CN116555233B - Thermostable beta-xylosidase mutant E179GD182G and application thereof - Google Patents
Thermostable beta-xylosidase mutant E179GD182G and application thereof Download PDFInfo
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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Abstract
The invention discloses a thermally unstable beta-xylosidase mutant E179GD182G and application thereof, and relates to the technical field of genetic engineering, wherein the amino acid sequence of the mutant E179GD182G is shown as SEQ ID NO. 1. Compared with the recombinant wild beta-xylosidase JB13GH39P28 with the amino acid sequence shown as SEQ ID NO.7, the mutant E179GD182G provided by the invention has the advantages that the optimal pH value is 4.5, the optimal temperature is 50 ℃, the thermal property of the mutant E179GD182G is changed, the activity of the mutant E179GD182G is lower at 60 ℃ and the mutant E179GD182G is rapidly inactivated, and the mutant E179GD182G is beneficial to the safe use of enzymes and the application to the biotechnology field under the low-temperature environment requirement.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a thermally unstable beta-xylosidase mutant E179GD182G and application thereof.
Background
Hemicellulose is the most abundant polysaccharide other than cellulose, and is also the second largest renewable resource in nature, one of the main components being xylan. Xylan is a rich biomass resource, has wide sources, exists in agricultural wastes such as corn straw, wheat straw, bagasse and the like, and is an important renewable resource. Complete hydrolysis of xylan requires a series of enzymatic synergists, beta-xylosidase being one of the key enzymes for degrading xylan, capable of hydrolyzing the non-reducing end of xylooligosaccharides and releasing xylose (Naidu, dset. Carbohydrate Polymers,2018, 179:28-41). Xylose is a raw material for producing xylitol, and can be used as a food sweetener, and can be converted into ethanol through microbial fermentation.
The enzyme has high heat stability and is widely applied to industrial environment under high temperature requirement, but part of enzyme has residual activity even after being treated at ultra-high temperature, and particularly has great influence on the flavor, quality and preservation of food. For example, psychrophilic bacteria in milk secrete enzymes with high heat resistance at low temperatures, which remain active after pasteurization or UHT treatment, can seriously affect the flavor of the milk during storage, destroy the texture of the milk and shorten shelf life (Deng Yingwang et al. Food industry, 2021,42 (06): 373-377).
Thermolabile enzymes have high utility in the field of low temperature biotechnology, especially when the catalytic substrate contains heat sensitive materials. Beta-xylosidase is generally used as maceration enzyme, and is mainly used for extracting and clarifying fruit juice to improve quality, but vitamins B and C in the fruit juice belong to heat-sensitive substances, and the thermolabile beta-xylosidase is used for reaction at low temperature, so that the fruit juice can be clarified to reduce viscosity and turbidity of the fruit juice, and nutrition can be ensured (ChenQet al, trends food science & Technology,2022, 125:126-135). When the enzymatic reaction efficiency needs to be regulated, the thermolabile enzyme has more remarkable advantages than the enzyme with better stability, the thermolabile enzyme is easy to denature, the enzyme is easy to degrade after denaturation, the catalytic reaction of the enzyme is more beneficial to control, the enzyme is deactivated by simply increasing the temperature of the enzymatic reaction system, the operation is simple and safe, and meanwhile, the microbial pollution can be prevented by heating. Therefore, the obtained heat-labile beta-xylosidase is more beneficial to the application of the enzyme in the industries of food, wine making, washing and the like, and is particularly suitable for the biotechnology field under the low-temperature environment requirement.
Disclosure of Invention
The invention aims to provide a thermally unstable beta-xylosidase mutant E179GD182G and application thereof, compared with recombinant wild beta-xylosidase JB13GH39P28, the mutant E179GD182G has the advantages of lower activity at 60 ℃ and more rapid inactivation, and is beneficial to safe use of enzymes and application to the biotechnology field under the low-temperature environment requirement.
In order to achieve the aim, the invention provides a heat-labile beta-xylosidase mutant E179GD182G, the amino acid sequence of which is shown in SEQ ID NO.1, and the mutant E179GD182G is inactivated after being treated for 10min at 60 ℃.
The invention also provides a coding gene of the thermally unstable beta-xylosidase mutant E179GD182G, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.
The invention also provides a recombinant plasmid containing the coding gene.
Preferably, the recombinant plasmid is selected from pET-28a (+).
The invention also provides a recombinant bacterium containing the coding gene.
Preferably, the recombinant bacterium is selected from E.coli BL21 (DE 3).
The heat-unstable beta-xylosidase mutant E179GD182G provided by the invention can be used in industries such as food, wine making or washing, and has greater applicability when the heat stability is relatively poor and the heat is easy to inactivate in the low-temperature biotechnology field.
The heat-labile beta-xylosidase mutant E179GD182G provided by the invention widens the applicability of the beta-xylosidase under different temperature requirements, and has the following advantages:
Compared with the recombinant wild beta-xylosidase JB13GH39P28 with the amino acid sequence shown as SEQ ID NO.7, the mutant E179GD182G provided by the invention has the advantages that the thermal property is changed, the activity at 60 ℃ is low, and the inactivation is more rapid. The optimum temperature of the recombinant wild enzyme JB13GH39P28 is 50 ℃, and the recombinant wild enzyme JB13GH39P28 has 10.67%, 23.94%, 43.61%, 69.58% and 77.97% of enzyme activities at 10 ℃,20 ℃, 30 ℃, 40 ℃ and 60 ℃ respectively; the mutant E179GD182G also had an optimal temperature of 50℃and an enzyme activity of 10.18%, 22.98%, 47.89%, 67.64% and 15.93% at 10 ℃,20 ℃, 30 ℃, 40 ℃ and 60 ℃, respectively; after 60min of 50 ℃, the enzyme activity of wild enzyme JB13GH39P28 was reduced to 83.17%, while the enzyme activity of mutant E179GD182G was reduced from 62.50%; the half-life of wild-type enzyme JB13GH39P28 at 60℃was about 10min, whereas mutant E179GD182G was inactivated after 10min of treatment at 60 ℃. The beta-xylosidase mutant E179GD182G which is unstable in heat and easy to inactivate by heat can be applied to industries such as food, wine making, washing and the like, and is particularly suitable for the biotechnology field under the low-temperature environment requirement.
Drawings
FIG. 1 shows the result of SDS-PAGE analysis of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G according to the present invention.
FIG. 2 shows the comparison of the activities of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G in the present invention at different pH values.
FIG. 3 shows the comparison of the thermal activities of the group wild-type enzyme JB13GH39P28 and mutant E179GD182G of the present invention.
FIG. 4 shows the comparison of the thermostability of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G at 50 ℃.
FIG. 5 shows the comparison of the thermal stability of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G at 60 ℃.
FIG. 6 shows the comparison of the activities of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G in KCl according to the present invention.
FIG. 7 shows the comparison of the stability of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G in KCl according to the present invention.
FIG. 8 shows the comparison of the activities of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G in Na 2SO4 according to the present invention.
FIG. 9 shows comparison of the stability of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G in Na 2SO4 according to the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Some of the experimental materials and reagents used in the present invention:
1) Strains and vectors: coli EscherchiacoliBL (DE 3) was purchased from Beijing Bomaide Gene technologies Co., ltd; the pET-28a (+) expression vector is from Hongsu biosciences, inc.
2) Enzymes and other biochemical reagents: pNPX available from Sigma company; the Nickel-NTA protein purification resin was purchased from QIAGEN; quickMutation TM gene site-directed mutagenesis kit was purchased from Biyun Tian Biotechnology company; dpn1 digestive enzymes were purchased from Takara Biotechnology; the other are all domestic reagents (all are available from common biochemical reagent companies).
3) Culture medium: LB medium: peptone10g, yeastextract g. NaCll0g, distilled water was added to 1000mL and the pH was natural (about 7). The solid medium was supplemented with 2.0% (w/v) agar.
Description: the molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.
Experimental example 1 construction and transformation of recombinant wild beta-xylosidase JB13GH39P28 expression vector
1) The beta-xylosidase JB13GH39 (with the number of AZC 12019.1) with the amino acid sequence shown in SEQ ID NO.3 and the coding gene JB13GH39 (with the number of MG 838204.1) with the nucleotide sequence shown in SEQ ID NO.4 are downloaded from GenBank, the sequence (nucleotide 1-57) of JB13GH39 coding signal peptide is removed, and then the sequence (nucleotide 58-1614) of JB13GH39 coding mature peptide is entrusted to the Suzhou biological technology Co., ltd.) for codon optimization, so that the GC content of the sequence is reduced from 60% to 48%, the target fragment obtained after codon optimization is named JB13GH39p28, the nucleotide sequence of which is shown in SEQ ID NO.5, and the length of the target fragment is 1557bp, and JB13GH39p28 can be obtained through gene synthesis.
2) Restriction sites NcoI (5 'CCATGG 3') and XhoI (5 'CTCGAG 3') are respectively introduced into the 5 ' and 3 ' ends of jb13gh39p28 by a PCR method to obtain a sequence nxjb gh39p28, the nucleotide sequence of which is shown as SEQ ID NO.6, the product and an expression vector pET-28a (+) are subjected to restriction enzyme digestion, and the digested products of nxjb gh39p28 and pET-28a (+) are connected by a ligase to obtain a recombinant plasmid nxjb containing nxjb gh39p28-pET-28a (+).
3) Using nxjb gh39p28-pET-28a (+) as a template, 2 mutant primers (F1 and R1) were designed, and specific sequences are shown below. The 1 leucine and 1 glutamic acid sequences introduced before the C-terminal histidine tag were removed by PCR, and the PCR reaction parameters were: denaturation at 95℃for 30sec; then denaturation at 95℃for 15sec, annealing at 70℃for 15sec, elongation at 72℃for 3min30sec,30 cycles; the temperature is kept at 72 ℃ for 5min. Finally, a recombinant plasmid JB13GH39P28-pET-28a (+) containing JB13GH39P28 is obtained, the nucleotide sequence formed by the recombinant JB13GH39P28 is shown as SEQ ID NO.8, and the amino acid sequence of the encoded recombinant wild enzyme JB13GH39P28 is shown as SEQ ID NO. 7.
The sequence of the mutant primer is as follows (5 '. Fwdarw.3'):
F1(SEQ ID NO.9)：
GAACGTAAACACCACCACCACCACCACTGAGAT
R1(SEQ ID NO.10)：
GTGGTGGTGTTTACGTTCTTTCGGTGCAATACT
4) The recombinant plasmid jb13gh39p28-pET-28a (+) was transformed into E.coli BL21 (DE 3) by heat shock to obtain a recombinant strain BL21 (DE 3)/jb 13gh39p28 comprising jb13gh39p28.
Experimental example 2 construction and transformation of mutant E179GD182G expression vector
1) The recombinant strain containing the recombinant plasmid jb13gh39p28-pET-28a (+) obtained in Experimental example 1 was inoculated into LB medium (containing 50. Mu.g/mL kanamycin) at a content of 0.1%, cultured overnight at 37℃and the plasmid was extracted by the kit.
2) 2 Mutant primers (F2 and R2) were designed using the recombinant plasmid jb13gh39p28-pET-28a (+) as a template, and specific sequences are shown below. The QuickMutation TM gene site-directed mutagenesis kit is used for mutation, and the PCR reaction parameters are as follows: denaturation at 95℃for 30sec; then denaturation at 95℃for 15sec, annealing at 70℃for 15sec, elongation at 72℃for 3min30sec,30 cycles; the temperature is kept at 72 ℃ for 5min. The PCR amplification is carried out to obtain the linearization recombinant plasmid E179GD182G-pET-28a (+) containing the coding gene E179GD182G with the nucleotide sequence shown as SEQ ID NO.2, the coding gene coding mutant is E179GD182G, and the amino acid sequence is shown as SEQ ID NO. 1. e179gd182g and e179gd182g-pET-28a (+) can also be obtained by gene synthesis.
The sequence of the mutant primer is as follows (5 '. Fwdarw.3'):
F2(SEQ ID NO.11)：
TTTTGGGGAGGCGCAGGTCAGAAAGCATATTTTGAACTGTACGA
R2(SEQ ID NO.12)：
ACCTGCGCCTCCCCAAAAACCAGACAGATTCGGT
3) The PCR product was digested with Dpn1 enzyme at 37℃for 3 hours.
4) Transferring the digested product in 3) into escherichia coli BL21 (DE 3) by a heat shock mode to obtain a recombinant strain BL21 (DE 3)/E179 GD182G containing E179GD182G encoding gene E179GD182G.
Experimental example 3 preparation of recombinant wild beta-xylosidase JB13GH39P28 and mutant E179GD182G
1) Recombinant strains BL21 (DE 3)/jb 13gh39p28 and BL21 (DE 3)/e 179gd182g obtained in Experimental example 2 were inoculated into LB (containing 50. Mu.g/mL kanamycin) culture medium at an inoculum size of 0.1%, respectively, and activated by shaking in a shaker at 37℃and 180rpm/min for 16 hours.
2) The bacterial solutions activated in 1) were inoculated into fresh LB (containing 50. Mu.g/mL kanamycin) culture medium at 1% inoculum size, and after shaking culture in a shaker at 37℃and 180rpm/min for about 2-3 hours (OD 600 reached 0.6-1.0), induction was performed by adding IPTG at a final concentration of 0.7mM, and shaking culture was continued at 20℃and 160rpm/min for about 20 hours to induce recombinant protein production.
3) The cells were collected by centrifugation at 6000rpm/min at 4℃for 8 min. After the cells were suspended with an appropriate amount of McIlvaine buffer at ph=7.0, the cells were sonicated in a low-temperature water bath. After the above intracellular concentrated crude enzyme solution was centrifuged at 12000rpm/min for 1.0 min, the supernatant was aspirated and the target protein was affinity purified with Nickel-NTAAgarose and 0-500mM imidazole, respectively. The SDS-PAGE results of the two purified proteins are shown in FIG. 1, wherein M is protein Marker, W is wild-type enzyme, and E179GD182G is mutant. The result shows that the recombinant wild enzyme JB13GH39P28 and mutant E179GD182G are expressed and purified, and the product is a single band.
4) A volume of 100 times the sample volume of dialysate (ph=7.0 McIlvainebuffer) was added to the dialysis device. Cutting a dialysis bag (mw: 14000) into small sections with proper length (10-20 cm), boiling in boiling water for 30min, thoroughly cleaning the dialysis bag with distilled water, respectively filling the purified recombinant wild enzyme JB13GH39P28 and mutant E179GD182G obtained in 3) into the dialysis bag, reserving the lengths of 3-5cm at the two ends of the dialysis bag, and clamping by using a dialysis clamp. The dialysis sample was placed in a dialysis buffer, dialyzed at 4℃and the dialysis solution was changed 3 times every 2 hours.
Experimental example 4 determination of the Properties of recombinant wild beta-xylosidase JB13GH39P28 and mutant E179GD182G
1) Activity analysis of recombinant wild enzyme JB13GH39P28 and mutant E179GD182G
The activity determination method adopts a PNP method, and uses P-nitrophenyl-beta-D-xylopyranoside (pNPX) as a substrate to determine the activity of the purified recombinant wild enzyme JB13GH39P28 and mutant E179GD 182G. pNPX was dissolved in buffer to a final concentration of 2mM; the reaction system contained 50. Mu.L of enzyme solution, 450. Mu.L of substrate-containing buffer; preheating a substrate at a reaction temperature for 5min, adding an enzyme solution, reacting for 10min, adding 2mL1MNA 2 CO3 to terminate the reaction, cooling to room temperature, and measuring the amount of released pNP at a wavelength of 405 nm; 1 enzyme activity unit (U) is defined as the amount of enzyme required to break down the substrate to produce 1. Mu. MolpNP per minute.
2) Activity determination of recombinant wild enzyme JB13GH39P28 and mutant E179GD182G in different pH values
The enzyme purified in experimental example 3 was subjected to enzymatic reaction at 37℃in pH=3.0 to 7.0 (3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0) McIlvainebuffer, and the enzyme activities of purified recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G were measured.
The comparison of the activities of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G at different pH values is shown in FIG. 2, and shows that the optimal pH of purified recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G is 4.5.
3) Thermal activity determination of recombinant wild enzyme JB13GH39P28 and mutant E179GD182G
The enzymatic reaction was carried out at 0-70 ℃ in a buffer at ph=4.5. And (3) taking pNPX as a substrate, reacting for 10min, and measuring the enzyme activities of the purified recombinant wild enzyme JB13GH39P28 and mutant E179GD 182G.
The thermal activity comparison of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G is shown in FIG. 3, which shows that: the optimal temperature for the purified recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G was 50 ℃. Wild-type enzyme JB13GH39P28 has 10.67%, 23.94%, 43.61%, 69.58%, 77.97% and 19.50% enzyme activities at 10 ℃, 20 ℃,30 ℃,40 ℃, 60 ℃ and 70 ℃, respectively; mutant E179GD182G had 10.18%, 22.98%, 47.89%, 67.64%, 15.93% and 2.70% enzyme activities at 10 ℃, 20 ℃,30 ℃,40 ℃, 60 ℃ and 70 ℃, respectively. Compared with the wild-type enzyme JB13GH39P28, the activity of the mutant E179GD182G is drastically reduced at 60 ℃ and is about 62% lower than that of the wild-type enzyme.
4) Thermal stability measurement of recombinant wild enzyme JB13GH39P28 and mutant E179GD182G
The enzyme solution of the same enzyme amount was subjected to treatment at 50℃for 60min at 60℃for measuring the enzyme activity every 10min, and the enzymatic reaction was carried out at pH=4.5 and 37℃with the untreated enzyme solution as a control. And (3) taking pNPX as a substrate, reacting for 10min, and measuring the enzyme activities of the purified recombinant wild enzyme JB13GH39P28 and mutant E179GD 182G.
The comparison result of the thermal stability of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G at 50 ℃ is shown in FIG. 4, and the comparison result of the thermal stability at 60 ℃ is shown in FIG. 5, and the result shows that the enzyme activity of the wild enzyme JB13GH39P28 is reduced to 83.17% and the enzyme activity of the mutant E179GD182G is reduced to 62.50% after the treatment at 50 ℃ for 60 min; the half-life of wild-type enzyme JB13GH39P28 at 60℃was about 10min, whereas the thermal properties of mutant E179GD182G were changed, which rapidly deactivated after treatment at 60℃for 10 min.
5) Activity determination of recombinant wild enzyme JB13GH39P28 and mutant E179GD182G in KCl
Adding 3.0-30.0% (w/v) KCl into the enzymatic reaction system, and performing enzymatic reaction at pH=4.5 and 37 ℃. And (3) taking pNPX as a substrate, reacting for 10min, and measuring the enzyme activities of the purified recombinant wild enzyme JB13GH39P28 and mutant E179GD 182G.
The comparison result of the activities of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G in KCl is shown in FIG. 6, and the result shows that the enzyme activity of the wild enzyme JB13GH39P28 is almost not lost and the activity is up to 111.32% when KCl with the concentration of 3.0-30.0% (3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0) (w/v) is added into a reaction system; the mutant E179GD182G has little change in enzyme activity in KCl of 3.0-25.0% (w/v), and the enzyme activity is reduced to 77.92% after adding KCl of 30.0% (w/v).
6) Determination of stability of recombinant wild enzyme JB13GH39P28 and mutant E179GD182G in KCl
The purified enzyme solution was placed in 3.0 to 30.0% (3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0) (w/v) aqueous KCl, treated at 37 ℃ for 60min, and then subjected to enzymatic reaction at ph=4.5 and 37 ℃ with untreated enzyme solution as a control. And (3) taking pNPX as a substrate, reacting for 10min, and measuring the enzyme activities of the purified recombinant wild enzyme JB13GH39P28 and mutant E179GD 182G.
The comparison result of the stability of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G in KCl is shown in FIG. 7, and the result shows that the enzyme activity of the wild enzyme JB13GH39P28 is almost not lost after the KCl treatment of 3.0-30.0% (3.0, 5.0, 10.0, 15.0, 20.0, 25.0 and 30.0) (w/v) for 60min, and the enzyme activity of the mutant E179GD182G is still kept above 90% after the KCl treatment of 3.0-25.0% (w/v) for 60min, and the enzyme activity is remained at 86.69% after the KCl treatment of 30.0% (w/v) for 60 min.
7) Activity determination of recombinant wild enzyme JB13GH39P28 and mutant E179GD182G in Na 2SO4
3.0 To 30.0% (3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0) (w/v) Na 2SO4 is added into the enzymatic reaction system, and the enzymatic reaction is carried out at pH=4.5 and 37 ℃. And (3) taking pNPX as a substrate, reacting for 10min, and measuring the enzyme activities of the purified recombinant wild enzyme JB13GH39P28 and mutant E179GD 182G.
The comparison of the activities of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G in Na 2SO4 is shown in FIG. 8, which shows that Na 2SO4 has an enhancing effect on the activities of wild-type enzyme JB13GH39P28 and mutant E179GD 182G. When 3.0-30.0% (w/v) of Na 2SO4 is added into the reaction system, the highest activity energy of the wild enzyme JB13GH39P28 is increased to 149.52%, and the highest activity energy of the mutant E179GD182G is increased to 149.82%.
8) Determination of stability of recombinant wild enzyme JB13GH39P28 and mutant E179GD182G in Na 2SO4
The purified enzyme solution was placed in 3.0-30.0% (w/v) aqueous Na 2SO4 solution, treated at 37 ℃ for 60min, and then subjected to enzymatic reaction at ph=4.5 and 37 ℃ with untreated enzyme solution as control. And (3) taking pNPX as a substrate, reacting for 10min, and measuring the enzyme activities of the purified recombinant wild enzyme JB13GH39P28 and mutant E179GD 182G.
The comparison result of the stability of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G in Na 2SO4 is shown in FIG. 9, and the result shows that Na 2SO4 has an enhancement effect on the stability of the wild enzyme JB13GH39P28 and the mutant E179GD182G, and the enzyme activity of the wild enzyme JB13GH39P28 is enhanced by 19.67% at the maximum and the enzyme activity of the mutant E179GD182G is enhanced by 18.67% at the maximum after the enzyme solution is treated by 3.0-30.0% (w/v) of Na 2SO4 for 60 min.
In summary, the mutant E179GD182G provided by the invention has the optimal pH value of 4.5 and the optimal temperature of 50 ℃, and compared with the recombinant wild beta-xylosidase JB13GH39P28 with the amino acid sequence shown as SEQ ID No.7, the mutant E179GD182G has the advantages of changing the thermal property, lower activity at 60 ℃ and more rapid inactivation, and is beneficial to the safe use of enzymes and application in the biotechnology field under the low-temperature environment requirement. And Na 2SO4 can enhance the enzyme activity and stability of mutant E179GD182G, and can increase the activity to 149.82% at the highest. The heat-unstable beta-xylosidase mutant E179GD182G provided by the invention can be applied to industries such as food, brewing, washing and the like, and is particularly suitable for the biotechnology field under the low-temperature environment requirement.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (8)
1. A heat-labile beta-xylosidase mutant E179GD182G is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO.1, and the mutant E179GD182G is inactivated after being treated for 10min at 60 ℃.
2. The gene encoding the thermostable beta-xylosidase mutant E179GD182G according to claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.
3. A recombinant plasmid comprising the coding gene of claim 2.
4. The recombinant plasmid according to claim 3, wherein said plasmid is selected from the group consisting of pET-28a (+).
5. A recombinant bacterium comprising the coding gene according to claim 2.
6. The recombinant bacterium according to claim 5, wherein said bacterium is selected from BL21 (DE 3).
7. Use of the thermostable β -xylosidase mutant E179GD182G according to claim 1 in the food or washing industry.
8. The use of claim 7, wherein the food industry comprises wine brewing.
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| CN116497005B (en) * | 2023-03-10 | 2024-06-04 | 云南师范大学 | Beta-xylosidase mutant K130GK137G with reduced heat tolerance and application thereof |
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