CN116555233B - Thermostable beta-xylosidase mutant E179GD182G and application thereof - Google Patents

Abstract

The invention discloses a thermolabile β-xylosidase mutant E179GD182G and its application, and relates to the technical field of genetic engineering, wherein the amino acid sequence of the mutant E179GD182G is shown in SEQ ID NO.1. The mutant E179GD182G provided by the invention has an optimum pH of 4.5 and an optimum temperature of 50°C, and compared with the recombinant wild β-xylosidase JB13GH39P28 with an amino acid sequence as shown in SEQ ID NO.7, the mutant E179GD182G has a change in thermal properties, and its activity is lower at 60°C and it is rapidly inactivated, which is beneficial to the safe use of the enzyme and its application in the field of biotechnology under low temperature environment requirements.

Description

Heat-labile β-xylosidase mutant E179GD182G and its application

Technical Field

The invention relates to the technical field of genetic engineering, and in particular to a thermally unstable beta-xylosidase mutant E179GD182G and an application thereof.

Background technique

Hemicellulose is the most abundant polysaccharide besides cellulose, and is also the second largest renewable resource in nature. One of its main components is xylan. Xylan is a rich biomass resource with a wide range of sources. It exists in agricultural wastes such as corn stalks, wheat straw, and sugarcane bagasse, and is an important renewable resource. The complete hydrolysis of xylan requires the synergistic action of a series of enzymes. As one of the key enzymes for degrading xylan, β-xylosidase can hydrolyze the non-reducing ends of oligoxylose and release xylose (Naidu, D Setal. Carbohydrate Polymers, 2018, 179: 28-41). Xylose is the raw material for the production of xylitol, which can be used as a food sweetener or converted into ethanol through microbial fermentation.

Enzymes are highly thermally stable and are widely used in industrial environments with high temperature requirements. However, some enzymes still retain activity even after ultra-high temperature treatment, which has a significant impact on the flavor, quality, and preservation of food. For example, the enzymes secreted by psychrophilic bacteria in milk at low temperatures are highly heat-resistant and can still maintain a certain level of activity after pasteurization or UHT treatment. During storage, they will seriously affect the flavor of milk, destroy the texture of milk, and shorten the shelf life (Deng Yingwang et al. Food Industry, 2021, 42(06): 373-377).

Heat-labile enzymes have a high application value in the field of low-temperature biotechnology, especially when the catalytic substrate contains heat-sensitive substances. β-Xylosidase is generally used as an impregnation enzyme, mainly used for extracting and clarifying juice to improve quality, but vitamins B and C in juice are heat-sensitive substances. Using heat-labile β-xylosidase, reacting at low temperatures, can not only clarify the juice to reduce juice viscosity and turbidity, but also ensure nutrition (Chen Q et al. Trends in Food Science & Technology, 2022, 125: 126-135). When it is necessary to regulate the efficiency of the enzymatic reaction, heat-labile enzymes have more significant advantages than enzymes with better stability. Heat-labile enzymes are easy to denature, and after denaturation, the enzyme is easily degraded, which is more conducive to controlling the catalytic reaction of the enzyme. The enzyme is inactivated by simply increasing the temperature of the enzymatic reaction system. The operation is simple and safe, and heating can prevent microbial contamination. Therefore, obtaining heat-labile β-xylosidase is more conducive to the application of enzymes in industries such as food, winemaking, and washing, and is particularly suitable for the field of biotechnology under low temperature environment requirements.

Summary of the invention

The purpose of the present invention is to provide a thermally unstable β-xylosidase mutant E179GD182G and an application thereof. Compared with the recombinant wild β-xylosidase JB13GH39P28, the mutant E179GD182G has changed thermal properties, has lower activity at 60°C and is inactivated more rapidly, which is beneficial to the safe use of the enzyme and its application in the field of biotechnology under low temperature environment requirements.

In order to achieve the above object, the present invention provides a heat-labile β-xylosidase mutant E179GD182G, the amino acid sequence of the mutant is shown in SEQ ID NO.1, and the mutant E179GD182G is inactivated after being treated at 60°C for 10 minutes.

The present invention also provides a gene encoding the thermally unstable β-xylosidase mutant E179GD182G. The nucleotide sequence of the gene encoding is shown in SEQ ID NO.2.

The invention also provides a recombinant plasmid comprising the above encoding gene.

Preferably, the above-mentioned recombinant plasmid is selected from pET-28a(+).

The present invention also provides a recombinant bacterium comprising the above encoding gene.

Preferably, the above-mentioned recombinant bacteria is selected from Escherichia coli BL21 (DE3).

The thermolabile β-xylosidase mutant E179GD182G provided by the present invention can be used in the food, brewing or washing industries, especially in the field of low-temperature biotechnology. When poor thermal stability and easy inactivation by heat are required, the mutant E179GD182G has greater applicability.

The thermolabile β-xylosidase mutant E179GD182G provided by the present invention broadens the applicability of β-xylosidase under different temperature requirements and has the following advantages:

Compared with the recombinant wild β-xylosidase JB13GH39P28 whose amino acid sequence is shown in SEQ ID NO.7, the mutant E179GD182G provided by the present invention has changed thermal properties, and its activity at 60°C becomes lower and inactivation is more rapid. The optimum temperature of the recombinant wild enzyme JB13GH39P28 is 50°C, and the enzyme activities at 10°C, 20°C, 30°C, 40°C and 60°C are 10.67%, 23.94%, 43.61%, 69.58% and 77.97%, respectively; the optimum temperature of the mutant E179GD182G is also 50°C, and the enzyme activities at 10°C, 20°C, 30°C, 40°C and 60°C are 10.18%, 22.98%, 43.61%, 69.58% and 77.97%, respectively. The enzyme activities of the wild enzyme JB13GH39P28 were 47.89%, 67.64% and 15.93% respectively; after being treated at 50°C for 60 minutes, the enzyme activity of the wild enzyme JB13GH39P28 decreased to 83.17%, while the enzyme activity of the mutant E179GD182G decreased to 62.50%; the half-life of the wild enzyme JB13GH39P28 at 60°C was about 10 minutes, while the mutant E179GD182G was inactivated after being treated at 60°C for 10 minutes. The heat-labile and heat-inactivated β-xylosidase mutant E179GD182G of the present invention can be applied to the food, wine making, washing and other industries, and is particularly suitable for the biotechnology field under low temperature environment requirements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the SDS-PAGE analysis result of the recombinant wild enzyme JB13GH39P28 and mutant E179GD182G in the present invention.

FIG. 2 is a comparison result of the activities of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G in different pH conditions in the present invention.

FIG3 is a comparison result of the thermal activities of the wild-type enzyme JB13GH39P28 and the mutant E179GD182G in the present invention.

FIG. 4 is a comparison of the thermal stability of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G at 50° C. in the present invention.

FIG5 is a comparison of the thermal stability of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G at 60° C. in the present invention.

FIG6 is a comparison result of the activities of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G in KCl in the present invention.

FIG. 7 is a comparison of the stability of the recombinant wild-type enzyme JB13GH39P28 and the mutant E179GD182G in KCl in the present invention.

FIG8 is a comparison result of the activities of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G in Na ₂ SO ₄ of the present invention.

FIG. 9 is a comparison result of the stability of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G in Na ₂ SO ₄ of the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Some experimental materials and reagents used in this invention:

1) Strains and vectors: Escherichia coli BL21 (DE3) was purchased from Beijing Biomed Gene Technology Co., Ltd.; pET-28a (+) expression vector was from Jiangsu Suzhou Hongxun Biotechnology Co., Ltd.

2) Enzymes and other biochemical reagents: pNPX was purchased from Sigma; Nickel-NTA protein purification resin was purchased from QIAGEN; QuickMutation ^TM gene site-directed mutagenesis kit was purchased from Beyotime Biotechnology; Dpn1 digestive enzyme was purchased from Takara Biotechnology; other reagents were domestically produced (all available from common biochemical reagent companies).

3) Culture medium: LB culture medium: Peptone 10g, Yeast extract 5g, NaCl 110g, add distilled water to 1000mL, pH natural (about 7). Solid culture medium plus 2.0% (w/v) agar.

Note: The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd Edition) by J. Sambrook, or in accordance with the kits and product instructions.

Experimental Example 1 Construction and transformation of recombinant wild β-xylosidase JB13GH39P28 expression vector

1) The amino acid sequence of β-xylosidase JB13GH39 (numbered AZC12019.1) as shown in SEQ ID NO.3 and the nucleotide sequence of the coding gene jb13gh39 (numbered MG838204.1) as shown in SEQ ID NO.4 were downloaded from GenBank. After removing the sequence encoding the signal peptide of jb13gh39 (nucleotides 1-57), Suzhou Hongxun Biotechnology Co., Ltd. was commissioned to perform codon optimization on the sequence encoding the mature peptide of jb13gh39 (nucleotides 58-1614). As a result, the GC content of the sequence was reduced from 60% to 48%. The target fragment obtained after codon optimization was named jb13gh39p28, and its nucleotide sequence was shown in SEQ ID NO.5. Its length was 1557 bp. jb13gh39p28 can also be obtained by gene synthesis.

2) Restriction enzyme sites NcoⅠ (5'CCATGG3') and XhoⅠ (5'CTCGAG3') were introduced into the 5 ^' and 3 ^' ends of jb13gh39p28 by PCR, respectively, to obtain the sequence nxjb13gh39p28, whose nucleotide sequence is shown in SEQ ID NO.6. The above product and the expression vector pET-28a(+) were digested with restriction enzymes, and the digestion products of nxjb13gh39p28 and pET-28a(+) were connected by ligase to obtain the recombinant plasmid nxjb13gh39p28-pET-28a(+) containing nxjb13gh39p28.

3) Using nxjb13gh39p28-pET-28a(+) as a template, two mutant primers (F1 and R1) were designed, and the specific sequences are shown below. One leucine and one glutamic acid sequence introduced before the C-terminal histidine tag during recombination were removed by PCR, and the PCR reaction parameters were: denaturation at 95°C for 30 seconds; then denaturation at 95°C for 15 seconds, annealing at 70°C for 15 seconds, extension at 72°C for 3min30sec, 30 cycles; insulation at 72°C for 5min. Finally, the recombinant plasmid jb13gh39p28-pET-28a(+) containing jb13gh39p28 was obtained. The nucleotide sequence formed by the recombinant jb13gh39p28 is shown in SEQ ID NO.8, and the amino acid sequence encoding the recombinant wild enzyme JB13GH39P28 is shown in SEQ ID NO.7.

The mutation primer sequences are as follows (5'→3'):

F1 (SEQ ID NO.9):

GAACGTAAACACCACCACCACCACCACTGAGAT

R1 (SEQ ID NO.10):

GTGGTGGTGTTTACGTTCTTTCGGTGCAATACT

4) The recombinant plasmid jb13gh39p28-pET-28a(+) was transformed into Escherichia coli BL21(DE3) by heat shock to obtain the recombinant strain BL21(DE3)/jb13gh39p28 containing 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 μg/mL kanamycin) at a concentration of 0.1%, cultured overnight at 37°C, and the plasmid was extracted using a kit.

2) Using the recombinant plasmid jb13gh39p28-pET-28a(+) as a template, two mutation primers (F2 and R2) were designed, and the specific sequences are shown below. The mutation was performed using the QuickMutation ^TM gene site-directed mutagenesis kit, and the PCR reaction parameters were: denaturation at 95°C for 30 seconds; then denaturation at 95°C for 15 seconds, annealing at 70°C for 15 seconds, extension at 72°C for 3min30sec, 30 cycles; and insulation at 72°C for 5 minutes. PCR amplification obtained a linearized recombinant plasmid e179gd182g-pET-28a(+) containing the coding gene e179gd182g with a nucleotide sequence as shown in SEQ ID NO.2. The mutant encoded by the coding gene is E179GD182G, and its amino acid sequence is shown in SEQ ID NO.1. e179gd182g and e179gd182g-pET-28a(+) can also be obtained by gene synthesis.

The mutation primer sequences are as follows (5'→3'):

F2 (SEQ ID NO.11):

TTTTGGGGAGGCGCAGGTCAGAAAGCATATTTTGAACTGTACGA

R2 (SEQ ID NO.12):

ACCTGCGCCTCCCCAAAAACCAGACAGATTCGGT

3) The PCR product was digested with Dpn1 enzyme at 37°C for 3 h.

4) The digestion product in 3) was transformed into Escherichia coli BL21 (DE3) by heat shock to obtain a recombinant strain BL21 (DE3) / e179gd182g containing the E179GD182G encoding gene e179gd182g.

Experimental Example 3 Preparation of recombinant wild β-xylosidase JB13GH39P28 and mutant E179GD182G

1) The recombinant strains BL21(DE3)/jb13gh39p28 and BL21(DE3)/e179gd182g obtained in Experimental Example 2 were inoculated at an inoculum size of 0.1% in LB (containing 50 μg/mL kanamycin) culture medium and activated by shaking at 37°C and 180 rpm/min for 16 h.

2) The activated bacterial solution in 1) was inoculated into fresh LB (containing 50 μg/mL kanamycin) culture medium at a 1% inoculum size, and cultured in a shaker at 37°C and 180 rpm/min for about 2-3 h ( _OD600 reached 0.6-1.0), and then IPTG was added at a final concentration of 0.7 mM for induction, and the culture was continued in a shaker at 20°C and 160 rpm/min for about 20 h to induce the production of recombinant protein.

3) Centrifuge at 4°C and 6000rpm/min for 8min to collect the bacteria. After suspending the bacteria with an appropriate amount of pH=7.0McIlvaine buffer, ultrasonically disrupt the bacteria in a low-temperature water bath. After the above intracellular concentrated crude enzyme solution was centrifuged at 12000rpm/min for 10min, the supernatant was aspirated and the target protein was affinity and purified with Nickel-NTAAgarose and 0-500mM imidazole respectively. The SDS-PAGE results of the two purified proteins are shown in Figure 1, where M is the protein marker, W is the wild enzyme, and E179GD182G is the mutant. The results show that the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G were expressed and purified, and the product was a single band.

4) Add 100 times the volume of the sample to the dialysate (pH = 7.0 McIlvaine buffer) into the dialysis device. Cut the dialysis bag (mw: 14000) into small pieces of appropriate length (10-20cm), boil it in boiling water for 30 minutes, and then thoroughly wash the dialysis bag with distilled water. Put the purified recombinant wild enzyme JB13GH39P28 and mutant E179GD182G obtained in 3) into the dialysis bag respectively, and clamp the two ends of the dialysis bag with 3-5cm of length reserved with dialysis clips. Place the dialyzed sample in the dialysis buffer and dialyze at 4℃. Change the dialysis solution every 2 hours, and change it 3 times.

Experimental Example 4 Determination of properties of recombinant wild β-xylosidase JB13GH39P28 and mutant E179GD182G

1) Activity analysis of the recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G

Activity determination method The PNP method was used to determine the activity of the purified recombinant wild enzyme JB13GH39P28 and mutant E179GD182G using p-nitrophenyl-β-D-pyranose (pNPX) as a substrate. pNPX was dissolved in a buffer solution to a final concentration of 2 mM; the reaction system contained 50 μL of enzyme solution and 450 μL of substrate-containing buffer solution; the substrate was preheated at the reaction temperature for 5 min, then the enzyme solution was added and the reaction was continued for another 10 min, and then 2 mL of 1 M Na ₂ CO 3 was added to terminate the reaction. After cooling to room temperature, the amount of pNP released was measured at a wavelength of 405 nm; 1 enzyme activity unit (U) was defined as the amount of enzyme required to decompose the substrate to produce 1 μmol pNP per minute.

2) Activity determination of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G at different pH

The purified enzyme in Experimental Example 3 was placed in McIlvaine buffer at pH = 3.0-7.0 (3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0), and the enzymatic reaction was carried out at 37°C to measure the enzyme activities of the purified recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G.

The results of the activity comparison of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G at different pH are shown in Figure 2. The results show that the optimal pH of the purified recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G is 4.5.

3) Thermal activity assay of the recombinant wild-type enzyme JB13GH39P28 and its mutant E179GD182G

The enzymatic reaction was carried out in a buffer solution at pH = 4.5 at 0-70° C. pNPX was used as a substrate, the reaction lasted for 10 min, and the enzyme activities of the purified recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G were determined.

The results of the thermal activity comparison of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G are shown in Figure 3. The results show that the optimum temperature of the purified recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G is 50°C. The wild enzyme JB13GH39P28 has 10.67%, 23.94%, 43.61%, 69.58%, 77.97% and 19.50% enzyme activities at 10°C, 20°C, 30°C, 40°C, 60°C and 70°C, respectively; the mutant E179GD182G has 10.18%, 22.98%, 47.89%, 67.64%, 15.93% and 2.70% enzyme activities at 10°C, 20°C, 30°C, 40°C, 60°C and 70°C, respectively. Compared with the wild enzyme JB13GH39P28, the activity of the mutant E179GD182G was sharply reduced at 60°C, which was about 62% lower than that of the wild enzyme.

4) Thermal stability determination of the recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G

The same amount of enzyme solution was placed at 50℃ and 60℃ for 60min, and the enzyme activity was measured every 10min. The enzymatic reaction was carried out at pH=4.5 and 37℃, and the untreated enzyme solution was used as a control. pNPX was used as a substrate, and the reaction lasted for 10min. The enzyme activity of the purified recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G was measured.

The comparative results of thermal stability of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G at 50°C are shown in Figure 4, and the comparative results of thermal stability at 60°C are shown in Figure 5. The results show that after treatment at 50°C for 60 minutes, the enzyme activity of the wild enzyme JB13GH39P28 decreased to 83.17%, while the enzyme activity of the mutant E179GD182G decreased to 62.50%; the half-life of the wild enzyme JB13GH39P28 at 60°C is about 10 minutes, while the thermal properties of the mutant E179GD182G change, and it is rapidly inactivated after treatment at 60°C for 10 minutes.

5) Activity determination of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G in KCl

3.0-30.0% (w/v) KCl was added to the enzymatic reaction system, and the enzymatic reaction was carried out at pH=4.5 and 37° C. pNPX was used as a substrate, and the reaction lasted for 10 min, and the enzyme activities of the purified recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G were determined.

The comparative results of the activities of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G in KCl are shown in Figure 6. The results show that when 3.0-30.0% (3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0) (w/v) of KCl is added to the reaction system, the enzyme activity of the wild enzyme JB13GH39P28 is almost not lost, and the highest activity can reach 111.32%; while the enzyme activity of the mutant E179GD182G is almost unchanged in 3.0-25.0% (w/v) of KCl, and the enzyme activity drops to 77.92% after adding 30.0% (w/v) KCl.

6) Stability determination of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G in KCl

The purified enzyme solution was placed in a 3.0-30.0% (3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0) (w/v) KCl aqueous solution and treated at 37°C for 60 min, and then the enzymatic reaction was carried out at pH = 4.5 and 37°C, with the untreated enzyme solution as a control. pNPX was used as a substrate, and the reaction was carried out for 10 min to determine the enzyme activity of the purified recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G.

The stability comparison results of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G in KCl are shown in Figure 7. The results show that after being treated with 3.0-30.0% (3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0) (w/v) KCl for 60 min, the enzyme activity of the wild enzyme JB13GH39P28 was almost not lost, while the enzyme activity of the mutant E179GD182G was still maintained above 90% after being treated with 3.0-25.0% (w/v) KCl for 60 min, and the enzyme activity remained 86.69% after being treated with 30.0% (w/v) KCl for 60 min.

7) Activity determination of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G in Na ₂ SO ₄

3.0-30.0% (3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0) (w/v) Na ₂ SO ₄ was added to the enzymatic reaction system, and the enzymatic reaction was carried out at pH=4.5 and 37° C. pNPX was used as a substrate, and the reaction lasted for 10 min. The enzyme activities of the purified recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G were determined.

The results of the comparison of the activities of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G in Na ₂ SO ₄ are shown in Figure 8. The results show that Na ₂ SO ₄ has an enhancing effect on the activities of the wild enzyme JB13GH39P28 and the mutant E179GD182G. When 3.0-30.0% (w/v) Na ₂ SO ₄ is added to the reaction system, the activity of the wild enzyme JB13GH39P28 can be increased to 149.52% at most, and the activity of the mutant E179GD182G can be increased to 149.82% at most.

8) Stability determination of recombinant wild-type enzyme JB13GH39P28 and mutant E179GD182G in Na ₂ SO ₄

The purified enzyme solution was placed in a 3.0-30.0% (w/v) Na ₂ SO ₄ aqueous solution and treated at 37°C for 60 minutes, and then the enzymatic reaction was carried out at pH = 4.5 and 37°C, with the untreated enzyme solution as a control. pNPX was used as a substrate, and the reaction lasted for 10 minutes to determine the enzyme activity of the purified recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G.

The stability comparison results of the recombinant wild enzyme JB13GH39P28 and the mutant E179GD182G in Na ₂ SO ₄ are shown in Figure 9. The results show that Na ₂ SO ₄ has an enhancing effect on the stability of the wild enzyme JB13GH39P28 and the mutant E179GD182G. After the enzyme solution was treated with 3.0-30.0% (w/v) Na ₂ SO ₄ for 60 minutes, the enzyme activity of the wild enzyme JB13GH39P28 was enhanced by up to 19.67%, and the enzyme activity of the mutant E179GD182G was enhanced by up to 18.67%.

In summary, the mutant E179GD182G provided by the present invention has an optimum pH of 4.5 and an optimum temperature of 50°C. Compared with the recombinant wild β-xylosidase JB13GH39P28 with an amino acid sequence as shown in SEQ ID NO.7, the mutant E179GD182G has a change in thermal properties, and its activity is lower at 60°C and inactivated more rapidly, which is conducive to the safe use of enzymes and their application in the field of biotechnology under low temperature environment requirements. And Na ₂ SO ₄ can enhance the enzyme activity and stability of the mutant E179GD182G, and its activity can be increased to 149.82% at most. The thermally unstable β-xylosidase mutant E179GD182G provided by the present invention can be used in the food, winemaking, washing and other industries, and is particularly suitable for the field of biotechnology under low temperature environment requirements.

Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended 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.