Beta-1,3-glucanase mutant n54w, gene and use thereof

Abstract

The present application relates to the field of agricultural biotechnology, and particularly relates to a beta-1,3-glucanase mutant N54W and a gene and application thereof. The amino acid sequence of the beta-1,3-glucanase N54W mutant is shown as SEQ ID NO:2. Compared with the wild-type beta-1,3-glucanase, the specific activity of the beta-1,3-glucanase mutant is increased by 3.6 times, reaches 902.1 U / mg, the catalytic activity is greatly improved, k cat / K m reaches 888.9 ml·mg ‑1 ·min ‑1 , reaches 5.5 times of the wild type. The present application provides the application of the beta-1,3-glucanase mutant with high specific activity, and the beta-1,3-glucanase mutant can be particularly applied in brewing, food and feed fields, and has a very broad application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, in particular to a beta-1,3-glucanase mutant N54W and its gene and application. BACKGROUND

[0002] Fungal contamination is an important problem in the food industry, because they can not only cause food to spoil and produce mycotoxins, but also indirectly cause human disease risk, so it is urgent to prevent and control fungal contamination. The main components of fungal cell wall are glycoprotein and polysaccharide, among which polysaccharide is mainly beta-1,3-glucan and beta-1,6-glucan, and polysaccharides such as glucan and chitin have structural functions, providing tough and rigid properties for the cell wall. Beta-1,3-glucanase is a class of enzymes that can hydrolyze beta-1,3-glucan linked by beta-1,3-glucoside bond, which can directly degrade the cell wall of pathogenic fungi, thereby achieving the effect of inhibiting fungal growth. Therefore, reasonable development and utilization of beta-1,3-glucanase has great significance for inhibiting feed mold and food preservation, and is also crucial for application in the fields of brewing, production of oligosaccharides, and degradation of biomass waste.

[0003] At present, for food contaminated by pathogenic fungi, methods such as heat treatment, ozone irradiation and application of chemical drugs can be selected to solve the problem, but these technologies also have safety problems, such as whether high ozone concentration or strong radiation treatment has adverse effects on human health, and the changes of food nutritional quality or sensory quality need to be verified. The use of enzyme preparations is becoming a trusted strategy for fungal control. Compared with traditional chemical methods of hydrolyzing fungal cell wall, enzyme degradation is a non-toxic green natural method with obvious advantages. However, the specific activity of beta-1,3-glucanase on the market is low, and the effect of degrading cell wall is poor, so the means of protein engineering is used to improve the catalytic activity of beta-1,3-glucanase to obtain mutant that can meet the application of efficient degradation of fungi and reduce the problem of fungal contamination in food industry, and reduce the production cost of enterprises. SUMMARY

[0004] The purpose of the present application is to provide a mutant obtained by point mutation of beta-1,3-glucanase derived from Streptomyces sp. S27 as a parent.

[0005] Another purpose of the present application is to provide a gene encoding the mutant.

[0006] Another purpose of the present application is to provide a recombinant vector comprising the mutant gene.

[0007] Another purpose of the present application is to provide a recombinant strain comprising the mutant gene.

[0008] Still another object of the present application is to provide an application of the mutant.

[0009] Still another object of the present application is to provide a method of preparing a β-1,3-glucanase having high specific activity.

[0010] According to the detailed embodiment of the present application, a wild-type β-1,3-glucanase having an amino acid sequence as shown in SEQ ID NO: 1 is subjected to site-directed mutagenesis.

[0011] SEQ ID NO: 1

[0012] GPAGETAGRTVQKAAQGAEAAPAAVLFEENFDGPAGSAVDSRRWQLETGDNSGN

[0013] NHERQYYTPGNANAALDGNGNLVITARKENPGNYQCWYGRCEYTSARMNTAGK

[0014] FTTTYGHIEARMKLPRGQGMWPAFWMLGHDIGSVGWTNSGEIDIMENVGYEPGT

[0015] VHGTLHGPGYSGGEGIGAGYTLPGGRAFADDFHTFAVDWSPNSITWSVDGQVYQ

[0016] RRTPADLGGDRWVFDKPFFLILNLAVGGDWPGLPDSSTVFPQKLVVDYVRVTSGG

[0017] DSGGGGGGRTGTITGLAGKCLDVAWADTANGTPVQIHDCNGNAAQQWTVGTDG

[0018] TIRALGKCLDVSGAGKADGTPVQIWDCNGTAAQQWVVTGARDIVNPNADKCLDVRDNNSANGTKTQIWTCSGTANQKWNTP.

[0019] According to the detailed embodiment of the present application, a wild-type β-1,3-glucanase having an amino acid sequence as shown in SEQ ID NO: 1 is subjected to site-directed mutagenesis.

[0020] The β-1,3-glucanase mutant having high specific activity according to the present application has an amino acid sequence as shown in SEQ ID NO: 2, which consists of 404 amino acids.

[0021] SEQ ID NO:2:

[0022] GPAGETAGRTVQKAAQGAEAAPAAVLFEENFDGPAGSAVDSRRWQLETGDNSGW

[0023] NHERQYYTPGNANAALDGNGNLVITARKENPGNYQCWYGRCEYTSARMNTAGK

[0024] FTTTYGHIEARMKLPRGQGMWPAFWMLGHDIGSVGWTNSGEIDIMENVGYEPGT

[0025] VHGTLHGPGYSGGEGIGAGYTLPGGRAFADDFHTFAVDWSPNSITWSVDGQVYQ

[0026] RRTPADLGGDRWVFDKPFFLILNLAVGGDWPGLPDSSTVFPQKLVVDYVRVTSGG

[0027] DSGGGGGGRTGTITGLAGKCLDVAWADTANGTPVQIHDCNGNAAQQWTVGTDG

[0028] TIRALGKCLDVSGAGKADGTPVQIWDCNGTAAQQWVVTGARDIVNPNADKCLDVRDNNSANGTKTQIWTCSGTANQKWNTP.

[0029] According to the specific embodiment of the present application, a gene encoding the above-mentioned β-1, 3-glucanase mutant with high specific activity is also provided, and the nucleotide sequence is shown in SEQ ID NO:3, which is 1119 bp in total.

[0030] GGCCCCGCCGGTTCGGCGGTCGACAGCCGCAGATGGCAGCTGGAGACCGGCG

[0031] ACAACAGCGGCAACAACCACGAGCGGCAGTACTACACGCCGGGCAACGCCAA

[0032] CGCCGCGCTCGACGGCAACGGCAACCTGGTGATCACCGCCCGCAAGGAGAAC

[0033] CGGGGCAACTACCAGTGCTGGTACGGGCGTTGCGAGTACACCTCGGCCCGTAT

[0034] GAACACCGCGGGGAAGTTCACCACCACCTACGGCCACATCGAGGCCCGGATGA

[0035] AGCTCCCGCGCGGACAGGGCATGTGGCCCGCGTTCTGGATGCTCGGCCACGAC

[0036] ATCGGCAGCGTCGGCTGGACCAACAGCGGCGAGATCGACATCATGGAGAACGT

[0037] CGGCTACGAGCCCGGCACCGTGCACGGCACCCTGCACGGCCCCGGATACTCCG

[0038] GCGGCGAGGGCATCGGCGCCGGTTACACCCTCCCCGGCGGCCGGGCGTTCGCC

[0039] GACGACTTCCACACCTTCGCCGTCGACTGGTCCCCGAACTCCATCACCTGGTC

[0040] GGTCGACGGCCAGGTCTACCAGCGCCGCACCCCGGCCGACCTGGGCGGCGAC

[0041] CGCTGGGTGTTCGACAAGCCGTTCTTCCTGATCCTCAACCTCGCGGTGGGCGG

[0042] CGACTGGCCGGGCCTGCCGGACTCGAGCACCGTCTTCCCGCAGAAGCTCGTCG

[0043] TCGACTACGTCCGCGTCACCAGCGGCGGCGACAGCGGCGGCGGTGGCGGCGG

[0044] GCGCACCGGCACCATCACCGGTCTGGCGGGCAAGTGCCTGGACGTGGCCTGG

[0045] GCGGACACCGCCAACGGCACCCCCGTGCAGATCCACGACTGCAACGGCAACG

[0046] CCGCCCAGCAGTGGACCGTCGGCACGGACGGCACCATACGGGCGCTCGGCAA

[0047] GTGCCTGGACGTCTCCGGCGCGGGCAAGGCGGACGGCACCCCCGTCCAGATCT

[0048] GGGACTGCAACGGCACGGCCGCCCAGCAGTGGGTCGTCACCGGGGCCCGGGA

[0049] CATCGTCAACCCCAACGCCGACAAGTGCCTGGACGTGAGGGACAACAACTCC

[0050] GCCAACGGCACGAAGACGCAGATCTGGACCTGCTCGGGCACCGCCAACCAGAAGTGGAACACGCCCTGA。

[0051] According to the specific embodiment of the present application, the recombinant expression vector containing the mutant gene of β-1, 3-glucanase is also provided, and the starting vector of the recombinant expression vector is specifically pET-22b(+).

[0052] According to the specific embodiment of the present application, the recombinant strain containing the mutant gene of β-1, 3-glucanase is also provided, and the starting strain of the recombinant strain is specifically E. coli BL21 (DE3).

[0053] The method for preparing β-1, 3-glucanase with high heat resistance according to the present application comprises the following steps:

[0054] 1) preparing a recombinant vector containing the mutant gene;

[0055] 2) transforming a host with the recombinant vector;

[0056] 3) fermenting and culturing the host, and separating the β-1, 3-glucanase.

[0057] Compared with the wild-type β-1, 3-glucanase, the specific activity of the mutant β-1, 3-glucanase is increased by 3.6 times, reaching 902.1 U / mg, and the catalytic activity is greatly improved, k cat / K m reaching 888.9 ml·mg -1 ·min -1, which is 5.5 times of the wild type. The application of the mutant β-1, 3-glucanase with high specific activity is provided, and can be applied in the fields of brewing, food and feed, and has a very wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 SDS-PAGE electrophoresis detection results of the wild type and the mutant of β-1, 3-glucanase after expression in E. coli BL21 (DE3) are shown;

[0059] Figure 2 Optimal action temperature of the wild type and the mutant of β-1, 3-glucanase after purification is shown;

[0060] Figure 3 Optimal pH of the wild type and the mutant of β-1, 3-glucanase after purification is shown;

[0061] Figure 4 pH stability of the wild type and the mutant of β-1, 3-glucanase after purification is shown;

[0062] Figure 5 Thermal stability of the wild type and the mutant of β-1, 3-glucanase after purification is shown;

[0063] Figure 6 Catalytic efficiency of the wild type and the mutant of β-1, 3-glucanase after purification is shown. DETAILED DESCRIPTION

[0064] Test materials and reagents

[0065] 1. Strains and vectors: expression host E. coli BL21 (DE3), expression plasmid vector pET-22b (+).

[0066] 2. Enzymes and other biochemical reagents: endonuclease, ligase, substrate soluble starch;

[0067] 3. E. coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCL, pH natural).

[0068] Description: In the following examples, the molecular biology experimental methods not specifically described are carried out according to the specific methods listed in the book of J. Sambrook, Molecular Cloning Experiment Guide (third edition), or according to the reagent kit and product instructions.

[0069] Example 1, preparation of recombinant strain BL21 (pET-22b (+) -bgls27)

[0070] 1. Construction of recombinant strain BL21 (pET-22b (+) -bgls27)

[0071] The recombinant plasmid pET-22b(+)-bgls27 containing the β-1,3-glucanase gene derived from Streptomyces sp. S27 was constructed, and the cloning host Escherichia coli XL10 was transformed to obtain the recombinant Escherichia coli strain X10 (pET-22b(+)-bgls27). The colonies were plated on LB (containing 100 μg / mL Amp) for screening. After the correct plasmid was verified by nucleic acid gel electrophoresis, the colonies were inoculated into 50 mL LB medium and cultured overnight in a shaker (37°C), and the plasmid was extracted using a plasmid miniprep kit. After the plasmid was transformed into the expression host Escherichia coli BL21 (DE3), the recombinant Escherichia coli strain BL21 (pET-22b(+)-bgls27) was obtained.

[0072] Example 2, Preparation of Recombinant Strain BL21 (pET-22b(+)-bgls27-N54W)

[0073] 1. Construction of Recombinant Plasmid pET-22b(+)-bgls27-N54W

[0074] The optimized mutation site was designed to mutate the asparagine at position 54 to tryptophan, and the mutation site was introduced by the method of a point mutation kit and sequenced for verification. Finally, the β-1,3-glucanase mutant plasmid was successfully obtained. The primers used are as follows:

[0075] N54W-F (SEQ ID NO: 1) AACAGCGGCTGGAACCACGAGCGGCAGTACTACA;

[0076] N54W-R (SEQ ID NO: 2) TGGTTCCAGCCGCTGTTGTCGCCGGTCTCCAG;

[0077] 2. Construction of Recombinant Strain BL21 (pET-22b(+)-bgls27-N54W)

[0078] The single colonies with correct sequencing were inoculated into 50 mL LB medium and cultured overnight in a shaker (37°C), and the plasmid was extracted using a plasmid miniprep kit. After the plasmid was transformed into the expression host Escherichia coli BL21 (DE3), the colonies were plated on LB (containing 100 μg / mL Amp) for screening. The recombinant Escherichia coli strain BL21 (pET-22b(+)-bgls27-N54W) was obtained.

[0079] Example 3, Obtaining of β-1,3-glucanase Protein Wild Type BglS27 and Mutant N54W

[0080] 1. Induced expression of protein BglS27 and N54W

[0081] The obtained recombinant expression strains BL21 (pET-22b(+)-BglS27) and BL21 (pET-22b(+)-bgls27-N54W) were inoculated into 50 ml of LB medium for seed culture, and after 16 h of culture at 200 rpm and 37 °C, they were transferred into 400 ml of LB medium at an inoculation amount of 1%, and after 2-4 h of culture at 200 rpm and 37 °C, the bacterial concentration was determined, and the absorbance value at a wavelength of 600 nm was read by an enzyme marker. When the value reached 0.6-0.8, IPTG was added to a final concentration of 1 mM, and the expression was induced at 200 rpm and 25 °C.

[0082] 2. Purification of protein BglS27 and N54W

[0083] After induction, the bacterial solution was centrifuged at 12000 rpm for 10 min, the bacterial cells were collected, and then resuspended with 10 mM Tris-HCl solution (pH 7.6), followed by ultrasonic disruption and centrifugal collection of the supernatant. The protein was purified by nickel affinity chromatography, and the eluent was 1 M imidazole, 20 mM Tris-HCl, and 0.5 M NaCl. The eluent was collected, and SDS-PAGE was performed. The protein purification results of BglS27 and N54W are shown in Figure 1 .

[0084] Example 4: Detection of the optimum temperature of β-1,3-glucanase BglS27 and N54W

[0085] After induction, BglS27 and N54W were purified and the enzyme activity was determined.

[0086] The enzyme activity determination method (DNS (3,5-dinitrosalicylic acid) method): The prepared 10% laminarin was diluted with a buffer solution (0.1 M HAc-NaAc) at pH 5.5 to a final concentration of 1% laminarin solution as a substrate. The measurement system included 450 μL of substrate and 50 μL of appropriately diluted enzyme solution, which was reacted in a water bath at 20, 30, 40, 50, 60, 65, 70, and 80 °C for 10 min. After adding 1.5 mL of DNS reagent to terminate the reaction, it was treated in a boiling water bath for 5 min, quickly cooled to room temperature, and then 250 μL of the mixture was taken and the absorbance value at a wavelength of 540 nm was read by an enzyme marker. Each group of reactions was set with one blank control and three parallels. The results are shown in Figure 2 , and the optimum temperature of BglS27 and N54W is about 65 °C.

[0087] Enzyme activity unit (U) definition: The amount of enzyme required to hydrolyze 1 μmoL of glucose per minute under optimal conditions is one enzyme activity unit.

[0088] Example 5, Determination of the optimum pH of β-1,3-glucanase BglS27 and N54W

[0089] The method of measuring enzyme activity is the same as in Example 4. The prepared 10% soluble starch was diluted to a final concentration of 1% laminarin solution with 0.1 M buffer solution at pH 2 to pH 11 as substrate. Glycine-hydrochloric acid was used at pH 1-2, citric acid-disodium hydrogen phosphate at pH 3-8, Tris-HCl at pH 9-10, and glycine-sodium hydroxide at pH 11-12. The results are shown in Figure 3 The optimum pH of BglS27 and N54W is 5.5 and 5, respectively.

[0090] Example 6, Determination of the pH stability of β-1,3-glucanase BglS27 and N54W

[0091] The enzyme solution was first diluted 5 times with 0.05 M buffer solution at pH 3-11 (citric acid-disodium hydrogen phosphate at pH 3-8, Tris-HCl at pH 9-10, and glycine-sodium hydroxide at pH 11-12), and then incubated at 37°C in a water bath for 1 h before measuring the enzyme activity. The method of measuring enzyme activity is the same as in Example 4. The results are shown in Figure 4 N54W has almost the same stability as BglS27. Both can maintain more than 80% of the activity at pH 5-9.

[0092] Example 6, Determination of the pH stability of β-1,3-glucanase BglS27 and N54W

[0093] 100 μL of the purified enzyme solution was heat-treated at 60°C for 0, 10, 20, 30, and 60 min, respectively, and then cooled on ice. The residual enzyme activity was then measured at the optimum temperature of each enzyme. The measurement system and method are the same as in Example 4. The results are shown in Figure 5 BglS27 only has about 60% of the activity after heat treatment at 60°C for 60 min, and only about 40% of the activity after heat treatment at 80°C for 60 min. N54W is still relatively stable after heat treatment at 80°C for 60 min, with more than 60% of the enzyme activity remaining.

[0094] Example 7, Determination of the catalytic efficiency of β-1,3-glucanase BglS27 and N54W

[0095] Laminarin at concentrations of 0.1, 0.5, 1.0, 2, 3, 4, 5, 6, 7, and 8 mM was prepared with buffer solution at pH 5.5 (0.1 M HAc-NaAc) as substrate. The kinetic parameters were measured at the optimum pH and temperature of each enzyme, respectively. The reaction time was 5 min. The results are shown in Figure 6 and Table 1. The catalytic efficiency of N54W is significantly improved compared with that of BglS27.

[0096] Table 1 Catalytic efficiency

[0097]

[0098] The above examples are only used to explain the technical solutions of the present application, and do not limit the protection scope of the present application.

Claims

1. A β-1,3-glucanase N54W mutant having a high specific activity, characterized in that, The amino acid sequence of the β-1,3-glucanase N54W mutant is shown as SEQ ID NO:

2.

2. A β-1,3-glucanase gene, characterized in that, The β-1,3-glucanase N54W mutant with high specific activity of claim 1.

3. The beta-1,3-glucanase gene according to claim 2, characterized in that, The nucleotide sequence of the β-1,3-glucanase gene is shown as SEQ ID NO:

3.

4. A recombinant vector comprising the β-1,3-glucanase gene of claim 2.

5. A recombinant strain comprising the β-1,3-glucanase gene of claim 2.

6. A method for preparing a β-1,3-glucanase having a high specific activity, characterized in that, The method comprises the following steps: 1) preparing a recombinant vector comprising the β-1,3-glucanase gene of claim 2; 2) transforming a host cell with the recombinant vector obtained in step 1); 3) fermenting the host cell and isolating the β-1,3-glucanase.

7. Use of the β-1,3-glucanase N54W mutant with high specific activity of claim 1 for hydrolyzing starch.

8. Use of the β-1,3-glucanase N54W mutant with high specific activity of claim 1 for inhibiting bacteria in food and feed.

9. A method for increasing the specific activity of a β-1,3-glucanase, characterized in that, The method comprises the following steps: performing N54W point mutation on the wild-type β-1,3-glucanase BglS27 with the amino acid sequence shown as SEQ ID NO: 1.

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