CN105039285A - Expression and application of high-sugar-resistant alkali-resistant beta-glucosidase - Google Patents
Expression and application of high-sugar-resistant alkali-resistant beta-glucosidase Download PDFInfo
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- IFBHRQDFSNCLOZ-UHFFFAOYSA-N 2-(hydroxymethyl)-6-(4-nitrophenoxy)oxane-3,4,5-triol Chemical compound OC1C(O)C(O)C(CO)OC1OC1=CC=C([N+]([O-])=O)C=C1 IFBHRQDFSNCLOZ-UHFFFAOYSA-N 0.000 description 3
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a beta-glucosidase glA2, the gene sequence of which is shown as SEQ ID NO: 1 is shown in the specification; the amino acid sequence coded by the gene sequence is shown as SEQIDNO: 2, respectively. Compared with the existing beta-glucosidase, the beta-glucosidase provided by the invention has the following characteristics: the glucose can obviously improve the enzyme activity. Glucose with the concentration lower than 1.75mol/L can enhance the enzyme activity; when the concentration of the glucose is 0.4mol/L, the highest enzyme activity reaches 136.8U/mg, which is 2.2 times of that in the absence of the glucose; (2) the enzyme has good alkali stability, and the enzyme activity of 92%, 99% and 97% can be maintained after 24h incubation at pH8.0, 9.0 and 10.0. The enzyme can be applied in a catalytic environment where high concentrations of glucose are present or where both are alkaline.
Description
Technical field
The present invention relates to biological technical field, specifically a kind of beta-glucosidase, it can be significantly improved enzyme by glucose and live, and can keep excellent stability in the basic conditions.
Background technology
Beta-glucosidase (β-glucosidase, EC3.2.1.21), also known as β-D-Glucose glycosides glucose hydrolysis enzyme, is a kind of circumscribed hydrolase.It can be hydrolyzed the β-D-Glucose glycosidic bond being incorporated into end irreducibility, is applied in widely in the release of food flavor substances, cellulose degradation and daily-use chemical industry industry.In these application, glucose is usually one of hydrolysate, but glucose is also the inhibitor of most of beta-glucosidase.Therefore, the beta-glucosidase excavating high glucose tolerance is the focus of research always.
More than 40 microbe-derived beta-glucosidase of having an appointment altogether in current bibliographical information has glucose tolerance in various degree.Glucose reaches more than 1.0mol/L, the enzyme beta-glucosidase that still can not be suppressed alive only has 3, respectively from BacillushaloduransC-125 (Xuetal.CurrMicrobiol2011,62:833-839) He 2 different grand genomic library (Uchiyamaetal.JBiolChem2013,288:18325-18334; Biveretal.JIndMicrobiolBiotechnol2014,41:479-488).But the beta-glucosidase of Uchiyama report has efficient transglycosylation, and the high glucose tolerance of this enzyme is because glucose can be converted to other product simultaneously.The beta-glucosidase and mutant that have 7 sections to disclose in China's patent to tolerate glucose, but do not have to live still impregnable by enzyme when 1.0mol/L glucose.
Yellow hot anaerobic spore-bearing bacilli Yunnan subspecies (Anoxybacillusflavithermussubsp.yunnanensis) is the new subspecies (Daietal.FEMSMicrobiolLett2011,320:72-78) of this laboratory isolation identification.From bacterial strain E13
tbeta-glucosidase gene is obtained with cloning respectively in PGDY12.The amino acid sequence similarity of these two enzymes is 98.4%.60% is only with there being beta-glucosidase (Brevesetal.ApplEnvironMicrobiol1997,63:3902-3910) the highest serial similarity of bibliographical information.Wherein bacterial strain E13
tthe aminoacid sequence of beta-glucosidase be AGS46809.1 at the sequence number of NCBI.But the wild type gene of these two beta-glucosidases is all beyond expression out activated enzyme in intestinal bacteria.Due to source bacterial strain E13
tbeta-glucosidase gene be do not observe band of expression completely, and the beta-glucosidase gene of the bacterial strain PGDY12 that originates is the inclusion body giving expression to non-activity, therefore selects the beta-glucosidase of source bacterial strain PGDY12 to carry out further optimizing research.
The present invention is by optimized gene sequence and explore abduction delivering condition, obtains an energy and goes out active beta-glucosidase gene at expression in escherichia coli, illustrate the core technology main points of activity expression.The glucose tolerance of this beta-glucosidase is very good, and does not have transglycosylation.Glucose lower than 1.75mol/L can strengthen enzyme and live, and when there is the glucose of 2.0mol/L, enzyme is lived and still remained 76%.The alkaline stability of enzyme is also fine, and pH9.0 and 10.0 still keeps the enzyme of 99% and 97% to live after hatching 24h.
Summary of the invention
The object of this invention is to provide the beta-glucosidase that a kind of resistance to high sugar is alkaline-resisting, to make up the weak point that prior art exists.
The invention provides the gene order of described beta-glucosidase of encoding, sequence is SEQIDNO:1; The aminoacid sequence of this gene sequences encode is SEQIDNO:2.
Beta-glucoside enzyme amino acid sequence (SEQIDNO:2) provided by the invention is from Huang hot anaerobic spore-bearing bacilli Yunnan subspecies PDGY12, with the Huang announced on NCBI hot anaerobic spore-bearing bacilli Yunnan subspecies E13
tthe aminoacid sequence (NCBI sequence number AGS46809.1) of middle beta-glucosidase has 7 amino acid not identical, and the sequence similarity of both is 98.4%.In addition, yellow hot anaerobic spore-bearing bacilli Yunnan subspecies E13
tmiddle beta-glucosidase does not make Function Identification.
Beta-glucosidase gene provided by the invention (SEQIDNO:1) is optimized according to e. coli codon Preference, synthesized by biotech firm.This gene order and Huang hot anaerobic spore-bearing bacilli Yunnan subspecies E13
tthe gene order similarity of middle beta-glucosidase is only 74.9%.In addition, the wild gene order do not optimized of this beta-glucosidase to be beyond expression out activated albumen intestinal bacteria.
The invention provides the recombinant plasmid of expressing above-mentioned beta-glucosidase, is be that the nucleotide fragments of SEQIDNO:1 is inserted in expression vector pET22b by gene order.
The invention provides the Escherichia coli recombinant strain of carrying and expressing above-mentioned beta-glucosidase.
The invention provides the low dissolved oxygen abduction delivering condition that above-mentioned recombinant bacterium gives expression to beta-glucosidase, under this condition, the beta-glucosidase with catalytic activity of solubility can be given expression to.In the expression condition provided, low dissolved oxygen (DO≤10%) is critical technical parameter.The control of low dissolved oxygen can be realized, such as: reduce rotating speed, increase liquid amount, reduce air flow etc. by number of ways.
The invention provides optimum reaction condition and the Reaction conditions range of above-mentioned beta-glucosidase.The temperature range of this enzyme is 0-70 DEG C, and optimum temperuture is 50 DEG C.Temperature stability is very good, and transformation period when 50 DEG C is 13h.The pH scope of this enzyme is 4.5-10.5, when optimal pH is 7.5, pH8.0 and 9.0 keep optimal pH time 93 and 53% enzyme live.The beta-glucosidase specific activity of purifying is 62.2U/mg.
Compared with known beta-glucosidase, the characteristic of beta-glucosidase provided by the invention is: (1) can strengthen enzyme work lower than the glucose of 1.75mol/L; When glucose concn is 0.4mol/L, enzyme is lived and is reached the highest, 2.2 times during for existing without glucose; (2) alkaline stability of enzyme is fine, at pH8.0,9.0 and 10.0 hatch 24h after still keep the enzyme of 92%, 99% and 97% to live.
Accompanying drawing explanation
Fig. 1 illustrates the pET22b-glA2 plasmid map of beta-glucosidase of the present invention.
After Fig. 2 illustrates the expression and purification of beta-glucosidase of the present invention, the SDS-PAGE of albumen schemes.1 is the whole bacterial protein of e. coli bl21 (DE3) ghost; 2 for containing pET22b-glA2 plasmid e. coli bl21 (DE3) abduction delivering after whole bacterial protein; 4 is the beta-glucosidase glA2 of purifying.
Fig. 3 illustrates the temperature of reaction curve of beta-glucosidase of the present invention.
Fig. 4 illustrates the reaction pH curve of beta-glucosidase of the present invention.
Fig. 5 illustrates beta-glucosidase of the present invention at the temperature-stable linearity curve of 50 DEG C.
Fig. 6 illustrates beta-glucosidase of the present invention beta stability line in the basic conditions.
Fig. 7 illustrates the enzyme of beta-glucosidase of the present invention under different glucose concn curve alive.
Embodiment
Following embodiment is to explain the present invention in more detail, and unrestricted the present invention.
Embodiment 1
The acquisition of the aminoacid sequence of beta-glucosidase glA2
The genomic dna of yellow hot anaerobic spore-bearing bacilli Yunnan subspecies PGDY12 extracts according to the operation instruction of Ezup pillar bacterial genomes DNA extraction agent box (Sheng Gong bio tech ltd, Shanghai).According to hot anaerobic spore-bearing bacilli Yunnan subspecies E13 yellow in NCBI
tthe upstream primer of the gene order design PCR of middle beta-glucosidase and downstream primer, its sequence is respectively: 5 '-ATGCTTCAGTTTCCGAAA-3 ' and 5 '-TTTAACTCCATGATTCATG-3 '.With the genome extracted for template, carry out product that pcr amplification obtains to serve Hai Sheng work bio tech ltd and carry out gene sequencing, obtain aminoacid sequence and the wildtype gene sequence of beta-glucosidase glA2 in yellow hot anaerobic spore-bearing bacilli Yunnan subspecies PGDY12.
Embodiment 2
The gene chemical synthesis of beta-glucosidase glA2
Due to the wildtype gene sequence of this beta-glucosidase be the inclusion body of the precipitation forms of non-activity at the albumen that escherichia coli expression goes out, therefore, wildtype gene sequence be optimized.The principle optimized mainly has redesigned the gene order of glA2 according to colibacillary codon preference, also the secondary structure of the mRNA of N end has been carried out to the optimization of 6 Nucleotide simultaneously, the final gene order obtained as SEQIDNO:1 display.This gene order and wildtype gene sequence similarity are only 75.2%, with Huang hot anaerobic spore-bearing bacilli Yunnan subspecies E13
tthe gene order similarity of middle beta-glucosidase is 74.9%.NdeI and XhoI restriction site is added at these gene two ends.The text sequence of this gene is delivered to biotech firm (Sheng Gong bio tech ltd, Shanghai) synthesis.The glA2 gene order of synthesis is cloned on carrier pUC57, and insertion point is T-A clone, and recipient bacterium is e.colistraindh5α.Wherein the nucleotides sequence of beta-glucosidase glA2 of the present invention is classified as SEQIDNO:1, and aminoacid sequence is SEQIDNO:2.
Embodiment 3
The structure of the expression vector pET22b-glA2 of beta-glucosidase glA2
Cloning vector pUC57 containing glA2 gene and expression plasmid pET22b is carried out double digestion with NdeI and XhoI respectively, after digestion products electrophoretic separation, reclaims glA2 gene fragment and expression plasmid pET22b.Add T4DNA ligase enzyme 16.DEG C to spend the night connection.Connect product conversion in e. coli bl21 (DE3) competent cell, filter out positive colony, obtain recombinant expression plasmid pET22b-glA2.
Embodiment 4
The low dissolved oxygen abduction delivering of beta-glucosidase glA2
Realize the expression of the solubility of the activity of beta-glucosidase glA2, the low dissolved oxygen in Induction Process controls very crucial.E. coli bl21 (DE3) containing pET22b-glA2 is seeded in the LB liquid nutrient medium containing 50ug/ml penbritin, 37 DEG C of cultivations.Rotating speed when shaking flask or fermentor cultivation controls as 200-220r/min.When the OD600 of E. coli broth reaches about 0.5, add 0.8mMIPTG (isopropyl-beta D-thio galactopyranoside), then rotating speed is reduced to 80-100r/min, induction 6-8h.In whole abduction delivering process, the oxygen dissolving value (DO) of substratum is less than or equal to 10%.In addition, also can realize oxygen dissolving value (DO) equally by methods such as increase liquid amount, minimizings air flow (during fermentor cultivation) and be less than or equal to 10%.
Embodiment 5
The purifying of beta-glucosidase glA2
By the centrifugal 5min of E. coli broth 6000g after abduction delivering, collecting precipitation cell.The BindingBuffer adding 0.5 times of volume is resuspended, ultrasonic disruption cell.Then the centrifugal 5min of 12000g, supernatant is crude enzyme liquid.Crude enzyme liquid is hatched 20min at 60 DEG C, a large amount of intestinal bacteria itself not resistant against high temperatures albumen can sex change precipitation.Then at the centrifugal 15min of 12000g, the metaprotein of precipitation is removed.Gained supernatant fluid is carried out purifying through Ni-NTA column chromatography.In elutriant, imidazole concentration is 60mM, wash-out 10 column volumes.The protein liquid SDS-PAGE electroresis appraisal purity of collecting, result as shown in Figure 2.
Embodiment 6
The determination of activity of beta-glucosidase glA2
The activity determination method of beta-glucosidase is that the p-NP (pNP) that substrate hydrolysis discharges can directly measure at 415nm with pNPG (p-nitrophenyl-β-D-glucopyranoside) for substrate carries out enzymolysis.Typical curve take pNP as standard test.A standard reaction system is 300 μ l, comprises 10 μ l enzyme liquid, the substrate pNPG (5mM) of 15 μ 1 and 275 μ l Sodium phosphate dibasic-citric acid (50mM, pH7.5) damping fluids.Enzyme, at 50 DEG C of reaction 5min, adds the Na of 300 μ l1mol/L
2cO
3termination reaction, measures light absorption value under 415nm.Standard enzyme unit (U) that lives is defined as per minute and discharges enzyme amount needed for l μm of olpNP.The beta-glucosidase specific activity of purifying is 62.2U/mg.
Embodiment 7
The zymologic property of beta-glucosidase glA2
Optimal reactive temperature and optimal reaction pH
According to the method that embodiment 6 describes, in 0-80 DEG C of temperature range, measure the activity of glA2 respectively, result as shown in Figure 4.GlA2 works at 0-70 DEG C, and optimum temperuture is 50 DEG C.
What pH4.0-8.0 adopted is 50mM Sodium phosphate dibasic-citrate buffer solution; What pH8.0-10.5 adopted is Glycine-NaOH damping fluid.In the method that 50 DEG C describe according to embodiment 6, measure optimal reaction pH, result as shown in Figure 3.The pH scope of glA2 is 4.5-10.5, and keeping when optimal pH is 7.5, pH8.0 and 9.0 has 93% and 53% enzyme to live during optimal pH.
The stability of temperature stability and alkaline pH
GlA2, in 50mM Sodium phosphate dibasic-citrate buffer solution (pH7.5), hatches 14h for 50 DEG C, and period takes out 10 μ l enzyme liquid at regular intervals, and the method mensuration residual enzyme described according to embodiment 6 is lived, and result as shown in Figure 5.The temperature stability of glA2 is very good, and transformation period when 50 DEG C is 13h.
Under room temperature, glA2 is incubated in respectively pH7.0 (50mM Sodium phosphate dibasic-citrate buffer solution), pH8.0,9.0 and 10.0 (50mM Glycine-NaOH damping fluid) 24h.Period takes out 10 μ l enzyme liquid at regular intervals, and the method mensuration residual enzyme described according to embodiment 6 is lived, and result as shown in Figure 6.The alkaline stability of glA2 is fine, at pH8.0,9.0 and 10.0 hatch 24h after still keep the enzyme of 92,99 and 97% to live.
Glucose is on the impact of glA2 activity
In standard reaction system, add the glucose of 0.2,0.4,0.6,0.8,1.0,1.25,1.5,1.75 and 2.0mol/L respectively, the method mensuration enzyme described according to embodiment 6 is lived, and result as shown in Figure 7.Glucose lower than 1.75mol/L can strengthen enzyme and live; When glucose concn is 0.4mol/L, enzyme is lived and is up to 136.8U/mg, 2.2 times during for existing without glucose.When there is the glucose of 2.0mol/L, enzyme is lived and is still remained 76%.This result illustrates that glA2 is a beta-glucosidase that can tolerate the sugar of high concentration.
Claims (7)
1. the beta-glucosidase glA2 that resistance to high sugar is alkaline-resisting, its gene order is as shown in SEQIDNO:1, and its aminoacid sequence is as shown in SEQIDNO:2.
2. the beta-glucosidase recombinant expression vector containing gene described in claim 1.
3. the recombinant expression vector pET22b-glA2 containing gene described in claim 1.
4. produce a recombinant bacterium for beta-glucosidase, in this recombinant bacterium, import gene according to claim 1.
5. recombinant bacterium according to claim 4, is characterized in that this recombinant bacterium is e. coli bl21 (DE3) bacterial strain.
6. the low dissolved oxygen derivational expression method of recombination bacillus coli BL21 (DE3) bacterial strain according to claim 5.It is characterized in that, LB substratum, 37 DEG C, in whole abduction delivering process, the oxygen dissolving value (DO) of substratum is less than or equal to 10%.
7., according to the application of beta-glucosidase in sugar degraded that claim 6 obtains, it is characterized in that there is high concentration glucose in catalytic environment, or catalytic environment is meta-alkalescence.
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| CN109880813A (en) * | 2019-03-20 | 2019-06-14 | 安徽大学 | A kind of beta-glucosidase and its expression bacterial strain and application with galactooligosaccharide synthesis capability |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109880813A (en) * | 2019-03-20 | 2019-06-14 | 安徽大学 | A kind of beta-glucosidase and its expression bacterial strain and application with galactooligosaccharide synthesis capability |
| CN109880813B (en) * | 2019-03-20 | 2022-06-07 | 安徽大学 | Beta-glucosidase with galactooligosaccharide synthesis capacity and expression strain and application thereof |
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