CN103031290A

CN103031290A - High-sugar-tolerant beta-glucosaccharase Bg14, and expressed gene and application thereof

Info

Publication number: CN103031290A
Application number: CN2012105412935A
Authority: CN
Inventors: 赵林果; 谢静聪; 裴建军; 王飞; 庞倩
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2012-12-14
Filing date: 2012-12-14
Publication date: 2013-04-10
Anticipated expiration: 2032-12-14
Also published as: CN103031290B

Abstract

The invention relates to high-sugar-tolerant beta-glucosaccharase Bg14, and an expressed gene and application thereof. The amino acid sequence is shown as SEQ ID NO.1. The gene of the high-sugar-tolerant beta-glucosaccharase Bg14 of aspergillusniger is cloned, recombined and expressed for the first time; and the glucose-tolerant coefficient Ki of the recombinase Bg14 is 2 mol/L.

Description

A kind of beta-glucosidase Bgl4 and expressing gene and application of anti-high sugar

Technical field

The invention belongs to genetically engineered and technical field of enzyme engineering, be specifically related to the sugared beta-glucosidase Bgl4 gene clone of novel anti-height and recombinant expressed and application to the aspergillus niger source.

Background technology

Beta-glucosidase (β-glucosidase, EC 3.2.1.21) belongs to circumscribed hydrolase, claim again β-D-Glucose glycosides lytic enzyme, it is a class shifts glucosides in the intermolecular catalysis of nucleophilic enzyme, can be inner at the oligosaccharides of short chain or cellobiose, between the residue with the carbohydrate of aromatic group or alkyl, interrupt β-1,4-glycosidic link, thereby the field (Science 311:484-489.) such as the cellulose hydrolysis that is widely used.One of main biological organism that Mierocrystalline cellulose produces as green plants photosynthesis, with hemicellulose and xylogen as the supporting tissue of plant along with agriculture and forestry organic waste material is constantly regenerated, be renewable energy source the abundantest on the earth.In the cellulose hydrolysis process, beta-glucosidase usually not with the Mierocrystalline cellulose direct effect, but it can be degraded and cellulose hydrolysis be played the cellobiose of strongly inhibited effect, along with the increase of glucose content in the hydrolyzed solution, equally also can produce strong restraining effect to beta-glucosidase simultaneously.So excavate and to have high glucose tolerance beta-glucosidase to add in the existing cellulolytic enzyme system be one of effective ways that improve enzymolysis efficiency.

The beta-glucoside enzyme source is quite extensive, plant, animal and microorganism can both separation and purification out, and wherein Aspergillus is considered to the best bacterial classification, the output with aspergillus niger is the highest especially.Most of Aspergillus bacterial strain can be secreted the beta-glucosidase of various ingredients, and its zymologic property is also different, has at least 4 kinds of beta-glucosidases to be purified with qualitative such as A.tubingensis by different culture condition.According to the tolerance classification of beta-glucosidase to glucose, beta-glucosidase can be divided into anti-high sugar and not anti-high sugared two kinds, be respectively: (a) molecular weight is the beta-glucosidase that belongs to hydrolase family 3 of 130-100kDa, its secretion is more, activity is higher, but its activity is subjected to glucose to suppress (the COEFFICIENT K i of anti-glucose generally is no more than 30mM); (b) another kind of molecular weight is the beta-glucosidase of 40-55kDa, its secretion is less, activity is lower, but can tolerate the glucose of high density, be the beta-glucosidase of 49kDa such as the molecular weight that derives from A.niger CCRC31494, the Ki of its anti-glucose is 543mM, and the molecular weight that derives from A.oryzae is the beta-glucosidase of 43kDa, the Ki of its anti-glucose is 1360mM(Applied Environmemt Microbiology, 64:3607-3614.).Although have the beta-glucosidase of the anti-high sugar in aspergillus source to be purified, relevant gene does not also have bibliographical information.

The present invention separates the beta-glucosidase that obtains a kind of novel anti-high sugar from Aspergillus niger strain (Aspergillus.niger) fermented liquid, and has extremely good glucose-tolerant ability (the Ki value reaches 2mol/L), can satisfy in existing cellulose degradation system the requirement to beta-glucosidase glucose-tolerant ability fully, through protein purification and protein sequencing, obtained its N terminal sequence, comparison finds that it belongs to GH72 family through aminoacid sequence, so far there is not report to find that there is the beta-glucosidase that can be hydrolyzed β-Isosorbide-5-Nitrae-glycosidic link in this family.

Summary of the invention

The technical problem that solves: thus the technical problem that the present invention mainly solves is the problem that causes production cost to improve that beta-glucosidase in the present cellulose degradation technology can't enduring high-concentration glucose, and then a kind of beta-glucosidase Bgl4 and expressing gene and application of anti-high sugar are provided.

Technical scheme:

A kind of beta-glucosidase Bgl4 of anti-high sugar, its aminoacid sequence is shown in SEQ ID NO.1.

The expressing gene of the beta-glucosidase Bgl4 of described anti-high sugar, its nucleotide sequence is shown in SEQ ID NO.2.

The recombinant plasmid that comprises above-mentioned nucleotide sequence.

The recombinant plasmid pPICZ alpha A-Bgl4 that comprises above-mentioned nucleotide sequence.

Comprise the preparation method of the recombinant plasmid of the described gene order of SEQ ID NO.2, step is:

(1) design primer P1 and P2 take the cDNA of the mRNA reverse transcription of Aspergillus niger strain Aspergillus niger as template, take P1 and P2 as primer carries out pcr amplification, obtains the pcr amplification product of the sugared beta-glucosidase Bgl4 of anti-height gene, and described primer is:

P1:5 '-CCG GAATTCATGAAGGGCACCGCTGTCG-3 ', underscore represent EcoR I site;

P2:5 '-GC TCTAGATTACAACAGGACGAGTCCGGCA-3 ', underscore represent Xba I site;

(2) step (1) gained pcr amplification product is spent the night under 16 ℃ with the pMD-19T cloning vector be connected; To connect product and transform intestinal bacteria Top10F ' competent cell, screening positive clone carries out sequential analysis; Select the correct clone of sequence and extract plasmid, obtain to contain the recombinant plasmid pMD-19T-Bgl4 of the sugared beta-glucosidase gene of anti-height;

(3) signal peptide is predicted and removed to the protein sequence that obtains by the translation to the Bgl4 complete sequence, and signal peptide primer P3:5 '-CC is removed in design GGAATTCGCGCCTTCGTCGACCATCAAG-3 ', underscore represent EcoR I site, and take P3 and P2 as primer, template is that pMD-19T-Bgl4 carries out pcr amplification, obtains removing the Bgl4 gene fragment of signal peptide;

(4) pcr amplification product and the pPICZ α A with step (3) gained uses respectively EcoR I and Xba I double digestion, and rubber tapping is reclaimed respectively, the connection of spending the night; To connect product and transform intestinal bacteria Top10F ' competent cell, screening positive clone carries out sequential analysis; Select the correct clone of sequence and extract plasmid, obtain the recombinant plasmid pPICZ alpha A-Bgl4 of the sugared beta-glucosidase gene of anti-height.

The pichia spp host cell that comprises above-mentioned recombinant plasmid.

The restructured Pichia pastoris in expression recombinase that comprises recombinant plasmid pPICZ alpha A-Bgl4, recombinant plasmid pPICZ alpha A-Bgl4 is transformed into the transformant that filters out multiple copied among the pichia spp Host Strains GS115 by the YPD flat board that is added with different concns gradient bleomycin by electric shock after the Bln I linearizing, use the BMGY culture medium culturing, transfer to again in the BMMY substratum methyl alcohol that adds 0.5%wt with every 24h and carry out abduction delivering, collect the enzyme liquid that supernatant liquor is the sugared beta-glucosidase Bgl4 of anti-height.

The application of beta-glucosidase Bgl4 in cellulose degradation of described anti-high sugar.

The concrete scheme content is:

Recombinant expressed and enzyme qualitative that comprises clone, the gene of genes involved.

1. the order-checking of N end is carried out in the albumen of the sugared beta-glucosidase of the anti-height of Aspergillus niger strain (Aspergillus.niger) that obtains, obtain its gene order with comparison in the full genome database of the N terminal sequence of measuring Aspergillus niger strain (Aspergillus.niger) in ncbi database again, according to the gene order design primer that obtains, cDNA take Aspergillus niger strain (Aspergillus.niger) carries out PCR as template, obtains the complete genome sequence of the sugared beta-glucosidase of the anti-height of Aspergillus niger strain (Aspergillus.niger);

2. the complete genome sequence that obtains is analyzed, removed signal peptide by the design primer, and be cloned into the expression vector pPICZ α A of yeast, obtain recombinant plasmid pPICZ alpha A-Bgl4;

3. recombinant plasmid pPICZ alpha A-Bgl4 is transformed into the transformant that filters out multiple copied among the pichia spp Host Strains GS115 by the YPD flat board that is added with different concns gradient bleomycin by electric shock after the Bln I linearizing, use the BMGY culture medium culturing, transfer to again in the BMMY substratum and to add 0.5% methyl alcohol with every 24h and carry out abduction delivering, collect the enzyme liquid that supernatant liquor is the sugared beta-glucosidase of anti-height.

Beneficial effect: the present invention has cloned and the recombinant expressed gene of the sugared beta-glucosidase Bgl4 of the anti-height of Aspergillus niger strain (Aspergillus.niger) first, and the anti-glucose COEFFICIENT K i of recombinase Bgl4 is 2mol/L.

Description of drawings

Fig. 1 sugared beta-glucosidase Bgl4 of anti-height optimal reaction pH value that represents to recombinate;

Fig. 2 sugared beta-glucosidase Bgl4 of anti-height optimal reactive temperature that represents to recombinate;

Fig. 3 represent to recombinate Ki coefficient of the sugared beta-glucosidase Bgl4 of anti-height.

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.Used material comprises in the embodiments of the invention: intestinal bacteria (Escherichia coli) Top10F '; PMD-19T cloning vector test kit, (available from Takara companies) such as restriction enzyme, modifying enzyme, ligase enzymes; PPICZ α Avector(is available from Invitrogen company), p-NPG is available from Sigma company.

The acquisition of the total RNA of embodiment 1. Aspergillus niger strains (Aspergillus niger)

1.1 the cultivation of Aspergillus niger strain (Aspergillus niger)

Aspergillus niger strain (Aspergillus niger) can obtain from occurring in nature screening, also can buy from commercial channels and obtains (for example can available from Chinese industrial microbial strains preservation administrative center).

The culture medium prescription of Aspergillus niger strain (Aspergillus.niger) is: glucose 30g/L, K ₂HPO ₄3H ₂O 1g/L, KCl 0.5g/L, MgSO ₄0.5g/L, FeSO ₄0.01g/L, NaNO ₃0.2g/L pH transfers to 4.8, inoculates fresh aspergillus niger spore suspension, 37 ℃ of lower 180rpm cultivate to filter in 3-4 days and collect mycelium.

1.2 the extraction of the total RNA of Aspergillus niger strain (Aspergillus niger)

After the mycelium that gets the Aspergillus niger strain (Aspergillus.niger) of collection cleans once with PBS buffered soln, be used in and grind mycelium under the liquid nitrogen to Powdered, collect in the 2mL centrifuge tube, concuss behind the adding 1mL Trizol, shifting supernatant liquor to 2mL centrifuge tube behind 4 ℃ of centrifugal 10min of lower 12000g adds behind the 200 μ L chloroforms concussion 15s mixing and hatches 2-3min under 30 ℃, 4 ℃ of centrifugal 10min of lower 12000g get the upper strata stillness of night, add 4 ℃ of centrifugal 10min of lower 12000g behind 0.8 times of volume Virahol mixing, (DEPC processed with the 75%wt aqueous ethanolic solution after removing supernatant, removed the mRNA enzyme) clean 2 times after 4 ℃ of centrifugal 5min of lower 7500g be precipitated, after making its seasoning, add an amount of DEPC water dissolution RNA precipitation, as template ribonucleic acid ,-20 ℃ of lower preservations.

The clone of embodiment 2. beta-glucosidase Bgl4 encoding genes

1.1 the acquisition of aspergillus niger (Aspergillus.niger) cDNA

Take the total RNA of aspergillus niger Aspergillus niger bacterial strain as template, utilize reverse transcription to synthesize cDNA the first chain (following each reverse transcription agents useful for same all comes from test kit " PrimeScriptTM1stStrand cDNA Synthesis Kit ", available from Takara company).

Preparation following template ribonucleic acid/Primer reaction solution in Eppendorf tube:

50μM Oligo dT 1μL，

10mM dNTP Mixture 1μL，

Total RNA 1 μ g,

DEPC-H ₂O 7μL；

Place 1min on ice behind 65 ℃ of lower insulation 5min behind the mixing

The following cDNA synthesis reaction solution of preparation in above-mentioned Eppendorf tube:

Above-mentioned RNA/Primer mixed solution 10 μ L,

5×PrimeScript Buffer 4μL，

40U/μL RNase Inhibiter 0.5μL，

200U/μL PrimeScript RTase 1μL

RNase free H ₂O 4.5μL;

Under 50 ℃, be incubated 1h behind the above-mentioned reaction solution mixing, cooled on ice behind 70 ℃ of lower insulation 15min, the reaction solution that obtains is used for synthesizing of cDNA the second chain immediately.

2.2 design of primers and the clone of beta-glucosidase Bgl4 gene

Aspergillus niger (Aspergillus.niger) is carried out fermentation culture, and (fermention medium is: glucose 10g/L, cellobiose 10g/L, K ₂HPO ₄3H ₂O 1g/L, KCl 0.5g/L, MgSO ₄0.5g/L, FeSO ₄0.01g/L, NaNO ₃0.2g/L pH transfers to 4.8), 37 ℃ of lower 180rpm cultivated 3-4 days.The ammonium sulfate precipitation supernatant liquor of 80%wt; obtain crude enzyme liquid; (elution requirement is that 7.5Tris cushions with membrane filtration (aperture of 50kDa) and DEAE anion column chromatography after the dialysis; the concentration gradient of NaCl is 0-0.5mol/L; volume is 500mL) carry out purifying; obtain the pure enzyme of the sugared beta-glucosidase of original anti-height, the pure enzyme that obtains is carried out albumen n end order-checking (Jikang Biotechnology Co Ltd, Shanghai).The N terminal sequence that obtains (VIAITVKGNAF-) is being compared in the aspergillus niger of genome sequencing (Aspergillus.niger) CBS 513.88 albumen databases, obtain the highest albumen of similarity, obtain the gene order (sequence number: XM_001402396.2) of this albumen, take the gene order of this albumen as template design primer P1 and P2, primer P1:5 '-CCG GAATTCATGAAGGGCACCGCTGTCG-3 ', underscore represent EcoR I site.Primer P2:5 '-GC TCTAGATTACAACAGGACGAGTCCGGCA-3 ', underscore represents the XbaI site, take cDNA the first chain as template, with P1, P2 is upstream and downstream primer amplification Bgl4 gene fragment, uses Ex Taq polysaccharase (available from Takara company) to prepare 50 μ L reaction solutions with the recommendation ratio and carries out fragment amplification, the PCR reaction conditions is 94 ℃, 5min; Time out adds Ex Taq polysaccharase, adds the sealing of 40 μ L paraffin oils; 35 circulations (94 ℃, 50s; 58 ℃, 90s; 72 ℃, 90s); 72 ℃, 10min; Reaction stops, 4 ℃ of insulations.Reclaim test kit by gel pcr amplification product is carried out purifying.Obtain the sugared beta-glucosidase Bgl4 of the anti-height of aspergillus niger (Aspergillus.niger) gene order, shown in SEQ ID NO.2.

The fragment that reclaims is connected with pMD-19T simple carrier, product is transformed among the intestinal bacteria E.coli Top10F ', it is dull and stereotyped that converted product is coated blue hickie screening, 37 ℃ of incubated overnight, after inoculating single bacterium colony and cultivating 8-10h in LB (add penbritin to the final concentration 100mg/L) liquid nutrient medium, extract plasmid and carry out sequencing, obtain recombinant plasmid pMD-19T-Bgl4.

The construction and expression of embodiment 3. beta-glucosidase Bgl4 expression vectors

3.1 the structure of beta-glucosidase Bgl4 expression vector

The expression vector plasmid that uses is pPICZ α A, with α-Factor signal peptide, so need to remove the signal peptide of protogene, therefore the protein sequence of the gene Bgl4 that obtains being translated carries out signal peptide prediction, predictor adopts at sequence of threads SingalP4.0(http: //www.cbs.dtu.dk/services/SignalP/) carry out, obtain the signal peptide of the sugared beta-glucosidase Bgl4 of anti-height, remove signal peptide primer P3:5 '-CC according to this design that predicts the outcome GGAATTCGCGCCTTCGTCGACCATCAAG-3 ', underscore represent EcoR I site, remove the Bgl4 fragment of signal peptide take pMD-19T-Bgl4 as template amplification with primer P2, and the PCR reaction conditions is 94 ℃, 5min; Time out adds Ex Taq polysaccharase, adds the sealing of 40 μ L paraffin oils; 35 circulations (94 ℃, 50s; 58 ℃, 90s; 72 ℃, 90s); 72 ℃, 10min; Reaction stops, 4 ℃ of insulations.PPICZ α A plasmid and amplified fragments divided be used EcoR I and Xba I enzyme is cut, rubber tapping is reclaimed, spend the night with 16 ℃ of connections of T4 ligase enzyme again, connecting product is transformed among the intestinal bacteria E.coli Top10F ', converted product is applied to LB(and adds bleomycin to final concentration 25mg/L) 37 ℃ of incubated overnight on the solid medium, inoculate several single bacterium colonies and add bleomycin to final concentration 25mg/L to LB() cultivate after 8-10 hour in the liquid nutrient medium, collect thalline and extract plasmid, enzyme is cut checking and is removed unloaded plasmid, recombinant plasmid is carried out determining nucleic acid sequence, obtain correct recombinant expression vector pPICZ α A-Bgl4, the transformant that will contain this recombinant plasmid is inoculated into 30mL LB (adding bleomycin to final concentration 25mg/L) liquid bulk substratum, cultivate and extract plasmid after 8-10 hour, obtain a large amount of recombinant plasmid pPICZ alpha A-Bgl4.

3.2 conversion and the screening of beta-glucosidase Bgl4 expression vector

Get recombinant plasmid pPICZ alpha A-Bgl4 42 μ L, 10 * K buffer, 5 μ L, 37 ℃ of enzymes are cut the 3h rear electrophoresis and are reclaimed the goal gene band behind the Bln I 3 μ L mixings, are dissolved in the 10 μ L sterilized waters after concentrating, and obtain linearizing recombinant plasmid.

The linearizing recombinant plasmid of about 4 μ g is added in the competent cell of 80 μ L Pichia pastoris GS115s (the competence preparation is according to the operation of Pichia anomala expression handbook), be transferred to behind the mixing in the electric revolving cup, adjust the parameter (according to the operation of Pichia anomala expression handbook) of electric shock, electric shock transforms, the precooling Sorbitol Solution USP that adds 1mol/L, conversion fluid is behind 30 ℃ of placement 1h, coat YPDS flat board (adding bleomycin to final concentration 100mg/L), cultivate until grow single bacterium colony, obtained to contain the recombinant yeast pichia pastoris of gene Bgl4 for 30 ℃.

3.3 the expression of beta-glucosidase recombination engineering bacteria pPICZ α A-Bgl4/GS115

Recombination microzyme lined on the YPD flat board activate, cultivate 2d for 28 ℃, after growing single bacterium colony, be inoculated in the 10mLBMGY liquid nutrient medium, in 28 ℃, the 180rpm shaking table is cultivated 24h, and to OD600=2-6, the centrifugal 5min of 3000rpm collects thalline, abandon supernatant, wash thalline 1-2 time with sterile purified water.Thalline is diluted to OD600=1 with the BMMY inducing culture, replaces cotton plug with 4 layers of gauze, in 28 ℃, the 180rpm shaking table is cultivated.Adding methyl alcohol to final concentration every day is 0.5% (v/v), takes out 1mL bacterium liquid from the BMMY substratum in the 1.5mL centrifuge tube every 24h, and the centrifugal 5min of 3000rpm gets supernatant liquor and is used for enzyme biopsy survey and analysis of protein.

The zymologic property of embodiment 4. restructuring beta-glucosidases

4.1 enzyme activity determination method

Reaction system 100 μ L, add 85 μ L 100mmol/L citric acids-Sodium phosphate dibasic damping fluid (pH 6.0) in 5 μ L 20mmol/L p-nitrophenyl β-D glucosides (pNPG), first hatch 5min at 50 ℃, add again 10 μ L enzyme liquid reaction 10min, add again the sodium carbonate solution 600 μ L termination reactions of 1mol/L after the colour developing.Under 405nm, measure light absorption value.Enzyme activity unit (U) is defined as: under condition determination, it is 1 enzyme activity unit that per minute produces the used enzyme amount of 1 μ mol p-NP.

4.2 the mensuration of optimal reactive temperature

In 35-80 ℃ of scope, every 5 ℃, measure respectively enzyme and live.Buffering is 100mmol/L citric acid-Sodium phosphate dibasic damping fluid, and pH 6.0, finds that the optimal reactive temperature of the sugared beta-glucosidase of the anti-height of restructuring is 50 ℃, such as Fig. 2.

4.3 optimal reaction pH

At different pH(4.0-8.2,100mmol/L citric acid-Sodium phosphate dibasic damping fluid) under the condition, measures respectively enzyme for 50 ℃ and live, find that the optimal reaction pH of the sugared beta-glucosidase of the anti-height of restructuring is 6.0, such as Fig. 1.

The anti-sugared coefficient of the sugared beta-glucosidase of anti-height 4.4 recombinate

Measure the glucose concn (0mmol/L of different concns, 100mmol/L, 200mmol/L, 400mmol/L, 600mmol/L, 800mmol/L, 1000mmol/L, 1200mmol/L, 1400mmol/L, 1600mmol/L, 1800mmol/L, 2000mmol/L) on the sugared beta-glucosidase Bgl4 of the anti-height enzyme impact alive of recombinating, find that the sugared beta-glucosidase of the anti-height of restructuring still has 48.5% enzyme activity compared with the control under the 2000mM glucose concn, the anti-sugared COEFFICIENT K i of glucose is about 2000mmol/L, such as Fig. 3.

SEQUENCE LISTING

＜110〉Nanjing Forestry University

＜120〉a kind of beta-glucosidase Bgl4 and expressing gene and application of anti-high sugar

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Claims

1. the beta-glucosidase Bgl4 of an anti-high sugar, its aminoacid sequence is shown in SEQ ID NO.1.

2. the expressing gene of the beta-glucosidase Bgl4 of the described anti-high sugar of claim 1, its nucleotide sequence is shown in SEQ ID NO.2.

3. the recombinant plasmid that comprises the described nucleotide sequence of claim 2.

4. the recombinant plasmid pPICZ alpha A-Bgl4 that comprises the described nucleotide sequence of claim 2.

5. comprise the preparation method of the recombinant plasmid of the described gene order of SEQ ID NO.2, it is characterized in that:

(1) design primer P1 and P2, take the cDNA of the mRNA reverse transcription of Aspergillus niger strain (Aspergillus.niger) as template, take P1 and P2 as primer carries out pcr amplification, obtain the pcr amplification product of the sugared beta-glucosidase Bgl4 of anti-height gene, described primer is:

P1:5 '-CCG GAATTCATGAAGGGCACCGCTGTCG-3 ', underscore represent EcoR I site;

P2:5 '-GC TCTAGATTACAACAGGACGAGTCCGGCA-3 ', underscore represent Xba I site;

6. the pichia spp host cell that comprises recombinant plasmid claimed in claim 4.

7. the application of beta-glucosidase Bgl4 in cellulose degradation of the described anti-high sugar of claim 1.