CN102220302B - Beta-glucosidase mutant, recombined expression plasmid and converted engineering strain - Google Patents
Beta-glucosidase mutant, recombined expression plasmid and converted engineering strain Download PDFInfo
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Abstract
The invention relates to a beta-glucosidase mutant, a recombined expression plasmid and a converted engineering strain. In the invention, the gene, the mutant plasmid and the engineering strain of beta-glucosidase with improved glucose tolerant concentration by a method of gene site-directed mutagenesis, and after engineering strain inducible expression, the beta-glucosidase with the improved glucose tolerant concentration can be obtained. In the invention, beta-glucosidase from ocean uncultured microorganism sources is taken as the foundation, and through PCR (Polymerase Chain Reaction) site-directed mutagenesis, the mutant gene can be obtained. Compared with the wild beta-glucosidase protein, the glucose tolerant concentration of the obtained mutain is improved by thirteen times, in addition, other zymologic properties of the mutain are not changed, and the mutain has an industrial value, can improve the fermentation process of methyl cellulose, and can be used for producing fruit juice with concentration of 20-30%.
Description
Technical field
The present invention relates to biological technical field, specifically, the method that relates to by site-directed point mutation obtains beta-glucosidase gene, mutant plasmid, engineering bacteria and the mutant enzyme that glucose tolerance concentration improves.
Background technology
Beta-glucosidase (EC 3.2.1.21) belongs to circumscribed hydrolase, it is a class shifts glucosides in the intermolecular catalysis of nucleophilic enzyme, 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 is a class important industrial enzymes, and potential being widely used has important use and be worth in numerous biotechnology production processes.It is food flavor enzyme important in the foodstuffs industry, and flavour precursors thing---the β-glucosides in can hydrolysis fruit discharges the volatility aglycone, fills the post of the role of fruit-like flavour flavouring enzyme; Beta-glucosidase can be for the synthesis of new carbohydrate ligands with the activity of FscM; Beta-glucosidase can act synergistically with other several cellulose degrading enzymes, and cellulose degradation is become glucose, and the latter can be that the production capacity microorganism utilizes, and then refining generates alcohol fuel equal energy source material.
Generally speaking, beta-glucosidase is responsive unusually to its hydrolysate glucose, the activity that the glucose of extremely low concentration just can the feedback inhibition beta-glucosidase.Beta-glucosidase is to the inhibition constant K of glucose
iBe generally 0.5mM-100mM (Watanabe T, Sato S, Yoshioka T, et al.Purification and properties of Aspergillus niger β-glucosidase.J.Biochem.1992,209:651-659; Woodward J and Wiseman A.Fungal and other β-D-glucosidases:their properties and applications.Enzyme Microb.Technol.1982,4:73-79.), only there is the beta-glucosidase of only a few to have higher glucose tolerance concentration (Christine R, Jean-Michel S, Marie-Jose V, et al.Purification, characterization, and subatrate specificity of a novel highly glucose-tolerant β-glucosidase from Aspergillus niger.Applied and Environmental Microbiology.1998,64 (10): 3607-3614.).In beta-glucosidase industrial application process, it is found that the speed of the beta-glucosidase degraded cellulose of non-glucose-sensitive is faster, can improve the zymotechnique of lignocellulose; And, utilize the beta-glucosidase of glucose tolerance, can produce 20-30% strength juice (Waldron CR, Becker-Vallone CA, and Eveleigh DE.Isolation and characterization of a cellulolytic actinomycete Microbispora bispora.Appl.Microbiol.Biotechnol.1986,24:477-486.).
Summary of the invention
Primary and foremost purpose of the present invention is to provide the beta-glucosidase mutant of the selective mutation that a kind of method of using rite-directed mutagenesis obtains.
Second purpose of the present invention is to provide dna sequence dna and the mutant plasmid thereof of a kind of coding as above-mentioned beta-glucosidase mutant.
The beta-glucosidase that the 3rd purpose of the present invention is to provide a kind of conversion that above-mentioned recombinant expression plasmid is arranged is produced bacterial strain.
The 4th purpose of the present invention is to provide a kind of mutant that utilizes above-mentioned beta-glucosidase to produce the beta-glucosidase of bacterial strain production.
Beta-glucosidase mutant of the present invention, aminoacid sequence is as described in following any one:
A), the 184th Histidine sports phenylalanine in the aminoacid sequence of the beta-glucosidase shown in the SEQ ID No.1;
B), shown in the SEQ ID No.1 shown in the aminoacid sequence of beta-glucosidase in the 409th leucine sport L-glutamic acid.
C), the 184th Histidine sports phenylalanine in the aminoacid sequence of the beta-glucosidase shown in the SEQ ID No.1, the 409th leucine sports L-glutamic acid.
A kind of beta-glucosidase mutant of the present invention, the aminoacid sequence of described mutant can also comprise nonsense mutation in the sequence or the combination of same sense mutation.
The invention provides a kind of mutant plasmid, comprise the dna sequence dna that above-mentioned coding has described beta-glucosidase mutant.
The present invention also provides a kind of engineering bacteria with beta-glucoside enzymic synthesis performance, and described engineering bacteria contains mutant plasmid recited above, and described beta-glucosidase mutant is obtained by engineering bacterium fermentation.
Introduce the acquisition of beta-glucosidase mutant of the present invention down below
At first with from the structure of the beta-glucosidase of bacillus polymyxa (Paenibacillus polymyxa) and glucose mixture as template, utilize the not beta-glucosidase (Bgl1A) in culturing micro-organisms source of Swiss-Model modeling ocean, and pass through the structure that stack obtains Bgl1A and glucose mixture, thereby obtain corresponding amino-acid residue site of being combined with glucose.
According to the ocean gene order of the beta-glucosidase (Bgl1A) in culturing micro-organisms source not, design and synthesize mutant primer, again with comprise the ocean not the recombinant plasmid of the beta-glucosidase gene in culturing micro-organisms source be template plasmid, be primer with above-mentioned synthetic mutant primer, the method of PCR-based is carried out rite-directed mutagenesis, thus the mutator gene of the beta-glucosidase that the acquisition glucose tolerance can improve.
Mutant DNA is connected with the pMD18-T plasmid and transformed into escherichia coli DH5 α competent cell, and the picking positive colony carries out determined dna sequence to mutator gene.Select the correct clone of sequence and extract plasmid, obtain to contain the pMD18-T recombinant plasmid of the beta-glucosidase gene that suddenlys change, with pMD18-T recombinant plasmid and the expression plasmid carrier of Nde I and Xho I double digestion gained, use T then
4Mutator gene after dna ligase is cut enzyme is connected with the expression plasmid carrier, obtains connecting product; Connect product and transform the host bacterium, screening positive clone obtains containing the engineering strain of mutator gene of the present invention.
The expression plasmid carrier refers to pCold, pET15, pET22 or pET28 etc. described in the above-mentioned construction process.
Host bacterium described in the above-mentioned construction process refers to E.coli BL21 (DE3), E.coli DH5 α, E.coli JM109 or E.coli Rosetta etc.
The engineering bacteria that contains mutator gene that obtains is carried out fermentation culture, and purifying obtains beta-glucosidase mutant enzyme albumen.
Glucose tolerance energy, optimal pH, pH stability, optimum temperuture and the temperature stability etc. of mutant enzyme albumen and original wild-type protein are measured and compared.
Measurement result shows, the relative wild-type Bgl1A of glucose tolerance concentration of the mutant enzyme that the present invention obtains has improved 13 times, and other zymologic properties such as optimal pH, optimum temperuture etc. do not change, and other zymologic properties do not change, has a using value industrial, it can improve the zymotechnique of lignocellulose, and can produce the strength juice up to 20-30%.
Description of drawings
Fig. 1 is the electrophoretogram of pcr amplification product of the present invention;
1 is the 409S2 fragment of pcr amplification among Fig. 1, and 2 is the 409S1 fragment, and 3 is the 184S2 fragment, and 4 is the 184S1 fragment, and M is DNA marker.
Fig. 2 is combined as the electrophoretogram of template pcr amplification product respectively with 409S1 and 409S2,184S1 and 184S2 for the present invention.
1 for 184S1 and 184S2 are combined as the template pcr amplification product among Fig. 2, and 2 for 409S1 and 409S2 are combined as the template pcr amplification product, and M is DNA marker.
Fig. 3 is the mutain of purifying of the present invention and the electrophoretogram of wild-type protein
1 is the H184F mutain among Fig. 3, and 2 is the L409E mutain, and 3 is wild-type rBgl1A, and M is albumen marker.
Fig. 4 can compare for the glucose tolerance of mutain of the present invention and wild-type protein.
Fig. 5 is that mutain of the present invention and wild-type protein part zymologic property compare.
Wherein Fig. 5 A is that optimal pH compares, and Fig. 5 B is that optimum temperuture compares, and Fig. 5 C is that pH stability compares, and Fig. 5 D is that temperature stability compares.
Embodiment
Implementation method in the following example if no special instructions, is ordinary method.
(1), the structure that contains the expression strain of beta-glucoside enzyme mutant gene of the present invention
1, the selection in beta-glucosidase gene mutational site
With from the structure of the beta-glucosidase of bacillus polymyxa (Paenibacillus polymyxa) and glucose mixture as template, utilize Swiss-Model (
Http:// swissmodel.expasy.org/Kiefer F, Arnold K, K ü nzli M, Bordoli L, Schwede T.The SWIS S-MODEL Repository and associated resources.Nucleic Acids Research.2009,37, D387-392.) the modeling ocean beta-glucosidase (Bgl1A) in culturing micro-organisms source not, and obtain the structure of Bgl1A and glucose mixture by stack.
According to the structural information of modeling, the site of determining rite-directed mutagenesis is the Histidine H in 184 sites and the leucine L in 409 sites, and the sudden change direction is that the Histidine H in 184 sites is substituted by phenylalanine F, and the leucine L in 409 sites is substituted by L-glutamic acid E.
2, the pcr amplification of the design of mutant primer and mutator gene
According to the ocean gene order of the beta-glucosidase in culturing micro-organisms source: SEQ ID No:2 (its aminoacid sequence such as SEQ ID No:1) not, and selected mutational site 184H and 409L, design following 6 rite-directed mutagenesis primers:
With comprise the ocean not the recombinant plasmid of beta-glucosidase (Bgl1A) gene in culturing micro-organisms source be template plasmid, above-mentioned synthetic mutant primer is matched, be respectively: bgl1AS and 184A, 184S and bgl1AA, bgl1AS and 409A, 409S and bgl1AA, right as the PCR primer, amplified production is called after 184S1,184S2,409S1,409S2 successively; Reclaim amplified fragments in 4,184S1 and 184S2 are added in the same reaction system, 409S1 and 409S2 add in another reaction system, as the PCR reaction template in the reaction system separately, and utilizing bgl1AS, bgl1AA primer to as amplimer, amplification bgl1B mutator gene full length sequence also reclaims standby.
3, the structure of expression vector
To obtain pcr amplification product in the step 2, be connected (TaKaRa) with the pMD18-T plasmid, set up following enzyme and cut system: 25ng pMD18-T carrier, the 50ng pcr amplification product, 5ul ligation mix, moisturizing is to 10ul, and 16 ℃ connect 1h.Connect product heat shock transformed into escherichia coli DH5 α competent cell, the transformant that obtains, whether sequence verification suddenlys change; Select the correct clone of sequence and extract plasmid, obtain to contain the pMD18-T recombinant plasmid of beta-glucoside enzyme mutant gene of the present invention; With pMD18-T recombinant plasmid and pET22b (+) carrier of Nde I and Xho I double digestion gained, use T then
4Mutator gene after dna ligase is cut enzyme is connected with the expression plasmid carrier, sets up following enzyme and cuts system: 25ng pET22b (+) carrier, 50ng mutator gene endonuclease bamhi, 3ul 10 * ligation buffer, 1ul T
4Dna ligase (TaKaRa), moisturizing are to 30ul, and 16 ℃ connect 8h, obtain connecting product; Connect product and transform the host bacterium, whether the transformant sequence verification that obtains suddenlys change, and selects engineering strain E.coli BL21 (DE3)/pET22b (+)-bgl1A/H184 and E.coli BL21 (DE3)/pET22b (+)-bgl1A/L409E that the correct transformant of sequence obtains containing mutator gene of the present invention.
Bacterial strain e. coli bl21 of the present invention (DE3)/pET22b (+)-bgl1A/H184F Escherichia coli BL21 (DE3)/pET22b (+)-bgl1A/H184F has been sent to Chinese typical culture collection center (China Center for Type Culture Collection, CCTCC) preservation, depositary institution address: Chinese Wuhan Wuhan University; The bacterial strain deposit number is CCTCC NO:M 2010159, preservation date: on June 25th, 2010.
(2), the expression and the protein purification that contain beta-glucoside enzyme mutant gene engineering bacteria of the present invention
Engineering strain E.coli BL21 (DE3)/pET22b (+)-bgl1A/H184F and E.coli BL21 (DE3)/pET22b (+)-bgl1A/L409E of gene of obtaining in (one) are inoculated in the 200ml LB liquid nutrient medium that contains penbritin, are positioned under 37 ℃, 250rpm condition and are cultured to OD
600=0.6 (UNICO UV2102 ultraviolet-visible pectrophotometer is blank to cultivate the LB substratum); The IPTG that adds final concentration and be 0.75mM induces, and continues to cultivate 5 hours under 30 ℃, 180rpm condition; 4 ℃, the centrifugal collection thalline of 8000g add the long-pending Binding buffer of 0.1 times of bacteria liquid, ultrasonic 40min smudge cells under the 350W condition of ice bath, and the centrifugal collection supernatant of 30000g obtains crude enzyme liquid.Whole bacterial protein SDS-PAGE shows that expressing quantity accounts for more than 50% of whole bacterial protein.
Crude enzyme liquid carries out purifying through the Ni-NTA column chromatography.Imidazole concentration is 60mM in the elutriant, 10 column volumes of wash-out.The albumen that obtains reaches SDS-PAGE purity through detecting.
Be substrate with pNPG, the optimal pH of the beta-glucosidase mutain of purifying is 6.5, and enzyme can show the enzyme activity more than 80% in the pH5.5-7.5 scope.Enzyme has advantages of higher stability in the pH6.0-9.0 scope, insulation 1h still can keep more than 60% of former enzyme activity under 30 ℃ of conditions.Mutain all can show catalytic activity in 15 ℃ of-55 ℃ of scopes, its optimum temperuture is 40 ℃.
Relative wild-type beta-glucosidase albumen, the glucose tolerance concentration of the mutain of acquisition has improved 13 times, the K that glucose suppresses bgl1A/H184E
iBe 400mM (Fig. 4), and other zymologic properties not do not change, have a using value industrial.
Claims (5)
1. beta-glucosidase mutant is characterized in that: the aminoacid sequence of described mutant is as described in following any one:
A), the 184th Histidine sports phenylalanine in the aminoacid sequence of the beta-glucosidase shown in the SEQ ID No. 1;
B), the 409th leucine sports L-glutamic acid in the aminoacid sequence of the beta-glucosidase shown in the SEQ ID No. 1.
2. the encode dna sequence dna of a kind of beta-glucosidase mutant as claimed in claim 1.
3. a mutant plasmid is characterized in that, contains the dna sequence dna of the described beta-glucosidase mutant of claim 2.
4. the engineering bacteria with beta-glucoside enzymic synthesis performance is characterized in that, contains the described mutant plasmid of claim 3.
5. a beta-glucosidase is characterized in that, is obtained by the described engineering bacterium fermentation of claim 4.
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| CN102517307B (en) * | 2011-12-29 | 2013-01-30 | 山东大学 | Beta-glucosidase Mut1b as well as expressed gene and application thereof |
| CN103160483B (en) * | 2013-04-11 | 2015-03-18 | 山东大学 | Beta-glucosidase, as well as expression gene and application thereof |
| CN103602646B (en) * | 2013-11-20 | 2015-08-26 | 菏泽学院 | The beta-glucoside enzyme mutant that a kind of optimal reactive temperature improves and application thereof |
| CN104232606B (en) * | 2014-08-29 | 2017-12-05 | 山东大学 | The β glucuroides and its expressing gene of a kind of transformation and application |
| CN104560917B (en) * | 2014-10-30 | 2018-12-18 | 中国科学院天津工业生物技术研究所 | A kind of beta-glucosidase and β-glucosidase mutant and application |
| CN105039285A (en) * | 2014-11-16 | 2015-11-11 | 安徽大学 | Expression and application of high-sugar-resistant alkali-resistant beta-glucosidase |
| CN104726432B (en) * | 2014-12-22 | 2017-12-22 | 江苏大学 | A kind of D types β glucosides enzyme mutant and its expression plasmid and recombinant bacterium |
| CN104726435B (en) * | 2015-04-13 | 2018-05-29 | 安徽大学 | β -glucosidase mutant, recombinant expression plasmid thereof and transformed engineering strain |
| CN105524903B (en) * | 2016-01-28 | 2019-03-12 | 中国农业科学院饲料研究所 | A kind of beta-glucosidase improvement mutant E168Q and its encoding gene and application |
| CN105524902B (en) * | 2016-01-28 | 2019-03-12 | 中国农业科学院饲料研究所 | A kind of high catalytic efficiency β-glucosidase mutants M36E and its encoding gene and application |
| CN110438136B (en) * | 2019-08-30 | 2020-10-30 | 中国水产科学研究院黄海水产研究所 | Beta-glucosidase and mutant gene, amino acid sequence and application thereof |
| CN114426961B (en) * | 2022-03-10 | 2024-01-26 | 安徽大学 | Beta-glucosidase mutant, encoding gene, expression strain and application thereof |
| CN115896071B (en) * | 2022-09-07 | 2024-07-02 | 中国科学院青岛生物能源与过程研究所 | Beta-glucosidase mutant with high enzyme activity and high glucose tolerance and application thereof |
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| Fang,Z.M..ACCESSION No. ACY09072.《Gene Bank》.2009,全文. * |
| 房伟 等.新型海洋微生物β-葡萄糖苷酶基因的克隆、表达及重组酶性质.《生物工程学报》.2009,第25卷(第12期),第1914页-1920页. * |
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