Chitosan enzyme mutant with capacity of hydrolyzing chitosan to produce chitobiose per unit

Abstract

The invention discloses a chitosanase mutant which is derived from bacillus subtilis and has the capacity of hydrolyzing chitosan to produce chitosan disaccharide per unit and belongs to a GH46 family, and application of the chitosanase mutant in hydrolyzing chitosan to produce chitosan oligosaccharide. Compared with wild type chitosanase capable of producing chitodisaccharide and chitotriose, the chitosanase mutant has the advantage that the chitodisaccharide can be produced per unit when the chitodisaccharide is hydrolyzed by the chitosanase mutant. The invention provides eight kinds of chitosanase mutants, and the mutants are obtained by carrying out site-specific mutagenesis on 21st-site glycine on the basis of wild type chitosanase and respectively mutating the 21st-site glycine into lysine, arginine, leucine, histidine, methionine, tyrosine, proline and glutamine. The mutant can hydrolyze chitosan under mild conditions to produce chitosan oligosaccharide with single polymerization degree, and can greatly relieve the difficulty in separation and purification in chitosan oligosaccharide production.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and particularly relates to a chitosanase mutant capable of hydrolyzing chitosan to produce chitobiose and an application thereof. Background Art

[0002] Chitosan (CS) is the most important chitin derivative and the only natural cationic polysaccharide found in nature. Chitosan is composed of D-glucosamine and N-acetyl-D-glucosamine linked by β-1,4-glycosidic bonds and is primarily found in the cell walls of crustaceans, fungi, and plants.

[0003] Chitooligosaccharides (COS) are homo- or hetero-oligomers of N-acetyl-D-glucosamine and D-glucosamine obtained by enzymatic or chemical degradation of CS, with a DP range of 2-20. COS is one of the few known basic oligosaccharides and possesses antibacterial, antioxidant, antiviral, and immunomodulatory bioactivities, offering significant applications in food, healthcare, and agriculture. Therefore, converting chitosan into chitooligosaccharides with high solubility and broad bioactivity is highly beneficial for promoting their commercial application. Currently, the main methods for producing chitooligosaccharides include physical, chemical, and enzymatic hydrolysis. Chemical methods are associated with significant pollution and difficulty in precisely controlling the degree of polymerization (DP). While non-polluting, physical methods produce products with relatively high DP and low yield. In comparison, enzymatic hydrolysis offers mild conditions and a controllable DP. Therefore, enzymatic hydrolysis is a current research hotspot.

[0004] Chitosanase is a chitosan-specific hydrolase. Based on amino acid sequence similarity, chitosanases are primarily distributed in the GH8, GH46, GH75, and GH80 families. Currently, chitosanases from the GH46 family have attracted considerable research attention due to their high catalytic activity and stability. However, the chitosan oligosaccharides produced by chitosan hydrolysis using chitosanase are mostly mixtures, and studies have shown that chitosan oligosaccharides with different degrees of polymerization have different biological activities. However, the properties of chitosan oligosaccharides with different degrees of polymerization are not very different, making their separation and purification difficult. Therefore, obtaining a mutant that can produce chitosan oligosaccharides with a single degree of polymerization would greatly alleviate these separation and purification difficulties. Summary of the Invention

[0005] The purpose of the present invention is to obtain a chitosanase mutant capable of producing chitosan oligosaccharides with a single degree of polymerization.

[0006] The invention provides a chitosanase mutant. Compared with a wild chitosanase which hydrolyzes chitobiose and chitotriose, the chitosanase mutant can hydrolyze chitosan to produce only chitobiose.

[0007] The present invention provides amino acid sequences of chitobiose mutants that can be obtained by hydrolysis as shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, and SEQ ID NO: 17. The chitosanase mutants are obtained by subjecting the parent sequence SEQ ID NO: 2 to site-directed mutagenesis at position 21, whereby the glycine at position 21 is mutated to lysine, arginine, leucine, histidine, methionine, tyrosine, proline, and glutamine, respectively.

[0008] The present invention provides nucleotide sequences encoding chitobiose mutants that can be hydrolyzed to obtain chitobiose, as shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18.

[0009] The present invention provides an expression vector, which comprises the mutant nucleotide sequence and is constructed using pET-28a(+).

[0010] The present invention provides a genetically engineered bacterium, which comprises the above-mentioned expression vector and is constructed using Escherichia coli BL21 (DE3).

[0011] The invention provides a preparation method of a chitosanase mutant. The method comprises the following steps: inducing expression in genetically engineered bacteria, collecting the bacteria by centrifugation, resuspending the bacteria, performing ultrasonic disruption, collecting the supernatant by centrifugation, and separating and purifying the chitosanase mutant.

[0012] The present invention provides a method for converting chitosan into chitobiose by utilizing the genetically engineered bacteria.

[0013] Specifically, a chitosanase mutant is added to a reaction system containing chitosan to catalyze a hydrolysis reaction to produce chitobiose.

[0014] The hydrolysis products of wild chitosanase include chitobiose and chitotriose. Compared with wild chitosanase, the chitosanase mutant provided by the present invention can hydrolyze chitosan to produce chitobiose alone. This mutant can hydrolyze chitosan under mild conditions to produce chitosan oligosaccharides with a single degree of polymerization, greatly alleviating the difficulties in separation and purification during chitosan oligosaccharide production. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 1 is a thin layer chromatogram of the chitosan hydrolysis products of the wild-type chitosanase and its mutants in the examples of the present invention. DETAILED DESCRIPTION

[0016] The present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0017] The experimental methods or experimental operations not otherwise specified in the following examples of the present invention were all performed using conventional experimental methods, or those skilled in the art can understand and easily perform them based on the product instructions and basic knowledge in the field, and therefore will not be described in detail. The culture media or other reagent materials involved in the following examples of the present invention were all conventionally prepared or purchased unless otherwise specified.

[0018] Example 1: Obtaining a mutation vector Using the pET-28a(+) plasmid containing the wild-type chitosanase gene (SEQ ID NO: 2) as a template, inverse PCR amplification was performed to generate mutant expression vectors using the primer sequences shown in Table 1. Mutants are represented by "amino acid substituted at the original amino acid position." For example, G21K indicates that the glycine at position 21 has been mutated to lysine.

[0019] Table 1: Primer sequences used in the present invention .

[0020] Table 2: Inverse PCR system .

[0021] The inverse PCR amplification conditions were as follows: pre-denaturation at 95 °C for 3 min; 16 cycles of denaturation at 95 °C for 30 s, annealing at 64 °C for 30 s, and extension at 68 °C for 12.5 min; and insulation at 12 °C.

[0022] Digest the PCR product with 0.7 μL DpnI restriction enzyme for 1.5 h to remove the template plasmid. Take 20 μL of the reaction and transform it into E. coli. E. coli DH5α competent cells. Positive clones were screened on kanamycin resistance plates, and transformants were picked and sent to Jinweizhi Biotechnology Co., Ltd. for sequencing. The plasmids of the correctly sequenced recombinant bacteria were extracted and then transformed into Escherichia coli. E. coli BL21(DE3) competent cells. Store genetically engineered bacteria at -80°C.

[0023] Example 2: Expression of mutants The recombinant bacteria were inoculated into 10 mL of LB liquid medium at a 1% inoculum size for activation. 1 mL of the activated bacterial solution was then transferred to 50 mL of LB liquid medium for expansion. When the OD value of the bacterial solution reached about 0.4, IPTG (isopropyl-β-D-thiogalactopyranoside) was added at a final concentration of 1 mM and cultured overnight to induce the synthesis of the target protein.

[0024] Example 3: Purification of mutants Aliquot 50 mL of bacterial suspension into two 50 mL centrifuge tubes and collect the cells by centrifugation at 8000 rpm at 4°C for 5 min. Wash the cells twice with binding buffer M0 (50 mM Tris-HCl, 0.5 mM NaCl, pH 8.0, 10% glycerol), combine the tubes, and resuspend the cells in 5 mL of M0. Disrupt the crude enzyme solution using a sonicator. After disruption, centrifuge at 10,000 rpm at 4°C for 30 min to collect the supernatant. Purify the crude enzyme solution using Ni-NTA affinity chromatography. Apply the entire supernatant to a Ni-NTA affinity column and thoroughly elute unbound proteins with 100 mL of M0 binding buffer. Elute the target protein with elution buffer (50 mM Tris-HCl, 0.5 mM NaCl, 0.1 M imidazole, pH 8.0, 10% glycerol). Collect the eluted protein sample and store at -20°C. The purity of the separated protein was analyzed by SDS-PAGE protein electrophoresis, and the protein content in the purified enzyme solution was determined by the Coomassie Brilliant Blue method.

[0025] Example 4: Determination of enzyme activity The catalytic activity of the purified enzyme was measured using the DNS method. The reaction system was as follows: 1475 μL of buffer, 500 μL of 1% colloidal chitosan solution, 18 μL of 100 mM MnCl₂, and 25 μL of appropriately diluted purified enzyme solution. The reaction was incubated at 55°C for 5 minutes, and immediately terminated by the addition of 1.5 mL of DNS reagent. The reaction was then incubated in a boiling water bath for 5 minutes to develop color. Finally, the volume was adjusted to 25 mL with distilled water. The reaction mixture was cooled and allowed to stand for approximately 1 hour, and the absorbance at 520 nm was measured. The reaction mixture, supplemented with 25 μL of water, served as a blank control, and the enzyme activity was calculated using a glucosamine standard curve.

[0026] Chitosanase activity was defined as the amount of enzyme required to generate 1 μmol of reducing sugar per minute (1 μmol / mL·min).

[0027] Table 3: Enzyme activity .

[0028] Example 5: Hydrolysis reaction To 1 mL of the hydrolysis reaction system, 450 μL of 1% colloidal chitosan solution, 50 U of purified enzyme solution, 500 μL of pH 6.6 phosphate buffer solution, and 9 μL of 100 mM MnCl2 were added. The reaction was incubated in a constant temperature shaker at 37 °C and 180 rpm for 24 h. The mixture was then removed and the reaction was terminated by boiling in a water bath for 10 min. The supernatant was collected by centrifugation at 10,000 rpm and 4 °C for 10 min and stored at -20 °C.

[0029] Example 6: Thin layer chromatography Using GlcN2-4-chitosan oligosaccharide and D-glucosamine hydrochloride as mixed standards, 2 μL of the mixed standard and hydrolyzed sample were spotted onto a high-performance silica gel plate. After the spotted plate was thoroughly dried, it was placed in a chromatography tank filled with developing solvent and fully developed. When the developing solvent reached 2 cm from the top of the plate, the plate was removed and blown dry with a hair dryer until there was no ammonia odor. The plate was evenly sprayed with a color developer (0.3% ninhydrin in ethanol) and then placed in a 110°C oven for approximately 10 minutes until clear purple-red spots appeared.

[0030] The thin layer chromatograms of chitosan hydrolysis products of wild-type chitosanase and its mutants are shown in Figure 2. Figure 1 As shown. Figure 1 It can be clearly seen that the hydrolysis products of the original wild-type chitosanase include chitobiose and chitotriose, while the chitosanase mutants G21K, G21R, G21L, G21H, G21M, G21Y, G21P, and G21Q produce a relatively simple product under the same hydrolysis conditions, which is only chitobiose.

[0031] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A chitosanase mutant capable of producing chitobiose, characterized in that: The amino acid sequence of the chitosanase BsCsn46A of Bacillus subtilis is mutated at glycine 21 as shown in SEQ ID NO:

1. The amino acid sequence of the chitosanase mutant producing only chitobiose is any one of the following: (1) mutated to lysine, the amino acid sequence of which is shown in SEQ ID NO: 3; (2) mutated to arginine, the amino acid sequence of which is shown in SEQ ID NO:5; (3) mutated to leucine, the amino acid sequence of which is shown in SEQ ID NO:7; (4) mutated to histidine, the amino acid sequence of which is shown in SEQ ID NO:9; (5) mutated to methionine, the amino acid sequence of which is shown in SEQ ID NO: 11; (6) mutated to tyrosine, the amino acid sequence of which is shown in SEQ ID NO: 13; (7) mutated to proline, the amino acid sequence of which is shown in SEQ ID NO: 15; (8) mutated to glutamine, the amino acid sequence of which is shown in SEQ ID NO:

17.

2. A gene encoding the chitosanase mutant capable of producing chitobiose alone according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is as follows: The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3 is shown in SEQ ID NO:4; The nucleotide sequence encoding the amino acid sequence SEQ ID NO:5 is shown in SEQ ID NO:6; The nucleotide sequence encoding the amino acid sequence SEQ ID NO:7 is shown in SEQ ID NO:8; The nucleotide sequence encoding the amino acid sequence of SEQ ID NO:9 is shown in SEQ ID NO:10; The nucleotide sequence encoding the amino acid sequence SEQ ID NO:11 is shown in SEQ ID NO:12; The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13 is shown in SEQ ID NO: 14; The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 15 is shown in SEQ ID NO: 16; The nucleotide sequence encoding the amino acid sequence SEQ ID NO:17 is shown in SEQ ID NO:

18.

4. A recombinant vector expressing the chitosanase mutant protein according to claim 1, characterized in that: The recombinant vector comprises the gene according to claim 2 or 3.

5. A recombinant bacterium expressing the chitosanase mutant according to claim 1, characterized in that: The recombinant bacteria comprises the gene according to claim 2 or 3, or the recombinant vector according to claim 4.

6. Use of the chitosan mutant according to claim 1 or the chitosanase mutant recombinant bacterium according to claim 4 in catalyzing the hydrolysis of chitosan to produce chitooligosaccharides.

7. The use according to claim 6, characterized in that The application is to catalyze the hydrolysis of chitosan to produce chitobiose.

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