CN102827820B - Beta-glucosidase and application thereof - Google Patents
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a beta-glucosidase and application thereof, comprising a) a beta-glucosidase with a sequence of SEQ ID NO: 1; ) An enzyme derived from a) wherein the amino acid sequence in a) is substituted, deleted or added with one or several amino acids and has the activity of the enzyme described in a. The beta-glucosidase provided by the invention has the resistance of deoxyglucose and hygromycin and also has the transglycosylation function, under the transglycosylation function, oligosaccharides such as cellobiose and the like can generate inducers such as sophorose and the like, and then enter cells through a constitutive permease system on cell membranes to start the synthesis of cellulase in the cells. Therefore, the production of the cellulase can be induced by catalyzing and synthesizing the sophorose by using the beta-glucosidase, and the method has great commercial value.
Description
Technical field
The invention belongs to enzyme gene and technical field of enzyme engineering, be specifically related to a kind of beta-glucosidase enzyme and application thereof.
Background technology
The English name of beta-glucosidase enzyme (EC 3.2.1.21) is β-glucosidase, claims β-D-glucoside lytic enzyme again, another name gentiobiase, cellobiase and amygdalase.Belong to circumscribed hydrolase, being a class shifts the enzyme of glucosides in the intermolecular catalysis of nucleophilic, can be in oligosaccharides or the cellobiose inside of short chain, having between the residue of carbohydrate of aromatic group or alkyl and interrupting β-1, the 4-glycosidic link.Beta-glucosidase enzyme is a kind of in the cellulase system, is the rate-limiting enzyme of cellulose degradation, in the cellulase hydrolysis process various oligosaccharides and cellobiose is degraded to glucose, is last key enzyme, and hydrolysis has key effect to saccharification of cellulose.Beta-glucosidase enzyme also has transglycosylation, under certain conditions can be by transglycosylation with the synthetic sophorose molecule of two glucose molecules.Found that sophorose is the strong inductor that cellulose enzyme gene is expressed.It is generally acknowledged that the synthetic abduction mechanism of cellulase in the filamentous fungus is: the small number of groups that is present in conidium and the mycelia surface cellulase that becomes second nature, at first degraded cellulose generates oligosaccharides such as cellobiose, then under the special glycosides effect of the glucuroide of plasma membrane combination, generate inductors such as sophorose, composition permease system on cytolemma enters in the cell, starts the synthetic of cellulase.Utilizing beta-glucosidase enzyme to catalyze and synthesize sophorose induces the production of cellulase to have huge commercial value and realistic meaning.
Beta-glucosidase enzyme extensively is present in plant, animal and microorganism.In microorganism, aspergillus is generally believed it is the excellent species that produces beta-glucosidase enzyme, and is wherein the highest with aspergillus niger and wooden mould output especially.But beta-glucosidase enzyme liquid state fermentation enzyme is lived generally on the low side at present; Can make complicated component and produce beta-glucosidase enzyme by solid state fermentation, should not extract.So adopt genetic engineering technique to make up the engineering strain of high yield beta-glucosidase enzyme, have important practical significance.
Paper shape Stachybotrys atra has the fibering of having a liking for, and its that report cellulase-producing has wideer pH adaptability, and for alkaline condition tolerance is preferably arranged also.Therefore, the novel beta-glucosidase enzyme gene of clone from paper shape Stachybotrys atra, except can studying its enzymatic property, the in-vitro directed transformation that also can be research glycoside hydrolysis enzyme family feature and enzyme provides theoretic guidance.
Summary of the invention
The purpose of this invention is to provide a kind of beta-glucosidase enzyme and application thereof, i.e. a kind of novel beta-glucosidase enzyme with industrial application value, thus remedy the deficiencies in the prior art.
The gene of the beta-glucosidase enzyme that the present invention obtains includes the DNA of 2508bp, the protein of being made up of 817 amino acid of encoding.
Beta-glucosidase enzyme of the present invention includes:
A) sequence is the enzyme of SEQ ID NO:1;
B) aminoacid sequence in a) is through replacing, lack or add one or several amino acid and have the activity of enzyme described in a, the enzyme of being derived by a.
The encode Nucleotide of above-mentioned beta-glucosidase enzyme, its sequence is SEQ ID NO:2.
The present invention also is provided for expressing the recombinant expression plasmid of above-mentioned beta-glucosidase enzyme, is to be that the nucleotide fragments of SEQ ID NO:2 is inserted in the prokaryotic expression carrier and makes up with sequence.
Described prokaryotic expression carrier is pGm.
The invention still further relates to the reorganization bacterium that carries the recombinant expression plasmid of expressing above-mentioned xylosidase.
Beta-glucosidase enzyme of the present invention is used for the production of alcohol fuel equal energy source material, the food flavor enzyme of foodstuffs industry, the production of feed etc.
Beta-glucosidase provided by the invention, when fermentation proceeded to the 5th day, the fermentation broth enzyme amount reached maximum value 5313.82U/ml; Its optimum temperuture is 50 ℃, and optimal pH is 5.8.And this enzyme also has very high xylosidase activity, is 58.20U/ml.In addition, beta-glucosidase of the present invention also has deoxyglucose and hygromycin resistance and transglycosylation.Under transglycosylation, can make inductors such as oligosaccharides generation sophorose such as cellobiose, the composition permease system on cytolemma enters in the cell again, starts the synthetic of cell inner cellulose enzyme.So can utilize beta-glucosidase to catalyze and synthesize the production that sophorose is induced cellulase, have more huge commercial value.
Description of drawings
Fig. 1: beta-glucosidase enzyme gene DNA electrophoretogram of the present invention;
Fig. 2: the design of graphics of plasmid pGm-g10158 of the present invention;
Fig. 3: the relative vigor of Polyglucosidase of the present invention under differing temps;
Fig. 4: the relative vigor of Polyglucosidase of the present invention under different pH values;
Fig. 5: the thermostability of Polyglucosidase of the present invention under differing temps.
Embodiment
Below in conjunction with drawings and Examples, describe in further detail of the present invention.Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.
One, the screening of beta-glucosidase enzyme and detection
1, paper shape Stachybotrys atra bacterium (in Chinese microorganism strain board of trustees common micro-organisms, CGMCC 3.5365) total DNA extraction
(1) with paper shape Stachybotrys atra bacterium CMC liquid nutrient medium, shaking table was cultivated 48 hours under 30 ℃, 200rpm condition;
(2) with sterile gauze filter mycelium, mycelium is clayed into power in liquid nitrogen.
(3) extracting method reference reagent box (Fungal DNA kit).
2, the amplification of beta-glucosidase enzyme gene
The beta-glucosidase enzyme gene order that obtains according to the full genome analysis of paper shape Stachybotrys atra bacterium is the nucleotide sequence shown in the SEQ NO.1, designs a pair of Oligonucleolide primers P1 and P2, and its sequence is:
P1:5′-CCATTACGTAAGAATGCGGACCTCACTGTCAGTCG-3′
P2:5′-CTAGTCTAGACTAAGCCTGCACGGGCTGAT-3′
The genomic dna that extracts with embodiment 1 is masterplate, amplification beta-glucosidase enzyme gene.Response procedures is as follows: 95 ℃ of fs sex change, 3min; 94 ℃ of subordinate phase sex change, 30s anneals 58 ℃, and 30s extends 68 ℃ of 3min, carries out 25 circulations altogether; Phase III is extended 72 ℃, 10min.The PCR product that obtains detects with agarose gel electrophoresis, the results are shown in Figure 1.
3, the structure of expression vector
The pcr amplification product and the pGm plasmid (having ethanamide and ammonia benzyl selective marker) that obtain with SnaB1 and Xba1 double digestion embodiment 2, after glue reclaims, g10158 after with the T4 ligase enzyme enzyme being cut then and pGm plasmid place 16 ℃ to spend the night and be connected, and obtain connecting product; To connect product transformed into escherichia coli DH5 α (Trans5 α Chemically Competent Cell, Beijing Quanshijin Biotechnology Co., Ltd) and experience too cell, screening positive clone carries out sequential analysis; Select the correct clone of sequence and extract plasmid, obtain to contain the recombinant plasmid of beta-glucosidase enzyme gene (g10158).Sequencing result shows that the nucleotides sequence of the beta-glucosidase enzyme that filters out classifies SEQ ID NO:2 as, and the aminoacid sequence that translates is SEQ ID NO:2.
4, conversion aspergillus niger and recombinant screen are firm
(1) with aspergillus niger G1 spore CMC liquid nutrient medium, 30 ℃ of overnight incubation.
(2) thalline that obtains in (1) is filtered with 6 layers of sterile gauze, and wash with SolutionA
(3) mycelium that obtains in (2) is put into the aseptic triangular flask that is added with 0.6g Lysing Enzymes and 40ml Solution B, mixing, 30 ℃, 150rpm after 1 hour, changes rotating speed into 80rpm, 1 hour.
(4) filter with two-layer aseptic magical filter cloth then, installed in the aseptic centrifuge tube of 50ml filtrate branch, add Solution B to 45ml, 4000rpm, centrifugal 10min.Abandon supernatant; Add 20ml Solution B mixing, 4000rpm, centrifugal 5min abandons supernatant; Add 20ml Solution B mixing again, 4000rpm, centrifugal 5min abandons supernatant.
(5) add 100ul Solution B mixing, add the recombinant plasmid that 10ul embodiment 3 obtains; Add 12.5ul Solution C mixing again, leave standstill 20min on ice.
(6) add 2ml Solution B, 1ml Solution C and 8ml adms upper strata substratum, mixing is poured in 3 adms lower floor culture medium flat plates, and 30 ℃ of thermostat containers are cultivated.After 3-4 days, observation has or not transformant to grow.
(7) with in the transformant switching adms secondary checking culture medium flat plate that grows, 30 ℃ of thermostat containers are cultivated, and carry out bacterium colony PCR checking, obtain positive strain.
5, the expression of the cultivation of recombinant bacterial strain and beta-glucosidase enzyme
The positive strain that obtains is inoculated in the Promosoy Special medium liquid nutrient medium, cultivates in 30 ℃ of 200rpm shaking tables.
Two, the determination of activity of beta-glucosidase enzyme
Be the activity that substrate is measured beta-glucosidase with right-nitrophenols-β-D-glucoside (pNPG), with per minute catalysis pNPG generate 1 micromole right-the required enzyme amount of nitrophenols (p-nitrophenol) is the enzyme unit (IU) that lives.
1, beta-glucosidase enzyme optimal reactive temperature, pH and thermal stability determination
1). measure the ratio vigor of Polyglucosidase of the present invention under differing temps:
Reaction system is: the crude enzyme liquid 50ul that adds suitable weaker concn in the 100ul damping fluid adds 5mMpNPG 50ul again.Mixing places 30-80 ℃ of water-bath respectively, accurately timing 15min; Reaction adds 1M sodium carbonate solution 100ul after finishing rapidly, exactly, shakes up, and measures light absorption value then under 410nm.Measurement result is referring to Fig. 3.As can be seen from the figure, the optimal reactive temperature of xylosidase is 50 ℃
2, measure the ratio vigor of Polyglucosidase of the present invention under different pH:
Reaction system is identical with the ratio vigor of mensuration Polyglucosidase of the present invention under differing temps, and the pH of damping fluid is respectively 2.4-7.8, and temperature of reaction is 50 ℃.Measurement result is referring to Fig. 4.As can be seen from the figure, the optimal reaction pH of Polyglucosidase is 4.8
3, measure the thermostability of Polyglucosidase of the present invention
Certain density crude enzyme liquid is put into 40,50 and 60 ℃ of water-bath temperature bathe, took out a part of enzyme every one hour and do enzyme reaction alive.
Reaction system is: the crude enzyme liquid 50ul that adds suitable weaker concn in the 100ul damping fluid adds 5mMpNPG 50ul again.Mixing places 50 ℃ of water-baths, accurately timing 15min; Reaction adds 1M sodium carbonate solution 100ul after finishing rapidly, exactly, shakes up, and measures light absorption value then under 410nm.Measurement result is referring to Fig. 5.
Three, the application of beta-glucosidase enzyme
In the Erlenmeyer flask of 50ml, add 30ml, the crude enzyme liquid of the glucose solution of 400mg/ml, 3ml beta-glucosidase of the present invention, at 50 ℃, 4.5 times reactions of pH 100h, the heating termination reaction, the compound liquid glucose of prepared one-tenth can be used for inducing cellulase production.
Dress liquid 50ml in the 250ml Erlenmeyer flask, inoculation 10% at 30 ℃, is cultivated under the 180r/min, measures filter paper enzyme activity and reducing sugar content in the fermented liquid.The result is presented at and utilizes the mixture of glucuroide preparation to compare with the contrast that with glucose is carbon source for the product enzyme result under the situation of carbon source, and cellulase activity has improved 20 times, has shown favorable industrial application prospect.
Claims (6)
1. a beta-glucosidase enzyme is characterized in that, the aminoacid sequence of described beta-glucosidase enzyme is shown in SEQ ID NO:1.
2. a Nucleotide is characterized in that, described Nucleotide is used for the described beta-glucosidase enzyme of coding claim 1.
3. Nucleotide as claimed in claim 2, the sequence that it is characterized in that described Nucleotide is SEQ ID NO:2.
4. a recombinant expression plasmid of be used for expressing the described beta-glucosidase enzyme of claim 1 is characterized in that, described recombinant expression plasmid is to be that the nucleotide fragments of SEQ ID NO:2 is inserted in the prokaryotic expression carrier and makes up with sequence.
5. a reorganization bacterium is characterized in that described reorganization bacterium carries the described recombinant expression plasmid of claim 4.
6. the application of the described beta-glucosidase enzyme of claim 1 in energy substance production, food flavor enzyme and fodder production.
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| US10066237B2 (en) | 2011-11-22 | 2018-09-04 | Novozymes Inc. | Polypeptides having beta-xylosidase activity and polynucleotides encoding same |
| DK2782998T3 (en) | 2011-11-22 | 2018-04-16 | Novozymes Inc | POLYPEPTIDES WITH BETA-XYLOSIDASE ACTIVITY AND POLYNUCLEOTIDES CODING THEM |
| CN104145016B (en) * | 2011-11-22 | 2018-08-21 | 诺维信股份有限公司 | Polynucleotides with the active polypeptide of xylobiase and the coding polypeptide |
| CN103695393B (en) * | 2013-12-17 | 2015-07-15 | 宁夏夏盛实业集团有限公司 | Method for producing cellulase by using beta-glucosidase and application of cellulase |
| CN107099565B (en) * | 2017-06-23 | 2020-09-04 | 江南大学 | Preparation method of gentiooligosaccharide |
| CN111690629B (en) * | 2020-05-29 | 2022-04-19 | 浙江工业大学 | Endoglucanase mutant, gene, engineering bacterium and application thereof |
| CN112553181B (en) * | 2020-12-29 | 2022-09-02 | 宜昌东阳光生化制药有限公司 | Production method of glucoamylase |
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| US9441214B2 (en) * | 2010-06-29 | 2016-09-13 | Dsm Ip Assets B.V. | Polypeptide having beta-glucosidase activity and uses thereof |
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