CN104130988B - 1,3-1,4-Beta-glucanase mutant - Google Patents

Abstract

The invention discloses a 1,3-1,4-β-glucanase mutant and belongs to the fields of genetic engineering and enzyme engineering. The present invention mutates the lysines at positions 20, 117 and 165 of 1,3-1,4-β-glucanase derived from Bacillus terquilensis by overlapping extension PCR For serine, single mutants K20S, K117S and K165S were obtained, respectively. The three mutation sites were integrated and mutated to obtain the K20S/K117S/K165S triple mutant enzyme. The four mutant enzymes showed higher catalytic activity and better thermal stability. Compared with wild enzymes, these mutant enzymes are more favorable for their industrial application.

Description

A 1,3-1,4-β-glucanase mutant

technical field

The present invention relates to a 1,3-1,4-β-glucanase mutant, especially a 1,3-1,4-β-glucanase with higher catalytic activity and thermostability A mutant belongs to the fields of genetic engineering and enzyme engineering.

Background technique

β-glucan is a kind of non-starch polysaccharide existing in the cell wall of gramineous plants, and its content is very high in economical grains such as barley, wheat, and rice. It is composed of thousands of β-D-glucose residues arranged linearly through β-1,3 or β-1,4 glycosidic bonds, and has a very high molecular weight. It can be dissolved in water, and the resulting solution has a high viscosity, which brings many disadvantages to the beer industry and the feed industry. Malt, the main raw material of the beer industry, contains a large amount of β-glucan. The presence of undegraded β-glucan in the wort will cause the viscosity of the wort to be too high, resulting in difficulty in filtration, prolonging the filtration time of wort mash, and reducing the extract. Content, for the finished beer will affect its non-biological stability. When producing pure draft beer, too much β-glucanase will cause the membrane pores of the filter membrane to be clogged and the filtration capacity to decrease. In the feed industry, the β-glucan in wheat feed cannot be directly digested and absorbed in the intestines of humans or animals, and must undergo enzymatic degradation before it can be utilized, and it prevents the active ingredients from The absorption in the intestinal tract of animals reduces the conversion rate of active ingredients in the feed. It is an anti-nutritional factor and provides rich nutrition for the colonization and reproduction of microorganisms, especially pathogenic bacteria, causing a large number of harmful microorganisms to multiply in the intestinal tract of animals, causing Livestock and poultry have diarrhea, and at the same time, they will consume a large amount of substances competitively and reduce feed utilization.

1,3-1,4-β-glucanase derived from Bacillus terquilensis CGX5-1, referred to as β-glucanase. 1,3-1,4-β-glucanase is a class of β-glucans that can specifically cleave β-glucans into trisaccharides and tetrasaccharides at the 3-O-glucopyranose site, which is its industrial application Base. In the process of wort saccharification in the beer industry, the temperature is increased from 48°C to 78°C, while the baking temperature in the feed industry is also above 65°C. However, the optimum temperature of most wild 1,3-1,4-β-glucanase screened at present is mainly concentrated at 45°C and 55°C, which cannot meet the needs of industry. The low catalytic activity is also an important reason why it cannot be popularized and used. At present, the β-glucanase preparations developed and produced by several enzyme preparation companies such as Novozymes and DMS in the market are expensive, and at present, some domestic enzyme preparation companies produce β-glucanase preparations, but their level is far lower than that of Foreign enzyme preparation companies, and the production strains and processes of β-glucanase preparations are commercial secrets, and the domestic research and development speed is slow, so the domestic market still largely relies on imports. Therefore, if the catalytic activity and thermostability of wild-type 1,3-1,4-β-glucanase can be improved, and a highly active and highly thermostable β-glucanase can be obtained, then the cost can be reduced and its Applications in industry.

In order to improve the thermal stability of 1,3-1,4-β-glucanase, some studies have been carried out at home and abroad. Current studies have found that the only pair of disulfide bonds in 1,3-1,4-β-glucanase has little effect on protein thermal stability. Mutations of Gln1, Thr2, Ser5 and Phe7 greatly improved the thermostability of β-glucanase. The hybridization results of β-glucanase showed that the hybridase H obtained by replacing the first 16 amino acids from B. macerans with the first 16 amino acids from B. amyloliquefaciens (A16-M) and the first 12 amino acids between the two are replaced, or the hybrid enzyme H(A12-M)-△13 formed by deleting Tyr13 has good stability at high temperature.

The optimum temperature of the wild 1,3-1,4-β-glucanase derived from Bacillus tequilensis (B.terquilensis) CGX5-1 used in the present invention is 45°C, and the specific activity is 2490.1U/ mg, and cannot meet the requirements of industrial applications. Therefore, it is of great significance to further improve the catalytic activity and thermal stability of the enzyme for its industrial application and promotion.

Contents of the invention

The purpose of the present invention is to provide a 1,3-1,4-β-glucanase mutant, especially a 1,3-1,4-β-glucanase mutant with higher catalytic activity and thermostability Glycanase mutants.

The amino acid sequence of the mutant is shown in (1) or (2) or (3) or (4) or (5) below:

(1) the amino acid sequence shown in SEQ ID NO.1,

(2) the amino acid sequence shown in SEQ ID NO.2,

(3) the amino acid sequence shown in SEQ ID NO.3,

(4) the amino acid sequence shown in SEQ ID NO.4,

(5) Based on the amino acid sequence of (1) or (2) or (3) or (4), one or several amino acid substitutions, deletions or additions have been carried out, and have 1,3-1,4-β-glucan Amino acid sequence of carbohydrase activity.

The nucleotide sequence encoding the above mutant is preferably the nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.

Amino acid sequence such as the mutant of SEQ ID NO.1 is the lysine Lys at the 20th position of the wild 1,3-1,4-β-glucanase shown in SEQ ID NO.9 as the nucleotide sequence Mutated to serine Ser. The resulting mutant was named K20S.

Amino acid sequence such as the mutant of SEQ ID NO.2 is the 117th lysine Lys of the wild 1,3-1,4-β-glucanase shown in SEQ ID NO. Mutated to serine Ser. The resulting mutant was named K117S.

Amino acid sequence such as the mutant of SEQ ID NO.3 is the 165th lysine Lys of wild 1,3-1,4-β-glucanase shown in SEQ ID NO. Mutated to serine Ser. The resulting mutant was named K165S.

The mutant whose amino acid sequence is such as SEQ ID NO.4 is the 20th, 117th, and 165th positions of the wild 1,3-1,4-β-glucanase whose nucleotide sequence is shown in SEQ ID NO.9. Both lysine Lys are mutated to serine Ser. The resulting mutant was named K20S/K117S/K165S.

Another object of the present invention is to provide a method for constructing the 1,3-1,4-β-glucanase mutant, which is to introduce the wild enzyme gene (SEQ ID NO .9) as a template, obtained by site-directed mutagenesis by the method of overlap extension PCR; the nucleotide sequences of the genes encoding β-glucanase mutants are obtained as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO .7 or shown in SEQ ID NO.8. After sequencing, it was identified that all mutation sites had been successfully mutated according to the preset target, and then the gene fragment encoding the mutant enzyme was recombined and expressed to obtain mutants.

The recombinant expression preferably uses pET28a(+) as the expression vector and Escherichia coli as the expression host.

For the construction method of the three β-glucanase single mutant enzymes K20S, K117S and K165S, it is preferable to use the wild enzyme gene as a template, and respectively use the lysine Lys at positions 20, 117 and 165 by overlapping extension PCR Mutation to serine Ser.

For the construction method of triple mutant enzyme K20S/K117S/K165S, it is preferable to use K20S single mutant enzyme as a template to mutate lysine Lys at positions 117 and 165 to serine Ser in two steps by overlapping extension PCR.

The third technical problem to be solved by the present invention is to provide a method for fermentatively producing the 1,3-1,4-β-glucanase mutant, preferably using pET28a(+) as the expression vector and Escherichia coli as the expression vector. Host. Using TB liquid medium as the fermentation medium, the recombinant bacteria were cultured at 37°C and 200 rpm until the OD ₆₀₀ was about 1.0, and the expression was induced by adding 0.06 mM final concentration of IPTG and 8 mM final concentration of α-lactose, and cultured at 24°C and 150 rpm for 6 hours. After the obtained bacterial liquid was centrifuged, the bacterial cells were discarded to collect the supernatant. The supernatant was purified by Ni-NTA affinity chromatography column and imidazole was removed by GEPD-10 column to obtain 1,3-1,4-β-glucanase mutant.

The method for activating the recombinant bacteria is preferably to pick a single colony on a plate and place it in LB liquid medium containing 100 μg/mL kanamycin sulfate, and culture it at 200 rpm at 37° C. for 10-12 hours.

Beneficial effects of the present invention: the specific activity of the four β-glucanase mutants provided by the present invention is increased by 98.2% compared with the wild enzyme; the optimum temperature is increased from 45°C to 55-60°C; the T ₅₀ value is increased by 62°C increased to about 76°C; the half-life at 50°C was extended from 95min to 214min, which was much higher than that of the wild enzyme, an increase of 125.3%; the half-life at 60°C was extended from 32.5min to 59min at most, an increase up 81.5%. It can be seen that the catalytic activity and thermal stability of the mutant enzyme are greatly improved, which is more conducive to industrial application.

Description of drawings

Figure 1 Comparison of optimum temperature between wild enzyme and four β-glucanase mutants; □: wild enzyme, ○: K20S, △: K117S, ▽: K165S, ◇: K20S/K117S/K165S.

Figure 2 Comparison of T ₅₀ values between wild enzyme and four β-glucanase mutants; □: wild enzyme, ○: K20S, △: K117S, ▽: K165S, ◇: K20S/K117S/K165S.

Figure 3 Comparison of the half-life of wild enzyme and four β-glucanase mutants at 50°C; □: wild enzyme, ○: K20S, △: K117S, ▽: K165S, ◇: K20S/K117S/K165S.

Figure 4 Comparison of the half-life of four β-glucanase mutants from wild fish at 60°C; □: wild enzyme, ○: K20S, △: K117S, ▽: K165S, ◇: K20S/K117S/K165S.

detailed description

Example 1 Selection of mutation sites

Submit the deduced amino acid sequence of β-glucanase to the I-TASSER online server for homology modeling, and input the PDB file obtained from the modeling into Voronoia software to calculate the position of lysine in the structure. It can be seen from Table 1 that in the three-dimensional structure of β-glucanase, the average packing values of all 12 lysines are between 0.47 and 0.62, all of which are on the surface of the protein and have the highest solution accessibility. This means that lysine located on the surface of the protein may have an effect on the thermostability of β-glucanase.

Table 1 The average packing density of 12 lysines in β-glucanase

Using the genome of Bacillus tequila CGX5-1 as a template, the bglt gene encoding β-glucanase was amplified by PCR, and its nucleotide sequence was shown in SEQ ID NO.9. Using the overlap extension PCR technique and using the expression vector pET28a(+)-bglt as a template, the 12 sites in Table 1 were mutated to serine respectively.

The deduced amino acid sequences of twelve mutant strains were submitted to the I-TASSER online server for homology modeling. And input the PDB file obtained by modeling into Gromacs software to calculate its total energy. In order to determine its catalytic properties, 12 mutant enzymes were recombinantly expressed in Escherichia coli. It can be seen from Table 2 that the specific activities of K20S, K117S and K165S have been greatly improved compared with wild enzymes, while the specific activities of the other nine mutant enzymes have decreased to a certain extent. For the total energy, except K58S, K80S and K214S mutant enzymes, the total energy of the other mutants were lower than the wild enzyme. The lower the total energy, the better the thermal stability of the enzyme. Combining the above two points, three mutant strains K20S, K117S and K165S were selected for the determination of subsequent properties. However, the specific activity of the K20S/K117S/K165S triple mutant enzyme obtained by mutating the three sites at the same time has been greatly improved compared with the wild enzyme, and the total energy is also lower than the wild enzyme, which indicates that it may also have Better thermal stability.

Table 2 Comparison of specific activity and total energy between wild enzyme and mutant enzyme

Example 2 Preparation of β-glucanase mutants K20S, K117S, K165S and K20S/K117S/K165S

(1) Site-directed mutation

1) Construction of vector pET28a(+)-bglt

Using the genome of Bacillus tequila CGX5-1 as a template, the bglt gene was amplified by PCR. Primers are as follows:

Forward primer: 5'-C GGATCC ATGAAACGAGTGTTGCTAATT-3', the underline is the restriction site of BamHI,

Reverse primer: 5'-T CTCGAG gTATTTTTTTGTATAGCGCAC-3', the underline is the XhoI restriction site, and the lowercase letter is the mutation site;

The PCR reaction system is: 5U/μL rTaq 1μL, 10×rTaq Buffer 5μL, 2.5mM dNTPs 4μL, 100μM forward primer 1μL, 100μM reverse primer 1μL, second-step PCR product 20μL, add double distilled water to make up to 50μL;

PCR reaction amplification conditions: pre-denaturation at 94°C for 5 minutes; followed by 35 cycles of 94°C for 1min, 56°C for 50s, and 72°C for 50s;

The PCR amplified product was recovered by gel cutting, and then double-digested with restriction endonucleases BanHI and XhoI, then ligated with the pET28a(+) plasmid digested with the same restriction enzymes, and transformed into Escherichia coli BL21( DE3) in competent cells. The resulting recombinant plasmid pET28a(+)-bglt was used as a template for site-directed mutagenesis studies. The nucleotide sequence of the bglt gene is shown in SEQ ID NO.9 (including the signal peptide sequence).

2) Site-directed mutation of single mutant enzymes K20S, K117S and K165S; using overlap extension PCR technology, using the expression vector pET28a(+)-bglt as a template,

The primers for site-directed mutagenesis introducing the K20S codon are:

Forward primer: 5'-GGTTTTTGGCAA AGT GCAGATGGTTATT-3', the underline is the mutant base,

Reverse primer: 5'-AATAACCATCTGC ACT TTGCCAAAAACC-3', underlined is the mutated base; the primer for site-directed mutagenesis introducing the K117S codon is:

Forward primer: 5'-AAAAGACACAACG AGT GTTCAATTTAAC-3', the underline is the mutant base,

Reverse primer: 5'-AGTTAAATTGAAC ACT CGTTGTGTCTTTT-3', the underline is the mutation base; the site-directed mutation primer introducing the K165S codon is:

Forward primer: 5'-ACGGGCAATTA AGT CATACTGCAACA-3', the underline is the mutant base,

Reverse primer: 5'-TGTTGCAGTATG ACT TAATTGCCCGTCGA-3', the underline is the mutant base;

3) Site-directed mutation of the triple mutant enzyme K20S/K117S/K165S: using the overlap extension PCR method, using the single mutant enzyme K20S gene as a template, the primers for site-directed mutation introducing the K117S codon are:

Forward primer: 5'-AAAAGACACAACG AGT GTTCAATTTAAC-3', the underline is the mutant base,

Reverse primer: 5'-AGTTAAATTGAAC ACT CGTTGTGTCTTTT-3', the underline is the mutant base; then use the K20S/K117S double mutant enzyme gene as a template to introduce K165S site-directed mutation primers:

Forward primer: 5'-ACGGGCAATTA AGT CATACTGCAACA-3', the underline is the mutant base,

Reverse primer: 5'-TGTTGCAGTATG ACT TAATTGCCCGTCGA-3', the underline is the mutant base;

Overlap extension PCR is divided into three steps, and the specific implementation conditions of the three steps are as follows:

The first step PCR reaction system is: 2×PrimeSTAR max premix 25 μL, 100 μM forward primer 1 μL, 100 μM reverse primer 1 μL, template DNA 1 μL, double distilled water to make up to 50 μL;

The amplification conditions of the first step PCR reaction: pre-denaturation at 94°C for 5 minutes; followed by 30 cycles of 94°C for 1min, 56°C for 50s, and 72°C for 50s; and finally stored at 4°C.

The second-step PCR reaction system is: 2×PrimeSTAR max premix 10 μL, the first-step PCR product 14 μL, the first-step PCR product 2 4 μL, double-distilled water to make up to 20 μL;

The amplification conditions of the second-step PCR reaction: pre-denaturation at 94°C for 5 minutes; followed by 15 cycles of 94°C for 1min, 56°C for 50s, and 72°C for 50s; and finally stored at 4°C.

The third-step PCR reaction system is: 5U/μL rTaq 1μL, 10×rTaq Buffer 5μL, 2.5mM dNTPs 4μL, 100μM forward primer 1μL, 100μM reverse primer 1μL, the second-step PCR product 20μL, add double distilled water to make up to 50 μL;

The third step of PCR reaction amplification conditions: pre-denaturation at 94°C for 5min; followed by 35 cycles of 94°C for 1min, 56°C for 50s, and 72°C for 50s; and finally stored at 4°C.

The above-mentioned fragments amplified by PCR were double-digested with restriction endonucleases BanHI and XhoI, then ligated with the pET28a(+) plasmid digested with the same restriction enzymes, and transformed into Escherichia coli BL21( DE3) in competent cells.

(2) Expression and purification of mutants

Pick a single colony of Escherichia coli containing the above-mentioned recombinant plasmid on the plate and place it in LB liquid medium containing 100 μg/mL kanamycin sulfate, culture it at 200 rpm at 37°C for 10-12 hours, and transfer it to a medium containing 100 μg/mL TB broth in kanamycin sulfate. The recombinant bacteria were cultured at 200 rpm at 37°C until the OD ₆₀₀ was about 1.0, and the expression was induced by adding 0.06mM final concentration of IPTG and 8mM final concentration of α-lactose, and cultured at 24°C at 150rpm for 6h. Centrifuge the expressed bacterial solution at 4°C and 9000rpm for 20min, discard the bacterial cells and collect the supernatant. Add the obtained supernatant to a Ni-NTA affinity chromatography column, and use 1×BindingBuffer to elute until the absorbance value is stable, and then add 50mM, 100mM and 250mM imidazole solutions at final concentrations to elute the target protein.

Through enzyme activity determination and SDS-PAGE analysis, it was found that the mutant enzyme mainly appeared in the eluate of 100mM imidazole, and the band was single. Pass the eluate containing the target protein through a GEPD-10 desalting column, and use 20 mM phosphate buffer (pH6.5) to wash down the target protein. Then use protein ultrafiltration centrifuge tubes to concentrate to obtain single mutant enzymes K20S, K117S, K165 and triple mutant enzymes K20S/K117S/K165S products.

Example 3 Analysis of Enzyme Activity and Protein Concentration

(1) Enzyme activity assay method:

3,5-Dinitrosalicylic acid (DNS) method combined with improved AZO assay method for the determination of β-glucanase activity:

Enzyme activity definition: 1 mL of enzyme solution under the conditions of 40°C and pH value of 6.5, the amount of hydrolyzing β-glucan per minute to produce glucose reducing substances equivalent to 1 μmol is 1 enzyme activity unit, expressed in U/mL.

Determination of the enzyme activity of the fermentation broth: after the fermentation broth is centrifuged, the supernatant is diluted to an appropriate multiple to measure its enzyme activity.

Drawing of glucose standard curve: draw 1% glucose standard solution 2.0, 3.0, 4.0, 5.0, 6.0mL respectively into 50mL volumetric flask, dilute to the mark with distilled water, and make each milliliter contain glucose 200, 400, 600, 800 , 1000, 1200μg dilute standard solution. Take 0.5mL of dilute standard solutions of different concentrations in test tubes, add 1.5mL of pH 6.5 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, then add 3.0mL of DNS reagent, boil in a boiling water bath for 7min, take it out and cool it down quickly After reaching room temperature, add 10 mL of distilled water and shake well. Use 0.5mL of distilled water instead of dilute glucose standard solution as a control, and use a 10mm cuvette to measure its absorbance with a spectrophotometer at a wavelength of 550nm. With the absorbance as the ordinate and the corresponding glucose concentration as the abscissa, draw a standard curve.

Determination of sample enzyme activity: Accurately draw 0.5mL of the diluted enzyme solution to be tested (3 parallel test tubes for each sample), and 1.0mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 6.5, and preheat in a water bath at 40°C for 5 minutes , add 0.5mL of preheated 1.0% β-glucan solution, start timing immediately, react accurately in 40°C water bath for 10min, immediately add 3.0mlDNS solution to stop the reaction, then put it in boiling water bath for 7min, take it out and cool it quickly, then add 10mL of deionized water, shake well, measure the absorbance of the reaction solution at 550nm. At the same time, carry out the blank control measurement, the steps are to draw 0.5mL of the diluted enzyme solution to be tested, add 1.0mL of pH5.0 disodium hydrogen phosphate-citric acid buffer solution, then first add 3.0mL DNS solution to inactivate the enzyme, and pre-heat in a water bath at 40℃. heat, then add 0.5mL of the same preheated 1.0% β-glucan solution, bathe in water at 40°C for 10min, then place in a boiling water bath for 7min, the following steps are the same as the sample measurement, the absorbance value is obtained from the sample measurement, according to the standard The corresponding enzyme activity units can be obtained from the curve.

(2) Determination of protein concentration:

Method for determining protein concentration in solution by Bradford method:

Take 200μL of the sample to be tested and add 2mL of Bradford reagent, after mixing, quickly measure the absorbance value at 595nm, and the blank is pH6.5 phosphate buffer. Each group of samples has three parallels, and the obtained absorbance value is compared with the standard curve equation y=0.0042x+0.0082 to obtain the protein concentration in the solution.

(3) Comparison of specific activity: the experimental results are listed in Table 3. Comparing wild enzyme preparations with mutant enzyme preparations, it can be found that the specific activities of the four mutant enzymes are greatly improved compared with wild enzymes. Among them, the specific enzyme activities of K20S, K117S and K165S single mutant enzymes reached 4675.1U/mg, which were respectively increased by 88.3%, 59.6% and 84.1% compared with wild enzymes. The specific enzyme activity of the triple mutant enzyme K20S/K117S/K165S reached 4936.4U/mg, which was 98.2% higher than that of the wild enzyme. Obviously, the specific enzyme activity of the three single-mutant enzymes and one triple-mutant enzyme has been greatly improved compared with the wild enzyme.

Table 3 Comparison of specific enzyme activity between wild enzyme and four β-glucanase mutants

The thermostability of embodiment 4 wild enzyme and mutant enzyme

(1) Optimum temperature determination method for wild enzyme and mutant enzyme:

Take 100 μL of the obtained enzyme preparation, and measure the enzyme activity at different temperatures (35, 40, 45, 50, 55, 60, 65, 70° C.). The maximum value of enzyme activity is taken as 100% relative enzyme activity, and the percentage obtained by dividing the enzyme activity value at other temperatures by the maximum value is the relative enzyme activity at this temperature. See Figure 1 for the optimum temperature of wild enzyme and mutant enzyme. It can be seen from Figure 1 that the optimum temperature of the wild enzyme is 45°C, the optimum temperature of K165S reaches 55°C, and the optimum temperature of K20S, K117S and K20S/K117S/K165S reaches 60°C.

(2) Determination method of _T50 value of wild enzyme and mutant enzyme:

Take 2 mL of the obtained enzyme product, treat it at different temperatures (40, 45, 50, 55, 60, 65, 70, 75, 80°C) for 15 minutes, take it out immediately and place it on ice for 10 minutes, take 100 μL to measure β-glucan Enzyme activity. The maximum value of enzyme activity is taken as 100% relative enzyme activity, and the percentage obtained by dividing the enzyme activity value at other temperatures by the maximum value is the relative enzyme activity at this temperature. The T ₅₀ value is defined as the temperature at which the enzyme activity is reduced to half of the initial enzyme activity after the above treatment. It can be seen from Figure 2 that the inactivation curves of the mutant enzymes are all gentler than those of the wild enzymes. The T ₅₀ value of the wild enzyme was 62°C, the T ₅₀ values of the K20S, K117S and K165S single mutant enzymes were 76°C, 76°C and 75°C, respectively, and the T ₅₀ values of the triple mutant enzymes K20S/K117S/K165S were 76°C. The T ₅₀ values of the mutant enzymes were higher than those of the wild enzymes.

(3) Method for determining the half-life of wild enzyme and mutant enzyme at 50°C:

Take 2 mL of the obtained enzyme product, treat it at 50°C for different time (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240 min), take it out immediately and place it on ice for 10 min, After diluting the treatment solution to a reasonable multiple, take 100 μL to measure the activity of β-glucanase. The enzyme activity value of the fermentation broth before treatment is taken as 100% relative enzyme activity, and the percentage obtained by dividing the enzyme activity value under different treatment times by the maximum value is the relative enzyme activity under this condition. The half-life is defined as the time required for the enzyme to lose half of its activity after the above treatment. It can be seen from Figure 3 that the inactivation curve of the mutant enzyme at 50°C is relatively flat compared with that of the wild enzyme. The half-life of the wild enzyme at 50°C is 95 minutes, while the half-lives of the three single mutant enzymes K20S, K117S and K165S reach 149 minutes, 210 minutes and 102 minutes respectively, and the half-lives of the triple mutant enzymes K20S/K117S/K165S reach 214 minutes, far 125.3% higher than wild enzyme.

(4) Determination of half-life of wild enzyme and mutant enzyme at 60°C:

Take 2mL of the obtained enzyme product, treat it at 60°C for different times (10, 20, 30, 40, 50, 60, 70min), take it out immediately and place it on ice to cool for 10min, dilute the treatment solution by a reasonable multiple and take 100μL to measure β - Glucanase activity. The enzyme activity value of the fermentation broth before treatment is taken as 100% relative enzyme activity, and the percentage obtained by dividing the enzyme activity value under different treatment times by the maximum value is the relative enzyme activity under this condition. It can be seen from Figure 4 that the inactivation curve of the mutant enzyme at 60°C is more gentle than that of the wild enzyme. The half-life of the wild enzyme at 60°C was determined to be 32.5min, while the half-lives of the three single mutant enzymes K20S, K117S and K165S reached 46min, 58min and 37min, respectively. The half-life of the triple mutant enzyme K20S/K117S/K165S reached 59min, which was 81.5% higher than that of the wild enzyme.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can, without departing from the spirit and scope of the present invention, go through an amino acid sequence based on the present invention. Alternatively, several amino acid substitutions, deletions or additions result in an amino acid sequence having 1,3-1,4-β-glucanase activity. Therefore, the protection scope of the present invention should be defined by the claims.

Claims (10)

1. one kind 1,3-1,4- beta glucan enzyme mutant is it is characterised in that the aminoacid sequence of described mutant for example following (1) Or shown in (2) or (3) or (4)：

(1) aminoacid sequence shown in SEQ ID NO.1,

(2) aminoacid sequence shown in SEQ ID NO.2,

(3) aminoacid sequence shown in SEQ ID NO.3,

(4) aminoacid sequence shown in SEQ ID NO.4.

2. the gene of mutant described in coding claim 1.

3. carry carrier or the reconstitution cell of gene described in claim 2.

4. a kind of method obtaining 1,3-1,4- beta glucan enzyme mutant described in claim 1 is it is characterised in that be with wild The gene of 1,3-1,4- 1,4 beta-glucanase is template, carries out rite-directed mutagenesises using the method for Overlap extension PCR and obtains encoding mutant The genetic fragment of encoding mutant body is subsequently carried out recombinant expressed acquisition mutant by the gene of body.

5. method according to claim 4 it is characterised in that for aminoacid sequence respectively as SEQ ID NO.1, The structure of 1,4 beta-glucanase single mutant K20S, K117S and the K165S shown in SEQID NO.2, SEQ ID NO.3, with wild Enzyme gene is template, respectively by Overlap extension PCR, the lysine of the 20th, 117 and 165 is sported serine.

6. method according to claim 4 it is characterised in that for aminoacid sequence as shown in SEQ ID NO.4 three The lysine of the 20th, 117 and 165 is successively passed through Overlap extension PCR by the structure of mutant K20S/K117S/K165S Sport serine.

7. according to the arbitrary described method of claim 4-6 it is characterised in that with pET28a (+) as expression vector, with large intestine bar Bacterium is expressive host.

8. method according to claim 7 is it is characterised in that with TB fluid medium as fermentation medium, recombinant bacterium exists Cultivate to OD for 37 DEG C₆₀₀=0.8-1.0, adds IPTG and alpha-lactose abduction delivering, abandons thalline after gained bacterium solution is centrifuged, and collects Supernatant containing 1,3-1,4- beta glucan enzyme mutant.

9. method according to claim 8 it is characterised in that supernatant through Ni-NTA affinity chromatograph column purification and Imidazoles sloughed by GEPD-10 post, obtains 1,3-1,4- beta glucan enzyme mutant.

10. application in Beer Production for the mutant described in claim 1.