Chitosanase mutants producing chitotetraose

Abstract

The present application relates to chitosanase mutants producing chitotetraose and application thereof, and belongs to the field of enzyme engineering. Specifically, the chitosanase is derived from GH46 family glycoside hydrolase (BsCsn46A) of Bacillus subtilis, the chitosanase mutant is that the aspartic acid at the 151th position of the amino acid sequence of BsCsn46A is mutated into serine, the mutant is D151S; or mutated into threonine, the mutant is D151T; or mutated into tyrosine, the mutant is D151Y. The mutant has the ability of hydrolyzing chitosan to produce chitotetraose.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, specifically relating to a chitosanase mutant that produces chitosan tetrasaccharides. The mutant with chitosan tetrasaccharide production ability is obtained by site-directed mutagenesis at position 151 of the amino acid sequence of Bacillus subtilis chitosanase BsCsn46A. Background Technology

[0002] Chitosan (CS) is a deacetylated product obtained by chitin deacetylase catalyzing the GlcNAc bond in chitin. It is the only naturally occurring basic polysaccharide in nature, composed of D-glucosamine and N-acetyl-D-glucosamine linked by β-1,4-glycosidic bonds. It is mainly found in the cell walls of crustaceans, fungi, and plants. Chitin is the second largest biomass resource after cellulose, with an estimated annual production of approximately 100 billion tons. Due to its good biocompatibility and biofunctionality, chitosan has significant potential applications in the food, medical, environmental, and cosmetic industries. However, its high molecular weight and strong intramolecular and intermolecular hydrogen bonding result in dense, water-insoluble molecules and weak biocompatibility, which significantly limits its applications.

[0003] Chitosan oligosaccharide (COS) is a hydrolysis product of chitosan. It possesses low molecular weight, high water solubility, biocompatibility, and non-allergenicity. Due to its excellent biological activity, it is widely used in food, medicine, industry, and agriculture, and is one of the most promising bioactive functional oligosaccharides. COS exhibits good absorption in the human body, especially COS with a degree of polymerization between 3 and 7, which demonstrates particularly outstanding biological activity. COS possesses strong antioxidant capacity, antitumor activity, immunomodulatory functions, lipid-lowering effects, and excellent antibacterial, antiviral, and antiparasitic properties in plant protection.

[0004] The raw materials for COS preparation are inexpensive and readily available. Based on these, COS can be prepared through physical, chemical, enzymatic, or combined techniques. Physical methods include ultrasonication, hydrothermal methods, eddy cavitation, gamma rays, irradiation, or microwave methods. Physical methods are easy to control and pollution-free, but the products have uneven molecular weight and low yields. Chemical methods include acid hydrolysis and oxidative degradation. Acid hydrolysis is the most commonly used method in large-scale industrial production, involving the use of hydrochloric acid, nitrous acid, phosphoric acid, or hydrogen fluoride; oxidative degradation is also widely used, typically using oxidants such as hydrogen peroxide, persulfate, or sodium nitrite. Chemical reactions are difficult to control, costly, and polluting. Enzymatic preparation of COS can be divided into non-specific enzyme hydrolysis and specific enzyme hydrolysis. Many non-specific enzymes, such as glycosidases (e.g., cellulase, hemicellulase, pectinase, lysozyme), catalyze the β-1,4 glycosidic bonds in chitosan. Non-specific enzymes are inexpensive and readily available, but have limited catalytic activity; specific enzymes, such as chitosanase, are specific.

[0005] Chitosanase (EC3.2.1.132) is a type of enzyme that specifically catalyzes the hydrolysis of β-1,4-glycosidic bonds in chitosan, producing COS with different degrees of polymerization (DP). COS with a high degree of polymerization exhibits greater biological activity than COS with a low degree of polymerization. Summary of the Invention

[0006] To overcome the limitations of the degree of polymerization of existing chitosanase products, the enzyme was modified to obtain a chitosanase mutant with improved degree of polymerization.

[0007] In Bacillus subtilis ( Bacillus subtilis Analysis of the substrate channel of chitosanase BsCsn46A revealed a saturation mutation at position 151 of its amino acid sequence, which screened for mutants with the potential to produce chitosan tetrasaccharides.

[0008] The chitosanase mutant of the present invention is a site-directed mutation at position 151 of the amino acid sequence of Bacillus subtilis chitosanase BsCsn46A, which mutates aspartic acid to serine (labeled D151S), to threonine (labeled D151T), and to tyrosine (labeled D151Y). 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 D151S is SEQ ID NO:3, and the nucleotide sequence is SEQ ID NO:4; the amino acid sequence of the chitosanase mutant D151T is SEQ ID NO:5, and the nucleotide sequence is SEQ ID NO:6; the amino acid sequence of the chitosanase mutant D151Y is SEQ ID NO:7, and the nucleotide sequence is SEQ ID NO:8.

[0009] SEQ ID NO:1

[0010] 1 AGLNKDQKRRAEQLTSIFEN

[0011] 21 GTTEIQYGYVERLDDGRGYT

[0012] 41 CGRAGFTTATGDALEVVEVY

[0013] 61 TKAVPNSKLKKYLPELRRLA

[0014] 81 KEESDDTSNLKGFASAWKSL

[0015] 101 ANDKEFRAAQDKVNDHLYYQ

[0016] 121 NAMKRSDNAGLKTALARAVM

[0017] 141 YDTVIQHGDGDDPDSFYALI

[0018] 161 KRTNKKAGGSPKDGIDEKKW

[0019] 181 LNKFLDVRYDDLMNPANHDT

[0020] 201 RDEWRESVARVDVLRSIAKE

[0021] 221 NNYNLNGPIHVRSNEYGNFV

[0022] 241 IP。

[0023] SEQ ID NO:2

[0024] 。

[0025] SEQ ID NO:3

[0026] 1 AGLNKDQKRR AEQLTSIFEN

[0027] 21 GTTEIQYGYV ERLDDGRGYT

[0028] 41 CGRAGFTTAT GDALEVVEVY

[0029] 61 TKAVPNSKLK KYLPELRRLA

[0030] 81 KEESDDTSNL KGFASAWKSL

[0031] 101 ANDKEFRAAQ DKVNDHLYYQ

[0032] 121 NAMKRSDNAG LKTALARAVM

[0033] 141 YDTVIQHGDG SDPDSFYALI

[0034] 161 KRTNKKAGGS PKDGIDEKKW

[0035] 181 LNKFLDVRYD DLMNPANHDT

[0036] 201 RDEWRESVAR VDVLRSIAKE

[0037] 221 NNYNLNGPIH VRSNEYGNFV

[0038] 241 IP。

[0039] SEQ ID NO:4

[0040] 。

[0041] SEQ ID NO:5

[0042] 1 AGLNKDQKRR AEQLTSIFEN

[0043] 21 GTTEIQYGYV ERLDDGRGYT

[0044] 41 CGRAGFTTAT GDALEVVEVY

[0045] 61 TKAVPNSKLK KYLPELRRLA

[0046] 81 KEESDDTSNL KGFASAWKSL

[0047] 101 ANDKEFRAAQ DKVNDHLYYQ

[0048] 121 NAMKRSDNAG LKTALARAVM

[0049] 141 YDTVIQHGDG TDPDSFYALI

[0050] 161 KRTNKKAGGS PKDGIDEKKW

[0051] 181 LNKFLDVRYD DLMNPANHDT

[0052] 201 RDEWRESVAR VDVLRSIAKE

[0053] 221 NNYNLNGPIH VRSNEYGNFV

[0054] 241 IP。

[0055] SEQ ID NO:6

[0056] 。

[0057] SEQ ID NO:7

[0058] 1 AGLNKDQKRR AEQLTSIFEN

[0059] 21 GTTEIQYGYV ERLDDGRGYT

[0060] 41 CGRAGFTTAT GDALEVVEVY

[0061] 61 TKAVPNSKLK KYLPELRRLA

[0062] 81 KEESDDTSNL KGFASAWKSL

[0063] 101 ANDKEFRAAQ DKVNDHLYYQ

[0064] 121 NAMKRSDNAG LKTALARAVM

[0065] 141 YDTVIQHGDG YDPDSFYALI

[0066] 161 KRTNKKAGGS PKDGIDEKKW

[0067] 181 LNKFLDVRYD DLMNPANHDT

[0068] 201 RDEWRESVAR VDVLRSIAKE

[0069] 221 NNYNLNGPIH VRSNEYGNFV

[0070] 241 IP。

[0071] SEQ ID NO:8

[0072] .

[0073] This invention provides a site-directed mutagenesis-modified recombinant chitosanase, expressed in Escherichia coli BL21(DE3). The mutated amino acid site is obtained by mutating aspartic acid at position 151 of the chitosanase (BsCsn46A) to serine (D151S), threonine (D151T), or tyrosine (D151Y).

[0074] The present invention provides a recombinant vector carrying the gene encoding the chitosanase mutant described above.

[0075] The present invention provides a recombinant vector carrying the gene encoding the above-mentioned chitosanase mutant and recombinant bacteria transformed / transfected with the recombinant vector.

[0076] The specific mutation method and chitosanase mutant preparation method of this invention are as follows:

[0077] 1. The spatial structure of chitosanase (BsCsn46A) was obtained using Swiss-Model online software; then, the key site (D151) affecting the degree of hydrolysis of the product was obtained through substrate channel simulation and molecular docking analysis.

[0078] 2. Design primers for site-directed mutagenesis, amplify the chitosanase gene by PCR, and construct a recombinant vector with the expression vector pET-28a, which is then transformed into E. coli BL21(DE3);

[0079] 3. The above recombinant bacteria were cultured and induced to express by IPTG. The bacterial cells were collected, the cells were disrupted by sonication, the supernatant was collected by centrifugation, and the protein was purified by Ni-NTA affinity chromatography to obtain the purified chitosanase mutant.

[0080] The present invention further provides the industrial application of the above-mentioned chitosanase mutant, specifically, in addition to producing chitobiose and chitotriose, it can also produce chitotetraose. Attached Figure Description

[0081] Figure 1 Thin-layer chromatograms of chitosan degradation products from wild-type chitosanase and the D151E mutant.

[0082] Figure 2 Thin-layer chromatograms of chitosan degradation products from chitosanase D151R and D151H mutants.

[0083] Figure 3 Thin-layer chromatograms of chitosan degradation products from chitosanase D151K and D151A mutants.

[0084] Figure 4 Thin-layer chromatograms of chitosan degradation products from chitosanase D151N and D151C mutants.

[0085] Figure 5 Thin-layer chromatograms of chitosan degradation products from chitosanase D151Q and D151I mutants.

[0086] Figure 6 Thin-layer chromatograms of chitosan degradation products from chitosanase D151L and D151F mutants.

[0087] Figure 7 Thin-layer chromatograms of chitosan degradation products from chitosanase D151G and D151M mutants.

[0088] Figure 8 Thin-layer chromatograms of chitosan degradation products from chitosanase D151S and D151T mutants.

[0089] Figure 9 Thin-layer chromatograms of chitosan degradation products from chitosanase mutants D151W and D151Y.

[0090] Figure 10 Thin-layer chromatograms of chitosan degradation products from chitosanase D151V and D151P mutants. Detailed Implementation

[0091] The present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0092] In the embodiments of the present invention, unless otherwise specified, conventional molecular biology experimental methods were used. The PCR, plasmid construction, transfection / transformation and other processes involved in the embodiments can be understood and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore will not be described in detail. Example 1

[0093] Using chitosanase BsCsn46A with high enzyme activity preserved in the early stage of the experiment as the starting enzyme, potential sites affecting the degree of product polymerization were identified through substrate channel analysis, and a mutant library was obtained by saturation mutagenesis of key sites.

[0094] The amino acid sequence BsCsn46A (SEQ ID NO:1) was submitted to SWISS-MODEL to construct the three-dimensional structure of the enzyme. The obtained chitosanase three-dimensional structure was analyzed using substrate channel analysis technology to identify amino acid sites that can affect the enzyme's catalytic activity. The 151st site in the substrate channel was identified as a potential site that affects catalytic activity.

[0095] Specific method: Primer sequences were designed using the online software PrimerX as follows, and the mutant chitosanase gene was obtained through genetic engineering.

[0096] Table 1. Primers for saturated mutant libraries

[0097] Primer name Primer uses Primers (5–3') D151CF D151C <![CDATA[GTTATTCAGCATGGCGATGGT TGC GACCCTGACTCTTTTTATGC]]> D151CR D151C <![CDATA[GCATAAAAAGAGTCAGGGTC GCA ACCATCGCCATGCTGAATAAC]]> D151EF D151E <![CDATA[CATGGCGATGGT GAA GACCCTGACTC]]> D151ER D151E <![CDATA[GAGTCAGGGTC TTC ACCATCGCCATG]]> D151FF D151F <![CDATA[GTTATTCAGCATGGCGATGGT TTT GACCCTGACTCTTTTTATG]]> D151FR D151F <![CDATA[CATAAAAAGAGTCAGGGTC AAA ACCATCGCCATGCTGAATAAC]]> D151GF D151G <![CDATA[CATGGCGATGGT GGA GACCCTGACTC <!-- 5 -->]]> D151GR D151G <![CDATA[GAGTCAGGGTC TCC ACCATCGCCATG]]> D151HF D151H <![CDATA[CATGGCGATGGT CAT GACCCTGACTC]]> D151HR D151H <![CDATA[GAGTCAGGGTC ATG ACCATCGCCATG]]> D151IF D151I <![CDATA[GTTATTCAGCATGGCGATGGT ATC GACCCTGACTCTTTTTATGC]]> D151IR D151I <![CDATA[GCATAAAAAGAGTCAGGGTC GAT ACCATCGCCATGCTGAATAAC]]> D151KF D151K <![CDATA[GTTATTCAGCATGGCGATGGT AAA GACCCTGACTCTTTTTATGC]]> D151KR D151K <![CDATA[GCATAAAAAGAGTCAGGGTC TTT ACCATCGCCATGCTGAATAAC]]> D151LF D151L <![CDATA[GTTATTCAGCATGGCGATGGT TTG GACCCTGACTCTTTTTATGC]]> D151LR D151L <![CDATA[GCATAAAAAGAGTCAGGGTC CAA ACCATCGCCATGCTGAATAAC]]> D151MF D151M <![CDATA[GTTATTCAGCATGGCGATGGT ATG GACCCTGACTCTTTTTATGC]]> D151MR D151M <![CDATA[GCATAAAAAGAGTCAGGGTC CAT ACCATCGCCATGCTGAATAAC]]> D151NF D151N <![CDATA[GCATGGCGATGGT AAT GACCCTGACTC]]> D151NR D151N <![CDATA[GAGTCAGGGTCA TTA CCATCGCCATGC]]> D151PF D151P <![CDATA[GTTATTCAGCATGGCGATGGT CCT GACCCTGACTCTTTTTATG]]> D151PR D151P <![CDATA[CATAAAAAGAGTCAGGGTC AGG ACCATCGCCATGCTGAATAAC]]> D151QF D151Q <![CDATA[GTTATTCAGCATGGCGATGGT CAG GACCCTGACTCTTTTTATGC]]> D151QR D151Q <![CDATA[GCATAAAAAGAGTCAGGGTC CTG ACCATCGCCATGCTGAATAAC]]> D151SF D151S <![CDATA[GTTATTCAGCATGGCGATGGT AGC GACCCTGACTCTTTTTATGC]]> D151SR D151S <![CDATA[GCATAAAAAGAGTCAGGGTC GCT ACCATCGCCATGCTGAATAAC]]> D151TF D151T <![CDATA[GTTATTCAGCATGGCGATGGT ACA GACCCTGACTCTTTTTATGCC]]> D151TR D151T <![CDATA[GGCATAAAAAGAGTCAGGGTC TGT ACCATCGCCATGCTGAATAAC]]> D151VF D151V <![CDATA[CATGGCGATGGT GTA GACCCTGACTC]]> D151VR D151V <![CDATA[GAGTCAGGGTC TAC ACCATCGCCATG]]> D151YF D151Y <![CDATA[CATGGCGATGGT TAT GACCCTGACTC]]> D151YR D151Y <![CDATA[GAGTCAGGGTC ATA ACCATCGCCATG]]> D151AF D151A <![CDATA[CATGGCGATGGT GCC GACCCTGACTC]]> D151AR D151A <![CDATA[GAGTCAGGGTC GGC ACCATCGCCATG]]> D151WF D151W <![CDATA[GTTATTCAGCATGGCGATGGT TGG GACCCTGACTCTTTTTATGC]]> D151WR D151W <![CDATA[GCATAAAAAGAGTCAGGGTC CCA ACCATCGCCATGCTGAATAAC]]> D151RF D151R <![CDATA[GTTATTCAGCATGGCGATGGT CGC GACCCTGACTCTTTTTATGC]]> D151RR D151R <![CDATA[GCATAAAAAGAGTCAGGGTC GCG ACCATCGCCATGCTGAATAAC]]>

[0098] The chitosanase gene was obtained by PCR amplification and combined with the expression vector pET-28a to form a recombinant vector, which was then transformed into E. coli BL21(DE3). The PCR system is shown in Table 2. PCR conditions: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 30 s, 65 ℃ annealing for 1 min, 68 ℃ extension for 10 min, 15 cycles; incubation at 4 ℃.

[0099] Table 2. Reverse PCR System

[0100] Reagent Name Volume (μL) template 2 PCR Buffer 5 dNTPs (10 mM) 1 Upstream / downstream primers (100 mM) 0.3 each Pfu DNA polymerase 1.5 <![CDATA[ddH2O]]> 40 Total volume 50

[0101] PCR reactants were digested with DpnI for 1.5 h. The remaining product from the above reaction was added to the transformation reaction solution to form the transformation system. The system was incubated on ice for 30 minutes, then heat-shocked at 42 °C for 1.5 min, followed by an ice bath for 5 min. The plasmid was transformed into *E. coli* DH5α and cultured for 1 h. Transformants were plated on kanamycin-resistant plates and incubated overnight at 37 °C. Single colonies were picked and inoculated into liquid LB medium and incubated overnight at 37 °C. A suitable amount of bacterial culture was taken using an EP tube and sent to Shanghai Bioengineering Co., Ltd. for sequencing. For correctly sequenced colonies, the plasmid was extracted using a plasmid extraction kit and then transformed into the heterologous expression host *E. coli*. E. coli Glycerin bacteria were preserved in BL21 at -80 ℃.

[0102] The preserved strain was activated by inoculating it into 10 mL of liquid LB medium at a rate of 1 / 1000, and then transferred to 50 mL of liquid LB medium for expansion culture. IPTG (isopropyl-β-D-thiogalactopyranoside) was then added to induce protein production in the bacteria.

[0103] The bacterial culture of the induced expression strain was aliquoted into centrifuge tubes, balanced, and centrifuged using a refrigerated centrifuge to remove the supernatant, retaining the bacterial cells. An appropriate amount of Mg2+ (20 mM Tris-HCl, 0.5 M NaCl, 10% glycerol, pH 8.0) was used to resuspend the cells, followed by centrifugation to remove the supernatant. The cells were then resuspended a second time with Mg2+ and carefully pipetted to homogenize. The bacterial culture was then used to disrupt the cells using an ultrasonic cell disruptor to extract the protein. The supernatant was collected by centrifugation and stored on ice. The Ni-IDA column was removed from the 4 ℃ freezer and inverted to loosen the agarose packing. The column was first washed with pure water (Wahaha purified water), then the supernatant was passed through the column. Unbound proteins were eluted thoroughly with loading buffer, followed by elution buffer (20 mM Tris-HCl, 0.5 M NaCl, 0.08 M imidazole, 10% glycerol, pH 8.0). The eluted protein-containing solution was collected and stored frozen. The protein content in the enzyme solution can be determined using a protein quantification reagent. The kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. (Bradford Reagent: E211-01).

[0104] Enzyme activity was measured using the DNS method. The reaction system consisted of 1475 μL of pH buffer and 18 µL of 100 mM Mn. 2+ Add 25 μL of purified enzyme solution to 500 μL of 1% colloidal chitosan solution. Incubate the reaction system in a water bath for 5 min at optimal temperature and pH. Terminate the reaction by adding 1.5 mL of DNS solution, then boil in water for 5 min. Finally, bring the volume to 25 mL with distilled water and allow to cool and stand for 1 h. The blank group (without enzyme and water added) is used for zeroing. The absorbance of the samples at 520 nm was measured using a UV-Vis spectrophotometer. A standard curve was plotted using a 1 mg / mL N-acetylglucosamine standard solution. The content of the reducing end of the catalytic reaction product was calculated based on the standard curve. Chitosanase activity is defined as the amount of enzyme required to generate 1 μmol of reducing sugar per minute as one enzyme activity unit. The enzyme activities of the mutant enzyme are shown in the table below.

[0105] Table 3. Enzyme activity of mutant enzymes

[0106] mutant enzyme Optimal pH Optimal temperature (°C) Enzyme activity (U / mg) WT 6.0 50 15620.46 D151E 6.0 55 2507.79 D151R 6.0 55 9409.33 D151H 6.0 60 3176.52 D151K 6.0 60 2658.49 D151I 6.0 55 5627.81 D151L 6.0 55 5407.40 D151W 6.0 55 9166.95 D151Y 6.0 55 9416.38 D151V 6.0 55 8623.77 D151P 6.0 55 7398.48 D151A 6.0 55 9137.61 D151N 6.0 55 6454.30 D151C 6.0 55 4366.99 D151Q 6.0 55 3916.59 D151G 6.0 55 3252.92 D151S 6.0 55 3618.67 D151T 6.0 60 3918.82 D151F 6.2 55 9318.81 D151M 6.6 55 4117.38 Example 2

[0107] The hydrolysis reaction of chitosanase. The substrate is 450 μL of 1% colloidal chitosan solution, with 500 μL of pH 6.6 phosphate buffer added, and 9 μL of 100 mM Mn added. 2+Add 20 U of purified enzyme solution, place at 37 °C, and shake at 160 rpm for full reaction. Centrifuge the hydrolysis system at 5 min, 30 min, 1 h, 3 h, 5 h, 7 h, 9 h, and 11 h, respectively, and collect 20 μL of supernatant. Boil for 10 min and retain the supernatant.

[0108] TLC analysis. The procedure for thin-layer chromatography is as follows: Aspirate the hydrolysate sample using a capillary tube and spot it onto a high-performance silica gel plate. Use a mixture of GlcN2-5 standard and D-glucosamine hydrochloride as controls. After thoroughly drying the spotted silica gel plate, place it in the chromatography column for full development. Remove the plate and dry it with a hairdryer until no ammonia odor remains. Evenly spray the developing agent (0.5% ninhydrin-ethanol solution), dry it, and then place it in a 110 ℃ oven until clear purple-red spots appear.

[0109] TLC analysis showed that mutant enzymes D151S, D151T, and D151Y produced chitosan within a certain hydrolysis time, but ceased producing chitosan after 3 hours. Compared to the original enzymes, they exhibited better potential for producing high-polymerization-degree chitosan oligosaccharides. This invention, based on saturation mutation of the enzyme, screened for mutants with chitosan-producing potential. Studies have shown that chitosan oligosaccharides with a degree of polymerization of 3-7 have better biological activity. The obtained mutants possess the potential for chitosan production, which offers significant advantages in the preparation of bioactive oligosaccharides.

[0110] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A chitosanase mutant, characterized in that: The product of the chitosanase mutant hydrolyzing chitosan contains chitotetrasaccharide, and the amino acid sequence of the chitosanase mutant is any one of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:

7.

2. The gene encoding the chitosanase mutant of claim 1, characterized in that, The gene sequence encoding the chitosanase mutant as shown in SEQ ID NO:3 is shown in SEQ ID NO:4; the gene sequence encoding the chitosanase mutant as shown in SEQ ID NO:5 is shown in SEQ ID NO:6; and the gene sequence encoding the chitosanase mutant as shown in SEQ ID NO:7 is shown in SEQ ID NO:

8.

3. A recombinant vector, characterized in that, The recombinant vector contains one of the genes described in claim 2.

4. A recombinant bacterial strain, characterized in that, The recombinant strain is a host bacterium containing the recombinant vector as described in claim 3.

5. The method for preparing the chitosanase mutant according to claim 1, characterized in that, A site-directed mutation was performed at position 151 of the amino acid sequence of Bacillus subtilis chitosanase BsCsn46A, with the mutation being D151S, D151T, or D151Y. Primers for the site-directed mutation were designed, and the chitosanase gene was amplified by PCR. The gene was then combined with the expression vector pET-28a to form a recombinant vector, which was transformed into E. coli BL21(DE3). After IPTG induction, bacterial cells were collected, and the supernatant was collected by centrifugation after cell disruption by sonication. The protein was then purified by Ni-NTA affinity chromatography to obtain the purified chitosanase mutant. The amino acid sequence of Bacillus subtilis chitosanase BsCsn46A is shown in SEQ ID NO:

1.

6. The application of the chitosanase mutant of claim 1 or the recombinant strain of claim 4 in the catalytic hydrolysis of chitosan to produce chitotetrasaccharide.

7. The application according to claim 6, characterized in that, The reaction system for hydrolyzing chitosan is as follows: colloidal chitosan solution, phosphate buffer solution at pH 6.6, and Mn. 2+ The solution, along with the chitosanase mutant or the recombinant strain, was reacted in a shaker at 37°C for a reaction time of no more than 3 hours.

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