CN102676442A

CN102676442A - Construction of inrtaurethral beta-1, 4-glucanase gene engineering bacterium resisting surface active agent

Info

Publication number: CN102676442A
Application number: CN2012101358611A
Authority: CN
Inventors: 吴敬; 陈晟; 闫鹏安; 宿玲恰; 陈坚
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2012-04-26
Filing date: 2012-04-26
Publication date: 2012-09-19

Abstract

The invention relates to construction of an inrtaurethral beta-1, 4-glucanase gene engineering bacterium resisting a surface active agent, belonging to the field of gene engineering and enzyme engineering. The invention adopts the total DNA of thermoascus aurantiacus as a template, obtains the inrtaurethral beta-1, 4-glucanase gene ce15A (NCBI code: 3579455) by a PCR (Polymerase Chain Reaction) method, connects expression vectors, converts E.coli host bacteria and realizes the high-efficiency expression of the inrtaurethral beta-1, 4-glucanase gene engineering bacterium. The culture is carried out in an industrial fermentation culture medium, and by induced recombination, the activity of the inrtaurethral beta-1, 4-glucanase gene engineering bacterium reaches 638.51U/mL. The recombinase has good stability in the surface active agent commonly used by the washing agent, is very suitable for being applied to the washing industry, and is an ideal additive of the washing agent.

Description

A kind of inscribe β of surfactant-tolerant-1,4-glucanase gene engineering bacteria and construction process thereof

Technical field

The present invention relates to a kind of inscribe β-1 of surfactant-tolerant, the construction process of 4-glucanase gene engineering bacteria belongs to genetically engineered and enzyme engineering field.

Background technology

Cellulase is meant the general name of the class of enzymes of ability degraded cellulose, mainly comprises three types, is respectively inscribe β-1,4-LSD (EC3.2.1.4), cellobiohydrolase (EC3.2.1.91) and beta-glucosidase (EC3.2.1.21).Wherein, inscribe β-1,4-LSD are the important component in the cellulase system, ability random hydrolysis cellulosic β-1, and the 4-glucoside bond produces cellooligosaccharide, is widely used in industry such as weaving and washing.Inscribe β-1, the generation bacterium of 4-LSD is a lot, mainly contains filamentous fungus, bacterium etc.What research was maximum at present is wood mould (Trichoderma).

Dirt attached to clothing surface is prone to cleaned by the conventional washing agent, and the dirt in the clothing gap is sticky in clothing and is difficult to clean, and after the repetitive scrubbing, clothing just can send out yellowing old.With inscribe β-1, the 4-LSD adds in the washing composition, has changed traditional detergency mechanism; It makes the cellulosic structure of cotton fabric bulk; Be beneficial to detergent ingredients and advance people's fibre gap and fully contact, thereby improved washing effect greatly with dirt, and the clothing of washing with the washing composition that adds cellulase; Bright in colour, soft, little to the clothing damage.

Usually contain a large amount of tensio-active agents in the washing composition, be prone to cause partly or completely sex change inactivation of zymoprotein.Therefore the cellulase that requires to add in the washing composition has certain tolerance to tensio-active agent.Andre etc. from soil, screen Streptomyces drozdowiczii and separation of pure dissolves inscribe β-1 wherein, and the 4-LSD has been studied its stability in commercialization washing composition environment, and room temperature is placed enzyme reduction about 15% alive after a hour; Separation of pure from Staphylotrichum coccosporum such as Jinichiro dissolves inscribe β-1, and 4-LSD STCE1 discovers that STCE1 is a restriction endonuclease best to the tensio-active agent tolerance in 45 families.

In recent years, along with the washing industrial expansion, washing is with inscribe β-1; The demand of 4-LSD increases day by day; Therefore screen the inscribe β-1 of surfactant-tolerant, the 4-LSD, and the genetic engineering bacterium of the suitable large scale fermentation production of structure seems increasingly important.

Summary of the invention

The technical problem that the present invention will solve provides a kind of inscribe β-1 of surfactant-tolerant; 4-glucanase gene engineering bacteria; Be inscribe β-1 to carry the expression of recombinant plasmid surfactant-tolerant, the intestinal bacteria E.coli BL21 (DE3) of 4-glucanase gene.

Another technical problem that the present invention will solve provides a kind of method that makes up said genetic engineering bacterium; Through clone's inscribe β-1; 4-glucanase gene cel5A (NCBI coding: 3579455) to the pET-24a carrier; With E.coli BL21 (DE3) is expressive host, has realized inscribe β-1, and 4-LSD Cel5A efficiently expresses.

Said recombinant plasmid is a pUC series, pET series, pT7-7, or among the pGEX any one.

Recombinase provided by the invention is stability experiment in the tensio-active agent that washing composition is used always; Comprise the steps: to add respectively in the recombinase tensio-active agent commonly used in several kinds of washing composition that final concentration is 1mM; Under 25 ℃, be incubated and measure residual enzyme work after one hour.

Described tensio-active agent be in aliphatic alcohol polyethenoxy nine ethers, polyoxyethylene nonylphenol and AS linear alkylbenzene sulphonic acid, the sodium lauryl sulphate any.

Description of drawings

Fig. 1 recombined endo β-1,4-LSD Cel5A shake flask fermentation produces the enzyme curve.

Fig. 2 recombined endo β-1,4-LSD Cel5A protein SDS-PAGE figure.

1, fermented liquid supernatant; 2, broken wall supernatant; 3, inclusion body; M, standard protein molecular weight.

Fig. 3 recombined endo β-1,4-LSD Cel5A fed-batch fermentation produces the enzyme curve.

Fig. 4 recombined endo β-1,4-LSD Cel5A separation and purification SDS-PAGE figure.

1, sample behind the purifying; 2, fermented liquid; M, standard protein molecular weight.

Embodiment

Embodiment 1: the structure of genetic engineering bacterium

1, goes up the gene order (Genbank number 3579455) of the cel5A of login according to NCBI; Total DNA is a template with thermophilic ascomycete (Thermobifidafusca); Adopt the synthetic inscribe β-1 of polymerase chain reaction method, the gene order of 4-LSD Cel5A is connected to the pMD18-Tsimple carrier; Connect product transformed into escherichia coli JM109, the converted product coating contains the LB flat board of 100mg/L penbritin.Through 37 ℃ of overnight cultures, choosing colony, insert the LB liquid nutrient medium, extract plasmid behind 8～10h, called after pMD18-T simple-cel5A carries out sequencing with this plasmid.The result shows that the gene order of the cel5A that 1293 Nucleotide of this full length gene and NCBI upward login is identical.

2, the plasmid that is used to make up coli expression carrier is pET-24a, has the T7 promotor.PET-24a plasmid and pMD18-Tsimple-cel5A are carried out Nco I and HindIII double digestion respectively; Enzyme connects with the T4 ligase enzyme after cutting product rubber tapping recovery again, connects product Transformed E .coli JM109 competent cell; Cultivate 8h through 37 ℃; Choose transformant shaking culture in the LB that contains 30 μ g/mL kantlex, extract plasmid, enzyme is cut checking and is obtained expression plasmid pET-24a-cel5A.

3, with recombinant plasmid pET-24a-cel5A Transformed E .coli BL21 (DE3) host bacterium, coating contains on the LB flat board of kantlex (30 μ g/mL), cultivates 8h for 37 ℃.Choose single bacterium colony to liquid LB, 37 ℃ of overnight cultures obtain genetic engineering bacterium E.coli BL21 (DE3)/pET-24a-cel5A.

Embodiment 2: recombined endo β-1, the shake flask fermentation of 4-LSD Cel5A

Described genetic engineering bacterium 37 ℃ of liquid culture in the LB substratum are spent the night, and TB (glycerine 5g/L, peptone 12g/L, yeast extract paste 24g/L, K are inserted in the back ₂HPO ₄12.54g/L, KH ₂PO ₄2.31g/L) fermentation broth; 37 ℃ be cultured to OD and reach 2.0 after, use final concentration to induce as the 0.4mM isopropylthio-, be cooled to 25 ℃ and cultivate after 48 hours; Centrifugal thalline; Collect supernatant and measure enzyme activity, recombined endo β-1,4-LSD Cel5A shake flask fermentation vigor reaches 46.78U/mL.Recombined endo β-1,4-LSD Cel5A produce the enzyme curve and see Fig. 1, and the SDS-PAGE electrophorogram is seen Fig. 2.

Embodiment 3: recombined endo β-1,4-LSD Cel5A fed-batch fermentation

With described genetic engineering bacterium in technical grade LB substratum 37 ℃ cultivate 8 hours after; Insert NBS (New Brunswick Scientific) 3L automatic fermenter by 10% inoculum size; The fermentor tank 1.1L technical grade complex medium of initially packing into; 35 ℃ of ferment at constant temperature are kept dissolved oxygen about 30%, utilize ammoniacal liquor control pH about 7.0.After glycerine exhausts in the batch phase initial medium, dissolved oxygen and pH fast rise, start index flow feeding liquid, ratio growth velocity μ=0.2h ^-1As the dense OD of bacterium ₆₀₀Reach at 50 o'clock, the flow velocity stream with 2% adds lactose induces, and whole fermentation process is carried out controlled on-line and data gathering by fermentor tank system software.Recombined endo β-1,4-LSD Cel5A fed-batch fermentation produce the enzyme curve and see Fig. 3.

Embodiment 4: recombined endo β-1, the separation and purification of 4-LSD Cel5A

With fermented liquid in 4 ℃, the centrifugal thalline of removing of 12000rpm, the solid ammonium sulfate that in supernatant, adds 70% (w/v) is saltoutd and is spent the night.In 4 ℃, the centrifugal supernatant that removes of 12000rpm dissolves albumen precipitation and dialysed overnight in buffer A with 20mM phosphoric acid buffer A (pH 7.0) then.

DEAE-Sepharose FF post is used the buffer A balance in advance; The linear gradient mixed solution (5 column volumes) that after the last appearance, use buffer A (at least 2 column volumes) more in order respectively, contains buffer A and buffer B (containing 20mM potassiumphosphate, 1M sodium-chlor) carries out wash-out.Omnidistance flow velocity is 1mL/min; The ultraviolet detection wavelength is 280nm; In the gradient elution process, collect the elutriant that absorption peak is arranged, the mensuration enzyme is lived and is carried out protein electrophoresis and detect, and the vigor component goes up kind Superdex 200 posts after 20mM phosphoric acid buffer (pH 5.5) dialysis; Collection has the elutriant of absorption peak, and the mensuration enzyme is lived and carried out protein electrophoresis and detect.

The purge process parameter sees the following form, and the protein SDS-PAGE electrophorogram is seen Fig. 4.

Table?1.Summary?of?purification?of?the?recombinant?endoglucanase?Cel5A

Embodiment 5: recombined endo β-1,4-LSD Cel5A is in the stability of washing composition tensio-active agent commonly used

Tensio-active agent commonly used mainly contains two kinds of non-ionics and aniorfic surfactant in the washing composition; Ionic surfactant pack is drawn together aliphatic alcohol polyethenoxy 9 ethers, polyoxyethylene nonylphenol, and AS comprises linear alkylbenzene sulphonic acid, sodium lauryl sulphate.Adding final concentration in the recombinase respectively is the tensio-active agent of using always in the above-mentioned several kinds of washing composition of 1mM; Under 25 ℃; Be incubated and measure residual enzyme work after one hour; The relative enzyme activity of recombinant C el5A under above-mentioned each tensio-active agent environment be all more than 90%, recombined endo β-1, and 4-LSD Cel5A sees the following form in the stability of washing composition tensio-active agent commonly used.

Claims

1. the inscribe β of a surfactant-tolerant-1,4-glucanase gene engineering bacteria is characterized in that, this genetic engineering bacterium is to carry expression of recombinant plasmid surfactant-tolerant inscribe β-1, the intestinal bacteria of 4-glucanase gene.

2. make up the method for the said genetic engineering bacterium of claim 1; It is characterized in that cloning Cel5A encoding sox (Genbank accession number 3579455) to the pET-24a carrier; With E.coli BL21 (DE3) is expressive host, has realized the inscribe β-1 of surfactant-tolerant, the efficiently expressing of 4-LSD.

3. method according to claim 2 is characterized in that, said recombinant plasmid is a pUC series, pET series, pT7-7, or among the pGEX any one.

4. method according to claim 2 is characterized in that, said host e. coli be among E.coli BL21 (DE3), E.coli W3110, E.coli JM109, E.coli JM109 (DE3), the E.coli DH5 α any one.

5. the inscribe β-1 that produces of the said genetic engineering bacterium of claim 1, the 4-LSD has good stability in washing composition tensio-active agent commonly used.

6. method according to claim 5 is characterized in that, described tensio-active agent be in aliphatic alcohol polyethenoxy nine ethers, polyoxyethylene nonylphenol and AS linear alkylbenzene sulphonic acid, the sodium lauryl sulphate any.