Chitosan enzyme mutant for producing chitotetraose

Abstract

The invention relates to a chitotetraose-producing chitosanase mutant 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 mutant of the chitosanase is obtained by mutating the 151st aspartic acid of the BsCsn46A amino acid sequence into serine, and the mutant is D151S; or the mutant is mutated into threonine, and the mutant is D151T; or the mutant is mutated into tyrosine, and the mutant is D151Y. The mutant has the capability of hydrolyzing chitosan to produce chitotetraose.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and particularly relates to a chitosanase mutant capable of producing chitotetraose. The mutant having the ability of producing chitotetraose is obtained by performing site-directed mutation at position 151 of the amino acid sequence of Bacillus subtilis chitosanase BsCsn46A. Background Art

[0002] Chitosan (CS) is a deacetylated product of the GlcNAc bond in chitin catalyzed by chitin deacetylase. It is the only natural alkaline polysaccharide in nature. It is composed of D-glucosamine and N-acetyl-D-glucosamine connected by β-1,4-glycosidic bonds. It mainly exists in crustaceans, fungi and plant cell walls. Chitosan is the second largest biological organic resource after cellulose, with an estimated annual output of about 100 billion tons. Due to its good biosafety and biofunctionality, chitosan has great potential applications in the food, medical, environmental protection and cosmetics industries. However, chitosan has a high molecular weight and strong hydrogen bonding within and between its molecules, which leads to the formation of dense molecules that are insoluble in water and has weak biocompatibility, which greatly limits its application.

[0003] Chitosan oligosaccharide (COS) is a hydrolysis product of chitosan. It has low molecular weight, high water solubility, biocompatibility and non-sensitization. With its good biological activity, it is widely used in food, medical, industrial and agricultural fields. It is one of the most promising bioactive functional oligosaccharides. COS has good absorption performance in the human body, especially COS with a degree of polymerization between 3-7 has more outstanding biological activity. COS has strong antioxidant ability, anti-tumor, immunomodulatory function, lipid-lowering, and antibacterial, antiviral and antiparasitic effects on plant protection.

[0004] The raw materials for the preparation of COS are cheap and readily available. On this basis, COS can be prepared by physical, chemical, enzymatic or combined methods. Physical methods include ultrasound, hydrothermal, vortex cavitation, γ-ray, irradiation or microwave. The physical method is easy to control and pollution-free, but the product molecular weight is uneven and the yield is low. 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, usually using oxidants such as hydrogen peroxide, persulfate or sodium nitrite. Chemical reactions are not easy to control, costly and polluting. Enzymatic preparation of COS can be divided into two schemes: nonspecific enzymatic hydrolysis and specific enzymatic hydrolysis. Many nonspecific enzymes such as glycosidases, such as cellulase, hemicellulase, pectinase, lysozyme, etc., have catalytic effects on β-1,4 glycosidic bonds in chitosan. Nonspecific enzymes are cheap and readily available, with limited catalytic activity; specific enzymes are chitosanase, which has specificity.

[0005] Chitosanase (EC3.2.1.132) is a type of enzyme that specifically catalyzes the hydrolase of glycosidic bonds between chitosans, producing COS with different degrees of polymerization (DP) by hydrolyzing the β-1,4-glycosidic bonds in chitosan. COS with high DP has greater biological activity than COS with low DP. Summary of the invention

[0006] In order to solve the limitation of the polymerization degree of the product of the existing chitosanase, the enzyme is modified to obtain a chitosanase mutant with an improved polymerization degree of the product.

[0007] In Bacillus subtilis ( Bacillus subtilis ) The substrate channel of chitosanase BsCsn46A was analyzed and a saturation mutation was performed at position 151 of its amino acid sequence to screen out mutants with the potential to produce chitotetraose.

[0008] The chitosanase mutant of the present invention is a site-directed mutation of the 151st position of the amino acid sequence of the Bacillus subtilis chitosanase BsCsn46A, where aspartic acid is mutated to serine, marked as D151S; mutated to threonine, marked as D151T; mutated to tyrosine, marked as D151Y. The amino acid sequence of the Bacillus subtilis chitosanase BsCsn46A is SEQ ID NO:1, and the nucleotide sequence is SEQ ID NO:2, the chitosanase mutant D151S amino acid sequence is SEQ ID NO:3, and the nucleotide sequence is SEQ ID NO:4; the chitosanase mutant D151T amino acid sequence is SEQ ID NO:5, and the nucleotide sequence is SEQ ID NO:6; the chitosanase mutant D151Y amino acid sequence is SEQ ID NO:7, and the nucleotide sequence is SEQ ID NO:8.

[0009] SEQ ID NO:1 1 AGLNKDQKRRAEQLTSIFEN 21 GTTEIQYGYVERLDDGRGYT 41 CGRAGFTTATGDALEVVEVY 61 TKAVPNSKLKKYLPELRRLA 81 KEESDDTSNLKGFASAWKSL 101 ANDKEFRAAQDKVNDHLYYQ 121 NAMKRSDNAGLKTALARAVM 141 YDTVIQHGDGDDPDSFYALI 161 KRTNKKAGGSPKDGIDEKKW 181 LNKFLDVRYDDLMNPANHDT 201 RDEWRESVARVDVLRSIAKE 221 NNYNLNGPIHVRSNEYGNFV 241 IP。

[0010] SEQ ID NO:2 GCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTTGAAAACGGCACAACGGAGATCCAATATGGATATGTAGAGCGATTGGATGACGGGCGAGGCTATACATGCGGTCGGGCAGGCTTTACAACGGCTACCGGGGATGCATTGGAAGTAGTGGAAGTATACACAAAGGCAGTTCCGAATAGCAAACTGAAAAAGTATCTGCCTGAATTGCGCCGTCTGGCCAAGGAAGAAAGCGATGATACAAGCAATCTCAAGGGATTCGCTTCTGCCTGGAAGTCGCTTGCAAATGATAAGGAATTTCGCGCCGCTCAAGACAAAGTAAATGACCATTTGTATTATCAGAATGCCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTGATGTACGATACGGTTATTCAGCATGGCGATGGTGATGACCCTGACTCTTTTTATGCCTTGATTAAACGTACGAACAAAAAAGCGGGCGGATCACCTAAAGACGGAATAGACGAGAAGAAGTGGTTGAATAAATTCTTGGACGTACGCTATGACGATCTGATGAATCCGGCCAATCATGACACCCGTGACGAATGGAGAGAATCAGTTGCCCGTGTGGACGTGCTTCGCTCTATCGCCAAGGAGAACAACTATAATCTAAACGGACCGATTCATGTTCGTTCAAACGAGTACGGTAATTTTGTAATCCCTTAA。

[0011] SEQ ID NO:3 1 AGLNKDQKRR AEQLTSIFEN 21 GTTEIQYGYV ERLDDGRGYT 41 CGRAGFTTAT GDALEVVEVY 61 TKAVPNSKLK KYLPELRRLA 81 KEESDDTSNL KGFASAWKSL 101 ANDKEFRAAQ DKVNDHLYYQ <h2 style=";text-align:left;direction:ltr">121 Names of the People<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 141 YDTVIQHGDG SDPDSFYALI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 161 KRTNKKAGGS PKDGIDEKKW<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 181 LNKFLDVRYD DLMNPANHDT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 201 RDEWRESVAR VDVLRSIAKE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 221 NNYNLNGPIH VRSNEYGNFV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 241 IP.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0012] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:4<h2 style=";text-align:left;direction:ltr"> GCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTTGAAAACGGCACAACGGAGATCCAATATGGATATGTAGAGCGATTGGATGACGGGCGAGGCTATACATGCGGTCGGGCAGGCTTTACAACGGCTACCGGGGATGCATTGGAAGTAGTGGAAGTATACACAAAGGCAGTTCCGAATAGCAAACTGAAAAAGTATCTGCCTGAATTGCGCCGTCTGGCCAAGGAAGAAAGCGATGATACAAGCAATCTCAAGGGATTCGCTTCTGCCTGGAAGTCGCTTGCAAATGATAAGGAATTTCGCGCCGCTCAAGACAAAGTAAATGACCATTTGTATTATCAGAATGCCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTGATGTACGATACGGTTATTCAGCATGGCGATGGTAGCGACCCTGACTCTTTTTATGCCTTGATTAAACGTACGAACAAAAAAGCGGGCGGATCACCTAAAGACGGAATAGACGAGAAGAAGTGGTTGAATAAATTCTTGGACGTACGCTATGACGATCTGATGAATCCGGCCAATCATGACACCCGTGACGAATGGAGAGAATCAGTTGCCCGTGTGGACGTGCTTCGCTCTATCGCCAAGGAGAACAACTATAATCTAAACGGACCGATTCATGTTCGTTCAAACGAGTACGGTAATTTTGTAATCCCTTAA。

[0013] SEQ ID NO:5 1 AGLNKDQKRR AEQLTSIFEN 21 GTTEIQYGYV ERLDDGRGYT 41 CGRAGFTTAT GDALEVVEVY 61 TKAVPNSKLK KYLPELRRLA 81 KEESDDTSNL KGFASAWKSL 101 ANDKEFRAAQ DKVNDHLYYQ <h2 style=";text-align:left;direction:ltr">121 Names of the People<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 141 YDTVIQHGDG TDPDSFYALI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 161 KRTNKKAGGS PKDGIDEKKW<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 181 LNKFLDVRYD DLMNPANHDT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 201 RDEWRESVAR VDVLRSIAKE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 221 NNYNLNGPIH VRSNEYGNFV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 241 IP.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0014] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:6<h2 style=";text-align:left;direction:ltr"> GCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTTGAAAACGGCACAACGGAGATCCAATATGGATATGTAGAGCGATTGGATGACGGGCGAGGCTATACATGCGGTCGGGCAGGCTTTACAACGGCTACCGGGGATGCATTGGAAGTAGTGGAAGTATACACAAAGGCAGTTCCGAATAGCAAACTGAAAAAGTATCTGCCTGAATTGCGCCGTCTGGCCAAGGAAGAAAGCGATGATACAAGCAATCTCAAGGGATTCGCTTCTGCCTGGAAGTCGCTTGCAAATGATAAGGAATTTCGCGCCGCTCAAGACAAAGTAAATGACCATTTGTATTATCAGAATGCCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTGATGTACGATACGGTTATTCAGCATGGCGATGGTACAGACCCTGACTCTTTTTATGCCTTGATTAAACGTACGAACAAAAAAGCGGGCGGATCACCTAAAGACGGAATAGACGAGAAGAAGTGGTTGAATAAATTCTTGGACGTACGCTATGACGATCTGATGAATCCGGCCAATCATGACACCCGTGACGAATGGAGAGAATCAGTTGCCCGTGTGGACGTGCTTCGCTCTATCGCCAAGGAGAACAACTATAATCTAAACGGACCGATTCATGTTCGTTCAAACGAGTACGGTAATTTTGTAATCCCTTAA。

[0015] SEQ ID NO:7 1 AGLNKDQKRR AEQLTSIFEN 21 GTTEIQYGYV ERLDDGRGYT 41 CGRAGFTTAT GDALEVVEVY 61 TKAVPNSKLK KYLPELRRLA 81 KEESDDTSNL KGFASAWKSL 101 ANDKEFRAAQ DKVNDHLYYQ <h2 style=";text-align:left;direction:ltr">121 Names of the People<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 141 YDTVIQHGDG YDPDSFYALI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 161 KRTNKKAGGS PKDGIDEKKW<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 181 LNKFLDVRYD DLMNPANHDT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 201 RDEWRESVAR VDVLRSIAKE<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 221 NNYNLNGPIH VRSNEYGNFV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 241 IP.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0016] <h2 style=";text-align:left;direction:ltr"> SEQ ID NO:8<h2 style=";text-align:left;direction:ltr"> .

[0017] 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 to mutate the 151st aspartic acid of chitosanase (BsCsn46A) to serine (D151S), or to threonine (D151T), or to tyrosine (D151Y).

[0018] The invention provides a recombinant vector carrying the gene encoding the chitosanase mutant.

[0019] The invention provides a recombinant vector carrying the gene encoding the chitosanase mutant and a recombinant bacterium for transforming / transfecting the recombinant vector.

[0020] The specific mutation method and chitosanase mutant preparation method of the present invention are: 1. Use Swiss-Model online software to obtain the spatial structure of chitosanase (BsCsn46A); then obtain the key site (D151) that affects the degree of hydrolysis of the product through substrate channel simulation and molecular docking analysis; 2. Design primers for site-directed mutagenesis, obtain chitosanase gene by PCR amplification, and construct a recombinant vector with expression vector pET-28a, which is then transferred into E. coli BL21 (DE3); 3. The above recombinant bacteria were cultured and induced to express by IPTG, the bacteria were collected, the cells were broken by ultrasound and then centrifuged to collect the supernatant, and the protein was purified by Ni-NTA affinity chromatography to obtain the purified chitosanase mutant.

[0021] The present invention further provides industrial applications of the chitosanase mutant. Specifically, in addition to producing chitobiose and chitotriose, chitotetraose can also be produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Thin layer chromatograms of chitosan degradation products of wild-type chitosanase and D151E mutant.

[0023] Figure 2 Thin layer chromatograms of chitosan degradation products of chitosanase D151R and D151H mutants.

[0024] Figure 3 Thin layer chromatograms of chitosan degradation products of chitosanase D151K and D151A mutants.

[0025] Figure 4 Thin layer chromatograms of chitosan degradation products of chitosanase D151N and D151C mutants.

[0026] Figure 5 Thin layer chromatograms of chitosan degradation products of chitosanase D151Q and D151I mutants.

[0027] Figure 6 Thin layer chromatograms of chitosan degradation products of chitosanase D151L and D151F mutants.

[0028] Figure 7 Thin layer chromatograms of chitosan degradation products of chitosanase D151G and D151M mutants.

[0029] Figure 8Thin layer chromatograms of chitosan degradation products of chitosanase D151S and D151T mutants.

[0030] Fig. 9 Thin layer chromatograms of chitosan degradation products of chitosanase D151W and D151Y mutants.

[0031] Fig.10 Thin layer chromatograms of chitosan degradation products of chitosanase D151V and D151P mutants. DETAILED DESCRIPTION

[0032] The present invention is described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] In the embodiments of the present invention, conventional molecular biological experimental methods are used unless otherwise specified. The processes such as PCR, plasmid construction, transfection / transformation, etc. involved in the embodiments are understandable 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

[0034] The chitosanase BsCsn46A with high enzyme activity preserved in the early stage of the experiment was used as the starting enzyme. Through the analysis of the substrate channel, the potential sites that affect the polymerization degree of the product were found, and saturation mutations were performed on the key sites to obtain a mutant library.

[0035] The BsCsn46A amino acid sequence SEQ ID NO:1 was submitted to SWISS-MODEL to construct the three-dimensional structure of the enzyme. The obtained three-dimensional structure of chitosanase was analyzed using substrate channel analysis technology to obtain amino acid sites that can affect the catalytic activity of the enzyme, and the 151st site located in the substrate channel was determined to be a potential site affecting the catalytic activity.

[0036] Specific method: The primer sequences were designed by online software PrimerX as follows, and the mutant chitosanase gene was obtained by genetic engineering.

[0037] Table 1. Primers for saturation mutant library Primer name Primer usage Primer (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]]> The chitosanase gene was obtained by PCR amplification, and the recombinant vector was constructed with the expression vector pET-28a and transferred into E. coli BL21 (DE3). The PCR system is shown in Table 2. PCR conditions: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 30 s, 65 °C annealing for 1 min, 68 °C extension for 10 min, 15 cycles; 4 °C insulation.

[0038] Table 2. Inverse PCR system Reagent name Volume (μL) template 2 PCR Buffer 5 dNTPs (10 mM) 1 Upstream / downstream primers (100 mM) 0.3 each PfuDNA polymerase 1.5 <![CDATA[ddH 2 The]]> 40 Total volume 50 The PCR reaction was digested with DpnI for 1.5 h, and 10 μL of the remaining product of the above reaction was added to the transformation reaction solution to form a transformation system. Ice bath for half an hour, heat shock at 42 °C for 1.5 min, and then ice bath for 5 min. The plasmid was transformed into E. coli DH5α, and then post-cultured for 1 h, spread on a karanomycin-resistant plate, and cultured the transformants at 37 °C overnight. A single colony was picked and inoculated into liquid LB medium, cultured at 37 °C overnight, and an appropriate amount of bacterial solution was taken with an EP tube and sent to Shanghai Bioengineering Co., Ltd. for sequencing. The plasmids that were sequenced correctly were extracted with a plasmid extraction kit and then transformed into the heterologous expression host E. coli E. coli Glycerol culture was stored in BL21 at -80°C.

[0039] The preserved strain was inoculated into 10 mL of liquid LB medium at a rate of one thousandth for activation, and then transferred to 50 mL of liquid LB medium for expansion culture, and then the inducer IPTG (isopropyl-β-D-thiogalactopyranoside) was added to induce the bacteria to produce protein.

[0040] The bacterial solution of the above-mentioned induced expression strain was divided into centrifuge tubes and balanced, and the supernatant was removed by centrifugation in a refrigerated centrifuge, the bacteria were retained, and an appropriate amount of M0 (20 mM Tris-HCl, 0.5 M NaCl, 10% glycerol, pH8.0) was taken to resuspend the bacteria, and then the supernatant was removed by centrifugation, and M0 was used for the second resuspending, and the bacteria were carefully blown and sucked evenly with a gun. The above-mentioned bacterial solution was broken into cells by an ultrasonic cell disruptor, the protein was broken out, and the supernatant was collected by centrifugation and stored on ice. The Ni-IDA column was taken out of the 4 ℃ refrigerator, turned upside down to make the agarose filler contained in the column fluffy, and the column was washed with pure water (Wahaha pure water) first, and then the supernatant was passed through the column, and the unbound protein was fully eluted with the loading buffer, and then the eluted protein-containing solution was collected with the elution buffer (20 mM Tris-HCl, 0.5 M NaCl, 0.08M imidazole, 10% glycerol, pH 8.0) and stored in the refrigerator. The protein content in the enzyme solution can be determined by a protein quantitative reagent, and the kit was purchased from Nanjing Novezan Biotechnology Co., Ltd. (Bradford Reagent: E211-01).

[0041] The enzyme activity was measured by DNS method. The reaction system was 1475 μL pH buffer, 18 μL 100 mM Mn 2+, 500 μL 1% colloidal chitosan solution, add 25 μL purified enzyme solution, water bath the reaction system for 5 min at the optimal temperature and optimal pH conditions, add 1.5 mL DNS solution to terminate the reaction, boil it with boiling water for 5 min, and finally dilute it to 25 mL with distilled water, cool and stand for 1 h. The blank group was zeroed with water without enzyme. The absorbance of the sample was measured at a wavelength of 520 nm using a UV-visible spectrophotometer. A standard curve was drawn using a 1 mg / mL N-acetylglucosamine standard solution. The reducing end content of the product released by the catalytic reaction was calculated based on the standard curve, and the chitosanase activity was defined as: the amount of enzyme used to generate 1 μmol of reducing sugar per minute is one enzyme activity unit. The enzyme activity of the mutant enzyme is shown in the following table.

[0042] Table 3. Enzyme activities of mutant enzymes Mutant enzyme Optimum pH Optimum temperature (℃) Specific 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

[0043] The substrate is 450 μL of 1% colloidal chitosan solution, 500 μL of pH 6.6 phosphate buffer, and 9 μL of 100 mM Mn 2+ , then add 20 U of purified enzyme solution and place it at 37 ℃ and shaker 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, take 20 μL of the supernatant, boil it for 10 min and retain it.

[0044] TLC analysis. The operation method of thin layer chromatography is: use a capillary to absorb the hydrolyzate sample, spot it on a high-efficiency silica gel plate, and use the GlcN2-5 standard and D-glucosamine hydrochloride mixed solution as a control. After the spotted silica gel plate is fully dried, place it in the chromatography bar and fully unfold it. Take out the silica gel plate and blow it dry with a hair dryer until there is no ammonia smell, evenly spray the color developer (0.5% ninhydrin-ethanol solution), blow dry and place it in a 110 ℃ oven until clear purple-red spots appear.

[0045] According to TLC analysis, the mutant enzymes D151S, D151T, and D151Y produce chitosan within a certain hydrolysis time, and do not hydrolyze and produce chitosan after 3 hours. Compared with the original enzyme, it has a better potential for producing high-polymerization degree chitosan oligosaccharides. The present invention is based on the saturation mutation of the enzyme to screen out mutants with the potential for producing chitosan oligosaccharides. Studies have shown that chitosan oligosaccharides with a polymerization degree of 3-7 have better biological activity. The obtained mutant has the potential for producing chitosan oligosaccharides, which has obvious advantages in preparing active oligosaccharides.

[0046] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A chitosanase mutant, characterized in that: The chitosan hydrolyzed product of the chitosan mutant contains chitotetraose, and the amino acid sequence of the chitosanase is shown in SEQ ID NO:3, or shown in SEQ ID NO:5, or shown in SEQ ID NO:

7.

2. A gene encoding the chitosanase mutant according to claim 1, characterized in that: The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:3 is shown in SEQ ID NO:4; the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:5 is shown in SEQ ID NO:6; the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:7 is shown in SEQ ID NO:

8.

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

4. A recombinant strain, characterized in that: The recombinant strain is a host bacterium comprising the recombinant vector as claimed in claim 3.

5. The method for preparing the chitosanase mutant according to claim 1, characterized in that: The 151st position of the amino acid sequence of Bacillus subtilis chitosanase BsCsn46A is subjected to site-directed mutagenesis, primers for site-directed mutagenesis are designed, and the chitosanase gene is obtained by PCR amplification, and a recombinant vector is formed with the expression vector pET-28a, which is then transferred into Escherichia coli BL21 (DE3); after IPTG induction for expression, the bacteria are collected, the cells are broken by ultrasound and the supernatant is collected by centrifugation, and the protein is purified by Ni-NTA affinity chromatography to obtain a purified chitosanase mutant; the amino acid sequence of the Bacillus subtilis chitosanase BsCsn46A is shown in SEQ ID NO:1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

6. Use of the chitosanase mutant according to claim 1, the gene encoding the chitosanase mutant according to claim 2, the recombinant vector according to claim 3, or the recombinant strain according to claim 4 in catalyzing the hydrolysis of chitosan to produce chitotetraose.

7. The use according to claim 6, characterized in that: The reaction system for hydrolyzing chitosan is: colloidal chitosan solution, pH 6.6 phosphate buffer, Mn 2+ The solution and the chitosanase mutant were reacted at 37°C in a shaking incubator, and the reaction time was controlled within 3 hours.

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