

(4) adding 100% methyl alcohol to final concentration every 24h to substratum is 0.5% (about 1mL);

(5) detect the expression of cbh1 gene in yeast sampling in the 24th, 48,72,96,120,144,168 hour.

The proteic extracting method of embodiment 4. Yeast gene engineerings

(1), protein separating method

A. centrifugal collection supernatant liquor behind the inducing culture 150h

B. after using 70% saturated ammonium sulphate 3h, the centrifugal 10min of 5000r/min, collecting precipitation

The careful collecting precipitation of c is with its Tris-HCl damping fluid with an amount of (being generally about 50ml) (pH8.0,50mmol/L) fully dissolving

D. the dialysis 24h in same buffer (1000ml) that packs in the dialysis tubing, the stirring dialyzate that does not stop with magnetic stirring apparatus during dialysis is changed a damping fluid every 6h therebetween, so that reach the effect completely of dialysing

E. the solution in the dialysis tubing is collected, centrifugal 5min under the low temperature (10,000rpm, 4 ℃) gets supernatant liquor.During long storage, be divided into aliquot quick freezing in liquid nitrogen ,-80 ℃ of storages;

(2), SDS-PAGE electrophoretic analysis cbh1 expression of gene situation:

A. gel records

After 12% the separation gel solution for preparing mixed, in the rapid impouring glue of gelating soln sheet glass, to glue identity distance sheet glass groove 3cm, on the glue face, spread gently then and annotate the high water layer of 1cm, the vertical offset plate of placing, behind about 20-30min, glue can condense.Water on the sucking-off glue face then.3% concentrate the upper strata that glue is injected into separation gel rapidly with what prepare, concordant to liquid level with the groove of sheet glass, then insert comb, at room temperature place 20-30min, make its cohesion.Carefully extract comb and pour into electrode buffer.

B. go up sample and electrophoresis

Induce at yeast to add isopyknic sample loading buffer in the fermented liquid, in 100 ℃ of heating 3-5min sex change, application of sample advances separation gel with 100V electrophoresis to tetrabromophenol sulfonphthalein in order, improves voltage to 150V, when tetrabromophenol sulfonphthalein arrives bottom precontract 1cm place end electrophoresis;

C. dye and decolour

The offset plate that electrophoresis is finished takes out, and after rinsing well with deionized water, places about 100ml staining fluid to dye, and under the room temperature, 20min at least dyes on the platform shaking table.Discard staining fluid, flushing is placed in the 100-200ml high methanol destainer decolours.Change destainer when dark when the color of the color of destainer and glue is the same.So about 3-4 time, till extremely most of blueness takes off to the greatest extent usually.Gel places about 100ml standard (low methyl alcohol) destainer 4-6h or spends the night, and makes it to decolour fully.So far, the background of offset plate cleans substantially.After the decolouring, offset plate is stored in room temperature, in the deionized water.

D. electrophoresis result

Obtain the expression (Fig. 5) of cbh1 gene in Yeast expression carrier.

The proteic determination of activity of embodiment 5. genetically engineered cellobiohydrolase I

Take by weighing 4mg absorbent cotton, after the enzyme liquid that adding 0.1mL suitably dilutes, 0.1mL 50mmol/L pH 5.0 acetate buffer solutions mixed, 50 ℃ were incubated 24h down, added 0.1mL DNS reagent survey reducing sugar amount.An enzyme activity unit (U) is defined as: the per minute hydrocellulose produces the required zymoprotein amount of 1 μ mol glucose.

Claims (2)

1, a kind of Yeast gene engineering bacterial strain of expressing the cbh1 gene coded protein, it is characterized in that this bacterial strain is a kind of pichia spp, name is called Pichia pastoris GS-H, this project bacterium can efficiently express cellobiohydrolase I, expressing protein has higher thermostability, and enzymic activity can reach 21U/mL.

2, a kind of Yeast engineering bacterium strain of expressing the cbh1 gene coded protein according to claim 1, it is characterized in that gene order according to chaetomium cbh1, the application PCR method is a template with total RNA reverse transcription product of chaetomium thermophilum CT2, amplify the maturation protein encoding gene fragment of cbh1, be cloned on next carrier pMD18-T, after order-checking confirms, again the CT2 cellobiohydrolase I gene of being cloned into is inserted into the multiple clone site of pichia spp (Pichia pastoris) expression vector pPIC9K, transform among the importing pichia spp host Gs115 by electricity again, therefrom filter out the yeast gene engineering bacteria that efficiently expresses cellobiohydrolase I again.

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