Chitosanase mutant with improved catalytic activity by changing the acidity and alkalinity of the substrate channel

Abstract

The present invention belongs to the technical field of enzyme engineering, and specifically relates to a chitosanase mutant with improved catalytic activity by changing the acidity and alkalinity of the substrate channel. Based on the substrate channel-regulated Bacillus subtilis chitosanase BsCsn46A, the 49th alanine of the chitosanase with the amino acid sequence shown in SEQ ID NO: 1 is mutated to the basic amino acid lysine, and the mutant is A49K. Compared with the wild-type chitosanase, the chitosanase mutant of the present invention uses chitosan as a substrate, and its catalytic activity is 1.13 times higher than that of the wild-type.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and particularly relates to a chitosanase mutant with improved catalytic activity by changing the acidity and alkalinity of the substrate channel. Background Art

[0002] Chitosanase is a class of glycoside hydrolases that have high catalytic activity for chitosan and hardly hydrolyze chitin. It can convert high-molecular-weight chitosan into low-molecular-weight functional chitosan oligosaccharides. Chitosanase can specifically degrade chitosan, and the hydrolysis products of this type of enzyme are chitosan oligosaccharides with different degrees of polymerization, mainly chitobiose and chitotriose. Chitosanase mainly exists in four glycoside hydrolase families (GH), namely GH8, GH46, GH75, and GH80 families. Compared with other families, the research on chitosanase in the GH46 family is more in-depth.

[0003] Chitosan is a cationic natural basic polysaccharide formed by connecting glucosamine (GlcN) through β-1,4-glycosidic bonds. Its molecular chain contains abundant active amino groups and hydroxyl groups. It is the product of the deacetylation of chitin. Generally speaking, chitin with a deacetylation degree of more than 50% is called chitosan. Chitosan has good properties in antibacterial, biocompatibility, film-forming, and biodegradability, and is mainly used in chemical applications such as food packaging, active antibacterial agents, and water treatment. Chitosan widely exists in chitinous animals such as shrimps and crabs, algae plants, and large fungi such as mushrooms. It has a wide source and rich resources, and is the second largest macromolecular compound only after cellulose. Due to its large relative molecular mass and poor solubility, the application of chitosan is limited. Chitosan can be hydrolyzed to produce chitosan oligosaccharides (degree of polymerization 2-10) or glucosamine.

[0004] Chitosan oligosaccharide (COS) is the only oligosaccharide that is alkaline and positively charged among known oligosaccharides, so it is easily absorbed by the intestine. COS has good immunomodulatory, antioxidant, antimicrobial, and antitumor properties in the fields of biochemistry and medicine, which are better than those of chitosan in some aspects. Research shows that among chitosan oligosaccharides with different degrees of polymerization, chitobiose has the best effect on improving oleic acid-induced steatosis and can effectively relieve fatty liver. In addition, chitosan oligosaccharides have effects on treating diabetes, lowering blood sugar, blood pressure, and blood lipids. Chitosan oligosaccharides have good application prospects in the fields of medicine and health care, cosmetics, food, and agriculture.

[0005] The production of chito-oligosaccharides by enzymatic hydrolysis has the characteristics of high selectivity, high activity, high efficiency, etc. It has the advantages of high yield of chito-oligosaccharides production, obvious cost advantages, and little environmental pollution. In recent years, optimizing enzymatic hydrolysis has become a research hotspot. At present, the catalytic activity of chitosanase is relatively low. Therefore, obtaining chitosanase with high catalytic activity has important value in the industrial production of chito-oligosaccharides. Chinese Patent CN113755471A calculated the folding free energy of all amino acid residues of chitosanase BsCsn46A derived from Bacillus subtilis, screened a candidate residue Pro121, and obtained three mutants P121N, P121C, and P121V after saturation mutagenesis of it. Compared with the wild type, the specific enzyme activities of the mutants against colloidal chitosan were increased by 1.69, 1.97, and 2.15 times respectively. Among them, the thermal stability of P121N was comparable to that of the wild-type chitosanase, which provided a new strategy for improving the enzymatic properties of chitosanase. Chinese Patent CN115873831A, based on the mutation at position 121 of BsCsn46A, obtained a mutant K242P by saturation mutagenesis of the terminal amino acids. Its temperature stability was increased by 1.67 times, and the catalytic activity was about 1.74 times that of the wild type. Summary of the Invention

[0006] The present invention is a further improvement based on the above patents. For chitosanase BsCsn46A derived from Bacillus subtilis, the amino acid sequence is as shown in SEQ ID NO:1. After analysis on the substrate channel, potential mutation sites related to catalytic activity were found, and the channel sites were mutated into representative acidic or representative basic amino acids, and mutants with improved enzyme catalytic activity were screened by directed mutagenesis.

[0007] Site-directed mutagenesis was carried out at position 49 of the amino acid sequence of the above-mentioned Bacillus subtilis chitosanase BsCsn46A, that is, alanine was mutated into lysine, a basic amino acid, and aspartic acid, an acidic amino acid. The mutants were labeled as A49K and A49D. The amino acid sequence of Bacillus subtilis chitosanase BsCsn46A is SEQ ID NO:1, and the nucleotide sequence is SEQ ID NO:2. The amino acid sequence of the chitosanase mutant A49K is SEQ ID NO:3, the nucleotide sequence of the chitosanase mutant is SEQ ID NO:4, the amino acid sequence of the chitosanase mutant A49D is SEQ ID NO:5, and the nucleotide sequence of the chitosanase mutant is SEQ ID NO:6.

[0008] The DNS method was used to determine the enzyme activity. The results showed that compared with the wild-type chitosanase, the chitosanase mutant A49K, using chitosan as the substrate, had a catalytic activity 1.13 times higher than that of the wild type.

[0009] The present invention provides a mutant recombinant chitosanase modified by site-directed mutagenesis and expressed in Escherichia coli BL21(DE3). The amino acid site where the mutation occurs is the mutation of alanine at position 49 of chitosanase (BsCsn46A) to lysine (A49K).

[0010] The present invention provides a recombinant vector carrying the gene encoding the chitosanase mutant described above and a recombinant bacterium transformed / transfected with the recombinant vector.

[0011] The present invention also provides the application of the above chitosanase mutant in the catalytic hydrolysis of chitosan to produce chitosan oligosaccharides. Specifically, during the catalytic hydrolysis of chitosan by the mutant of the present invention, the catalytic activity is significantly improved.

[0012] Compared with the wild chitosanase, the chitosanase mutant provided by the present invention has broad application prospects in the hydrolysis to produce chitosan oligosaccharides. Brief Description of the Drawings

[0013] Figure 1 are the enzyme activities of the wild type and the mutant, where Wild-type is the chitosanase BsCsn46A of Bacillus subtilis;

[0014] Figure 2 is the structural diagram of the A49K chitosanase protein. Detailed Embodiments

[0015] In order to further improve the enzyme activity of chitosanase BsCsn46A derived from Bacillus subtilis, the present invention analyzed the substrate channel of BsCsn46A, found potential mutation sites related to catalytic activity, mutated the channel sites into acidic or basic amino acids, and screened for mutants with improved enzyme catalytic activity by site-directed mutagenesis.

[0016] The amino acid sequence SEQ ID NO:1 of BsCsn46A was submitted to SWISS-MODEL to construct the three-dimensional structure of the enzyme; the three-dimensional structure of the obtained chitosanase was analyzed using substrate channel analysis technology to obtain amino acid sites that can affect enzyme catalytic activity, and it was determined that the 49th site located in the substrate channel is a potential site affecting catalytic activity; this site was mutated into a representative basic amino acid (lysine K) and a representative acidic amino acid (aspartic acid D).

[0017] Using site-directed mutagenesis technology, the wild-type chitosanase BsCsn46A gene SEQ ID NO:2 was used as a template, and the primer sequences are shown in Table 1 for PCR amplification.

[0018] Table 1 Primer Sequences

[0019]

[0020] Inverse PCR system:

[0021]

[0022] Inverse PCR amplification conditions: Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 50 s, annealing at 65°C for 30 s, extension at 68°C for 12.5 min, 12 cycles; incubation at 4°C.

[0023] Digest the amplified PCR product with an additional 0.7 μL of DpnI for 2 hours to remove the plasmid template. Take 10 μL of the cooled reaction mixture and directly transform it into Escherichia coli DH5α. Then culture for 1 h, centrifuge, resuspend and spread. Culture the transformants overnight at 37°C, pick single colonies and transfer them to liquid LB medium for overnight culture. Send the obtained recombinant cells carrying the mutant plasmid to Shanghai Bioengineering Co., Ltd. for sequencing. The mutants containing the correct plasmid are used to extract the plasmid through a plasmid extraction kit, and then transformed into Escherichia coli BL21 cells to preserve glycerol bacteria.

[0024] Inoculate the preserved strain containing the mutant plasmid into 10 mL of LB liquid medium at an inoculation amount of one-thousandth, culture overnight at 37°C and 160 rpm on a shaker. Take 1 mL of the bacterial solution and transfer it to 100 mL of LB medium for expansion culture for 3 h, add the inducer IPTG, and culture overnight at 16°C and 160 rpm on a shaker.

[0025] After the induced mutants are cultured on a shaker, collect the bacterial cells and ultrasonically disrupt them. Purify the supernatant by Ni-IDA affinity chromatography. Load the supernatant onto the Ni-IDA column, first wash the unbound proteins thoroughly with the loading buffer, and then elute the proteins with the elution buffer (50 mM Tris-HCl, 0.5 mM NaCl, 0.1 M imidazole, pH 8.0). Collect the eluted protein samples and store them at -20°C. Determine the protein content in the enzyme solution by the Broadford method.

[0026] Enzyme activity assay of chitosanase BsCsn46A and its mutants: Use the DNS method to assay the enzyme activity. Add 1475 μL of pH buffer and 18 μL of 100 mM Mn 2+, 500 μL of 1% colloidal chitosan solution, 25 μL of the purified enzyme solution was added, incubated in a water bath at 55 °C for 5 min, 1.5 mL of DNS solution was added to terminate the reaction, then boiled in boiling water for 5 min, and finally made up to 25 mL with distilled water, cooled and allowed to stand. ddH2O was added instead of the enzyme solution as a blank control group for zero adjustment, and its absorbance at 520 nm was measured. Under these conditions, the amount of enzyme that catalyzes the formation of 1 μM reducing sugar per minute is defined as one enzyme activity unit (U).

[0027] Enzymatic properties of chitosanase BsCsn46A from Bacillus subtilis and its mutants

[0028] Optimal pH and pH stability: Under the temperature condition of 50 °C, the enzyme activities of wild-type chitosanase and chitosanase mutants were measured in phosphate buffer solutions with pH = 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2. Taking the highest enzyme activity point as 100%, the optimal pH is shown in Table 1. Under the condition of 4 °C, the chitosanase was stored in phosphate buffer solution with pH 6.2 for 2 h, and the enzyme activities at 0 h and 2 h were measured. Taking the chitosanase enzyme activity measured at 0 h as 100%.

[0029] Optimal temperature and temperature stability: Under the optimal pH condition, the reaction system was placed at 40 - 75 °C for reaction respectively, and the enzyme activity of chitosanase was measured. Taking the temperature at the highest enzyme activity point as the optimal temperature. The chitosanase was stored at 55 °C for 2 h, and the chitosanase enzyme activity measured at 0 h was taken as 100%.

[0030] The enzymatic properties of wild-type chitosanase and its mutants are as Figure 1 and shown in Table 2. The catalytic activity of mutant A49K is significantly higher than that of wild-type chitosanase BsCsn46A from Bacillus subtilis.

[0031] Table 2 Enzymatic properties of chitosanase BsCsn46A from Bacillus subtilis and its mutants

[0032]

[0033] It can be seen from the above results that the catalytic activity of chitosanase mutant A49K is significantly improved, its optimal pH is 6.2, and its optimal temperature is 50 °C.

[0034] The above are only the preferred specific embodiments of the present invention, and the protection scope of the present invention is not limited thereto. Any simple variations or equivalent replacements of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope disclosed by the present invention all fall within the protection scope of the present invention.

Claims

1. A chitosanase mutant, characterized in that: The chitosanase mutant is obtained by mutating alanine at the 49th position in the amino acid sequence of chitosanase BsCsn46A from Bacillus subtilis into lysine, while other amino acid residues remain unchanged; the amino acid sequence of chitosanase BsCsn46A from Bacillus subtilis is shown as SEQ ID NO:1; the amino acid sequence of the chitosanase mutant is shown as SEQ ID NO:

3.

2. A gene, characterized in that, The gene encoding the chitosanase mutant according to claim 1, the nucleotide sequence of which is shown as SEQ ID NO:

4.

3. A recombinant vector, characterized in that: The recombinant vector carries the gene according to claim 2.

4. A recombinant bacterium, characterized in that, The recombinant bacterium contains the recombinant vector according to claim 3.

5. Use of the chitosanase mutant according to claim 1, characterized in that, The application of the chitosanase mutant in enzymatic hydrolysis of chitosan.

6. Use of the chitosanase mutant according to claim 5, characterized in that, The optimum pH of the chitosanase mutant is 6.2 and the optimum temperature is 50 °C.

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