CN107828806A

CN107828806A - A kind of β alpha-glucosidase genes of new resistance to glucose and its application

Info

Publication number: CN107828806A
Application number: CN201710698739.8A
Authority: CN
Inventors: 李荷; 张雪玲; 夏玉林
Original assignee: Guangdong Pharmaceutical University
Current assignee: Guangdong Pharmaceutical University
Priority date: 2017-08-15
Filing date: 2017-08-15
Publication date: 2018-03-23

Abstract

The invention discloses a kind of β alpha-glucosidase genes of new resistance to glucose and its application, its nucleotide sequence is as shown in SEQ ID NO.1；Its amino acid sequence is as shown in SEQ ID NO.2.The recombinant plasmid pET32a Bgl2238 of β alpha-glucosidase genes the invention also discloses the preparation method of the β alpha-glucosidase genes of the new resistance to glucose and containing the new resistance to glucose, the invention also discloses a kind of recombinant beta glucuroide and preparation method thereof, by recombinant beta alpha-glucosidase gene overexpression in escherichia coli prokaryotic expression system, recombinant beta glucuroide solution expression with high efficiency in coli expression system.The invention also discloses hydrolysis application of the recombinant beta glucuroide to Resveratrol in Rhizoma Polygoni Cuspidati glycosides, illustrate the recombinant beta glucuroide obtained using methods described, with higher catalytic activity and resistance to glucose activity, there are very big industrialized production and application prospect.

Description

A kind of beta-glucosidase gene of new resistance to glucose and its application

Technical field

The invention belongs to genetic engineering field, is related to beta-glucosidase gene and the coding production of a kind of new resistance to glucose Thing, a kind of new β-glucose is obtained from the corn edaphon of Heilungkiang especially with the method for Metagenomic library screening Glycoside enzyme gene and coded product.

Background technology

Beta-glucosidase (taxology numbering is EC 3.2.1.21), also known as β-D-Glucose glycosides hydrolase, is three kinds of fibres One of plain enzyme is tieed up, the glycosidic bond generation glucose between alkyl or aromatic radical and glycosyl can be hydrolyzed, belong to glycoside hydrolase man Race, played a decisive role in cellulose degradation.Beta-glucosidase industrially has a wide range of applications, food service industry application In improvement flavour of food products；Pharmaceuticals industry is applied to conversion polydatin and soybean isoflavone glucoside compound, prepares oligomeric dragon Courage sugar, the detection of disease and development of medicine etc.；Bioenergy industry degraded cellulose produces bio-ethanol；In weaving, papermaking Industry also widely uses beta-glucosidase.The research of beta-glucosidase is carried out in a deep going way with important theory significance and wide Application prospect.

The beta-glucosidase overwhelming majority industrially applied at present is fitted from fungies such as trichodermas to conditions such as temperature and pH Answer scope narrower, and enzyme activity is also relatively low, causes fermenting and producing and use cost higher, and this turns into current beta-glucosidase The wide variety of bottleneck of enzyme.Therefore, how from various natural habitats especially in extreme environment screening with high temperature resistant, acidproof The excellent new beta-glucosidases of zymologic property such as alkali, organic solvent-resistant, high enzyme activity, substrate be wide, are currently in β-glucose The vital task faced in the research of glycosides enzyme.

Microorganism present in nature 99% is not educable, therefore micro- from being separated to traditional culture technique The method that new biocatalyst is screened in biology is very limited.Utilize Culture-independent method-technique of metagenome (Metagenomics) DNA, is directly extracted from environment, is cloned into different carriers, builds grand genomic library, then Therefrom screened.The technology shows in excavation and using Anticipated transient without scram resource and in terms of screening novel bioactive substance Go out huge potentiality, it has also become the Disciplinary Frontiers and focus of current microbiological research in the world.So far domestic and foreign scholars have been Such as amylase, zytase, cellulase, esterase new gene are cloned into by the technology, these enzymes have novel zymetology Characteristic and commercial application potentiality.Therefore this method provides new approaches for discovery beta-glucosidase gene.

Chinese medicine giant knotweed has expelling wind and removing dampness, relieving cough and reducing sputum, promoting blood circulation and stopping pain pharmacological action as the traditional Chinese medicine in China. Resveratrol is one of active component in giant knotweed, can reduce the concentration of blood fat, suppress hematoblastic aggregation, prevent angiocarpy The generation of disease；Each stage of cancer generation can also be acted on, suppresses the activity of tumour cell, kinds of tumors can be treated Disease.In addition resveratrol also has the effect such as anti-inflammatory, anti-oxidant, plays the curative effects such as antipyretic and analgesic, makes in recent years Study hotspot.But the resveratrol overwhelming majority is combined together with glucose in plant, polydatin is formed, this Part resveratrol can not be utilized in human body.Content of the resveratrol in giant knotweed is in the plant being currently known Highest, but 0.2%-0.4% is also only accounted for, even if the resveratrol to dissociate in other words in giant knotweed is extracted completely, Double centner giant knotweed also can only obtain most 0.4 kilogram of resveratrol, far can not meet the needs of market.But if tiger 1.5% -3.0% polydatin is hydrolyzed into resveratrol in vitro in cane, will largely improve the production of resveratrol Raw rate.Polydatin belongs to β-glycoside, and resveratrol can be hydrolyzed in the presence of beta-glucosidase.Therefore efficiently And the good beta-glucosidase of stability is the key of Resveratrol in Rhizoma Polygoni Cuspidati glycosides hydrolysis.But current β-glucose on the market Glycosides enzyme mostlys come from the fungies such as trichoderma, aspergillus, and enzyme activity is low and temperature and pH equistability scopes are narrower, causes β-glucose Glycosides enzyme it is expensive, be unfavorable for Resveratrol in Rhizoma Polygoni Cuspidati glycosides hydrolysis industrialized production.Present invention restructuring beta-glucosidase The problem of polydatin in enzyme hydrolysis giant knotweed can avoid the above.

The content of the invention

First purpose of the present invention is to solve the deficiencies in the prior art part and provides a kind of new resistance to glucose Beta-glucosidase gene is named as bgl2238.

Second object of the present invention is the preparation method for providing the beta-glucosidase gene of new resistance to glucose.

Third object of the present invention is to provide a kind of restructuring matter of the beta-glucosidase gene of new resistance to glucose Grain pET32a-bgl2238.

Fourth object of the present invention is the table for providing the DNA of the beta-glucosidase containing above-mentioned new resistance to glucose Up to carrier.

The 5th purpose of the present invention is to provide a kind of preparation method of the beta-glucosidase of resistance to glucose.

The 6th purpose of the present invention is to provide a kind of solution method for solving resveratrol shortage on the market.

First purpose of the present invention is achieved by the following technical solution：A kind of β-grape of new resistance to glucose The DNA of glycosidase, is named as bgl2238, comprising an ORFs, the beta-glucosidase base of the new resistance to glucose Because nucleotide sequence is as shown in SEQ ID NO.1：

The beta-glucosidase of above-mentioned new resistance to glucose provided by the invention, its amino acid sequence such as SEQ ID Shown in NO.2：

Second object of the present invention is achieved by the following technical solution：A kind of new beta-glucosidase it is grand Genomics cloning process and a kind of preparation method of the beta-glucosidase gene of new resistance to glucose.It is micro- to avoid tradition The not educable limitation of biology, is greatly improved the utilization of resources of microorganism in environment.Extract the total of Heilungkiang corn pedotheque DNA simultaneously uses OMEGA company Gel Extraction Kit (D2500-01) kits, and STb gene after purification is passed through 37 DEG C of digestion 1.2h of EcoR I, carrier pUC118/EcoR I (BAP) are connected to, it is electroporated to the super sense of bacillus coli DH 5 alpha Grand genomic library is established by state, by putting in the LB flat boards using aesculin as screening substrate, screening has the list of black hydrolysis circle Bacterium colony obtains positive clone molecule, is analyzed through sequencing, pBLAST and ORF Finder and designs primer, so as to be cloned into purpose piece Section.

Third object of the present invention is achieved by the following technical solution：The beta-glucosidase of new resistance to glucose Enzyme gene is named as bgl2238, according to the beta-glucosidase gene bgl2238 of new resistance to glucose primers, Using the plasmid pUC118-bgl2238 of extraction as template, enter performing PCR amplification, PCR primer is coagulated through gel electrophoresis and recovery product 1 Glue reclaim product 1, through restriction enzyme Xho I, EcoR I double digestions and recovery product 2, plasmid pET32a through Xho I, It is connected after EcoR I double digestions with recovery product 2, obtains connection product, i.e. recombinant plasmid pET32a-bgl2238；It is wherein described Primers sequence is as follows：

bgl2238-fw：5’-CCGGAATTCATGAAACACATCCTAAACCTATGCC-3’

(underscore part is EcoR I restriction enzyme sites)

bgl2238-rv：5’-CCGCTCGAGTTATCGTACGCTAAAAGTAAGGGCTTC-3’

(underscore part is Xho I restriction enzyme sites).

Fourth object of the present invention is achieved by the following technical solution：A kind of expression vector, containing above-mentioned new The expression vector of type beta-glucosidase gene.Structure gained with the following method：Using electric robin, by recombinant plasmid PET32a-bgl2238 is transformed into e. coli bl21 competent cell, by the bacterial suspension renewal cultivation after conversion and dilute Release to be coated on amicillin resistance culture plate and cultivate, picking positive transformant, β-glucose of the new resistance to glucose of acquisition The expression vector of glycoside enzyme gene is ETEC BL21-pET32a-bgl2238.

The preparation method of restructuring beta-glucosidase provided by the invention, including with above-mentioned recombinant plasmid pET32a- Bgl2238 converts host cell, cultivates transformant, and the method and steps of restructuring beta-glucosidase is obtained from culture.

In the preparation method of above-mentioned restructuring beta-glucosidase, host cell is e. coli bl21.

The preparation method of above-mentioned restructuring beta-glucosidase, its detailed process are：Recombinant plasmid pET32a-bgl2238 The e. coli bl21 of conversion is coated on the flat board containing amicillin resistance, 37 DEG C of inversion overnight incubations, picking monoclonal Be inoculated in LB fluid nutrient mediums, 37 DEG C, 180rpm shaken cultivations stay overnight, by volume 1：100 ratio is forwarded to fresh contain Amicillin resistance LB culture mediums, are cultivated to OD in 37 DEG C, 180rpm₆₀₀=0.8, IPTG Fiber differentiations are added, are obtained efficiently Solubility expression；The Ni-NTA Agerose nickel post affinity columns through Novagen companies enter to restructuring beta-glucosidase after culture Row purifying, produces restructuring beta-glucosidase.

The IPTG of the restructuring beta-glucosidase induces final concentration of 1.0~1.4mM, and inducing temperature is 25~37 DEG C.

The optimal IPTG of the restructuring beta-glucosidase induces final concentration of 1.0mM, and inducing temperature is 25 DEG C, during induction Between 11h.

The 6th purpose of the present invention is that the polydatin in giant knotweed is hydrolyzed by recombinating beta-glucosidase To realize：Giant knotweed powder is taken, recombinates ratio of the beta-glucosidase crude enzyme liquid than 10-100mL water than 2-10mL in 2g giant knotweeds powder, first Beta-glucosidase crude enzyme liquid is added into giant knotweed powder, is added water, is then placed within constant-temperature table and shakes enzymolysis, HPLC detections The conversion ratio of polydatin；Temperature is 37 DEG C -55 DEG C, and rotating speed is 100-200rpm, and enzymolysis duration is 4-24h.

Further, 2g giant knotweeds powder is than 100mL water, solid-liquid ratio than 4mL restructuring beta-glucosidase crude enzyme liquids during enzymolysis 1：50, enzymolysis time is 12h, and temperature is 44 DEG C, and rotating speed is 120rpm.

Beneficial effects of the present invention：The present invention obtains one from the grand genomic library of Heilungkiang corn pedotheque structure Individual new beta-glucosidase gene bgl2238, it is the complete beta-glucosidase gene of a 2238bp size.Utilize BLAST compares its nucleotide sequence progress homology searching analysis and shown online, the new beta-glucosidase gene There is relatively low similitude in bgl2238, be defined as new beta-glucosidase gene in a kind of Anticipated transient without scram with known.

1st, functional study is done to the new beta-glucosidase gene by technique for gene engineering, finds the sequence in large intestine Efficient soluble-expression in bacillus BL21, restructuring beta-glucosidase can hydrolyze the common substrate pNPG of beta-glucosidase.Through The Ni-NTA of Novagen companiesResins protein purifications and SDS-PAGE electrophoresis, respectively obtain a single albumen Band, it is about 80.67kDa to primarily determine that molecular weight.

2nd, restructuring beta-glucosidase is through nickel post parent after restructuring bgl2238 is expressed in escherichia coli prokaryotic expression system Purify to obtain with the method for chromatography, carry out amino acid identity than analysis through albumen, it is found that the restructuring beta-glucosidase contains There is the domain of GH3 superfamilies and GH3C superfamilies.

3rd, the DNA sequence dna shown in SEQ ID NO.1 is cloned on prokaryotic expression carrier by the present invention, is transformed into Escherichia coli BL21 (DE3) competent cell, beta-glucosidase is recombinated by obtaining recombinant protein to the induced expression of positive clone molecule, Its zymologic property is studied, it is as a result as follows：

(1) in coli expression system, the recombinant protein has solution expression with high efficiency.

(2) using pNPG as substrate, it is described restructuring beta-glucosidase optimal reactive temperature and pH be respectively 44 DEG C and 6.10。K⁺、Na⁺、Fe²⁺、Mg²⁺、Mn²⁺、Cu²⁺、Ca²⁺、Co²⁺、Zn²⁺And Ni²⁺There is larger facilitation to enzyme activity, wherein Fe²⁺Facilitation maximum to recombinating beta-glucosidase enzyme activity.Recombinate beta-glucosidase has necessarily resistance to NaCl By property.Urea, guanidine hydrochloride and guanidinium isothiocyanate have facilitation in 1mM to the enzyme activity for recombinating beta-glucosidase, although Disappeared with concentration rise facilitation, but in high concentration, restructuring beta-glucosidase enzyme activity still retains 97% respectively, 52%th, 48% or so.This phenomenon is rarely found in the beta-glucosidase having found.Organic solvent DMSO, methanol, ethanol, Isopropanol, TritonX-100 have facilitation in 1mM to the enzyme activity for recombinating beta-glucosidase, with solvent strength Rise, organic solvent DMSO, methanol, ethanol and isopropanol all have certain inhibitory action, but protein denaturant to enzyme When DMSO is at concentrations up to 100mM, the enzyme activity for recombinating beta-glucosidase still retains more than 85%, in summary, recombinant beta-Portugal Polyglycoside enzyme shows certain tolerance to organic solvent, has good application prospect in commercial Application.Recombinant beta-Portugal When polyglycoside enzyme is using pNPG as substrate, V_max=576uM/min, K_m=0.296mM, during using cellobiose as substrate, V_max= 572uM/min, K_m=0.543mM.Bgl2238 is distinguished less to the hydrolysis rate of two kinds of substrates, but has stronger parent to pNPG And property.Product of the glucose as beta-glucosidase enzymatic reaction, has certain inhibitory action to enzymatic reaction, works as glucose When concentration is 1.0M, restructuring beta-glucosidase can still keep 56% or so of maximum enzyme activity, show recombinant beta-grape Glycosidase has certain tolerance to glucose simultaneously.

The present invention provides a kind of new beta-glucosidase gene of resistance to glucose, and gene constructed this is arrived into plasmid pET32a In, then the recombinant plasmid is transferred in escherichia coli prokaryotic expression system and is expressed and is purified.The product of the gene expression With higher catalytic activity, there is the property of good organic solvent-resistant and resistance to glucose, there is larger industrialized production And application potential.

4th, restructuring beta-glucosidase Bgl2238 is hydrolyzed Resveratrol in Rhizoma Polygoni Cuspidati glycosides by the present invention, can be by content in giant knotweed About 1.5% -3.0% polydatin is hydrolyzed into resveratrol in vitro, can make the conversion of Resveratrol in Rhizoma Polygoni Cuspidati glycosides Rate reaches 95%, and the extracted amount of resveratrol also improves 6 times, largely improves the yield of resveratrol, and it is optimal Enzymolysis process is：Bgl2238 dosages are 1:4, solid-liquid ratio 1:25, enzymolysis time 12h, 44 DEG C of temperature, rotating speed 120rpm.To tiger Resveratrol and polydatin have carried out preliminary extraction research in cane solution, are 1 by liquid ratio:15 add 95% ethanol It is heated to reflux 2h.Content with rear resveratrol after HPLC detection enzymolysis is 6.87%, than improving 6 times before enzymolysis.

Brief description of the drawings

The conserved sequence region figure for the Bgl2238 that Fig. 1 speculates；

Fig. 2 is beta-glucosidase gene bgl2238PCR amplified production agarose gel electrophoresis figures；Wherein, M： DL5000；1：bgl2238；

Fig. 3 is restructuring beta-glucosidase gene Bgl2238 SDS-PAGE；Wherein, M is standard protein molecule Maker is measured, 1 is restructuring Bgl2238 protein crude extract administrations, and 2 be restructuring Bgl2238 purified products；

Fig. 4 is influence of the induction time to restructuring beta-glucosidase gene Bgl2238 expressions；

Fig. 5 is influence of the IPTG concentration to restructuring beta-glucosidase gene Bgl2238 expressions；

Fig. 6 is p-nitrophenol standard curve；

Fig. 7 is influence result line chart of the temperature to restructuring activity of beta-glucosidase；

Fig. 8 is influence result line chart of the temperature to restructuring beta-glucosidase stability；

Fig. 9 is influence result line charts of the pH to restructuring activity of beta-glucosidase；

Figure 10 is influence result line charts of the pH to restructuring beta-glucosidase stability；

Figure 11 is influence of the metal ion to restructuring activity of beta-glucosidase；

Figure 12 is the influence of Organic Acids on Heavy group activity of beta-glucosidase；

Figure 13 is influence of the organic solvent to restructuring activity of beta-glucosidase；

Figure 14 is influence of the glucose to restructuring beta-glucosidase vigor；

Figure 15 is influence of the restructuring beta-glucosidase enzyme concentration to polydatin hydrolysis efficiency；

Figure 16 is influence of the solid-liquid ratio to polydatin hydrolysis efficiency；

Figure 17 is influence of the enzymolysis time to polydatin hydrolysis efficiency；

Figure 18 is influence of the operative temperature to polydatin hydrolysis efficiency；

Figure 19 is influence of the rotating speed to polydatin hydrolysis efficiency；

Figure 20 is the HPLC results of resveratrol and polydatin before restructuring beta-glucosidase enzymolysis；

Figure 21 is the HPLC results of resveratrol and polydatin after restructuring beta-glucosidase enzymolysis.

Embodiment

The present invention will be further described with reference to the accompanying drawings and examples, but the invention is not limited in any way.

The foundation of 1 grand genomic library of embodiment and acquisition, the gene cloning and expression of positive clone molecule

1st, the extraction of STb gene：

(1) the 50ml centrifuge tubes after 4 autoclavings are taken, Heilungkiang Suihua Area milpa pedotheque is weighed, often manages 6g, respectively add DNA extraction buffer13.5ml, after the concussion that is vortexed mixes, put shaking table 220rpm, 37 DEG C of activation 30min.

(2) SDS of each pipe plus 1.5ml 20%, makes its final concentration reach 2% (m/V).

(3) the water-bath 2h in 65 DEG C of water-baths, gently overturn every 30mim and mix for several times up and down.

(4) 25 DEG C, 6,000rpm, centrifuge 10min.

(5) supernatant is transferred to autoclaved 50ml high speed centrifugations pipe, adds isometric chloroform：Isoamyl alcohol (24： 1), gently overturn and mix up and down.

(6) 25 DEG C, 11,000rpm, centrifuge 30min.

(7) supernatant is collected, the isopropanol for adding 0.6 times of volume gently overturns mixing up and down, is stored at room temperature more than 3h.

(8) 25 DEG C, 11,000rpm, centrifuge 30min.

(9) supernatant is abandoned, precipitation is washed 1 time with 75% ethanol of 4 DEG C of precoolings.

(10) 4 DEG C, 8,000rpm, 10min centrifugations, supernatant is abandoned, is put into 60 DEG C of oven for drying.

(11) ddH of often 65 DEG C of preheatings of pipe plus 0.2ml₂O, and 65 DEG C of warm bath 5min, dissolve genomic DNA.

The genomic DNA of (12) 1.0% agarose gel electrophoresis Detection and Extraction, and the genomic DNA of purifying recovery immediately.

2nd, RNA isolation kit purifying DNA：According to OMEGA companies of U.S. Gel Extraction Kit glue reclaim kit explanations Book is carried out, and step is as follows：

(1) electrophoresis：The soil genomic DNA of extraction electrophoresis (voltage 10V/cm) in 1% Ago-Gel, electrophoresis knot Shu Hou, 5min is dyed in EB solution；

(2) glue is cut：Ago-Gel after EB is dyed is observed in gel imager, and cuts genome containing soil DNA Ago-Gel band, then with deionized water rinsing, it is placed in 2ml centrifuge tubes；

(3) purify：UseExtraction Kit kits reclaim soil genomic DNA, specific steps It is as follows：

1. weigh the quality of the Ago-Gel of the genomic DNA containing soil；

2. corresponding to 1ml Binding Buffer ratio according to every 1g Ago-Gels, solution is added into centrifuge tube Binding Buffer。

3. the centrifuge tube of the gels of Buffer containing Binding is placed in 60 DEG C of water-baths, vibrates and mix every 2-3min, Melt completely to Ago-Gel；

4. HiBind DNA posts are placed in 2ml collecting pipes, and the solution that step 3 is obtained is transferred to HiBind DNA posts In, 14,000rpm centrifugation 1min after 2min are stood, abandon filtrate；

5. adding 700 μ l SPW Wash Buffer, 10,000rpm centrifugation 1min, filtrate is abandoned, and repeat this step；

6. 14,000rpm centrifuges 2min to dry column matrix；

7. HiBind DNA posts are placed in new 1.5ml centrifuge tubes, and appropriate ultra-pure water is added dropwise to post center, stands After 2min, 14,000rpm centrifugation 2min.Ttom of pipe liquid is soil genomic DNA fragment after purification, and -20 DEG C save backup.

3rd, grand genome electrophoresis detection：With the purity and quality of 1% agarose gel electrophoresis detection STb gene, ensure purifying DNA A afterwards₂₆₀/A₂₈₀Ratio is higher than 1.8.

4th, digestion STb gene：With the partially digested STb genes of restriction enzyme EcoR I, enzyme of the recovery clip size in 2-8kb Cut into slices section, specific method is the same as RNA isolation kit purifying DNA in above-mentioned 2.Endonuclease reaction system is as follows：37 DEG C, digestion 1.2h.

5th, the electrophoresis detection of endonuclease bamhi：Method is the same as grand genome electrophoresis detection.

6th, the connection of endonuclease bamhi：Obtained endonuclease bamhi will be reclaimed and pUC118/EcoR I (BAP) carrier uses TaKaRa T4DNA Ligase are attached processing, and in 16 DEG C of connections overnight, linked system is as follows；Added into connection product The isopropanol of 0.7 times of volume, -20 DEG C of precipitates overnights, 14000r/min centrifugation 20min, abandon supernatant and add 80% into precipitation Ethanol is washed 1 time, and room temperature is dried, and adds 10 μ l dd H₂The carrier DNA of O dissolving connections.

7th, Escherichia coli electricity converts the preparation of super competent cell, and step is as follows：

(1) E.coli DH5 α bacterium solutions are taken to line LB solid plates, 37 DEG C are incubated overnight；

(2) from one E.coli DH5 α single bacterium colony of picking on LB solid plates, the sterile LB fluid nutrient mediums of 30ml are inoculated in In, shaken cultivation is stayed overnight under the conditions of 37 DEG C, 180rpm；

(3) bacterium solution that transferase 12 ml is incubated overnight is shaken in 200ml LB fluid nutrient mediums under the conditions of 37 DEG C, 200rpm Culture is swung to thalline OD₆₀₀For 0.25-0.35；

(4) bacterium solution is placed in cooled on ice 30min, and transfer bacterium solution is into the 50ml sterile centrifugation tubes of precooling, 4 DEG C, 6,000g 10min is centrifuged, abandons supernatant；

(5) ultra-pure water of 30ml precoolings is added, cell is resuspended in soft piping and druming, and whole operation process is sterile and is grasped on ice Make；4 DEG C, 6,000g centrifugation 10min, abandon supernatant, repeat this step once, and most at last all thalline be incorporated in a 50ml without In bacterium centrifuge tube；

(6) 10% glycerine of the pre- cold sterilizations of 25ml is added, soft piping and druming is resuspended cell, 4 DEG C, 6,000g centrifugation 10min, abandoned Supernatant；

(7) cell is resuspended in 10% glycerine for adding 1ml precoolings, and cell is resuspended in soft piping and druming, according to the specification of 100 μ l/ pipes Dispensed, -80 DEG C of ultra low temperature freezers save backup.

8th, the conversion of connection product：

(1) shift to an earlier date 5min and take the μ l E.coli DH5 α electricity of a pipe 100 to turn competent cell from -80 DEG C of ultra low temperature freezers and put Slowly thawed on ice, add 5 μ l connection products, (operated after gently piping and druming mixes in super-clean bench), stand 5min on ice；

(2) mixture is transferred in sterile electric shock cup (0.2cm), is shocked by electricity under 2,500V voltages, is added immediately into electric shock cup Enter the SOC culture mediums of 500 μ l, 46 DEG C of preheatings；

(3) gently piping and druming is moved in EP pipes after mixing, 37 DEG C, 180rpm shaken cultivations 50min；

(4) take appropriate culture to be coated on LAXI culture mediums, 37 DEG C of overnight incubations, produce corn soil metagenome text Storehouse.

9th, the identification of library screening and positive clone molecule

In picking library all blank single bacterium colony with the pipette tips point to sterilize to screening and culturing medium (100 μ g/mlAmp, 0.1mM IPTG, 0.1% aesculin, 0.25% ferric citrate) in flat board, after 37 DEG C of cultures 2~3 days, see whether black Color hydrolysis circle, if so, then illustrating to be possible to containing beta-glucosidase gene in the clone.It is saturating that obvious black can be produced Bright circle is positive clone molecule.1 positive clone molecule is obtained by the screening technique.By positive colony from corresponding flat board Son is chosen and is seeded in LB fluid nutrient mediums of the 5mL containing ampicillin (100 μ g/ml), 37 DEG C, 180rpm shaking table culture mistakes At night, plasmid is extracted, concrete operations are carried out according to the Plasmid Mini Kit I specifications of OMEGA companies.Insert Fragment is surveyed Sequence, the sequence of measure is compared by NCBI BLAST software analysis, find bases of the DNA by an ORF size for 2238bp Because of composition, its nucleotide sequence is as shown in SEQ ID NO.1, and its amino acid sequence is as shown in SEQ ID NO.2.

10th, the clone of genetic fragment

Pair of primers is designed according to sequencing result, primer sequence is as follows：

bgl2238-fw：5’-CCGGAATTCATGAAACACATCCTAAACCTATGCC-3’

(underscore part is EcoR I restriction enzyme sites)

bgl2238-rv：5’-CCGCTCGAGTTATCGTACGCTAAAAGTAAGGGCTTC-3’

(underscore part is Xho I restriction enzyme sites)

Using the plasmid comprising pUC118-bgl2238 as template, using bgl2238-F and bgl2238-R as primer, use Prime STAR^TMMax Premix enter performing PCR amplification, and system is as follows:

The reaction condition of PCR cycle is as follows：

First stage：98 DEG C of denaturation 3min；

Second stage：98 DEG C of denaturation 5sec, 55 DEG C of annealing 5sec, 72 DEG C of extension 5sec, totally 30 circulate；

Phase III：72 DEG C of extension 7min, most after 4 DEG C.

PCR primer is purified with above-mentioned OMEGA glue reclaims kit, as shown in Figure 2.And with fermentas companies Restricted quick restriction endonuclease Xho I, EcoR I in 37 DEG C to bgl2238PCR amplified production double digestion 20min, with Xho I, PET-32a (+) expression vector of EcoR I double digestions carries out double digestion is handled using TaKaRa T4DNA Ligase PET-32a and PCR primer are attached, 16 DEG C of connection 12-16h.Take the above-mentioned recombinant plasmid transformed e. coli bl21s of 3 μ l (DE3), LB solid medium of the conversion fluid coating containing ampicillin (100 μ g/ml), 37 DEG C of overnight incubations, random 5 plants of picking Single bacterium colony inoculation extraction DNA, after double digestion checking, delivers sequencing.Above-mentioned digestion and coupled reaction system difference is as follows：

11st, the induced expression of the destination protein of embodiment 1 restructuring beta-glucosidase and purifying

Recombination engineering is rule into the LB solid mediums containing ampicillin (100 μ g/ml), 37 DEG C of overnight incubations Activation, random 1 recombinant bacterium of picking are seeded in the LB fluid nutrient mediums containing ampicillin (100 μ g/ml), 37 DEG C, 220r/ Min shaking table cultures are stayed overnight, by 1:100 inoculum concentration is forwarded to the 40mL LB Liquid Cultures containing ampicillin (100 μ g/ml) In base, when growing to OD₆₀₀IPTG to final concentration 1.0mM, 25 DEG C, 200r/min shaking table cultures 11h, 6000r/ are added when=0.8 Min centrifuges 10min, abandons supernatant, thalline is resuspended in into 40ml sterilized waters, with Ultrasonic Cell Disruptor (Sonics companies) smudge cells, 4 DEG C, 6000r/min centrifugation 20min, supernatant is collected, big and heavy histone is obtained, by big and heavy histone Ni-NTA Agerose (Novagen) nickel post affinity column purification of recombinant proteins, affinity column concrete operation step are entered by Novagen Products specifications OK.

Recombinant protein by the big and heavy histone of acquisition and after purification carries out PAGE gel electrophoresis (10%) by thick enzyme Each component of albumen separates in liquid, is dyed with coomassie brilliant blue R_250, and albumen maker estimates the size of zymoprotein.Pass through egg White Purification Kit zymoprotein, SDS-PAGE electrophoresis obtain a single protein band.SDS-PAGE electrophoresis result tables Bright, the polypeptide described in SEQ ID NO.1 coded by nucleotide sequence obtains high efficient expression in e. coli bl21 (DE3), and All recombinant proteins are solvable, and no inclusion body is formed, and recombinant protein Bgl2238 molecular weight is about according to a preliminary estimate 80.67kDa (as shown in Figure 3).

Embodiment 2 recombinates the optimization of beta-glucosidase induced expression condition

(1) optimization of induction time

Picking verifies correct recombinant bacterial strain in 20ml LB fluid nutrient mediums, 37 DEG C, 200rpm overnight incubations, as Seed liquor.1 is pressed respectively:100 ratio transfers seed liquor into 50ml LB fluid nutrient mediums, 37 DEG C, 220rpm shaken cultivations extremely Cell density OD₆₀₀During=0.8-1.2, addition IPTG makes its final concentration of 1.0mM, and under the conditions of 220rpm, 25 DEG C are cultivated respectively After 8h, 11h, 13h, 15h, 18h, 21h, crude enzyme liquid is prepared, quantitative determines beta-glucosidase enzyme activity, is defined with enzyme activity highest For 100, it is determined that optimal inducing temperature and induction time.(as shown in Figure 4).

(2) optimization of IPTG concentration

Recombinant bacterial strain after picking checking is in 20ml LB culture mediums, 37 DEG C, 200rpm overnight incubations, as seed liquor. 1 is pressed respectively:100 ratio transfers seed liquor into 50ml LB fluid nutrient mediums, 37 DEG C, 220rpm shaken cultivations it is close to thalline Spend OD₆₀₀During=0.8-1.2, add IPTG to final concentration be respectively 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM, 1.2mM, 1.4mM, 1.6mM, the Fiber differentiation 11h under 25 DEG C, 220rpm, crude enzyme liquid is prepared, quantitative determine beta-glucosidase enzyme activity, with Enzyme activity highest is defined as 100, it is determined that optimal IPTG induced concentrations.(as shown in Figure 5).

Embodiment 3 recombinates beta-glucosidase enzyme activity determination

(1) measure of enzyme activity

The present invention detects the enzymatic activity of beta-glucosidase using pNPG as substrate.Take the crude enzyme liquid 10 of certain extension rate μ l, the B-R buffer solutions of 80 μ l optimal pHs, the 50mM μ l of pNPG 10 are mixed, and water-bath 20min under optimum temperature, take 200 μ l 1M Na₂CO₃Solution terminates enzymatic reaction, takes the μ l of said mixture 200, determines OD₄₀₅.The parallel each enzymatic of measuring three times is anti- Should.The crude enzyme liquid through inactivation handled under the same conditions is done into blank control.One enzyme-activity unit (U) is defined as per minute point Solve the enzyme amount needed for 1 μm of olpNP of pNPG generations.

(2) drafting of p-nitrophenol standard curve

The accurate pNP for weighing 0.6956g, dissolved with 100ml PH 6.10 acid buffers of B-R tri-, dilute ten times, press The amount shown of table 3.7 is diluted to various concentrations again, determines OD₄₀₅, negative control is used as by 0.00mM samples of pNP concentration.With pNP Concentration is to OD₄₀₅Value makees pNP canonical plottings.(such as accompanying drawing 6).

The p-nitrophenol of table 1 (ρ NP) titer

Embodiment 4 recombinates the zymologic property research of beta-glucosidase

1st, influence of the temperature to restructuring beta-glucosidase vigor and stability

The pNPG of the crude enzyme liquid of the 10 certain dilution factors of μ l, 10 μ l 50mM is added to the B-R buffer solutions of 80 μ l optimal pHs In, it is respectively placed in 4 DEG C, 15 DEG C, 30 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 60 DEG C, 70 DEG C and 80 DEG C water-baths and reacts 20min, adds 200 μ l 1M Na₂CO₃Terminating reaction, determine OD₄₀₅Value, the optimum temperature range under one-level thermograde is obtained, then by temperature Gradient further reduces, to obtain enzyme optimal reactive temperature.By the crude enzyme liquid suitably diluted be put in respectively 4 DEG C, 15 DEG C, 30 DEG C, 4h is incubated in 40 DEG C, 45 DEG C, 50 DEG C, 60 DEG C, 70 DEG C and 80 DEG C water-baths, remaining enzyme activity is determined by 3.2.8.1 method, with not The enzyme activity of the enzyme liquid of thermally treated (4 DEG C) is set to 100, the remaining enzyme activity after treatment of different temperature is determined, with relative enzyme activity Power draws curve to temperature.Testing result is as shown in accompanying drawing 7,8.

2nd, influences of the pH to restructuring beta-glucosidase vigor and stability

Under the conditions of 44 DEG C, the vigor of the enzyme under condition of different pH is determined using the B-R buffer solutions of different pH value, by one-level PH gradient is set to 1.98,2.87,4.10,5.02,6.09,6.59,7.0,7.54,7.96,8.95,9.91,10.88,11.82. The diluted crude enzyme liquids of 10 μ l, 10 μ l 50mM pNPG is taken to be added to more than 80 μ l in different pH B-R buffer solutions, 44 DEG C of water 20min is bathed, adds 200 μ l 1M Na₂CO₃Terminating reaction, determine OD₄₀₅Value, obtains roughly optimal pH scope.Obtaining one-level After high vigor scope under pH gradient, pH gradient is further reduced, finally to obtain enzyme optimal reaction pH.By 10 μ l through pH 2.0~12.0 buffer solutions dilute 50 times of crude enzyme liquid, after 4 DEG C are placed 4h, determine remaining enzyme activity, will be incubated under optimal pH without 4h The highest enzyme activity educated is defined as 100, and pH is mapped with enzyme activity.Testing result is as shown in Figure 9.

3rd, influence of the metal ion to enzyme activity

It is diluted that 10 μ l are added in 80 μ l metal ion final concentrations are respectively 1mM or 10mM optimal pH buffer solution Crude enzyme liquid, after 4 DEG C are placed 4h, the pNPG for adding 10 μ l 50mM determines remaining enzyme activity under the conditions of optimum temperature.Will be not plus golden Belong to enzyme activity in the enzymatic reaction of ion and be defined as 100.Different metal ions are mapped with enzyme activity, detect different ions Influence to enzyme activity.As a result it is as shown in figure 11.

4th, influence of the different compounds to restructuring beta-glucosidase vigor

10 μ l warps are added in 80 μ l are respectively 1mM, 10mM or 100mM optimal pH buffer solution containing organic Reagents Final Concentration The crude enzyme liquid of dilution, after 4 DEG C are placed 4h, the pNPG for adding 10 μ l 50mM determines remaining enzyme activity under the conditions of optimum temperature, with The enzyme activity of the enzyme liquid treated without chemical reagent is set to 100, and different compounds are mapped with enzyme activity, and detection is different Influence of the organic compound to enzyme activity.As a result as shown in Figure 12,13.

5th, influence of the glucose to restructuring beta-glucosidase vigor

Glucose is the product of beta-glucosidase enzyme hydrolysis β-Isosorbide-5-Nitraes-glycosidic bond generation, can to the activity of beta-glucosidase Feedback inhibition or substrate facilitation can be produced, the glucose of various concentrations, measure are added in enzymatic reaction system Influence of the glucose to enzyme activity.By the crude enzyme liquid through suitably diluting respectively 0.2M, 0.5M final concentration of with glucose, 0.8M, 1.0M, 1.5M, 2.0M, 2.5M, 3.0M pH 6.10 B-R buffer solutions mix, and after being stored at room temperature 2h, determine remaining enzyme activity, It is set to 100 with the enzyme activity of the enzyme liquid without glucose.As a result it is as shown in figure 14.

Embodiment 5 recombinates research of the beta-glucosidase to Resveratrol in Rhizoma Polygoni Cuspidati glycosides hydrolysis process

(1) influence of the enzyme concentration to polydatin hydrolysis efficiency

5 100mL conical flask is taken, 2g giant knotweed powder is added, is separately added into 2mL, 4mL, 6mL, 8mL, 10mL restructuring Beta-glucosidase crude enzyme liquid, the ratio for making its giant knotweed powder and Bgl2238 is respectively 1：1、1：2、1：3、1：4、1：5.Add water To 20mL, after 120rpm shakes 12h on 44 DEG C of constant-temperature tables, the conversion ratio of polydatin is detected with HPLC.As a result such as Figure 15 It is shown；

(2) influence of the solid-liquid ratio to polydatin hydrolysis efficiency

Add the amount of 2g giant knotweeds powder and optimal restructuring beta-glucosidase in 6 100mL conical flasks, then add water respectively To 10mL, 20mL, 30mL, 50mL, 60mL, 100mL, it is respectively 1 to make solid-liquid ratio：5、1：10、1：15、1：25、1：30、1：50, After 120rpm shakes 12h on 44 DEG C of constant-temperature tables, the changing effect of polydatin is detected with HPLC.As a result it is as shown in figure 16.

(3) influence of the enzymolysis time to polydatin hydrolysis efficiency

6 be equipped with optimal enzymatic hydrolysis system conical flasks, on 44 DEG C of constant-temperature tables 120rpm hydrolyze respectively 4h, 8h, 12h, After 16h, 20h, 24h, recombinated with HPLC detections are different under beta-glucosidase action time to the changing effect of polydatin. As a result it is as shown in figure 17.

(4) influence of the temperature to polydatin hydrolysis efficiency

4 be equipped with optimal enzymatic hydrolysis system conical flasks, under conditions of optimal enzymolysis time, respectively at 37 DEG C, 44 DEG C, 50 DEG C, 120rpm concussion 12h on 55 DEG C of constant-temperature table, detected with HPLC and beta-glucosidase dialogue black false hellebore recombinated under different temperatures The changing effect of alcohol glycosides.As a result it is as shown in figure 18.

(5) influence of the rotating speed to polydatin hydrolysis efficiency

5 conical flasks that optimal enzymatic hydrolysis system is housed, under conditions of optimal enzymolysis time and optimal hydrolysis temperature, respectively Shaken with 100,120,150,200 rotating speed, detecting conversions of the Bgl2238 to polydatin under different rotating speeds with HPLC imitates Fruit.As a result it is as shown in figure 19.

3.3.9 the extraction of resveratrol and polydatin

The amount that resveratrol and polydatin only have 3% in water can be dissolved, so can not be extracted with water.But It is that they are soluble in organic solvent, as methanol, ethanol, acetone, ether, chloroform etc. may serve to extract resveratrol and white lamb's-quarters Reed alcohol glycosides, it is contemplated that practicality and the security of operating method economically, it is the most suitable to be extracted with ethanol.According to having reported Resveratrol and polydatin ethanol extraction process, the present invention using by 20mL in optimal enzymatic hydrolysis system and optimal enzymolysis Giant knotweed sample and reference substance of the condition after restructuring beta-glucosidase enzymolysis, 2h is heated to reflux with 300mL95% ethanol.With The content of polydatin and resveratrol after HPLC detection alcohol extractings.As a result as shown in Figure 20,21.

SEQUENCE LISTING

<110>Guangdong pharmaceutical university

<120>A kind of beta-glucosidase gene of new resistance to glucose and its application

<130> 2017

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 2238

<212> DNA

<213>It is unknown

<400> 1

atgaaacaca tcctaaacct atgcctgttg gccgttcttt gcgccgtcct ttcctgccag 60

gacccccgct gggggcgtat tgccgagggc agcggtgaag atacgtacct ggacacccga 120

ctgtcccgga tgaccctggc cgaaaaaata ggtcagatga accaggtctc ggccggcggc 180

gacgtgtccg ccttccagcg ctctatccgc aagggccagg tgggctctat cctcaacgag 240

gtggatccgg taaagatcaa caactatgcc gaactggccg tggaggagtc ccgtctgggg 300

attccccttc tgctgggacg cgacgtcatt ggtcagtttc atacggtttt ccccattccc 360

ctgggactgg cggccacttt tgaccccgac actttctcgc ccggggcccg tgtggcggct 420

gtggaggcca cgtcgcaggg tgtccgctgg ttccccattc ccatgctgga cattgcccgc 480

gacccccgct gggggcgtat tgccgagggc agcggtgaag atacgtacct ggacacccga 540

atggcggaag ccatggtata cggttatcag ggccgcacgg ccgattccac ctccatggca 600

gcctgcgaca agcatttcgt cggctacggt catgcggaag gcggccggga ctataacagt 660

acctacctca ccgagcgcca gttgcgcaac gtataccttc ctcctttcga ggcggcggac 720

aaggccggcg ccatgacgct gatgacgtct tttaatgaca acgacggcgt gccctccacg 780

ggcaaccatt tcgttgtgaa agacgtactg catggcgaat ggggcttcga cggcctggtg 840

gtcaccgact gggattccat gggagagatg atagcccacg gatttggcgt cgaccgcaag 900

gacgtggccg aaaaggcagc taacgccggc gtagacatgg acatgatgac tttcgacttc 960

ctctcccatc tggaagaact ggtgaagagc ggtgccgtga agcagaatac cattgacaat 1020

gccgtgcgca acatccttcg cgtgaagttc tcccatggcc tgtttgagaa cccttatgta 1080

gtagacatgg cgtcccaggc ggttcagtat gcccccgaac acctggctgc cgcccaaaag 1140

acggcggaag aatcggccat tctcctgaag aacgacggcg tactgcctct ggtagacatg 1200

tcccatatcc tggtcaccgg ccctatggcc gacgccccgc acgaccagct gggaacctgg 1260

gctttcgacg ggcagaaagc gcataccgtt actacttggg aggccttgca ggcccgtttc 1320

cccggcgccg tggactatgt ccccggactc acgtacagcc gtgagaagcg cagcggcgac 1380

tcggacgttg tggccgccgc ccgcagtgcc ttcgtagtgc tggcgttcct gggcgaggaa 1440

gccatcctct ccggagaggc tcacagtctg gccgatctta acgcccctga ttcccagagc 1500

gaattgctct cgacgctgaa gactgcgggc aagccggtgg tggccaccgt aatggaagcc 1560

cgcccgctta ccattgagcg cgacctcccc aacgtgaacg ccatgctgta tagcttccat 1620

cccggcacca tgggcggtcc ggctctggcc aacctcctct tcggcgatgt gaatccttcc 1680

ggcaagacgc ccatcacact gttccgcacc gtgggacagg cccccctgta ctacagccac 1740

aatatgacag gacgtcccta caagggagaa gctggtctgg acgacatccc ggcccagacc 1800

ggacagacct ctctgggcaa tcagacctat gctctggact atggtgccta tccggctctc 1860

cccttcggct tcggtctcag ctacacctct ctcgcttatt ccgacatcgc tctggataag 1920

gagagttatg cggcggacga tgtcctgcat gtaagtttca acctggccaa taccggcgct 1980

ctcgacggaa cggaagtggc ccaggtttat atccgtgacc tggtgggctc cgtgacccgt 2040

ccggtgaaag aactcaaggc attccggcgc gtgagcctga aggcgggtga aagccgcacc 2100

ctcacgctgg atattccggt ttctgagttg tcattctatg ggttagatat gcaaaaaaag 2160

gtagaaccgg ggcagttcca gctttgggtg cagaccgaca gctcctccgg tgaagccctt 2220

acttttagcg tacgataa 2238

<210> 2

<211> 745

<212> PRT

<213>It is unknown

<400> 2

Met Lys His Ile Leu Asn Leu Cys Leu Leu Ala Val Leu Cys Ala Val

1 5 10 15

Leu Ser Cys Gln Asp Pro Arg Trp Gly Arg Ile Ala Glu Gly Ser Gly

20 25 30

Glu Asp Thr Tyr Leu Asp Thr Arg Leu Ser Arg Met Thr Leu Ala Glu

35 40 45

Lys Ile Gly Gln Met Asn Gln Val Ser Ala Gly Gly Asp Val Ser Ala

50 55 60

Phe Gln Arg Ser Ile Arg Lys Gly Gln Val Gly Ser Ile Leu Asn Glu

65 70 75 80

Val Asp Pro Val Lys Ile Asn Asn Tyr Ala Glu Leu Ala Val Glu Glu

85 90 95

Ser Arg Leu Gly Ile Pro Leu Leu Leu Gly Arg Asp Val Ile Gly Gln

100 105 110

Phe His Thr Val Phe Pro Ile Pro Leu Gly Leu Ala Ala Thr Phe Asp

115 120 125

Pro Asp Thr Phe Ser Pro Gly Ala Arg Val Ala Ala Val Glu Ala Thr

130 135 140

Ser Gln Gly Val Arg Trp Phe Pro Ile Pro Met Leu Asp Ile Ala Arg

145 150 155 160

Asp Pro Arg Trp Gly Arg Ile Ala Glu Gly Ser Gly Glu Asp Thr Tyr

165 170 175

Leu Asp Thr Arg Met Ala Glu Ala Met Val Tyr Gly Tyr Gln Gly Arg

180 185 190

Thr Ala Asp Ser Thr Ser Met Ala Ala Cys Asp Lys His Phe Val Gly

195 200 205

Tyr Gly His Ala Glu Gly Gly Arg Asp Tyr Asn Ser Thr Tyr Leu Thr

210 215 220

Glu Arg Gln Leu Arg Asn Val Tyr Leu Pro Pro Phe Glu Ala Ala Asp

225 230 235 240

Lys Ala Gly Ala Met Thr Leu Met Thr Ser Phe Asn Asp Asn Asp Gly

245 250 255

Val Pro Ser Thr Gly Asn His Phe Val Val Lys Asp Val Leu His Gly

260 265 270

Glu Trp Gly Phe Asp Gly Leu Val Val Thr Asp Trp Asp Ser Met Gly

275 280 285

Glu Met Ile Ala His Gly Phe Gly Val Asp Arg Lys Asp Val Ala Glu

290 295 300

Lys Ala Ala Asn Ala Gly Val Asp Met Asp Met Met Thr Phe Asp Phe

305 310 315 320

Leu Ser His Leu Glu Glu Leu Val Lys Ser Gly Ala Val Lys Gln Asn

325 330 335

Thr Ile Asp Asn Ala Val Arg Asn Ile Leu Arg Val Lys Phe Ser His

340 345 350

Gly Leu Phe Glu Asn Pro Tyr Val Val Asp Met Ala Ser Gln Ala Val

355 360 365

Gln Tyr Ala Pro Glu His Leu Ala Ala Ala Gln Lys Thr Ala Glu Glu

370 375 380

Ser Ala Ile Leu Leu Lys Asn Asp Gly Val Leu Pro Leu Val Asp Met

385 390 395 400

Ser His Ile Leu Val Thr Gly Pro Met Ala Asp Ala Pro His Asp Gln

405 410 415

Leu Gly Thr Trp Ala Phe Asp Gly Gln Lys Ala His Thr Val Thr Thr

420 425 430

Trp Glu Ala Leu Gln Ala Arg Phe Pro Gly Ala Val Asp Tyr Val Pro

435 440 445

Gly Leu Thr Tyr Ser Arg Glu Lys Arg Ser Gly Asp Ser Asp Val Val

450 455 460

Ala Ala Ala Arg Ser Ala Phe Val Val Leu Ala Phe Leu Gly Glu Glu

465 470 475 480

Ala Ile Leu Ser Gly Glu Ala His Ser Leu Ala Asp Leu Asn Ala Pro

485 490 495

Asp Ser Gln Ser Glu Leu Leu Ser Thr Leu Lys Thr Ala Gly Lys Pro

500 505 510

Val Val Ala Thr Val Met Glu Ala Arg Pro Leu Thr Ile Glu Arg Asp

515 520 525

Leu Pro Asn Val Asn Ala Met Leu Tyr Ser Phe His Pro Gly Thr Met

530 535 540

Gly Gly Pro Ala Leu Ala Asn Leu Leu Phe Gly Asp Val Asn Pro Ser

545 550 555 560

Gly Lys Thr Pro Ile Thr Leu Phe Arg Thr Val Gly Gln Ala Pro Leu

565 570 575

Tyr Tyr Ser His Asn Met Thr Gly Arg Pro Tyr Lys Gly Glu Ala Gly

580 585 590

Leu Asp Asp Ile Pro Ala Gln Thr Gly Gln Thr Ser Leu Gly Asn Gln

595 600 605

Thr Tyr Ala Leu Asp Tyr Gly Ala Tyr Pro Ala Leu Pro Phe Gly Phe

610 615 620

Gly Leu Ser Tyr Thr Ser Leu Ala Tyr Ser Asp Ile Ala Leu Asp Lys

625 630 635 640

Glu Ser Tyr Ala Ala Asp Asp Val Leu His Val Ser Phe Asn Leu Ala

645 650 655

Asn Thr Gly Ala Leu Asp Gly Thr Glu Val Ala Gln Val Tyr Ile Arg

660 665 670

Asp Leu Val Gly Ser Val Thr Arg Pro Val Lys Glu Leu Lys Ala Phe

675 680 685

Arg Arg Val Ser Leu Lys Ala Gly Glu Ser Arg Thr Leu Thr Leu Asp

690 695 700

Ile Pro Val Ser Glu Leu Ser Phe Tyr Gly Leu Asp Met Gln Lys Lys

705 710 715 720

Val Glu Pro Gly Gln Phe Gln Leu Trp Val Gln Thr Asp Ser Ser Ser

725 730 735

Gly Glu Ala Leu Thr Phe Ser Val Arg

740 745

<210> 3

<211> 34

<212> DNA

<213>Manually

<400> 3

ccggaattca tgaaacacat cctaaaccta tgcc 34

<210> 4

<211> 36

<212> DNA

<213>Manually

<400> 4

ccgctcgagt tatcgtacgc taaaagtaag ggcttc 36

Claims

A kind of 1. beta-glucosidase gene of new resistance to glucose, it is characterised in that the beta-glucosidase of new resistance to glucose Enzyme gene is named as bgl2238, and its nucleotide sequence is as shown in SEQ IDNO.1：
A kind of 2. beta-glucosidase of new resistance to glucose, it is characterised in that：The beta-glucosidase of new resistance to glucose Amino acid sequence is as shown in SEQ ID NO.2：
3. a kind of preparation method of the beta-glucosidase gene of new resistance to glucose as claimed in claim 1, its feature exist In step is as follows：

(1) Heilungkiang corn soil metagenome storehouse is established, weighs Heilungkiang corn pedotheque extraction STb gene；Purify DNA； Digestion STb gene；The endonuclease bamhi that recovery obtains is connected with pUC118/EcoR I (BAP) carrier, obtains plasmid pUC118- Bgl2238, Heilungkiang corn soil metagenome text is established to the super competence of bacillus coli DH 5 alpha by the plasmid is electroporated Storehouse；

(2) positive clone molecule with black hydrolysis circle is screened from the corn soil metagenome library of Heilungkiang, is sequenced, Then gained sequence is analyzed with software, and designs primer, so as to be cloned into purpose fragment.
A kind of 4. preparation method of the recombinant plasmid of the beta-glucosidase gene comprising new resistance to glucose, it is characterised in that Step is：It is new in claim 1 according to the beta-glucosidase gene bgl2238 of new resistance to glucose primers The beta-glucosidase gene of the resistance to glucose of type is named as bgl2238, using the plasmid pUC118-Bgl2238 of extraction as template, enters Performing PCR expands, and PCR primer is through gel electrophoresis and recovery product 1, gel recovery product 1, through restriction enzyme Xho I, EcoR I double digestions and recovery product 2, plasmid pET32a are connected after Xho I, EcoR I double digestions with recovery product 2, obtain connection production Thing, i.e. recombinant plasmid pET32a-Bgl2238；Wherein described primers sequence is as follows：

bgl2238-fw：5’-CCGGAATTCATGAAACACATCCTAAACCTATGCC-3’

(underscore part is EcoR I restriction enzyme sites)

bgl2238-rv：5’-CCGCTCGAGTTATCGTACGCTAAAAGTAAGGGCTTC-3’

(underscore part is Xho I restriction enzyme sites).
5. a kind of expression vector of the beta-glucosidase gene containing new resistance to glucose as claimed in claim 1, it is special Sign is, builds the expression vector of the beta-glucosidase gene of new resistance to glucose with the following method：, will using electric robin Recombinant plasmid pET32a-Bgl2238 is transformed into e. coli bl21 competent cell, and the bacterial suspension after conversion is recovered Cultivate and dilution spread is cultivated on amicillin resistance culture plate, picking positive transformant, obtain new resistance to glucose The expression vector of beta-glucosidase gene is ETEC BL21-pET32a-Bgl2238.
6. a kind of preparation method for recombinating beta-glucosidase, it is characterized in that：Step is as follows：Recombinant plasmid pET32a-Bgl2238 It is e. coli bl21 competent cell to convert host cell；Transformant is cultivated, restructuring beta-glucosidase is obtained from culture Enzyme.
A kind of 7. preparation method for recombinating beta-glucosidase according to claim 6, it is characterised in that detailed process For：The recombinant plasmid pET32a-Bgl2238 Escherichia coli converted are coated on the flat board containing amicillin resistance, 37 DEG C Be inverted overnight incubation, picking monoclonal is inoculated in LB fluid nutrient mediums, 37 DEG C, 180rpm shaken cultivations stay overnight, by volume 1： 100 ratio is forwarded to the fresh culture mediums of LB containing amicillin resistance, is cultivated in 37 DEG C, 180rpm to OD₆₀₀=0.8, add Enter IPTG Fiber differentiations, obtain solution expression with high efficiency；The Ni-NTA Agerose nickel posts through Novagen companies are affine after culture Post purifies to restructuring beta-glucosidase, produces restructuring beta-glucosidase.
8. a kind of preparation method for recombinating beta-glucosidase according to claim 7, it is characterised in that step is as follows： Final concentration of 1.0~the 1.4mM of IPTG, inducing temperature are 25~37 DEG C, induction time 11h.
9. recombinate application of the beta-glucosidase in Resveratrol in Rhizoma Polygoni Cuspidati glycosides hydrolysis process, it is characterised in that take giant knotweed powder, press 2g giant knotweeds powder recombinates ratio of the beta-glucosidase crude enzyme liquid than 10-100mL water than 2-10mL, and β-Portugal is first added into giant knotweed powder Polyglycoside enzyme crude enzyme liquid, is added water, and is then placed within constant-temperature table and is shaken enzymolysis, and HPLC detects the conversion of polydatin Rate；Temperature is 37 DEG C -55 DEG C, and rotating speed is 100-200rpm, and enzymolysis duration is 4-24h.
10. the application according to claim 9 for recombinating beta-glucosidase in Resveratrol in Rhizoma Polygoni Cuspidati glycosides hydrolysis process, its It is characterised by, 2g giant knotweeds powder recombinates beta-glucosidase crude enzyme liquid than 100mL water than 4mL during enzymolysis, and solid-liquid ratio is 1：50, enzymolysis Time is 12h, and temperature is 44 DEG C, and rotating speed is 120rpm.