Beta-1,3-glucanase mutants with improved enzyme activity

Abstract

The application discloses a beta-1,3-glucanase mutant with improved enzyme activity, and belongs to the technical field of genetic engineering and enzyme engineering. Q71K , Bc16 G128M , Bc16 N198Q , Bc16 R106K , Bc16 M120L , Bc16 K87R , Bc16 A151T The beta-1,3-glucanase positive mutant obtained by the site-directed mutagenesis of the wild-type beta-1,3-glucanase Bc16 coding gene has an enzyme activity 1.38-1.62 times that of the wild enzyme, and the enzyme activity of the wild-type beta-1,3-glucanase is optimized and improved, so that better use conditions are created for the enzyme in practical application.

Description

Technical Field

[0001] This invention relates to β-1,3-glucanase mutants with enhanced enzyme activity, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] β-1,3-glucanase (EC3.2.1.39), abbreviated as β-1,3-GA, is a class of enzymes that specifically act on the β-1,3-glycosidic bonds in β-glucans and can be used in the production of β-1,3-glucan oligosaccharides. β-1,3-glucanase is widely found in bacteria, fungi, plants, and invertebrates. The molecular weight range, optimal pH, optimal temperature, and other enzymatic properties of β-1,3-GA from different sources vary significantly.

[0003] Currently, β-1,3-GA is mainly studied for the production of β-1,3-glucan oligosaccharides, offering advantages such as high conversion rate, simple process control, mild reaction conditions, and abundant raw materials. However, most β-1,3-glucanase-producing strains isolated from fungi, bacteria, and actinomycetes still exhibit low endonuclease activity and proportions. Therefore, finding β-1,3-glucanases with higher catalytic efficiency and greater safety and stability has become a key strategy for improving the application value of β-1,3-glucan oligosaccharides through efficient enzymatic preparation. Summary of the Invention

[0004] To solve the above problems, the present invention addresses the following issues: Alkalihalobacillus clausii The β-1,3-glucanase gene (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2) with accession number WP_095336276.1 was molecularly modified, and a β-1,3-glucanase mutant with significantly increased enzyme activity was obtained by site-directed mutagenesis.

[0005] The present invention provides a β-1,3-glucanase mutant, which is obtained by mutating one or more of the amino acids at positions 71, 128, 198, 106, 120, 87, and 151 of the β-1,3-glucanase amino acid sequence as shown in SEQ ID NO. 2.

[0006] In one embodiment, the mutant is obtained by mutating the amino acid sequence of β-1,3-glucanase as shown in SEQ ID NO.2, specifically glutamine at position 71, glycine at position 128, aspartic acid at position 198, arginine at position 106, methionine at position 120, lysine at position 87, or alanine at position 151.

[0007] In one embodiment, the mutant is obtained by mutating glutamine at position 71 of β-1,3-glucanase, as shown in SEQ ID NO.2, to lysine, to obtain mutant Q71K, with the amino acid sequence shown in SEQ ID NO.9.

[0008] In one embodiment, the mutant is obtained by mutating glycine at position 128 of β-1,3-glucanase, as shown in SEQ ID NO.2, to methionine, to obtain mutant G128M, with the amino acid sequence shown in SEQ ID NO.4.

[0009] In one embodiment, the mutant is obtained by mutating the 198th aspartic acid of β-1,3-glucanase, as shown in SEQ ID NO.2, to glutamine, to obtain mutant N198Q, with the amino acid sequence shown in SEQ ID NO.5.

[0010] In one embodiment, the mutant is obtained by mutating arginine at position 106 of β-1,3-glucanase, as shown in SEQ ID NO.2, to lysine, to obtain mutant R106K, with the amino acid sequence shown in SEQ ID NO.6.

[0011] In one embodiment, the mutant is obtained by mutating methionine at position 120 of β-1,3-glucanase, as shown in SEQ ID NO.2, to leucine, to obtain mutant M120L, with the amino acid sequence shown in SEQ ID NO.7.

[0012] In one embodiment, the mutant is obtained by mutating lysine at position 87 of β-1,3-glucanase, as shown in SEQ ID NO.2, to arginine, to obtain mutant K87R, with the amino acid sequence shown in SEQ ID NO.8.

[0013] In one embodiment, the mutant is obtained by mutating alanine at position 151 of β-1,3-glucanase, as shown in SEQ ID NO.2, to threonine, to obtain mutant A151T, with the amino acid sequence shown in SEQ ID NO.3.

[0014] The present invention also provides a gene encoding the mutant.

[0015] In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO.10~16.

[0016] The present invention also provides a recombinant plasmid carrying the said gene.

[0017] In one embodiment, the recombinant plasmid includes, but is not limited to, pET series plasmids.

[0018] In one embodiment, the pET series plasmids include pET-28a or pET-22b.

[0019] The present invention also provides recombinant microbial cells expressing the mutant.

[0020] In one embodiment, the recombinant microbial cells include, but are not limited to, bacteria or fungi.

[0021] In one embodiment, the microorganism is Escherichia coli.

[0022] In one embodiment, the *Escherichia coli* uses *Escherichia coli* BL21(DE3) as the host and pET-28a as the vector.

[0023] The present invention also provides a method for improving the activity of β-1,3-glucanase, wherein the method involves mutating the following amino acid sequences in the β-1,3-glucanase gene as shown in SEQ ID NO.2: glutamine at position 71, glycine at position 128, aspartic acid at position 198, arginine at position 106, methionine at position 120, lysine at position 87, or alanine at position 151.

[0024] In one embodiment, the method involves mutating glutamine at position 71 of β-1,3-glucanase, as shown in SEQ ID NO.2, to lysine, or mutating glycine at position 128 to methionine, or mutating aspartic acid at position 198 to glutamine, or mutating arginine at position 106 to lysine, or mutating methionine at position 120 to leucine, or mutating lysine at position 87 to arginine, or mutating alanine at position 151 to threonine.

[0025] The present invention also provides a method for preparing β-1,3-glucan oligosaccharides, wherein the method uses β-glucan as a substrate and utilizes the above-mentioned β-1,3-glucanase mutant to catalyze the generation of β-1,3-glucan oligosaccharides.

[0026] The present invention also provides the application of the β-1,3-glucanase mutant, the above-mentioned gene, the above-mentioned recombinant plasmid, the above-mentioned recombinant microorganism, or the above-mentioned method in the production of β-1,3-glucan oligosaccharides.

[0027] Beneficial effects: This invention is based on Alkalihalobacillus clausiiThe β-1,3-glucanase derived from this invention underwent molecular modification of the parent enzyme. Mutations were made at amino acid positions 71, 128, 198, 106, 120, 87, or 151, resulting in a series of mutants with increased enzyme activity. The β-1,3-glucanase mutants provided by this invention exhibit significantly higher enzyme activity compared to the wild-type enzyme. The mutant enzymes maintained a suitable pH and achieved activities of 1411.05 U / mg, 1438.09 U / mg, 1467.15 U / mg, 1500.72 U / mg, 1559.21 U / mg, 1614.46 U / mg, and 1653.71 U / mg, respectively, representing an increase of 1.38 to 1.62 times compared to the wild-type enzyme activity. This invention optimizes and improves the enzyme activity of wild-type β-1,3-glucanase, creating better conditions for its practical application. Attached Figure Description

[0028] Figure 1 This is a construction map of the recombinant plasmid.

[0029] Figure 2 The relative enzyme activities of wild-type enzyme BC16 and mutant enzyme are given. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0032] (a) Culture medium All culture media were prepared using ddH2O and sterilized at 121℃ for 15-20 min after preparation.

[0033] LB liquid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L.

[0034] LB solid medium: yeast extract 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L.

[0035] (II) Detection method for β-1,3-glucanase activity Enzyme activity assay conditions: The reaction system consisted of 50 μL of fermentation broth diluted 200-fold and purified or the supernatant of the lysate, and 950 μL of 10 mg / mL ketoran polysaccharide solution (pH=7.0). The reaction was carried out at 70℃ for 10 min, followed by boiling for 20 min to inactivate the enzyme. The product concentration was determined by the DNS method after centrifugation.

[0036] Enzyme activity is defined as the amount of glucose required to produce 1 μmol per minute under the above reaction conditions.

[0037] The formula for calculating the enzyme activity of β-1,3-glucanase is as follows: .

[0038] (iii) Buffer solution Phosphate buffer (PB): 50 mmol / L, pH 7.0; Binding Buffer: 50 mmol / L PB, 500 mmol / L NaCl, pH 7.0; Washing Buffer: 50 mmol / L PB, 500 mmol / L NaCl, pH 7.0, 20 mmol / L imidazole; Elution Buffer: 50 mmol / L PB, 500 mmol / L NaCl, pH 7.0, 500 mmol / L imidazole; Dialysis buffer: 50 mmol / L PB, pH 7.0, 10 mmol / L EDTA.

[0039] Materials and reagents: Restriction endonucleases, Dpn I enzyme PCR reagents, etc., were purchased from TaKaRa Biotechnology Co., Ltd.; primers were purchased from Anshengda Biotechnology Co., Ltd.; plasmid extraction kits, genome extraction kits, agarose purification kits, E. coil DH5α, and E. coil BL21(DE3) strains were purchased from Sangon Biotech (Shanghai) Co., Ltd.; other reagents were analytical grade reagents purchased domestically or internationally.

[0040] Example 1: Design of β-1,3-glucanase mutant sites Multiple sequence alignment was performed using the online server HotSpot Vizard (https: / / loschmidt.chemi.muni.cz / hotspotwizard / ) to select mutation sites. The tertiary structure model of β-1,3-glucanase was obtained by simulating the protein structure using SWISS-MODEL software. The amino acid sites to be mutated were identified as follows: glutamine at position 71, glycine at position 128, aspartic acid at position 198, arginine at position 106, methionine at position 120, lysine at position 87, alanine at position 151, glycine at position 149, and glutamate at position 181.

[0041] Example 2: Site-directed mutagenesis of β-1,3-glucanase and construction of recombinant plasmids and recombinant Escherichia coli (1) Constructing wild-type plasmids microorganism Alkalihalobacillus clausii The β-1,3-glucanase gene from which this origin is located has the accession number WP_095336276.1, and its nucleotide sequence is shown in SEQ ID NO.1, while its amino acid sequence is shown in SEQ ID NO.2.

[0042] The β-1,3-glucanase encoding gene with the nucleotide sequence shown in SEQ ID NO.1 was constructed between the NdeI and BamHI restriction sites of the plasmid vector pET-28a to obtain a recombinant plasmid, which was named pET-28a-Bc16.

[0043] (2) Constructing mutant plasmids According to SEQ ID NO.1 Alkalihalobacillus clausii Primers were designed from the source gene encoding Bc16, and a mutant plasmid was constructed by site-directed mutagenesis using pET-28a-Bc16 constructed in step (1) as a template: Q71K-F: GCAC AAG GAGACAGTTAGCGACC; Q71K-R: GTCTC CTT GTGCGCCTCGATGATCAG; G128M-F: ATCGCTACGGC ATG TGGGCAGCGAGCGGCGAG; G128M-R: CCA CAT GCCGTAGCGATCGTGCTGCGGCATCA; N198Q-F: GTGGACGGC CAA TTATATTTGACCCTGAATGATTGGTAC; N198Q-R: TATAA TTG GCCGTCCACATACCATCTGATCTC; R106K-F: AGCGCGTATG AAG TTGCCGGCGGGCCAGGGTT; R106K-R: GCAAC TTC ATACGCGCTTCAAAGCGGCCATAG; M120L-F: TTTGGATG CTG CCGCAGCACGATCGCT; M120L-R: GG CAG CATCCAAAACGCTGGCCAAA; K87R-F: GGTA GAG TTCTGACCGACGGTC; K87R-R: GTCAGAA CTC TACCAGAGGTGTAACCGTAC; A151T-F: TGGG ACC ATTCATTATGGTGGCCC; A151T-R: AATGAAT GGT CCCACCCACCTTGTGC; The underlined parts represent the codons corresponding to the mutant gene encoding the following positions: glutamine at position 71, glycine at position 128, aspartic acid at position 198, arginine at position 106, methionine at position 120, lysine at position 87, and alanine at position 151.

[0044] Table 1 PCR amplification system

[0045] After PCR amplification, 2 μL of Dpn I restriction endonuclease (10 U / μL) was added to the reaction solution, and the sample was incubated at 37°C for 2 hours to eliminate the template. The PCR product was then transformed into... E. coli In DH5α cells, LB plates were used to spread the cells, and single colonies were picked and transferred to LB liquid medium. Plasmids were extracted, and sequencing yielded the correct mutant plasmid pET-28a-Bc16. Q71K pET-28a-Bc16 G128M pET-28a-Bc16 N198Q pET-28a-Bc16 R106K pET-28a-Bc16 M120L pET-28a-Bc16 K87RpET-28a-Bc16 A151T The successfully constructed mutant plasmid and the wild-type plasmid from step (1) were transformed into [a specific technology / organization], respectively. E. coli From BL21(DE3), the mutant strain BL21(DE3) / pET-28a-Bc16 was obtained. Q71K BL21(DE3) / pET-28a-Bc16 G128M BL21(DE3) / pET-28a-Bc16 N198Q BL21(DE3) / pET-28a-Bc16 R106K BL21(DE3) / pET-28a-Bc16 M120L BL21(DE3) / pET-28a-Bc16 K87R BL21(DE3) / pET-28a-Bc16 A151T And the strain BL21(DE3) / pET-28a-Bc16 that expresses wild-type enzymes.

[0046] Example 3: Expression and purification of wild-type and mutant enzymes Single colonies of BL21(DE3) / pET-28a-Bc16 and each mutant strain prepared in Example 2 were picked and placed in LB liquid medium containing 50 μg / mL kanamycin. After incubation at 37°C and 200 r / min for 12 h, they were transferred to LB medium containing 50 μg / mL kanamycin and incubated at 37°C and 200 r / min until OD200. 600 Within the range of 0.5 to 0.7, add 1 mmol / L IPTG and induce fermentation at 16℃ and 200 r / min for 24 h, then collect the fermentation broth.

[0047] The enzyme activity of the fermentation broth was directly measured. The results showed that the fermentation enzyme activity of wild-type enzyme was 82.08 U / mL, that of Q71K was 71.51 U / mL, that of G128M was 93.20 U / mL, that of N198Q was 66.08 U / mL, that of R106K was 77.36 U / mL, that of M120L was 84.49 U / mL, that of K87R was 112.11 U / mL, and that of A151T was 85.70 U / mL.

[0048] The collected fermentation broth was centrifuged at 6000 r / min and 4℃ for 10 min, the supernatant was discarded, and the cells were washed twice with phosphate buffer. 15 mL of phosphate buffer was added to resuspend the cells, and the mixture was sonicated for 15 min (30% power, 1 s disruption, 2 s interval). The mixture was then centrifuged at 8000 r / min at 4℃ for 10 min, and the supernatant was collected as the crude enzyme solution. This solution was filtered through a 0.22 μm aqueous membrane. The Ni... 2+ The chelated agarose resin column was pre-equilibrated; crude enzyme solution was added and equilibrated with Binding Buffer and Washing Buffer respectively; the enzyme was eluted with Elution Buffer and recovered; the recovered enzyme solution was dialyzed in dialysis buffer and then stored in a refrigerator at 4 °C.

[0049] Under these conditions, the original enzyme activity was defined as 100%, and a graph was plotted against mutant types using the percentage of relative enzyme activity. The results of evaluating enzyme activity are as follows: Figure 2 Compared to the specific enzyme activity of wild-type enzyme Bc16 (1020.01 U / mg), the specific enzyme activities of the mutants were: Q71K (1411.05 U / mg), G128M (1438.09 U / mg), N198Q (1467.15 U / mg), R106K (1500.72 U / mg), M120L (1559.21 U / mg), K87R (1614.46 U / mg), and A151T (1653.71 U / mg).

[0050] Comparative Example 1: Following the same strategy as in Examples 1-3, conserved sites G149 and E181 were screened, and mutants G149L and E181T were constructed, respectively, along with plasmid pET-28a-ADI. G149L pET-28a-ADI E181T And construct the recombinant strain BL21(DE3) / pET-28a-ADI G149L BL21(DE3) / pET-28a-ADI E181T The recombinant bacteria were cultured using the same method as in Example 3, and the crude enzyme solution and the purified enzyme activity were tested respectively. The results showed that the crude enzyme activity and the enzyme activity of G149L and E181T were almost undetectable, indicating that the mutants G149L and E181T lost their enzyme activity. G149 and E181 are important residues that give the enzyme catalytic ability. After mutation, the enzyme activity was not improved but was lost.

[0051] G149L-F: TG CTT GGGGCCATTCATTATGGTGGCCCTTGGC; G149L-R: AATGAATGGCCCC AAG CACCTTGTGCGGGGTCGC; E181T-F: GCCACCGATTACCAT ACA TATGCAGTTGAATGGGAGCCG; E181T-R: TGT ATGGTAATCGGTGGCGTTCGTACCAGACGG.

[0052] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A β-1,3-glucanase mutant, characterized in that, Based on the β-1,3-glucanase with the amino acid sequence shown in SEQ ID NO.2, alanine at position 151 was mutated to threonine.

2. The gene encoding the mutant of claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. A microbial cell expressing the mutant of claim 1 or carrying the recombinant plasmid of claim 3.

5. The recombinant microbial cell according to claim 4, characterized in that, The recombinant microbial cells are bacteria or fungi.

6. The recombinant microbial cell according to claim 5, characterized in that, The recombinant microbial cells were Escherichia coli.

7. The recombinant microbial cell according to claim 6, characterized in that, The study used Escherichia coli BL21(DE3) as the host and pET-28a as the vector.

8. A method for increasing the activity of β-1,3-glucanase, characterized in that, The alanine at position 151 of the β-1,3-glucanase, as shown in SEQ ID NO.2, was mutated to threonine.

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