High-specific-activity and high-stability chitinase smchia mutant, and preparation method therefor and use thereof

Abstract

Provided are a high-specific-activity and high-stability chitinase SmchiA mutant, and a preparation method therefor and a use thereof, relating to the technical field of enzyme engineering. The chitinase SmchiA mutant has a fused ChBD sequence at the C-terminus of the mutant, enhancing chitin-binding ability. A GH18 catalytic domain forms the core region situated between a signal peptide and a ChBD. The mutant exhibits an optimal temperature of 45°C, good thermal stability, and high efficiency in chitin hydrolysis, degrading shrimp shells to generate chitin disaccharides and tetrasaccharides.

Description

Chitinase SmchiA mutant with high specific activity and high stability, and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of enzyme engineering, and particularly relates to a chitinase SmchiA mutant with high specific activity and high stability, and a preparation method and application thereof. BACKGROUND

[0002] Chitin, also known as chitin and chitin, is a linear polysaccharide formed by the connection of glucose monomers through beta-1,4 bonds, and its structural formula is (C8H 13 O5N) n Chitin is one of the most abundant natural polysaccharides on earth, accounting for more than 10% of the total biomass, and is the most abundant amino polysaccharide, with a production second only to cellulose among biological macromolecules, so it is a promising source of biological energy and polysaccharide. Chitin widely exists in nature, mainly in the cell wall of fungi, the exoskeleton of arthropods (such as shrimps, crabs, insects, scorpions, centipedes), the radula or rostral process of mollusks, fish scales, etc.

[0003] Chitin is an important biomass resource, and its degradation product chitooligosaccharide has multiple specific biological activities such as antioxidant, antibacterial, antitumor, moisturizing and water supplementing, and enhancing immune function of the body, and is widely used in agriculture, food industry and medical field. In the food industry, the degradation product of chitin can be used to develop health food to regulate intestinal flora, prevent tumors, regulate endocrine, lose weight, etc. It can also be used as an antibacterial agent, a clarifying agent, a preservative, etc. in food; in agriculture, the degradation product of chitin can be used as a plant growth regulator and a plant disease inhibitor to promote the development of plant roots and stems, and improve the yield and quality of plants, etc. In the biomedical field, chitin and its derivatives have good biocompatibility, no rejection reaction, and can be degraded by the human body, and are used as medical materials in large quantities, such as surgical sutures, materials for contact lenses, artificial skin.

[0004] In the native state, there are strong hydrogen bonding between and within chitin molecules, so that it exists in the form of dense and ordered crystal microfibers, compact structure, high degree of crystallinity. As a result, it is insoluble in water, dilute acid, alkali and other organic solvents, but soluble in concentrated hydrochloric acid, sulfuric acid, phosphoric acid and other acids. In concentrated acid, the glycosidic bond is easy to break. The traditional method for degrading chitin is chemical method, and in general industry, chitin is hydrolyzed by strong acid to degrade it into N-acetylglucosamine (N-Acetyl-D-Glucosamine, GlcNAc). But excessive acid load can lead to degradation of glucosamine, significantly reduce the yield, and cause product quality uneven, by-products, energy consumption, equipment corrosion and environmental pollution. The use of chitinase hydrolysis of chitin has mild reaction conditions, selectivity, can directly hydrolyze chitin, green and efficient and can produce chitooligosaccharide with high product value. Therefore, this method has wide industrial application prospect.

[0005] At present, the strains that can be used to produce chitinase mainly include Bacillus subtilis, Trichoderma, Candida and the like. Bacillus subtilis is the most common strain for producing chitinase, and among them, Bacillus subtilis C-1, Bacillus subtilis B-2, Bacillus subtilis B-1 and the like are the most common. Trichoderma is another important chitinase strain, and among them, Trichoderma T-1, Trichoderma T-2, Trichoderma T-3 and the like are the most common. Candida can also produce chitinase, and among them, Candida H-1, Candida H-2, Candida H-3 and the like are the most common. These strains have different chitinase activities, suitable culture conditions and the like, but the existing chitinase generally has low enzyme activity, long production cycle of chitinase, and due to protein modification and easy misfolding of protein, the protein expression needs to be completed through a complex eukaryotic expression system, which is more troublesome, and cannot realize large-scale production and use. Screening of chitinase mutants with good degradation capacity for chitin and capable of being expressed in E. coli prokaryotic system has a very important role in industrial, agricultural and environmental protection.

[0006] In recent years, with the development of biotechnology, great progress has been made in the research on chitinase strains. Through genetic engineering technology, the chitinase strains can be modified to improve the chitinase activity and stability, thereby providing a new way for the industrial production of chitinase.

[0007] SUMMARY

[0008] In order to solve the defects of the prior art, the purpose of the present application is to provide a high specific activity and high stability chitinase SmchiA mutant, a preparation method and application thereof. The chitinase produced by the mutant can hydrolyze chitin to generate chitooligosaccharide, thereby effectively solving the problem of preparation of chitooligosaccharide.

[0009] To achieve the above object, the present application provides the following technical solutions.

[0010] The present application provides a chitinase SmchiA mutant, wherein at least one of positions 16, 22, 23, 61, 106, 127, 213, 317, 328, 388, 406, 410, 462, 495, 496, 499, 509, 527, 531, 537, 538, 539, 541, 542, 543, 544 of the amino acid sequence shown in SEQ ID NO. 1 is mutated or one amino acid is added.

[0011] Based on the above technical solutions, further, the chitinase SmchiA mutant comprises any one of the following:

[0012] (a) the A at position 16 of the amino acid shown in SEQ ID NO. 1 is mutated to S, R, Q, most preferably S;

[0013] (b) the Q at position 22 of the amino acid shown in SEQ ID NO. 1 is mutated to R, I, N, most preferably R;

[0014] (c) the A at position 23 of the amino acid shown in SEQ ID NO. 1 is mutated to T, K, R, most preferably T;

[0015] (d) the K at position 61 of the amino acid shown in SEQ ID NO. 1 is mutated to Y, D, T, most preferably Y;

[0016] (e) the V at position 106 of the amino acid shown in SEQ ID NO. 1 is mutated to T, G, N, H, most preferably T;

[0017] (f) the K at position 127 of the amino acid shown in SEQ ID NO. 1 is mutated to R, E, A, most preferably R;

[0018] (g) the Q at position 213 of the amino acid shown in SEQ ID NO. 1 is mutated to E, L, G, most preferably E;

[0019] (h) the K at position 317 of the amino acid shown in SEQ ID NO. 1 is mutated to L, M, N, most preferably L;

[0020] (i) the T at position 328 of the amino acid shown in SEQ ID NO. 1 is mutated to G, N, H, most preferably G;

[0021] (j) the A at position 388 of the amino acid shown in SEQ ID NO. 1 is mutated to G, D, I, most preferably G;

[0022] (k) mutating A at position 406 of the amino acid set forth in SEQ ID NO. 1 to T, L, G, most preferably T;

[0023] (l) mutating K at position 410 of the amino acid set forth in SEQ ID NO. 1 to N, G, I, most preferably N;

[0024] (m) mutating Q at position 462 of the amino acid set forth in SEQ ID NO. 1 to N, Y, P, most preferably N;

[0025] (n) mutating A at position 495 of the amino acid set forth in SEQ ID NO. 1 to S, I, D, most preferably S;

[0026] (o) mutating R at position 496 of the amino acid set forth in SEQ ID NO. 1 to H, Q, T, E, most preferably H;

[0027] (p) mutating Q at position 499 of the amino acid set forth in SEQ ID NO. 1 to K, A, T, most preferably K;

[0028] (q) mutating Q at position 509 of the amino acid set forth in SEQ ID NO. 1 to D, R, E, most preferably D;

[0029] (r) mutating N at position 527 of the amino acid set forth in SEQ ID NO. 1 to D, G, V, most preferably D;

[0030] (s) mutating A at position 531 of the amino acid set forth in SEQ ID NO. 1 to D, R, I, most preferably D;

[0031] (t) mutating A at position 537 of the amino acid set forth in SEQ ID NO. 1 to T, R, I, most preferably T;

[0032] (u) mutating G at position 538 of the amino acid set forth in SEQ ID NO. 1 to D, V, T, most preferably D;

[0033] (v) mutating V at position 539 of the amino acid set forth in SEQ ID NO. 1 to I, E, H, most preferably I;

[0034] (w) adding an E to position 541 of the amino acid set forth in SEQ ID NO. 1;

[0035] (x) adding a G to position 542 of the amino acid set forth in SEQ ID NO. 1;

[0036] (y) adding a T to position 543 of the amino acid set forth in SEQ ID NO. 1;

[0037] (z) adding one I to the 544th amino acid of SEQ ID NO. 1.

[0038] Based on the above technical scheme, further, the amino acid sequence of the chitinase SmchiA mutant is A16S, Q22R, A23T, K61Y, V106T, K127R, Q213E, K317L, T328G, A388G, A406T, K410N, Q462N, A495S, R496H, Q499K, Q509D, N527D, A531D, A537T, G538D, V539I, 541E, 542G, 543T, 544I in SEQ ID NO. 1.

[0039] The present application provides a gene encoding the above-mentioned chitinase SmchiA mutant.

[0040] The present application provides a recombinant vector carrying the above-mentioned gene.

[0041] Based on the above technical scheme, further, the backbone vector of the recombinant vector is a pET series vector.

[0042] Based on the above technical scheme, further, the pET series vector includes pET-3a or pET-28a.

[0043] Based on the above technical scheme, further, the recombinant vector contains a T7 promoter.

[0044] The present application provides a recombinant strain carrying the above-mentioned recombinant vector.

[0045] Based on the above technical scheme, further, the host cell of the recombinant strain is bacteria, fungi or yeast.

[0046] Based on the above technical scheme, further, the host cell of the recombinant strain is one of Escherichia coli BL21 (DE3), Origami (DE3), Origami B (DE3) or Rosetta-gami (DE3).

[0047] The present application provides a method for preparing a chitinase SmchiA mutant, inoculating the recombinant strain into a fermentation medium for culture, inducing the expression of chitinase SmchiA mutant protein, and purifying to obtain chitinase SmchiA mutant.

[0048] Based on the above technical scheme, further, the inoculation amount is 1-10% of the volume of the fermentation medium; the culture conditions are 30-38 DEG C, 150-220 rpm for 3-24 h, induction culture for 2-20 h, and the inducer is IPTG (Isopropyl-beta-D-1-thiogalactopyranoside), with a final concentration of 0.05-5 mM.

[0049] The application also provides the use of the above chitinase SmchiA mutant, the gene, the recombinant vector and the recombinant strain in degrading materials containing chitin.

[0050] Based on the above technical scheme, further, the chitin is colloidal chitin or other polysaccharides, and the concentration of chitin is 0.01 g / ml-0.1 g / ml.

[0051] Based on the above technical scheme, further, the addition amount of the chitinase SmchiA mutant is 1-10 kU / l.

[0052] Based on the above technical scheme, further, the material containing chitin includes shrimp shells, and the substrate concentration is 0.1 g / ml-1 g / ml.

[0053] The technical scheme of the application has the following beneficial effects compared with the prior art:

[0054] (1) The chitinase mutant D399-5-9Ch obtained by the application has high activity on colloidal chitin, can catalyze the generation of chitin oligosaccharides, and has relatively mild reaction conditions without high temperature and harsh conditions, so it has high industrial application value;

[0055] (2) Compared with the wild-type SmchiA chitinase (2 U / mg), the chitinase mutant D399-5-9Ch has very high chitinase activity (800 U / mg), so it has broad application prospects;

[0056] (3) The chitinase has high salt ion tolerance, so it has broad application prospects;

[0057] (4) The chitinase has the ability to degrade shrimp shells and crab shells and generate chitin oligosaccharides, so it has broad application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0058] In order to more clearly illustrate the embodiments of the application, the drawings involved in the embodiments will be briefly introduced below.

[0059] Fig. 1 is a schematic diagram of the D399-5-9Ch chitinase plasmid constructed.

[0060] Figure 2 is a predicted structure of D399-5-9Ch chitinase domain.

[0061] Figure 3 is a predicted structure of D399-5-9Ch chitinase protein.

[0062] Figure 4 is a result of D399-5-9Ch chitinase protein purification, wherein 1: supernatant sample after crushing, 2: supernatant sample after denaturation, 3: precipitate sample after renaturation, 4: supernatant sample after renaturation, 5: Ni-flow-through sample, 6: Ni-elution sample, 7: anion flow-through sample, 8: anion elution sample.

[0063] Figure 5 is a result of D399-5-9Ch chitinase activity detection.

[0064] Figure 6 is a result of D399-5-9Ch chitinase activity detection, wherein A: result of D399-5-9Ch chitinase optimum temperature (with wild-type chitinase as positive control), B: result of D399-5-9Ch chitinase optimum pH (with wild-type chitinase as positive control), C: result of D399-5-9Ch chitinase temperature stability (with wild-type chitinase as positive control), D: result of D399-5-9Ch chitinase salt concentration stability (with wild-type chitinase as positive control).

[0065] Figure 7 is a result of D399-5-9Ch chitinase degradation of shrimp shells (with wild-type chitinase as positive control), wherein A: wild-type chitinase before shrimp shell degradation, B: wild-type chitinase after shrimp shell degradation, C: D399-5-9Ch chitinase before shrimp shell degradation, D: D399-5-9Ch chitinase after shrimp shell degradation.

[0066] Figure 8 is a result of HPLC analysis of D399-5-9Ch chitinase degradation products. DETAILED DESCRIPTION

[0067] The application will be described in detail below with reference to the embodiments. However, the embodiments of the application are not limited to the following description, and it is obvious that the embodiments described below are only part of the embodiments of the application, and other similar embodiments can be obtained by those skilled in the art without creative labor, which fall within the protection scope of the application.

[0068] Example 1

[0069] 1 Gene synthesis

[0070] The amino acid sequence of chitinase SmchiA (SEQ ID NO. 1) was obtained from the Uniport database. One chitinase SmchiA mutant was obtained by point mutation, which was A16S, Q22R, A23T, K61Y, V106T, K127R, Q213E, K317L, T328G, A388G, A406T, K410N, Q462N, A495S, R496H, Q499K, Q509D, N527D, A531D, A537T, G538D, V539I, 541E, 542G, 543T, 544I, respectively. The mutant chitinase D399-5-9Ch and wild-type chitinase gene were synthesized by Shanghai Shengong Bioengineering Co., Ltd.

[0071] The amino acid sequence of the above chitinase SmchiA mutant (SEQ ID NO. 2) was added with a secretion signal peptide (SEQ ID NO. 4) and a His tag, and was optimized by Shanghai Shengong Bioengineering Co., Ltd. according to the codon bias of E. coli. The nucleotide sequence is shown in SEQ ID NO. 3. The optimized gene was digested by BamH I and Nde I, and was inserted into the prokaryotic expression vector pET-3a to obtain a recombinant plasmid. The vector is an IPTG-induced expression vector, and the protein expressed has a 6×His tag, which is convenient for purifying the protein.

[0072] 2 Transformation of recombinant plasmid

[0073] The above recombinant plasmid was transformed into E. coli expression competent cell BL21 (DE3) to prepare for protein expression and purification, and the process was as follows: first, the competent cell BL21 (DE3) was thawed on ice, 1-2 μL of the recombinant plasmid (about 100 ng / μL) was taken in a clean bench, added to 100 μL of the competent cell, mixed gently, and placed on ice for 20 min; then, it was immediately placed in ice bath for 2 min after being heated in a 42℃ water bath for 90 s. 1000 μL of LB liquid medium without antibiotics was added to the competent cell into which the recombinant plasmid was transformed in the clean bench, and was cultured at 37℃ and 200 rpm for 1 h. 100 μL of the bacterial solution was spread on an LB solid culture medium containing ampicillin, and was cultured at 37℃ overnight.

[0074] 3 Expression and purification of protein

[0075] 3.1 Expression of protein

[0076] Single colony was picked from LB solid plate and inoculated into 50 mL liquid medium containing ampicillin, and incubated in 37°C constant temperature shaking incubator overnight. 1 mL overnight culture and 1 mL ampicillin were transferred into 1 L TB medium, and incubated at 37°C, 180 rpm for 15 h, and then 1 mL IPTG was added and incubated at 37°C, 180 rpm for 4 h.

[0077] 3.2 Purification of protein

[0078] The target protein was purified by urea denaturation, renaturation, and His-tag affinity purification. The purification process was as follows:

[0079] (1) The bacteria solution after induction was centrifuged at 4°C, 6000 rpm for 20 min, and the supernatant was removed and the bacterial pellet was collected. Resuspension solution (25 mM Tris-HCl) was added at one-third of the volume of the fermentation solution and mixed well.

[0080] (2) The resuspended cell solution was crushed by a homogenizer: the homogenizer was crushed several times at 4°C until the bacterial solution was clear and transparent. The crushed bacterial solution was centrifuged at 4°C, 12000 rpm for 30 min, and the supernatant was discarded, and the inclusion bodies were used.

[0081] (3) Inclusion body washing: 25 mM Tris-HCl, 300-500 mM NaCl, 0.1 mM EDTA, 5% glycerol (added before use), 0.1 mM PMSF (added before use), and 0.5% Triton-X100 (added before use) were added to the inclusion bodies, pH 8.0, mixed well for 1 h, and centrifuged at 4°C, 12000 rpm for 30 min, and the supernatant was discarded, and the inclusion bodies were used.

[0082] (4) Inclusion body denaturation: 25 mM Tris-HCl, 300-500 mM NaCl, 8 M urea, 0.1 mM PMSF (added before use), and 1 mM DTT / β-mercaptoethanol (added before use) were added to the inclusion bodies, pH 8.0, mixed well for 3 h, and centrifuged at 4°C, 12000 rpm for 30 min, and the supernatant was collected and used.

[0083] (5) Inclusion body renaturation: the supernatant was centrifuged at 4°C, 12000 rpm for 30 min, and the supernatant was collected and subjected to gradient renaturation (25 mM Tris-HCl, 100 mM NaCl + 4 M urea→25 mM Tris-HCl, 100 mM NaCl) / slow dropwise renaturation until the urea-free protein buffer (25 mM Tris-HCl, 100 mM NaCl);

[0084] (6) Protein Ni column purification: The target protein was purified using His-tag label affinity purification. Before use, the Ni affinity chromatography column was equilibrated with 10 times the column volume of purification equilibration solution, then loaded, washed with 5 times the column volume of cleaning solution to elute impurities, and then eluted with eluent to collect the target protein.

[0085] The composition of the purification equilibration solution was: 25 mM Tris-HCl, 100 mM NaCl + 30 mM imidazole, pH = 8.0.

[0086] The composition of the elution impurity buffer was: 25 mM Tris-HCl, 100 mM NaCl + 200 mM imidazole, pH = 8.0.

[0087] The composition of the eluent was: 25 mM Tris-HCl, 100 mM NaCl + 500 mM imidazole, pH = 8.0.

[0088] (7) Protein dialysis: The purified protein was dialyzed / replaced into 25 mM Tris (pH = 8.0) buffer.

[0089] 3.3 SDS-PAGE analysis

[0090] (1) Identification using protein precast gel (4-20%) from Baisheng Biotechnology Co., Ltd.

[0091] (2) Sample treatment: Mix the protein to be tested with 5 times the protein loading buffer at a ratio of 4:1, mix well, heat denature at 100 for 5 min, and centrifuge at 12000 rpm for 1 min.

[0092] (3) Loading: Place the precast protein gel in the electrophoresis tank, add the appropriate buffer, and after pulling out the hole comb, add 5 μL of sample to each gel well.

[0093] (4) Electrophoresis: Perform sample concentration at 120 V and protein separation at 140 V.

[0094] (5) Staining and destaining: Stain the gel with Coomassie Brilliant Blue pre-staining solution and destain the gel in destaining solution.

[0095] The amino acid sequence of chitinase SmchiA mutant D399-5-9Ch was analyzed using NCBI-Blast, and the results showed that the protein with a length of 578 amino acids belongs to GH18 family chitin. The prediction results are shown in Figure 2, the enzyme includes a fibronectin III module domain ChitinaseA_N (residues 29-159), a GH18 chitinase catalytic domain (residues 163-549), and a C-terminal fused ChBD domain sequence that can increase the binding capacity of chitin. Homology modeling of chitinase SmchiA mutant is shown in Figure 3, the catalytic domain is a barrel structure composed of 24 alpha-helices and 16 beta-strands.

[0096] The stability of wild-type chitinase and chitinase mutant D399-5-9Ch was predicted using the online protein physicochemical property prediction website ProtParam of Swiss Institute of Bioinformatics Expasy website. The lower the index, the more stable it is. A decrease in stability index means an increase in stability. The results show that the predicted stability index of wild-type chitinase is 16.11, and the stability index of chitinase mutant D399-5-9Ch after modification in this paper is 14.24, proving that the chitinase mutant D399-5-9Ch after modification is more stable.

[0097] The melting point of wild-type and mutant chitinase was analyzed online according to the protein Tm prediction website of Taiwan Tsinghua University (http: / / tm.life.nthu.edu.tw / ). The higher the melting point prediction index, the higher the protein melting point and the more heat-resistant. The results show that the predicted melting point of wild-type chitinase is 0.376, and the stability index of chitinase mutant D399-5-9Ch after modification in this paper is 1.149, proving that the melting point of chitinase mutant D399-5-9Ch after modification is higher and more heat-resistant.

[0098] The protein purification results of chitinase mutant D399-5-9Ch are shown in Figure 4. The purified chitinase mutant D399-5-9Ch shows a single band with an apparent molecular weight of about 63 kDa, which is consistent with the predicted protein molecular weight. The collected target protein was concentrated using an ultrafiltration tube and the buffer was replaced using dialysis. The final protein buffer composition is: 25 mM Tris-HCl (pH = 8.0).

[0099] Example 2

[0100] 1Characterization of chitinase mutant D399-5-9Ch enzymatic properties

[0101] A. Preliminary determination of chitinase mutant D399-5-9Ch enzyme activity

[0102] The chitinase activity of the wild-type and mutant chitinases was detected using a chitinase activity assay kit from Solabio Biotech Co., Ltd. (item number BC0825).

[0103] B. Determination of the optimum pH of chitinase mutant D399-5-9Ch

[0104] The activity of the wild-type and mutant chitinases was determined in 25 mM citric acid-sodium citrate buffer (pH 2-6) and 25 mM citric acid-Tris buffer (pH 7-12) at 37°C using 1% colloidal chitin (0.01 g / mL) as the substrate to determine the optimum pH of the chitinase.

[0105] C. Determination of the optimum temperature of chitinase mutant D399-5-9Ch

[0106] The activity of the wild-type and mutant chitinases was determined in 25 mM Tris buffer at pH 8 using 1% colloidal chitin as the substrate at different temperatures (20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C) to determine the optimum temperature of the chitinase.

[0107] D. Temperature stability study of chitinase mutant D399-5-9Ch

[0108] The wild-type and mutant chitinases were placed at different temperatures (20°C, 30°C, 40°C, and 50°C) for 0.5 h and then returned to room temperature, and the enzyme activity was determined.

[0109] E. Effect of salt concentration-NaCl on the enzyme activity of chitinase mutant D399-5-9Ch

[0110] The activity of the wild-type and mutant chitinases was determined under optimal reaction conditions (43°C, pH 8.0) by adding different concentrations of sodium salt (5%, 10%, 15%, and 20%) to the reaction system.

[0111] This example used 0.01 g / mL colloidal chitin as the substrate to detect chitinase activity, with the wild-type chitinase as the control. The activity of chitinase mutant D399-5-9Ch was 800 times that of the wild-type chitinase, as shown in Figure 5.

[0112] The optimum temperature and pH of chitinase were detected using 0.01 g / mL colloidal chitin as substrate, and the results are shown in Figures 6A, 6B. The enzyme activity of wild-type chitinase was maintained at 1-2 U / mg at each temperature, and the optimum reaction temperature was 35°C / 55°C. The optimum reaction temperature of chitinase mutant D399-5-9Ch was 45°C (enzyme activity was 829 U / mg), and the enzyme activity was higher than 60% of the highest enzyme activity at 30°C-45°C, and rapidly decreased after 50°C. The optimum reaction pH of wild-type chitinase was 6 (enzyme activity was 1.6 U / mg), and there was almost no chitinase activity at pH 2-3 and 8-12. The optimum reaction pH of chitinase mutant D399-5-9Ch was 7, and the enzyme activity was higher at pH 6-8.

[0113] The thermal stability of wild-type chitinase and chitinase mutant D399-5-9Ch is shown in Figure 6C. The enzyme activity of wild-type chitinase was almost unchanged after 0.5 h at 20°C, and the enzyme activity was 82% of the highest enzyme activity after 0.5 h at 40°C and 50°C. The enzyme activity of chitinase mutant D399-5-9Ch was almost the same at 20°C and 4°C, and the enzyme activity was the highest. The enzyme activity of chitinase mutant D399-5-9Ch was almost unchanged after 0.5 h at 30°C, and the enzyme activity of chitinase mutant D399-5-9Ch could still maintain 88% of the highest enzyme activity after 0.5 h at 40°C, and the enzyme activity of chitinase mutant D399-5-9Ch could still reach 34% of the highest enzyme activity after 0.5 h at 50°C.

[0114] Using the chitinase activity without NaCl as 100%, it can be seen from Figure 6D that the enzyme activity of wild-type chitinase solution could still maintain more than 90% in 5% NaCl solution, and the enzyme activity was almost 0 in 5-20% NaCl solution. The enzyme activity of chitinase mutant D399-5-9Ch solution could still maintain more than 50% in 0-20% NaCl solution, indicating that the enzyme has strong salt tolerance.

[0115] Example 3

[0116] Shrimp shell degradation experiment

[0117] In 1 g of shrimp shell, 10 kU of wild-type chitinase liquid / chitinase mutant D399-5-9Ch crude enzyme liquid was added, and the shrimp shell degradation experiment was carried out under the optimal reaction conditions (43°C, 200 rpm). After 7 days, the degradation supernatant was taken, and the polysaccharide content in the degradation liquid supernatant was determined (same as the above activity detection method) to characterize the chitinase activity; after centrifugation, the remaining residue was dried in an oven at 60°C until the weight was constant, and the mass of the residue was weighed to calculate the efficiency of shrimp shell degradation. The degradation before and after was photographed to record the different states before and after the degradation.

[0118] The results of the shrimp shell degradation efficiency experiment are shown in Figures 7A, B. Compared with before degradation, the addition of wild-type chitinase liquid after degradation reduced the shrimp shell part, and the residue weight was 0.94 g (1 g before degradation); as shown in Figures 7C, D, the addition of chitinase mutant D399-5-9Ch crude enzyme liquid after degradation significantly reduced the shrimp shell, and the residue weight was 0.78 g (1 g before degradation). It can be concluded that the wild-type chitinase and chitinase mutant D399-5-9Ch have chitinase activity, and the chitinase mutant D399-5-9Ch has better activity in degrading shrimp shell. Normal shrimp shell contains water, protein, ash, chitin, fat, total sugar, free fatty acid, amino acid, etc., and the chitin content accounts for 30%, and chitinase mainly degrades chitin. Therefore, it is concluded that the degradation efficiency of wild-type chitinase is 20%, and the degradation efficiency of chitinase mutant D399-5-9Ch is 73%.

[0119] After centrifugation of the chitinase mutant D399-5-9Ch degradation liquid, the supernatant was mixed with acetonitrile at a ratio of 1:1, filtered through a 0.22 μm organic membrane, and the degradation products were detected by high performance liquid chromatography. The enzyme degradation products were detected by 1260 Triple Quad LC / MS (Agilent) and 4.6 mm*250 mm amino liquid chromatography column (Yuxing, China). The mobile phase composition was V(acetonitrile):V(water)=7:3, the detector was selected as an ultraviolet detector, the wavelength was 195 nm, the flow rate was 0.5 mL·min -1 , the column temperature was 30°C, and the negative ion mode. (GlcNAc)1-6 was mixed with 50% acetonitrile to form a standard sample solution of 1 mg / mL, and a standard curve was drawn. The sample amount was 10 μL.

[0120] The results of the degradation product analysis are shown in Figure 8. The retention times of (GlcNAc)1-6 were 10.730, 13.638, 17.627, 23.325, 31.423, and 43.120, respectively. In the degradation sample, substances with almost the same retention times as (GlcNAc)2 and (GlcNAc)4 were detected. It is concluded that the products obtained after chitinase degrading shrimp shell are (GlcNAc)2 and (GlcNAc)4.

[0121] In conclusion, the chitinase mutant D399-5-9Ch has good catalytic activity and product specificity, and can efficiently catalyze colloidal chitin; and can also be applied to degrade shrimp shells, and has a wide application range, and has a strong industrial and agricultural application prospect.

[0122] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A chitinase SmchiA mutant, characterized in that, The mutant is a mutant of wild-type chitinase SmchiA shown in SEQ ID NO: 1 by mutating amino acids, and the mutant chitinase SmchiA includes any one of the following: (a) the alanine A at position 16 of the amino acid shown in SEQ ID NO. 1 is mutated to serine S, arginine R, glutamine Q, most preferably serine S; (b) the glutamine Q at position 22 of the amino acid shown in SEQ ID NO. 1 is mutated to arginine R, isoleucine I, asparagine N, most preferably arginine R; (c) the alanine A at position 23 of the amino acid shown in SEQ ID NO. 1 is mutated to threonine T, lysine K, arginine R, most preferably threonine T; (d) the lysine K at position 61 of the amino acid shown in SEQ ID NO. 1 is mutated to tyrosine Y, aspartic acid D, threonine T, most preferably tyrosine Y; (e) the valine V at position 106 of the amino acid shown in SEQ ID NO. 1 is mutated to threonine T, glycine G, asparagine N, histidine H, most preferably threonine T; (f) the lysine K at position 127 of the amino acid shown in SEQ ID NO. 1 is mutated to arginine R, glutamic acid E, alanine A, most preferably arginine R; (g) the glutamine Q at position 213 of the amino acid shown in SEQ ID NO. 1 is mutated to arginine E, leucine L, glycine G, most preferably glutamic acid E; (h) the lysine K at position 317 of the amino acid shown in SEQ ID NO. 1 is mutated to leucine L, methionine M, asparagine N, most preferably leucine L; (i) the threonine T at position 328 of the amino acid shown in SEQ ID NO. 1 is mutated to glycine G, asparagine N, histidine H, most preferably glycine G; (j) the alanine A at position 388 of the amino acid shown in SEQ ID NO. 1 is mutated to glycine G, aspartic acid D, isoleucine I, most preferably glycine G; (k) the alanine A at position 406 of the amino acid shown in SEQ ID NO. 1 is mutated to threonine T, leucine L, glycine G, most preferably threonine T; (l) the lysine K at position 410 of the amino acid shown in SEQ ID NO. 1 is mutated to asparagine N, glycine G, isoleucine I, most preferably asparagine N; (m) the glutamine Q at position 462 of the amino acid shown in SEQ ID NO. 1 is mutated to asparagine N, tyrosine Y, proline P, most preferably asparagine N; (n) the alanine A at position 495 of the amino acid shown in SEQ ID NO. 1 is mutated to serine S, isoleucine I, aspartic acid D, most preferably serine S; (o) the arginine R at position 496 of the amino acid shown in SEQ ID NO. 1 is mutated to histidine H, glutamine Q, threonine T, glutamic acid E, most preferably histidine H; (p) the glutamine Q at position 499 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to lysine K, alanine A, threonine T, most preferably lysine K; (q) the glutamine Q at position 509 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to aspartic acid D, arginine R, E, most preferably aspartic acid D; (r) the asparagine N at position 527 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to aspartic acid D, glycine G, valine V, most preferably aspartic acid D; (s) the alanine A at position 531 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to aspartic acid D, arginine R, isoleucine I, most preferably aspartic acid D; (t) the alanine A at position 537 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to threonine T, arginine R, isoleucine I, most preferably threonine T; (u) the glycine G at position 538 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to aspartic acid D, valine V, threonine T, most preferably aspartic acid D; (v) the valine V at position 539 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to isoleucine I, glutamic acid E, histidine H, most preferably isoleucine I; (w) one glutamic acid E is added at position 541 of the amino acid sequence shown in SEQ ID NO. 1; (x) one glycine G is added at position 542 of the amino acid sequence shown in SEQ ID NO. 1; (y) one threonine T is added at position 543 of the amino acid sequence shown in SEQ ID NO. 1; (z) one isoleucine I is added at position 544 of the amino acid sequence shown in SEQ ID NO.

1.

2. The chitinase SmChiA mutant of claim 1, wherein, The amino acid sequence of the chitinase SmchiA mutant is A16S, Q22R, A23T, K61Y, V106T, K127R, Q213E, K317L, T328G, A388G, A406T, K410N, Q462N, A495S, R496H, Q499K, Q509D, N527D, A531D, A537T, G538D, V539I, 541E, 542G, 543T, 544I in SEQ ID NO.

1.

3. A gene encoding the chitinase SmchiA mutant of claim 1 or 2.

4. A recombinant vector carrying the gene of claim 3.

5. The recombinant vector of claim 4, wherein, The backbone vector of the recombinant vector is pET series vector.

6. A recombinant strain carrying the recombinant vector of claim 4 or 5.

7. The recombinant bacterial strain of claim 6, wherein The host cell of the recombinant strain is bacteria, fungi or yeast.

8. A method for preparing a chitinase SmchiA mutant, inoculating the recombinant strain of claim 6 or 7 into a fermentation medium for culture, inducing the expression of chitinase SmchiA mutant protein, and purifying to obtain chitinase SmchiA mutant.

9. The method of claim 8, wherein, The inoculation amount of the recombinant strain is 1-10% of the volume of the fermentation medium; the culture conditions are 30-38℃, 150-220rpm for 3-24h, and the induction culture is 2-20h, the inducer is IPTG, and the final concentration is 0.05-5mM.

10. Use of the chitinase SmchiA mutant of claim 1 or 2, the gene of claim 3, the recombinant vector of claim 4 or 5, or the recombinant strain of claim 6 or 7 in degrading chitin-containing materials.

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