CN102936601A - Endoglucanase coding gene, recombinase and application - Google Patents

Abstract

The invention discloses an endoglucanase coding gene, a recombinase and an application thereof, belonging to the field of biotechnology. An endoglucanase coding gene, its sequence is shown in SEQ ID NO.1; the sequence of the endoglucanase recombinase is shown in SEQ ID NO.2. Experiments have proved that the recombinant endoglucanase of the present invention has very high carboxymethyl cellulase activity, reaching 2245U/mL, and the diameter of the transparent circle of the colony is more than 30 times that of the original strain. The endoglucanase coding gene and recombinant enzyme provided by the invention can be widely used in the degradation of cellulose.

Description

A kind of endoglucanase encoding gene and recombinase and application

Technical field

The invention belongs to biological technical field, relate to a kind of endoglucanase encoding gene and recombinase and application.

Background technology

Mierocrystalline cellulose is the abundantest a kind of carbohydrate resource of output in the biosphere, accounts for the 35%-50% of plant dry weight, and is widely distributed.Mierocrystalline cellulose is long-chain shape polymer, pass through β-1 by glucoside, 4 glycosidic links are formed by connecting, molecular weight is generally 50000-2500000, be equivalent to 300-15000 glucose unit, a kind of important storage form of glucose, but because degraded difficulty, very low of the utilization ratio that causes cellulose resource.Cellulose macromolecule can be degraded by cellulase.Cellulase is an enzyme system, according to the difference of its catalyzed reaction function, can be divided into endoglucanase (endo-1 to Mierocrystalline cellulose, 4-β-D-glucanase, EC3.2.1.4, i.e. C1 enzyme), exoglucanase (1,4-β-D-glucan cellobilhydrolase or exo-1, and beta-glucan glycosides enzyme (β-1 4-β-D-glucannase, EC.3.2.1.91),, 4-glucosidase, EC.3.2.1.21) abbreviation BG.It is generally acknowledged, endoglucanase is most important enzyme in the cellulase system, can act on the pars amorpha in the cellulosic molecule, the random hydrolysis glycosidic link, the long chain cellulose molecule is cut into the short chain shape, produce the small molecules Mierocrystalline cellulose, can provide a large amount of reaction ends for excision enzyme, can also be hydrolyzed micromolecular Mierocrystalline cellulose oligosaccharides simultaneously.

The research and development of cellulase are the bases of cellulose resource comprehensive utilization.The investigator is more to the cellulase research and development of fungi at present, and the research of exploitation high efficiency cellulose system will be lacked much in bacterium.The research of cellulolytic bacterium is the bacillus class mostly, and the research in Pantoea ananatis does not have report.Along with molecular biology, engineered fast development, all attempt using gene engineering technique both at home and abroad and make up highly effective cellulose degradation bacteria strains, the efficient cellulase system of development of new.Because the critical role of endoglucanase in cellulase system, therefore need to be by the research of biotechnological means increasing to the bacterium endoglucanase, make up recombined endo dextranase engineering bacteria, and carry out the research of zymologic property, enzyme product, to promote the development and use of Mierocrystalline cellulose application of enzymes and cellulose resource.

Summary of the invention

The object of the present invention is to provide gene order and the aminoacid sequence thereof of a kind of novel endoglucanase encoding gene eg.Adopt the PCR method clone to obtain the endoglucanase encoding gene.

A kind of novel endoglucanase encoding gene eg of the present invention, the gene order of described endoglucanase is SEQ ID NO.1.

The gene eg of described endoglucanase is a complete open reading frame (ORF), and this open reading frame begins with atg start codon ATG, finishes with terminator codon TGA, comprises altogether 1005bp.

A kind of novel restructuring endoglucanase of the present invention, the aminoacid sequence of described recombinase is SEQ IDNO.2.

Described endoglucanase is totally 334 aminoacid sequences.

Another object of the present invention is to provide a kind of novel restructuring endoglucanase EG.This recombinase has very high enzymic activity, reaches 2245U/mL.

The preparation method of above-mentioned recombined endo dextranase EG comprises the steps:

(1) structure of recombined endo dextranase bacterial strain EG/BL21 (DE3):

The primer of reaction take F:CATATGAGAGTTAAGCCAGTGT and R:CTCGAGACGCTTTTCCTGATAA as PCR; With PCR method from bacterial strain Pantoea ananatis P5(CGMCCNO.5356) clone obtains endoglucanase encoding gene eg total length the genome, after this gene cut by restricted endoenzyme NdeI and XhoI enzyme, be connected among the prokaryotic expression carrier pET258, construction recombination plasmid pETEG, transform colon bacillus E.coli BL21 (DE3) competent cell, utilize PCR reaction detection (primer is F and R) and restriction enzyme NdeI and XhoI double digestion to detect two kinds of methods, screening obtains to efficiently express the bacterial strain of recombined endo dextranase, i.e. recombinant escherichia coli EG/BL21 (DE3);

(2) fermentation culture and the abduction delivering of recombinant bacterial strain EG/BL21 (DE3):

Recombinant bacterial strain EG/BL21 (DE3) is transferred to 50mL LB(by 1% inoculum size contains 30 μ g/mL kantlex) cultivate in 37 ℃ of concussions in the liquid nutrient medium; Treat OD ₆₀₀Be 0.5 o'clock, add 0.1mM isopropyl-β-D-thiogalactoside(IPTG) (IPTG) and induce 4h to stop fermentation; Fermented liquid-80 ℃ of quick-frozens, is put in the frozen water and slowly melted fully, use the ultrasonic disruption cell, the centrifugal 5min of 10000r/min gets supernatant and is crude enzyme liquid;

(3) purifying of recombined endo dextranase:

Supernatant is added on the Ni-NTA post, the recombined endo dextranase with the His label is attached on the pillar; Use the elute soln of pH8.0 (to contain 50mM NaH ₂PO ₄, 300mM NaCl, 250mMimidazole) the recombined endo dextranase is eluted from pillar, and enzyme liquid is concentrated in the ultracentrifugation evaporating pipe, obtain purity and be higher than endoglucanase albumen more than 95%.

A further object of the present invention is to provide the application of above-mentioned novel restructuring endoglucanase EG.Described endoglucanase can be applied to the production of cellulose enzyme, is applied to cellulosic degraded.

The present invention compared with prior art has following advantage and beneficial effect:

(1) according to the diversity judgement of the characteristics such as molecular weight, iso-electric point and the enzymatic property of enzyme gene order BLAST analytical results desmoenzyme and known albumen, this recombined endo dextranase EG is a kind of novel β-Isosorbide-5-Nitrae-endo glucanase gene.This is to develop recombinant beta-Isosorbide-5-Nitrae-endo glucanase gene first from Pantoea ananatis bacterial strain.

(2) this recombined endo dextranase EG has wide, the anti-characteristics than strong acid alkalescence of adaptive temperature scope, has application potential in a plurality of industries such as bioenergy, laundry weaving, food-processing, fermentative production.

Description of drawings

Fig. 1 is the clone of endo glucanase gene and the agarose gel electrophoresis figure of detection.

1: endo glucanase gene pcr amplification result; 2: bacterium colony PCR identifies positive findings; The recombinant plasmid that 3:NdeI and Xho I double digestion cut, small band is the goal gene that cuts out, large band is empty carrier; The recombinant plasmid of 4:NdeI single endonuclease digestion; The recombinant plasmid of 5:Xho I single endonuclease digestion; 6: do not have the recombinant plasmid contrast of cutting; M1:1KB, once size is from top to bottom; 10kb, 8kb, 7kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1kb; M2:D2000, once size is 2000bp from top to bottom, 1000bp, 750bp, 500bp, 250bp, 100bp; The recombined endo glucanase gene marks with the left side arrow.

Fig. 2 is the transparent loop graph that recombinant bacterial strain EG/BL21 (DE3) forms at the Xylo-Mucine substratum.

Fig. 3 is the SDS-PAGE figure of the protein induced expression of recombinant bacterial strain EG/BL21 (DE3) endoglucanase.

Among the figure: 1: before inducing; 2: induce 3hr; 3: induce 4hr; 4: induce 5hr; M: albumen Marker, size is marked in the left side; Recombined endo dextranase albumen marks with right side arrow)

Fig. 4 is the SDS-PAGE figure of recombined endo dextranase purifying.

Among the figure: 1: before inducing; 2: induce 4hr; 3: the recombined endo dextranase albumen of purifying.

Embodiment

1, main raw

(1) substratum

LB liquid nutrient medium: contain peptone 10g in every L substratum, yeast powder 5g, NaCl 10g;

LB solid medium: contain peptone 10g in every L substratum, yeast powder 5g, NaCl 10g agar powder 12g;

Produce the transparent circle substratum: contain Xylo-Mucine 10g in every 1L substratum, peptone 10g, yeast powder 5g, KH ₂PO ₄1g, MgSO ₄0.2g, NaCl 10g, glucose 2g, agar powder 12g;

(2) experimental instrument and equipment

The rich Medical Equipment Plant of industry company limited that proves to be true after interrogation in YXQ-LS-75 vertical pressure steam sterilization pan---Shanghai

Pcr amplification instrument---American AB I company

FA2004 electronic balance---the more flat scientific instrument in Shanghai company limited

DNP-9162 type electro-heating standing-temperature cultivator---the upper grand experimental installation of Nereid company limited

YXJ-2 supercentrifuge---Changzhou Guohua Electric Appliance Co., Ltd.

Sigma3-16pk table-type high-speed refrigerated centrifuge---German SIGMA company

Low speed centrifuge---Changzhou Guohua Electric Appliance Co., Ltd.

Digital display type electric-heated thermostatic water bath---the Shanghai medical apparatus and instruments factory of making a leapleap forward

7230G visible spectrophotometer---Shanghai Precision Scientific Apparatus Co., Ltd

HYG type shaking table---the glad stamen automatic equipment in Shanghai company limited

Haier refrigerator---Qingdao Haier Group

Sanyo's Ultralow Temperature Freezer---SANYO GS Electronics Co., Ltd.

DYY-12 type electrophoresis apparatus---Beijing Liuyi Instrument Factory

Two vertical electrophoresis apparatus---the Beijing Liuyi Instrument Factory of DYCZ-24DN

Gel Doc XR+ gel imaging system---U.S. BIO-RAD company

Pipettor---Dragon Medical(Shanghai) Co., Ltd.

2, the clone of endo glucanase gene

Being used for clone P.ananatis endo glucanase gene primer sequence is:

F：AGAGTTTGATCCTGGCTCAG，R：GGTTACCTTGTTACGACTT。

The experimental technique of PCR reaction is: get three PCR tubules, add successively following reagent:

PCR reaction system (50 μ L)

The PCR reaction conditions is: 1. 95 ℃ of denaturation 5min; 2. 95 ℃ of sex change 30s; 3. 57 ℃ annealing 60s; 4. 72 ℃ are extended 90s, and repeating step 2-4 reacts 24 circulations; 5. 72 ℃ are extended 10min.The endo glucanase gene pcr amplification the results are shown in Figure 1 and gushes 1.

3, structure and the order-checking of restructuring order-checking plasmid

(1) gene is connected connection with sequencing vector

Use T4DNA Ligase that gene fragment eg is connected with sequencing vector pGMT.

The system that connects is as follows:

After each composition in the linked system added successively, mix, 10000r/min is centrifugal, and condition of contact is to connect in 16 ℃ of water-baths to spend the night to the plastic centrifuge tube bottom, makes up the sequencing vector of recombinating.Preserve for-20 ℃ and connect product.

(2) conversion of restructuring order-checking plasmid

5 μ L ligation reactions are added in the 50 μ L Dh5 α competent cells quick mixing gently, ice bath 30min.Then heat shock 90s in 42 ℃ of water-baths changes over to rapidly and places 2min in the ice bath, adds the LB substratum of 950 μ L, cultivates 1hr in 37 ℃ of shaking table 180r/min concussions.Get bacterium liquid and coat after centrifugal on the LB solid plate substratum that contains selection markers Amp in addition, be inverted overnight incubation for 37 ℃.Next day, according to blue hickie experimental principle, select white transformant.Be transferred to white transformant in the LB liquid nutrient medium of Amp afternoon, in 37 ℃ of shaking table 180r/min concussion overnight incubation.

(3) evaluation and the order-checking of restructuring order-checking plasmid

With the bacterium liquid preservation of bacteria strain of concussion overnight incubation, residue bacterium liquid adopts PCR method to identify.Concrete authentication method is: take bacterium liquid as template, carry out pcr amplification with F and two primers of R, if can amplify goal gene, illustrate tentatively that then the eg gene has been connected on the pGMT carrier.PCR test positive result's bacterium liquid is extracted plasmid, and the sequence on the pGMT carrier is as primer, serves the sea and gives birth to worker biotech firm and check order.

4, the structure of recombinant expression plasmid and checking

(1) double digestion of goal gene fragment and glue reclaim

The eg gene order that biotech firm's order-checking obtains is spliced, obtain the eg complete genome sequence.Utilize DNAMAN programanalysis sequence signature, determine the exactness of sequence.

Eg gene on the restructuring order-checking plasmid pGMTEG that order-checking is correct cuts down, and is connected on the expression vector pET258.Concrete grammar is:

Use NdeI, the XhoI enzyme is cut restructuring order-checking plasmid pGMTEG, obtains the eg gene.

It is as follows that the pGMTEG enzyme is cut system (20 μ L):

The pGMTEG enzyme is cut each composition of system mix gently, centrifugal to the centrifuge tube bottom, place 37 ℃ of warm water bath enzymes to cut 2hr.

Enzyme is cut product carry out electrophoresis.Under ultraviolet gel observation instrument, observation gel electrophoresis result.Determine that goal gene eg is digested and get off.Use DNA glue to reclaim test kit and reclaim goal gene eg.

(2) double digestion of pET258 plasmid, glue reclaim

Use NdeI and XhoI enzyme to cut the pET258 plasmid.Enzyme is cut system following (20 μ L):

Enzyme is cut each composition of system mix gently, centrifugal to the centrifuge tube bottom, place 37 ℃ of warm water bath enzymes to cut 2hr.The plasmid enzyme restriction product is carried out electrophoresis.Glue reclaims the plasmid after enzyme is cut.

(3) structure of recombinant expression plasmid pETEG and conversion

The goal gene eg that glue is reclaimed is connected on the pET carrier construction expression plasmid pETEG.Expression plasmid linked system (10 μ L) is:

After each composition in the linked system added successively, mix, 10000r/min is centrifugal, and condition of contact is to connect in 16 ℃ of water-baths to spend the night to the plastic centrifuge tube bottom, structure recombinant expression plasmid pETEG.Preserve for-20 ℃ and connect product.

(4) recombinant expression plasmid transformation receptor bacterium E.coli Dh5 ɑ

To connect product and transform among the intestinal bacteria E.coli Dh5 ɑ, and use the screening of kan LB solid medium to insert the recombinant bacterium pETEG/Dh5 ɑ of goal gene.Concrete transformation experiment method is: connecting fluid 5 μ L add competent cell 50 μ L → put 30min → 42 ℃ thermal shock 90s → put 2min in the ice-water bath in mixture of ice and water, add afterwards the LB liquid nutrient medium of 1ml → put 37 ℃ of shaking tables to shake 1h → coating kan LB solid medium.

(5) enzyme of recombinant expression plasmid pETEG is cut checking

Extract recombinant expression plasmid pETEG, adopt NdeI and XhoI double digestion electrophoresis detection plasmid.The enzyme system of cutting is (20 μ L):

Place 37 ℃ of warm water bath enzymes to cut 2hr.Electrophoresis detection pETEG enzyme is cut product.

5, make up recombined endo dextranase prokaryotic expression bacterial strain pETEG

Extract recombinant expression plasmid pETEG, be transformed among the E.coli BL21 (DE3), make up recombined endo dextranase EG prokaryotic expression bacterial strain.Coat on the Agar Plating that contains selection markers kan, be inverted overnight incubation for 37 ℃, select transformant, detect and NdeI with PCR, the XhoI double digestion comes screening positive clone (Fig. 1).The bacterial classification that preservation screens.

6, recombinant bacterial strain EG/BL21 (DE3) produces transparent circle

EG/BL21 (DE3) is inoculated into interpolation kan produces in the transparent circle substratum, cultivate in the incubators in 37 ℃ and cultivate 1d.With the 5min that dyes in 0.5% the Congo red product transparent circle substratum of pouring into after the cultivation, outwell Congo red after, use 5% NaCl solution soaking decolouring 1h.The result shows that it is more than 30 times (Fig. 2) of original strain that recombinant bacterial strain EG/BL21 (DE3) produces the transparent circle diameter.

7, the protein induced expression and purification of recombinant bacterial strain EG/BL21 (DE3) endoglucanase

Utilize recombinant bacterial strain EG/BL21 (DE3) to carry out the expression of recombinant protein.Derivational expression method is: evening, connect recombinant bacterial strain EG/BL21 (DE3) cell in LB (the Kan:30 μ g/mL) liquid nutrient medium 37 ℃ shake.Be transferred in 20mL LB (the Kan:30 μ g/mL) liquid nutrient medium by 1% inoculum size and cultivate.Treat OD ₆₀₀Be about at 0.5 o'clock, take out 5mL bacterium liquid and do control group.Adding an amount of IPTG of 0.1mM in the remaining bacterium liquid begins to induce.After inducing different time (3hr, 4hr and 5hr), the analysis of SDS-PAGE protein electrophoresis is done in sampling respectively.

The preparation of protein sample and electrophoresis: get lysate adding 3 * sample-loading buffer and mix, boil 5min, the centrifugal 10s of 6,000 * g gets the supernatant electrophoresis.Fill it up with electrophoretic buffer in electrophoresis chamber, use the pipettor loading, the about 1.5hr of voltage stabilizing 120V electrophoresis treats that tetrabromophenol sulfonphthalein migrates to gel lower edge stop electrophoresis.The careful gel that takes out placed staining utensil after electrophoresis finished, and poured staining fluid submergence gel into, at the horizontal shaking table 1hr that dyes.The evacuation staining fluid adds a small amount of distillation washing once, pours destainer submergence gel into again, disappears to blue background at the about 4hr of horizontal shaking table decolouring.Taking Pictures recording electrophoresis result after the decolouring, the result shows that endoglucanase albumen EG is successful abduction delivering (Fig. 3).

Recombined endo dextranase protein purification: bacterium liquid 10, the 000 * g centrifugation after 10ml induced, abandon supernatant, add 2ml Lysis buffer(in the precipitation and contain 50mM NaH ₂PO ₄, 300mM NaCl, 10mM imidazole, 1mg/mL N,O-Diacetylmuramidase), in frozen water, place 30min, it is complete to put subsequently-80 ℃ of quick-frozens.Solution after the quick-frozen is put slowly thawing in the frozen water, after melting fully, use ultrasonic disruption (power 300W 2s/ time, works every minor tick 15s 60 times).4 ℃ of centrifugal 20min of 10,000 * g abandon precipitation, and supernatant is added on the Ni-NTA post, and the recombinant protein with the His label is attached on the pillar.Add 4ml Wash buffer(to protein-bonded Ni-NTA post and contain 50mM NaH ₂PO ₄, 300mM NaCl, 20mMimidazole), clean pillar, repeat this step once.Add 2ml Elution buffer(and contain 50mMNaH ₂PO ₄, 300mM NaCl, 250mM imidazole) in connection with recombinant protein elute from pillar.The albumen that elutes is put into evaporating pipe concentrated (7,400 * g, 30min).The concentrated solution electrophoresis that takes a morsel is identified purity, and remaining adding equal-volume sterilization high-purity glycerin is put-80 ℃ and saved backup.SDS-PAGE shows that purifying has obtained purity at the recombined endo dextranase albumen (Fig. 4) more than 95%

8, recombined endo dextran enzyme activity determination

(1) glucose standard curve making

Get 6 tool plug scale test tubes of cleaning oven dry, after the numbering by 0,0.2,0.4,0.6,0.8,1.0mL adds standard glucose solution, and uses respectively distilled water to be settled to 1mL, is mixed with the glucose solution of a series of different concns.After fully shaking up, in each test tube, add 2mLDNS solution, shake up rear boiling water bath 5min, be settled to 25mL with distilled water after the taking-up cooling, fully mixing.Under the 540nm wavelength, as blank, zeroising is measured other and is respectively managed the optical density value of solution and record the result with the solution in No. 0 test tube.Take glucose content (mg) as X-coordinate, draw out the glucose typical curve take the absorbance of correspondence as ordinate zou.

(2) enzyme activity determination

Get 4 brace plug test tubes by 0 to 3 numbering, No. 0 pipe is as blank.Add 2mL damping fluid and 1mL enzyme liquid to each test tube, blank tube places boiling water bath inactivation 10min, and simultaneously preheating in 42 ℃ of water-baths of sample hose adds respectively the substrate solution of 2mL in test tube, take out behind the 20min.Add 2mL DNS nitrite ion to each test tube rapidly, shake up the rear boiling water bath that places rapidly, take out immediately behind the 10min, the flowing water cooling is settled to 25mL with distilled water after the cooling, and the solution in No. 0 test tube is as blank, in 540nm place survey OD value.

Enzyme work is calculated:

Will be within 42 ℃ of lower unit time of condition (1min) hydrolysis substrate produce the required crude enzyme liquid amount of 1 μ mol reducing sugar and be defined as the enzyme unit (U/mL) that lives.

The enzyme activity calculation formula is seen following formula:

$(U/\mathrm{mL})=\frac{\mathrm{OD}\×k\×n\×1000}{180\×t\×V}$

K in the formula---the slope of expression glucose typical curve;

N---expression extension rate;

1000---expression mg converts μ g to;

180---expression glucose standard molecular weight;

T---the expression reaction times (min);

V---expression participates in the crude enzyme liquid volume (mL) of reaction.

The result of above-mentioned experiment shows that it is active that recombined endo dextranase of the present invention has very high carboxymethylcelluloenzyme enzyme, reaches 2245U/mL, and the transparent circle diameter of bacterium colony is more than 30 times of original strain.Endoglucanase encoding gene provided by the present invention and recombinase can be widely used in cellulosic degraded.

Claims (4)

1. A recombinant endoglucanase coding gene, characterized in that: the enzyme gene sequence is SEQ ID NO.1. 2. A kind of endoglucanase coding gene according to claim 1, is characterized in that: the aminoacid sequence encoded by said recombinant endoglucanase gene is SEQ ID NO.2. 3. a kind of preparation method of a kind of recombinant endoglucanase described in claim 1 or 2, is characterized in that: (1) Construction of recombinant endoglucanase strain EG/BL21(DE3): Use primers F: CATATGAGAGTTAAGCCAGTGT, R: CTCGAGACGCTTTTCCTGATAA; clone the full-length endoglucanase coding gene from the genome of strain Pantoea ananatis P5 (CGMCC NO.5356) by PCR method, and pass the gene through restriction enzymes NdeI and XhoI After enzyme digestion, it was connected to the prokaryotic expression vector pET258, the recombinant plasmid pETEG was constructed, transformed into Escherichia coli E.coli BL21 (DE3) cells, and the strain that highly expressed the recombinant endoglucanase was obtained by screening, that is, the recombinant Escherichia coli Helicobacter EG/BL21(DE3); (2) Fermentation and induced expression of the recombinant strain EG/BL21(DE3): Transfer the recombinant strain EG/BL21(DE3) to 50mL LB (containing 30μg/mL kanamycin) liquid medium at 37°C with shaking at 1% inoculum; when the _OD600 was 0.5, add 0.1mM iso Propyl-β-D-thiogalactopyranoside (IPTG) induced 4h to stop the fermentation; quick-frozen the fermentation broth at -80°C, put it in ice water and slowly melted it completely, disrupted the cells with ultrasonic waves, centrifuged at 10000r/min for 5min, and took the supernatant That is the crude enzyme solution; (3) Purification of recombinant endoglucanase: Add the supernatant to the Ni-NTA column to bind the recombinant endoglucanase with His tag to the column; use the elution solution with pH8.0 (containing 50mM NaH ₂ PO ₄ , 300mM NaCl, 250mMimidazole) The recombinant endoglucanase is eluted from the column, and the enzyme solution is concentrated in an ultracentrifuge concentrator tube to obtain an enzyme protein with a purity higher than 90%. 4. An application of a recombinant endoglucanase, which can be used in the production of cellulase and in the degradation of cellulose.

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