CN114736891B - Chitinase mutant ChiM-SS and application thereof - Google Patents

Abstract

The invention belongs to the field of enzyme engineering, and particularly relates to a chitinase mutant ChiM-SS and application thereof. The chitinase mutant ChiM-SS provided by the invention is obtained by taking chitinase ChiM as a starting template through rational design of disulfide bond mutants and combined mutation, and the nucleotide sequence of the mutant ChiM-SS is shown as SEQ ID No. 1. The residual enzyme activities of the mutant ChiM-SS after heat treatment at 60 ℃, 65 ℃ and 70 ℃ for 60 minutes are 59.6 percent, 45.8 percent and 33.2 percent respectively, which are 2.71 times, 5.1 times and 4.7 times of the original template ChiM. The invention realizes the high-efficiency expression of the mutant ChiM-SS by combining gene copy number and molecular chaperone co-expression and taking pichia as a recombinant expression host, can be used for preparing the mutant ChiM-SS and further lays a foundation for the wide application of the mutant ChiM-SS.

Description

Chitinase mutant ChiM-SS and application thereof

Technical Field

The invention belongs to the field of enzyme engineering, and particularly relates to a chitinase mutant ChiM-SS and application thereof.

Background

Research statistics shows that the total amount of aquatic products produced in Guangdong province reaches 882 million tons in 2016, mainly shrimps and crabs, in processing industry, only the meat of shrimps and crabs is taken, and the rest is taken as processing byproducts, wherein the byproducts mainly comprise chitin, protein and inorganic salt. Chitin, a polysaccharide formed from N-acetylglucosamine linked by β -1,4 glycosidic bonds, has limited application value due to its poor solubility. The degradation product of the chitosan oligosaccharide has good solubility, so the chitosan oligosaccharide has great application value in many fields. In recent years, research and industrialization find that chitosan oligosaccharide can be applied to the field of agricultural planting as biostimulant. The chitosan oligosaccharide can promote the growth of plants and improve the stress resistance of the plants, and has great application potential in green agricultural planting. At present, the preparation method of the chitosan oligosaccharide is mainly divided into a chemical method and an enzymatic method. Compared with a chemical method, the enzyme method has the advantages of mild reaction conditions, complete product structure, greenness, no pollution and the like, so the enzyme method is the most advocated method at present. The main problem of the current method limiting the industrial application is that no suitable chitinase is found.

Chitinase ChiAm (patent application No. CN 202010795529.2), which is capable of decomposing colloidal chitin to produce chitooligosaccharides, was obtained by screening in our earlier studies. A mutant ChiM with improved catalytic activity is obtained through rational design (patent application No. CN202110762467. X). The specific activity of the mutant ChiM enzyme is 1.47 times that of chitinase ChiAm. In the previous research, the mutant ChiM is poor in thermal stability, and after the mutant ChiM is subjected to heat treatment at 60 ℃ for 60 minutes, the residual enzyme activity is only about 21%, so that the industrial application of the mutant ChiM is limited, and therefore the thermal stability of the mutant ChiM needs to be improved in an oriented mode. In the method, the mutant ChiM is used as a starting template, the mutant ChiM-SS with improved thermal stability is obtained through disulfide bond combined mutation, efficient preparation of the mutant ChiM-SS is realized through pichia pastoris, and the method lays a foundation for industrial application of the mutant ChiM-SS.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a chitinase mutant ChiM-SS and application thereof. The residual enzyme activity of the mutant ChiM-SS obtained by the invention after heat treatment at 60 ℃, 65 ℃ and 70 ℃ for 60 minutes is 2.71 times, 5.1 times and 4.7 times of that of the original template ChiM, and the thermal stability is effectively improved. In addition, the efficient expression of the mutant ChiM-SS is realized by combining high copy, molecular chaperone protein co-expression and high-density fermentation. The mutant ChiM-SS can efficiently hydrolyze and pretreat shrimp and crab shells to prepare the chitosan oligosaccharide for agricultural organic planting by optimizing reaction parameters.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention aims to provide a chitinase mutant ChiM-SS, wherein the amino acid sequence of the chitinase mutant ChiM-SS is shown as SEQ ID NO. 2.

ALSNNWCAAAPYLMPASNNPPDPVTVMNATGLKAFQLAFILAPNGGGCSPTWDGTSAVSSDTAVAGVISRIRGAGGDVSVSVGGYGGTKLGQTCGTVAATAAAYQQVITKYSLKAIDFDLEEPECENTAAIANELGAAKTLQANNPGLFVSVTMPGTAAGTGWFGTQCIDQAKSIGFSPNNFCIMPWDGGFNGGSSQVSALEAFHGLLMSHMGWDSATAYAHECFSGMNGKSDAAEMFYTSDFQTVYDYATSHGLGRFTFFSVNRDRACVGTTDNGVCSNVPQNDWDFTKFSVRFAGACPPQTTPPVTTTPTTPGNGSCTAAEWDRTKVYVKDNVVSHNSHKWTAKWWTQGEEPGTTGEWGVWQDNGAC（SEQ ID NO.2）。

Preferably, the sequence encoding the amino acid is a polynucleotide sequence, and the polynucleotide sequence is shown as SEQ ID No. 1.

GCTTTGTCTAACAACTGGTGCGCTGCTGCTCCATACTTGATGCCAGCATCTAACAACCCACCAGACCCAGTTACTGTTATGAACGCTACTGGTTTGAAGGCTTTCCAATTGGCTTTCATCTTGGCTCCAAACGGTGGTGGTTGTTCTCCAACTTGGGACGGTACTTCTGCTGTTTCTTCTGACACTGCTGTTGCTGGTGTTATCTCTAGAATCAGAGGTGCTGGTGGTGACGTTTCTGTTTCTGTTGGTGGTTACGGTGGTACTAAGTTGGGTCAAACTTGTGGTACTGTTGCTGCTACTGCTGCTGCTTACCAACAAGTTATCACTAAGTACTCTTTGAAGGCTATCGACTTCGACTTGGAAGAACCAGAATGTGAAAACACTGCTGCTATCGCTAACGAATTGGGTGCTGCTAAGACTTTGCAAGCTAACAACCCAGGTTTGTTCGTTTCTGTTACTATGCCAGGTACTGCTGCTGGTACTGGTTGGTTCGGTACTCAATGTATCGACCAAGCTAAGTCTATCGGTTTCTCTCCAAACAACTTCTGTATCATGCCATGGGACGGTGGTTTCAACGGTGGTTCTTCTCAAGTTTCTGCTTTGGAAGCTTTCCACGGTTTGTTGATGTCTCACATGGGTTGGGACTCTGCTACTGCTTACGCTCACGAATGTTTCTCTGGTATGAACGGTAAGTCTGACGCTGCTGAAATGTTCTACACTTCTGACTTCCAAACTGTTTACGACTACGCTACTTCTCACGGTTTGGGTAGATTCACTTTCTTCTCTGTTAACAGAGACAGAGCTTGTGTTGGTACTACTGACAACGGTGTTTGTTCTAACGTTCCACAAAACGACTGGGACTTCACTAAGTTCTCTGTTAGATTCGCTGGTGCTTGTCCACCACAAACTACTCCACCAGTTACTACTACTCCAACTACTCCAGGTAACGGTTCTTGTACTGCTGCTGAATGGGACAGAACTAAGGTTTACGTTAAGGACAACGTTGTTTCTCACAACTCTCACAAGTGGACTGCTAAGTGGTGGACTCAAGGTGAAGAACCAGGTACTACTGGTGAATGGGGTGTTTGGCAAGACAACGGTGCTTGTTAA（SEQ ID NO.1）。

The invention further aims to provide a recombinant expression vector pPICZ alpha A-chimss, which comprises the chitinase mutant ChiM-SS.

It is still another object of the present invention to provide a recombinant bacterium comprising the recombinant expression vector pPICZ α A-chimss as described above.

Preferably, the recombinant bacterium further comprises a chaperonin expression vector pGAPGA-pdi for expressing the chitinase mutant ChiM-SS, and the gene sequence information of the expression vector pGAPGA-pdi is shown as SEQ ID NO. 3.

AGATCTTTTTTGTAGAAATGTCTTGGTGTCCTCGTCCAATCAGGTAGCCATCTCTGAAATATCTGGCTCCGTTGCAACTCCGAACGACCTGCTGGCAACGTAAAATTCTCCGGGGTAAAACTTAAATGTGGAGTAATGGAACCAGAAACGTCTCTTCCCTTCTCTCTCCTTCCACCGCCCGTTACCGTCCCTAGGAAATTTTACTCTGCTGGAGAGCTTCTTCTACGGCCCCCTTGCAGCAATGCTCTTCCCAGCATTACGTTGCGGGTAAAACGGAGGTCGTGTACCCGACCTAGCAGCCCAGGGATGGAAAAGTCCCGGCCGTCGCTGGCAATAATAGCGGGCGGACGCATGTCATGAGATTATTGGAAACCACCAGAATCGAATATAAAAGGCGAACACCTTTCCCAATTTTGGTTTCTCCTGACCCAAAGACTTTAAATTTAATTTATTTGTCCCTATTTCAATCAATTGAACAACTATTTCGAAACGAGGAATTCATGCAATTCAACTGGAATATTAAAACTGTGGCAAGTATTTTGTCCGCTCTCACACTAGCACAAGCAAGTGATCAGGAGGCTATTGCTCCAGAGGACTCTCATGTCGTCAAATTGACTGAAGCCACTTTTGAGTCTTTCATCACCAGTAATCCTCACGTTTTGGCAGAGTTTTTTGCCCCTTGGTGTGGTCACTGTAAGAAGTTGGGCCCTGAACTTGTTTCTGCTGCCGAGATCTTAAAGGACAATGAGCAGGTTAAGATTGCTCAAATTGATTGTACGGAGGAGAAGGAATTATGTCAAGGCTACGAAATTAAAGGGTATCCTACTTTGAAGGTGTTCCATGGTGAGGTTGAGGTCCCAAGTGACTATCAAGGTCAAAGACAGAGCCAAAGCATTGTCAGCTATATGCTAAAGCAGAGTTTACCCCCTGTCAGTGAAATCAATGCAACCAAAGATTTAGACGACACAATCGCCGAGGCAAAAGAGCCCGTGATTGTGCAAGTACTACCGGAAGATGCATCCAACTTGGAATCTAACACCACATTTTACGGAGTTGCCGGTACTCTCAGAGAGAAATTCACTTTTGTCTCCACTAAGTCTACTGATTATGCCAAAAAATACACTAGCGACTCGACTCCTGCCTATTTGCTTGTCAGACCTGGCGAGGAACCTAGTGTTTACTCTGGTGAGGAGTTAGATGAGACTCATTTGGTGCACTGGATTGATATTGAGTCCAAACCTCTATTTGGAGACATTGACGGATCCACCTTCAAATCATATGCTGAAGCTAACATCCCTTTAGCCTACTATTTCTATGAGAACGAAGAACAACGTGCTGCTGCTGCCGATATTATTAAACCTTTTGCTAAAGAGCAACGTGGCAAAATTAACTTTGTTGGCTTAGATGCCGTTAAATTCGGTAAGCATGCCAAGAACTTAAACATGGATGAAGAGAAACTCCCTCTATTTGTCATTCATGATTTGGTGAGCAACAAGAAGTTTGGAGTTCCTCAAGACCAAGAATTGACGAACAAAGATGTGACCGAGCTGATTGAGAAATTCATCGCAGGAGAGGCAGAACCAATTGTGAAATCAGAGCCAATTCCAGAAATTCAAGAAGAGAAAGTCTTCAAGCTAGTCGGAAAGGCCCACGATGAAGTTGTCTTCGATGAATCTAAAGATGTTCTAGTCAAGTACTACGCCCCTTGGTGTGGTCACTGTAAGAGAATGGCTCCTGCTTATGAGGAATTGGCTACTCTTTACGCCAATGATGAGGATGCCTCTTCAAAGGTTGTGATTGCAAAACTTGATCACACTTTGAACGATGTCGACAACGTTGATATTCAAGGTTATCCTACTTTGATCCTTTATCCAGCTGGTGATAAATCCAATCCTCAACTGTATGATGGATCTCGTGACCTAGAATCATTGGCTGAGTTTGTAAAGGAGAGAGGAACCCACAAAGTGGATGCCCTAGCACTCAGACCAGTCGAGGAAGAAAAGGAAGCTGAAGAAGAAGCTGAAAGTGAGGCAGACGCTCACGACGAGCTTTAAGCGGCCGCCAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTTAGCCTTAGACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTTGCTAGATTCTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGCGGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTTCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAATTTTTTTTTTTAGTTTTTTTCTCTTTCAGTGACCTCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGTTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGGGCGGTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAACACGTCCGACGGCGGCCCACGGGTCCCAGGCCTCGGAGATCCGTCCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCAAGCTGGAGACCAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATGAGATC（SEQ ID NO.3）。

Preferably, the recombinant bacterium takes pichia pastoris engineering bacteria as a host, and the pichia pastoris engineering bacteria comprise pichia pastoris X33.

The invention also aims to provide application of the chitinase mutant ChiM-SS in preparation of chitosan oligosaccharide.

Preferably, the chitosan oligosaccharide is prepared by adopting chitinase mutant ChiM-SS to hydrolyze and pretreat shrimp and crab shells.

The invention finally aims to provide the application of the chitinase mutant ChiM-SS in agricultural organic planting.

Preferably, the chitosan oligosaccharide prepared by hydrolyzing and pretreating shrimp and crab shells by the chitinase mutant ChiM-SS is applied to agricultural organic planting.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a chitinase mutant ChiM-SS with improved thermal stability and application thereof. According to the invention, chitinase ChiM is taken as a starting template, and a mutant ChiM-SS with improved thermal stability is obtained by rationally designing disulfide bond mutants and combining mutations, so that the enzyme activity is improved, the capability of synthesizing chitosan oligosaccharide by the enzyme is enhanced, and an actual effective strategy is provided for industrial production. The experimental result shows that the residual enzyme activities of the mutant ChiM-SS after heat treatment at 60 ℃, 65 ℃ and 70 ℃ for 60 minutes are 59.6 percent, 45.8 percent and 33.2 percent respectively, which are 2.71 times, 5.1 times and 4.7 times of the original template ChiM. The invention combines gene copy number and molecular chaperone co-expression, realizes high-efficiency expression of the mutant ChiM-SS by taking pichia pastoris as a recombinant expression host, can be used for preparing the mutant ChiM-SS, and further lays a foundation for wide application of the mutant ChiM-SS.

Drawings

FIG. 1 is a three-dimensional conformation diagram of chitinase ChiM and a mutant ChiM-SS;

FIG. 2 is a graph of the optimization of the high-density fermentation reaction temperature of the recombinant engineering bacteria CM 33-P75;

FIG. 3 is a pH optimization curve diagram of a high-density fermentation reaction of a recombinant engineering bacterium CM 33-P75;

FIG. 4 is a diagram of the enzymology characteristics of the recombinant chitinase mutant ChiM-SS;

FIG. 5 is a diagram of the process optimization of the recombinant chitinase mutant ChiM-SS hydrolysis pretreatment of shrimp and crab shells;

FIG. 6 is an analysis diagram of a hydrolysate of recombinant chitinase mutant ChiM-SS.

Detailed Description

The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.

The molecular biology experiments, which are not specifically described in the following examples, were performed according to the specific methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions; the reagents and biomaterials, if not specifically indicated, are commercially available. Experimental materials and reagents involved in the present invention:

1. bacterial strains and vectors

Coli strains Top10 and Pichia pastoris X33 were all commercially available. The expression vector pPICZ alpha A-chim was constructed from a preliminary experiment (patent application No. 202110762467. X) and the expression vector pGAPGA-pdi was constructed from a preliminary experiment (patent application No. 202110762374.7).

2. Enzyme and kit

Q5 high fidelity Taq enzyme MIX was purchased from NEB; plasmid extraction kit (# DP 103-03), gel purification kit (# DP 209-02) was purchased from Tiangen Biochemical technology (Beijing) Ltd; taq enzyme MIX (emeraldAmp. MAX PCR Master MIX) purchased from Baozi Nissan physician technology (Beijing) Co., Ltd; zeocin was purchased from Invitrogen; g418 was purchased from mclin reagent, inc.

3. Culture medium

The E.coli medium was LB (1% (w/v) peptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, pH 7.0). LBZ is LB medium plus 25. mu.g/mL Zeocin (bleomycin).

The yeast medium was YPD (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose). The yeast screening medium is YPDZ (YPD + 300mg/L zeocin); YPDG (YPD + G418 at various concentrations).

Yeast induction medium BMGY (1% (w/V) yeast extract, 2% (w/V) peptone, 1.34% (w/V) YNB, 0.00004% (w/V) Biotin, 1% glycerol (V/V)), Note: YNB is Yeast Nitrogen source Base (Yeast Nitrogen Base); biotin is Biotin.

4. Reagent for measuring chitinase activity

Colloidal chitin: chitin (10 g) was weighed and added to concentrated hydrochloric acid (100 mL), and the mixture was stirred at 40 ℃ for 3 minutes to dissolve the chitin. The chitin precipitated as a colloidal suspension by adding 1 liter of cold water at 5 ℃. Filtering with coarse filter paper, and washing the filtered colloidal precipitate with distilled water to neutrality; DNS reagent (6.3 per mill (w/v) 3, 5-dinitrosalicylic acid, 18.2 percent (w/v) potassium sodium tetrahydrate, 5 per mill (w/v) phenol, 5 per mill (w/v) anhydrous sodium sulfite).

EXAMPLE 1 construction of disulfide bond mutant expression vectors

The chitinase ChiM was analyzed by molecular dynamics simulation software Gromacs 2019.06 using amber _ ff14SB force field for protein, GAFF force field for small molecule ligands and RESP potential in the GAFF force field, where the fit of RESP potential was done using Multiwfn 3.6 software in combination with Gaussian 16.0 software. The protein/ligand model was placed in a cubic box with the minimum distance of the protein from the box being 1.2 nm. The cartridge was filled with TIP3P water. The energy of the system is minimized by using a steepest descent method, molecular dynamics simulation of 100ns is carried out, the simulation step length is 2fs, and data are stored for 1 time every 10 ps. The chitinase ChiM is found to have a plurality of protein flexible regions such as a 42 th alanine to 54 th glycine region, an 274 th aspartic acid to 284 th asparagine region and the like through analysis and simulation results.

According to the simulation result, the rigidity of the chitinase ChiM is improved by directionally designing a disulfide bond in a flexible region of the chitinase ChiM three-dimensional conformation, so that the thermal stability of the chitinase ChiM is improved. By combining with online Disulfide Design software, disulifide by Design (reference website:

) And BRIDGED-Disulfide bond prediction (reference site:

) Analysis results, 9 pairs of disulfide bonds were finally selected for the experiment. The 9 disulfide bonds are Y7C-T299C, A10C-A34C, L41C-G54C, W52C-S56C, I71C-G76C, Y125C-L168C, S183C-G224C, G230C-V281C and Q283C-D287C, respectively.

Respectively designing primers to construct mutants, wherein the sequence information of 9 pairs of disulfide bond primers is shown in the following table 1, the primer sequences are shown in SEQ ID No. 4-33, and the construction process of the disulfide bond mutants is as follows:

TABLE 1 disulfide bond mutant primer sequence Listing

(taking mutant Y7C-T299C as an example, and the like):

(1) taking the constructed pPICZ alpha A-chim as a template, firstly carrying out PCR amplification by using upstream and downstream primers Y7C-fw and Y7C-rev, wherein the sequence information of the upstream primer is shown as SEQ ID NO.4, the sequence information of the downstream primer is shown as SEQ ID NO.5, the PCR reaction system is shown as the following table 2, and the PCR amplification result is detected by agarose electrophoresis;

TABLE 2 PCR reaction System

The PCR reaction procedure was as follows:

pre-denaturation at 98 ℃ for 30 seconds, denaturation at 98 ℃ for 5 seconds, annealing at 50 ℃ for 20 seconds, extension at 72 ℃ for 20 seconds, and amplification for 33 cycles;

(2) adding restriction enzyme DpnI into the PCR product successfully amplified for enzyme digestion, and removing a template vector pPICZ alpha A-chim, thereby reducing the false positive rate of a transformant;

(3) purifying and recovering the PCR product after the enzyme cutting, and transferring the PCR product into escherichia coli Top 10;

the PCR product purification and recovery process is as follows: cutting the target product into gel, and putting the gel into a 2mL centrifuge tube; adding sol solution, and reacting for 10 minutes at 60 ℃; adding the sol liquid in the second step into a collecting pipe, and centrifuging for 1 minute at 10000 rpm; washing with 75% ethanol twice, and air drying; add 50. mu.L of water and centrifuge for 3 minutes.

(4) The screening of the escherichia coli transformant adopts a bacteria liquid PCR method. Firstly picking recombinant transformants into LB culture medium in the form of single colony, culturing for 4 hours at 200rpm and 37 ℃, taking 2 microliter of bacterial liquid as a template, and carrying out PCR amplification, wherein a PCR reaction system is shown in the following table 3, primers used by bacterial liquid PCR are 5'AOX-fw and 3' AOX-rev, the sequence information of the primer of 5'AOX-fw is shown in SEQ ID NO.34, the sequence information of the primer of 3' AOX-rev is shown in SEQ ID NO.35, and the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 4 min, denaturation at 94 ℃ for 30 sec, annealing at 50 ℃ for 30 sec, extension at 72 ℃ for 90 sec, and amplification for 33 cycles. Sequencing the products which are verified to be correct, and determining a mutation site Q7C according to the sequencing result so as to obtain a mutation expression vector pPICZ alpha A-Y7C.

TABLE 3 bacterial liquid PCR reaction system

The sequence information of the primers 5'AOX-fw and 3' AOX-rev is:

5’AOX-fw：GACTGGTTCCAATTGACAAGC（SEQ ID NO.34）；

3’AOX-rev：GGCACCTGGCATTCTGACATCC（SEQ ID NO.35）。

(5) the expression vector pPICZ alpha A-Y7C is used as a template, and the disulfide bond mutant expression vector pPICZ alpha A-Y7C-T299C is obtained by replacing an amplification primer with T299C-fw and T299C-rev according to the same method for constructing the mutant Y7C.

Finally, 9 disulfide expression vectors were obtained by experiments, respectively pPICZ α A-chim1 (corresponding to mutant Y7C-T299C), pPICZ α A-chim2 (corresponding to mutant A10C-A34C), pPICZ α A-chim3 (corresponding to mutant L41C-G54C), pPICZ α A-chim4 (corresponding to mutant W52C-S56C), pPICZ α A-chim5 (corresponding to mutant I71C-G76C), pPICZ α A-chim6 (corresponding to mutant Y125C-L168C), pPICZ α A-chim7 (corresponding to mutant S183 7-G224 7), pPICZ α A-chim7 (corresponding to mutant G230-V281 7) and pPICZ α A-chim 36283 (corresponding to mutant D7-7).

Example 2 disulfide bond mutant screening

After the expression vectors of 9 pairs of different disulfide bonds are respectively linearized by restriction enzyme SacI, the linearized vectors are transferred into pichia pastoris X33 to obtain recombinant transformants.

In order to facilitate screening experiments, the expression frames of the recombinant strains are controlled in a single copy mode, and according to the previous research result, when the concentration of the transferred plasmid is controlled to be 80ng, the target genes corresponding to the grown recombinant engineering bacteria are all single copies. Therefore, the concentration of the transferred plasmid was controlled to 80ng when the transformation experiment was performed. Transformants were plated on YPDZ plates and cultured at 30 ℃ for 4 days, followed by screening. The transfer-in process is roughly as follows: (1) placing yeast competent cells on ice for 20 minutes; (2) adding 80ng of linearized expression vector, uniformly mixing, placing on ice for 5 minutes, and performing electric conversion under the conditions of 1.5 kilovolts and 400 ohms; (3) immediately adding 0.6mL of precooled 1M sorbitol into the cup after electric shock is finished, and transferring the content into a sterilized centrifuge tube; (4) standing at 30 deg.C for 2 hr, coating on YPDZ plate, culturing for 2-3 days, and observing transformant condition.

As the concentration of the transferred plasmid was controlled to 80ng, 2-4 positive transformants were grown on all the coated YPDZ plates. The screening of positive transformants adopts a 24-well plate method, and the specific steps are as follows: the recombinant transformants on the YPDZ plates were picked up one by one with a toothpick to 24-well plates containing 2mL of BMGY medium per well, incubated overnight at 30 ℃ and 200rpm for 24 hours, then centrifuged at 4000rpm to remove the supernatant, 2mL of BMMY medium was added, incubated at 30 ℃ and 200rpm for 24 hours, and the chitinase activity of the recombinant transformants was determined. And further determining the thermal stability of the enzyme according to the enzyme activity result.

The chitinase activity was determined as follows: firstly, preheating colloidal chitin and enzyme solution at 45 ℃; adding preheated 500 μ L enzyme solution into 10mL glass test tube, adding 500mL colloidal chitin solution, reacting at 45 deg.C for 30 min, adding 2mL DNS reagent to stop reaction, developing in 100 deg.C boiling water bath for 10 min, cooling, centrifuging, collecting supernatant, and measuring light absorption value at 540 nm. The definition of the enzyme activity unit is as follows: the amount of enzyme used to produce 1. mu. mol of acetylglucosamine per minute was defined as one activity unit.

The thermal stability test method is as follows: and (3) preserving the diluted enzyme solution in a water bath at 60 ℃ for 60 minutes, and then measuring the residual enzyme activity, wherein a sample without heat treatment is used as a reference.

The enzyme activity and the thermal stability of the recombinant bacteria corresponding to the 9 pairs of different disulfide bond expression vectors are obtained through screening and determination, and the experimental results are shown in table 4, and it can be known from table 4 that 3 pairs of disulfide bond mutants can improve the thermal stability of the starting template ChiM in the 9 pairs of disulfide bond mutants. Among them, mutant Y7C-T299C showed the best effect, and secondly mutant S183C-G224C and Y125C-L168C. After the mutants Y7C-T299C, S183C-G224C and Y125C-L168C are subjected to water bath heat preservation at 60 ℃ for 60 minutes, the residual enzyme activities are 35.5 percent, 26.5 percent and 25.6 percent respectively, which are 1.66 times, 1.24 times and 1.2 times of those of a control ChiM (21.3 percent).

TABLE 4 fermentation enzyme activity and thermal stability of recombinant bacteria of different disulfide bond mutants

Example 3 disulfide bond combining mutations

The effective disulfide bond mutants obtained in the following example 2, Y7C-T299, S183C-G224C and Y125C-L168C, were subjected to combinatorial mutation to further improve thermostability. The construction process of the mutant is basically the same as that of the disulfide bond mutant in the example 1, except that the PCR amplification template is replaced by an expression vector pPICZ alpha A-chim1, and the primers used in the experiment are respectively shown in the table 1 in the example 1. Finally obtaining expression vectors pPICZ alpha A-chim1-1 (containing mutation sites Y7C-T299C/S183C-G224C), pPICZ alpha A-chim1-2 (containing mutation sites Y7C-T299C/Y125C-L168C) and pPICZ alpha A-chim1-3 (containing mutation sites Y7C-T299C/S183C-G224C/Y125C-L168C) through experiments.

3 mutant expression vectors are respectively transformed into pichia pastoris X33 in a linear mode, and screening of recombinant transformants, enzyme activity determination and thermal stability determination are all consistent with those in the embodiment 2.

Through experimental screening, effective disulfide bond combinations are found to be capable of further improving the thermal stability, and table 5 shows that the 3-pair disulfide bond mutant Y7C-T299C/S183C-G224C/Y125C-L168C has the best effect, the residual enzyme activity after 60-minute water bath heat preservation at 60 ℃ is 56.5%, and compared with ChiM and Y7C-T299C, the thermal stability is respectively improved by 161.3% and 56.9%.

TABLE 5 disulfide bond combination mutants enzyme activity and thermostability

Example 4 high copy recombinant Pichia engineering construction

The gene copy number and the expression level of the recombinase have certain relevance, and the expression level of the mutant Y7C-T299C/S183C-G224C/Y125C-L168C (ChiM-SS for short) is improved by constructing high copies in the part. When the electric transformation is carried out, the mass of the linearized expression vector is more than 3 micrograms, and transformants after the electric transformation are plated on YPDZ plates (100 mg/L-500mg/L zeocin) with different concentrations. The screening method of the transformant is the same as that in the embodiment 2, and one enzyme activity dominant bacterium named as CM33 is obtained by screening.

The screened enzyme activity dominant strain CM33 is subjected to shake culture in a 250mL triangular flask, firstly, the corresponding recombinant engineering strain is inoculated into a 50mL centrifuge tube containing 5mLBMGY culture medium, the culture is carried out for about 24 hours at 30 ℃ and 220rpm, and the cultured recombinant yeast engineering strain is inoculated into the 250mL triangular flask containing 50mLBMMY culture medium according to the inoculation amount of 1% (v/v). The shake flask culture condition is 30 ℃, 220rpm, 1% (v/v) methanol is added every 24 hours for induction, simultaneously, samples are taken for testing the chitinase activity, and the enzyme activity test shows that after 120 hours of induction culture, the CM33 fermentation enzyme activity is 4.5U/mL.

In order to explore the correlation between the gene copy number and the enzyme activity, the copy number of the encoding gene chimss corresponding to the mutant ChiM-SS in the genome of the recombinant engineering bacteria CM33 is analyzed through fluorescent quantitative PCR. The experimental procedure was roughly as follows: the GAPDH gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control; designing primers aiming at target genes Chimss and gapdh by using Primer Express Software, wherein primers gap-fw and gap-rev are used for amplifying gapdh genes, and primers Chimss-fw and Chimss-rev are used for amplifying the Chimss genes, wherein the Primer sequence information of the gap-fw is shown as SEQ ID NO.36, the Primer sequence information of the gap-rev is shown as SEQ ID NO.37, the Primer sequence information of the Chimss-fw is shown as SEQ ID NO.38, and the Primer sequence information of the Chimss-rev is shown as SEQ ID NO. 39; all reactions and data collection were performed using a CFXConnect Real-Time Systems fluorescent quantitative PCR instrument, Burley, USA.

gap-fw：ACAAGGACTGGAGAGGTGGTAGAAC（SEQ ID NO.36）；

gap-rev：GAGACAACGGCATC TTCAGTGTAAC（SEQ ID NO.37）；

chimss-fw：CTGCTATCGCTAACGAAT（SEQ ID NO.38）；

chimss-rev：GAAAGTGAATCTACCCAAA（SEQ ID NO.39）。

The experimental procedure was roughly as follows: (1) firstly, connecting a target gene chimss and an internal reference gene gapdh to a vector pMD20T through experiments to obtain pMD20T-chimss and pMD20T-gapdh respectively; (2) establishing a double-standard curve, diluting the constructed vectors pMD20T-chimss and pMD20T-gapdh into copy numbers with different concentrations (108 to 103 copy numbers/. mu.L), respectively carrying out fluorescent quantitative PCR by using corresponding primers, wherein the reaction system is shown in Table 6, the reaction conditions are 95 ℃, and the pre-denaturation is carried out for 1 minute; denaturation at 95 ℃ for 15 seconds; annealing at 50 ℃ for 15 seconds; extension at 72 ℃ for 1 min; 40 cycles; (2) determining the copy number of the recombinant engineering bacteria, extracting the genome DNA of the target strain, diluting the extracted genome DNA according to 10-fold gradient (10-1, 10-2 and 10-3), performing fluorescence quantitative PCR on the diluted template, repeating each gradient for three times, and substituting the result obtained by the fluorescence quantitative PCR into a standard curve to calculate the corresponding copy number. The gene copy number is calculated by a relatively quantitative method because a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene exists as a housekeeping gene in a single copy form on a pichia pastoris genome, and thus the value of chimss relative to GAPDH is the gene copy number thereof in pichia pastoris. The copy number of chimss in the genome of the recombinant engineering bacterium CM33 is 4 by analysis.

TABLE 6 fluorescent quantitative PCR reaction System

Example 5 Co-expression of chaperone proteins

The enzyme activity dominant bacterium CM33 screened out in the embodiment 4 is taken as a host, and the expression quantity of the recombinant ChiM-SS is further improved by co-expressing molecular chaperone protein PDI. The experimental procedure was roughly as follows: (1) preparing enzyme activity dominant bacteria CM33 into competent cells; (2) extracting a molecular chaperone protein co-expression vector pGAPGA-pdi and linearizing the vector with a restriction enzyme BlnI; (3) purifying and recovering the purified and humanized molecular chaperonin expression vector pGAPGA-pdi, transferring the purified and humanized molecular chaperonin expression vector pGAPGA-pdi into enzyme activity dominant bacteria, uniformly coating transformants on a YPDG solid plate, and culturing for 3d to 4d at 30 ℃; (4) the selection of co-expressed chaperone protein transformants is in accordance with example 1.

The 3 enzyme activity dominant bacteria obtained by screening are respectively named as CM33-P6, CM33-P16 and CM 33-P75. The fermentation enzyme activities of the three recombinant engineering bacteria are further compared by shaking culture. After the induction culture in a shake flask, the highest fermentation enzyme activities of the recombinant bacteria CM33-P6, CM33-P16 and CM33-P75 are respectively 6.52U/mL, 6.23U/mL and 6.85U/mL.

Example 6 optimization of high Density fermentation Induction conditions

The recombinant engineered bacterium CM33-P75 obtained in example 5 was used for high-density fermentation. The high-density fermentation of the recombinant engineering bacteria is carried out in a 7L fermentation tank, and the specific process is approximately as follows: the single colony recombinant engineered yeast strain was inoculated into a 250mL Erlenmeyer flask containing 50mL YPG medium, and cultured overnight at 30 ℃ with shaking at 200 rpm. The overnight cultured recombinant engineered yeast was inoculated into a 500mL Erlenmeyer flask containing 100mL YPG medium at an inoculum size of 1% (v/v), and cultured overnight at 30 ℃ with shaking at 200rpm until OD600 was more than 10. The recombinant engineered yeast strain obtained by two overnight cultures was inoculated into a 7L fermentor containing 3L of BSM medium at an inoculum size of 10% (v/v). The culture conditions of the recombinant yeast engineering bacteria in a 7L fermentation tank are as follows: the temperature was 30 ℃, the pH was 5.0, the stirring speed was 500rpm, and the air flow rate was 40L/min. In the initial stage of culture, cells were grown using glycerol as a carbon source. When the wet weight of the cells reaches a certain amount (about 180 g/L), the glycerol feeding is stopped, and the induction with methanol is started after the glycerol is completely absorbed by the cells (the dissolved oxygen rises rapidly).

The induction temperature of the recombinant engineering bacteria is optimized, three induction temperatures (30 ℃, 27 ℃ and 24 ℃) are selected for experiments, and the fermentation pH is controlled to be 5. And measuring the enzyme activity and the total protein concentration every 24 hours in the fermentation process, and independently performing two batches of fermentation at each induction temperature. And (3) performing induction pH optimization on the basis of the optimization of induction temperature, wherein the induction temperature is 27 ℃, and the pH values are 4-7 respectively. The enzyme activity and the total protein concentration were measured every 24 hours during the fermentation.

As can be seen from FIG. 2 (A), the reduction of the induction temperature is beneficial to the secretion and expression of recombinant ChiM-SS by the recombinant engineering bacteria CM 33-P75. The highest fermentation enzyme activity is 165.2U/mL, 182.6U/mL and 196.3U/mL respectively at the induction temperature of 30 ℃, 27 ℃ and 24 ℃.

As can be seen from FIG. 2 (B), the maximum total protein concentrations at 27 ℃ and 24 ℃ were 3.31g/L and 3.52g/L, respectively, and were increased by 0.45g/L and 0.66g/L, respectively, relative to 30 ℃. Although the induction temperature of 24 ℃ has the best effect, in the actual fermentation production, the production cost is greatly increased due to the excessively low induction temperature, so that the induction temperature of 27 ℃ is selected for the next induction pH optimization.

As can be seen from FIG. 3 (A), the secretion expression of recombinant ChiM-SS is more facilitated when the pH is 6, and the activity of the fermentation enzyme after 144 hours of induction culture is 212.5U/mL, which is 1.36 times, 1.16 times and 1.01 times of the pH4, 5 and 7, respectively.

As can be seen from FIG. 3 (B), the maximum total protein concentration of the recombinant engineered bacteria was 4.1g/L at pH 6.

The section further improves the expression of the recombinant ChiM-SS in the pichia pastoris by optimizing the induced pH, and finally determines that the induced pH is 6 and the induced temperature is 27 ℃ by combining the induced temperature optimization result.

Example 7 purification of mutant ChiM-SS and determination of enzymatic Properties

And (3) determining the heat stability of the mutant ChiM-SS. The supernatant was obtained by first centrifugation and then concentrated and purified by passage through a10 kDa (Millipore, MWCO10 kD) ultrafiltration tube. And (3) respectively carrying out optimum reaction temperature and thermal stability determination on the purified supernatant enzyme solution, and taking chitinase ChiM obtained at the early stage as a control in the whole experimental process.

The purification process of the mutant ChiM-SS is as follows: (1) centrifuging fermentation liquor of a 7L fermentation tank, taking supernatant, purifying and recovering; (2) carrying out ultrafiltration concentration on the supernatant enzyme solution by using a10 kDa ultrafiltration tube; (3) purification was performed using Ni-IDA protein purification kit (purchased from Biotechnology engineering (Shanghai) Ltd., cat # C600292).

The optimum reaction temperature measurement method is roughly as follows: the enzyme activities of ChiM and the mutant ChiM-SS at 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃ are respectively determined, the enzyme activity at the highest temperature of the enzyme activity is determined to be 100%, and the relative enzyme activities at other temperatures are calculated, and the experimental result is shown in fig. 4 (A).

As is clear from FIG. 4 (A), the optimum reaction temperature for the starting template ChiM was 50 ℃ and the optimum reaction temperature for the mutant ChiM-SS was 60 ℃. In addition, the relative enzyme activity of the mutant ChiM-SS is obviously higher than that of ChiM in the range of 60-70 ℃.

The thermal stability measurement method is roughly as follows: and (3) carrying out water bath heat preservation for 60 minutes at 50 ℃, 55 ℃, 60 ℃, 65 ℃ and 70 ℃, then determining the residual enzyme activity, and calculating the relative residual enzyme activity at other temperatures by taking the enzyme activity of a sample which is not subjected to heat treatment as 100%. The results of the experiment are shown in FIG. 4 (B).

As can be seen from FIG. 4 (B), both ChiM and mutant ChiM-SS have better thermal stability and the residual enzyme activity is greater than 80% in the range of 50 ℃ to 55 ℃; when the heat treatment temperature is higher than 60 ℃, the ChiM residual enzyme activity is sharply reduced, and after heat treatment at 60 ℃, 65 ℃ and 70 ℃ for 60 minutes, the ChiM residual enzyme activity is 21.8%, 9.1% and 7.2% respectively. The residual enzyme activities of the mutant ChiM-SS after heat treatment at 60 ℃, 65 ℃ and 70 ℃ for 60 minutes are 59.6%, 45.8% and 33.2% respectively, which are 2.71 times, 5.1 times and 4.7 times of the original template ChiM.

Example 8 mutant ChiM-SS hydrolysis pretreatment of shrimp and crab shells

The pretreatment method of the shrimp and crab shells is as follows: (1) crushing shrimp and crab shells: crushing shrimp and crab shells, sieving with a 100-mesh sieve, and drying at 60 ℃ to constant weight; (2) removing inorganic salt from the shells of the shrimps and the crabs: weighing 10g of dried shrimp and crab shell powder, putting the dried shrimp and crab shell powder into a 500mL shake flask, adding citric acid (the concentration is 8%) according to the solid-liquid ratio of 1: 25, reacting for 3h, and centrifuging to remove supernatant; (3) removing shrimp and crab shell protein: putting the shrimp and crab shells without inorganic salt into a 500mL shake flask containing 200mL reaction liquid (pH9.0, the unit of activity of alkaline protease is 5 ten thousand U), reacting for 4 hours at 45 ℃, and centrifuging to remove the supernatant; (4) and (3) decoloring: soaking the shrimp and crab shell powder without protein and inorganic salt in 30% hydrogen peroxide solution for 2h, and centrifuging to remove supernatant to obtain chitin; (5) preparing colloidal chitin: the chitin prepared by the previous steps is firstly dissolved by concentrated hydrochloric acid, and then the pH value is adjusted to 5.0 by sodium hydroxide solution, so as to prepare the colloidal chitin.

The prepared colloidal chitin solution is used as a substrate to carry out hydrolysis experiments, and the hydrolysis experiments are mainly divided into reaction substrate concentration optimization, reaction temperature optimization, enzyme addition optimization and reaction temperature optimization. The experimental procedure was roughly as follows: (1) the total reaction system is 50mL, corresponding colloidal chitin and enzyme solution are added according to the experimental requirements, the reaction condition is 200rpm, and the experiment is respectively carried out at different temperatures and different reaction times according to the experimental requirements; (3) the hydrolysis effect is judged according to the hydrolysis rate of the colloidal chitin.

The hydrolysis rate was measured approximately as follows: (1) firstly, weighing the weights of centrifuge tubes with different specifications of 2mL respectively; (2) secondly, taking 2mL of hydrolysis samples with different reaction times; (3) centrifuging to remove supernatant, placing the centrifuge tube containing precipitate at 100 deg.C, and oven drying to constant weight; (4) the weight of the centrifuge tube subtracted from the dried weight is colloidal chitin without hydrolysis; (5) the hydrolysis rate was calculated as follows: hydrolysis rate = (theoretical colloidal chitin weight-weight without hydrocolloid)/theoretical colloidal chitin weight.

Firstly, optimizing the content of a substrate, wherein the content of colloidal chitin is respectively set to be 1%, 2%, 3% and 4%, the addition amount of enzyme is 5U/mL, the reaction temperature is 40 ℃, the reaction pH is 5.0, the reaction speed is 200rpm, and the reaction time is 3 hours.

As shown in FIG. 5A, as the substrate content was higher, the hydrolysis rate was lower, and the hydrolysis rates corresponding to 1%, 2%, 3% and 4% substrate contents were 78.2%, 63.3%, 53.2% and 42.1%, respectively, as shown in FIG. 5A. Considering the analysis, the substrate content of 2% is finally selected for the next experiment.

The enzyme addition amount was optimized based on the results of the substrate content experiment and was set to 2.5U/mL, 5U/mL, 7.5U/mL, 10U/mL, 12.5U/mL and 15U/mL, respectively. The substrate content was 2%, the reaction temperature was 40 ℃, the reaction pH was 5.0, 200rpm, and the reaction time was 3 hours, and the experimental results are shown in FIG. 5 (B). As shown in FIG. 5 (B), the production rate of the polypeptide gradually increased with the increase in the amount of the enzyme added, and the hydrolysis rate was 75.5% at the maximum when the amount of the enzyme added was 15U/mL. The hydrolysis rates of the enzyme addition amounts of 7.5U/mL, 10U/mL and 12.5U/mL were 69.5%, 70.5% and 72.5%, respectively. The final enzyme addition amount was 7.5U/mL in view of the cost of the enzyme.

The reaction temperature was optimized on the basis of the substrate content and the enzyme dosage, and was set to 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C and 55 deg.C, respectively. The substrate content was 2%, the enzyme addition amount was 7.5U/mL, the reaction pH was 5.0, 200rpm, and the reaction time was 3 hours, and the experimental results are shown in FIG. 5 (C). As can be seen from FIG. 5 (C), the reaction temperature was 50 ℃ and the hydrolysis rate was found to be 76.5%. Therefore, 50 ℃ was chosen for the next experiment.

The reaction time was optimized on the basis of the substrate content, the amount of enzyme used, and the optimum reaction temperature, and was set to 2 hours, 3 hours, 4 hours, and 5 hours. The substrate content was 2%, the enzyme addition amount was 7.5U/mL, the reaction pH was 5.0, the reaction temperature was 50 ℃ and 200rpm, and the results of the experiment are shown in FIG. 5 (D). As is clear from FIG. 5 (D), the effect was most excellent in 4 hours of the reaction, and the hydrolysis rate was 85.5%, and the polypeptide formation rate was 83.2% in the next 5 hours.

Example 9 analysis of mutant ChiM-SS hydrolysate and Chinese cabbage hydroponic planting experiment

The hydrolyzate obtained in example 8 was analyzed by thin layer chromatography and high performance liquid chromatography. The thin layer chromatography experimental procedure was as follows: (1) spotting 1. mu.L to 3. mu.L of the hydrolysis reaction product and 2. mu.L of the chitosan oligosaccharide standard mixture onto a Silica gel plate (Silica gel 60, Merck), respectively; placing the well-spotted silica gel plate in an expansion cylinder for expansion, wherein the expansion buffer solution is a mixture of n-butyl alcohol, methanol, ammonia water and water (the volume ratio is 5:4:2: 1); (2) taking the expanded silica gel plate out of the expansion cylinder, drying and spraying a display agent (the display agent is 0.4g of diphenylamine, 0.4mL of aniline and 3mL of 85% phosphoric acid dissolved in 20mL of acetone solution); (3) after drying, the silica gel plate is placed at 100 ℃ for high-temperature color development. As is clear from FIG. 6 (A), the hydrolyzate is mainly composed of chitobiose.

The hydrolysate was further subjected to high performance liquid chromatography, the experimental procedure was as follows: (1) filtering the hydrolysate with 0.22 μm filter membrane; (2) the hydrolysate composition was analyzed by hplc UltiMate 3000 with Zorbax carbonate analysis column (4.6 × 250 mm) and mobile phase acetonitrile: water = 7: 3, the flow rate is 0.7mL/min, the detection temperature is 30 ℃, and the detector is a differential detector. As is clear from FIG. 6 (B), the hydrolyzate is mainly composed of chitobiose.

The application effect of the hydrolysate on the rooting and growth promotion of the water culture pakchoi is researched by taking pakchoi as a research object. The experiment adopts a water culture method, the Chinese cabbage seeds are placed in a laboratory water culture flask, and the water culture is carried out by adopting a basic nutrient solution. The hydrolysis products were diluted 1000 times, 2000 times, 3000 times, 4000 times and 5000 times for experiments, and the root length and plant weight were measured periodically with the basic nutrient solution culture group as a control. The experimental results are shown in the following table 7, and it can be seen from the table 7 that the addition of the hydrolysate can promote the rooting and growth of the pakchoi, and after 30 days of culture, the root length and fresh weight of the pakchoi in the hydrolysate-added experimental group are higher than those in the control group. The effect of 4000 times dilution is most obvious, the fresh weight and the root length of the pakchoi are respectively 23.8g and 48.6 cm, and are respectively improved by 33.7 percent and 58.8 percent compared with the control group, which shows that the hydrolysate has better growth promoting and yield increasing effects on the pakchoi.

TABLE 7 hydrolyzate cabbage planting experiments

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and scope of the present invention as defined in the appended claims.

SEQUENCE LISTING

<110> Shenzhen Runkang ecological environment shares Limited

<120> chitinase mutant ChiM-SS and application thereof

<130> 2022.5.26

<160> 39

<170> PatentIn version 3.3

<210> 1

<211> 1110

<212> DNA

<213> Polynucleotide sequence encoding amino acid sequence of chitinase mutant ChiM-SS (Polynucleotide sequence encoding amino acid sequence of chitinase mutant ChiM-SS)

<400> 1

gctttgtcta acaactggtg cgctgctgct ccatacttga tgccagcatc taacaaccca 60

ccagacccag ttactgttat gaacgctact ggtttgaagg ctttccaatt ggctttcatc 120

ttggctccaa acggtggtgg ttgttctcca acttgggacg gtacttctgc tgtttcttct 180

gacactgctg ttgctggtgt tatctctaga atcagaggtg ctggtggtga cgtttctgtt 240

tctgttggtg gttacggtgg tactaagttg ggtcaaactt gtggtactgt tgctgctact 300

gctgctgctt accaacaagt tatcactaag tactctttga aggctatcga cttcgacttg 360

gaagaaccag aatgtgaaaa cactgctgct atcgctaacg aattgggtgc tgctaagact 420

ttgcaagcta acaacccagg tttgttcgtt tctgttacta tgccaggtac tgctgctggt 480

actggttggt tcggtactca atgtatcgac caagctaagt ctatcggttt ctctccaaac 540

aacttctgta tcatgccatg ggacggtggt ttcaacggtg gttcttctca agtttctgct 600

ttggaagctt tccacggttt gttgatgtct cacatgggtt gggactctgc tactgcttac 660

gctcacgaat gtttctctgg tatgaacggt aagtctgacg ctgctgaaat gttctacact 720

tctgacttcc aaactgttta cgactacgct acttctcacg gtttgggtag attcactttc 780

ttctctgtta acagagacag agcttgtgtt ggtactactg acaacggtgt ttgttctaac 840

gttccacaaa acgactggga cttcactaag ttctctgtta gattcgctgg tgcttgtcca 900

ccacaaacta ctccaccagt tactactact ccaactactc caggtaacgg ttcttgtact 960

gctgctgaat gggacagaac taaggtttac gttaaggaca acgttgtttc tcacaactct 1020

cacaagtgga ctgctaagtg gtggactcaa ggtgaagaac caggtactac tggtgaatgg 1080

ggtgtttggc aagacaacgg tgcttgttaa 1110

<210> 2

<211> 369

<212> PRT

<213> Amino acid sequence of chitinase mutant ChiM-SS (Amino acid sequence of chitinase mutant ChiM-SS)

<400> 2

Ala Leu Ser Asn Asn Trp Cys Ala Ala Ala Pro Tyr Leu Met Pro Ala

1 5 10 15

Ser Asn Asn Pro Pro Asp Pro Val Thr Val Met Asn Ala Thr Gly Leu

20 25 30

Lys Ala Phe Gln Leu Ala Phe Ile Leu Ala Pro Asn Gly Gly Gly Cys

35 40 45

Ser Pro Thr Trp Asp Gly Thr Ser Ala Val Ser Ser Asp Thr Ala Val

50 55 60

Ala Gly Val Ile Ser Arg Ile Arg Gly Ala Gly Gly Asp Val Ser Val

65 70 75 80

Ser Val Gly Gly Tyr Gly Gly Thr Lys Leu Gly Gln Thr Cys Gly Thr

85 90 95

Val Ala Ala Thr Ala Ala Ala Tyr Gln Gln Val Ile Thr Lys Tyr Ser

100 105 110

Leu Lys Ala Ile Asp Phe Asp Leu Glu Glu Pro Glu Cys Glu Asn Thr

115 120 125

Ala Ala Ile Ala Asn Glu Leu Gly Ala Ala Lys Thr Leu Gln Ala Asn

130 135 140

Asn Pro Gly Leu Phe Val Ser Val Thr Met Pro Gly Thr Ala Ala Gly

145 150 155 160

Thr Gly Trp Phe Gly Thr Gln Cys Ile Asp Gln Ala Lys Ser Ile Gly

165 170 175

Phe Ser Pro Asn Asn Phe Cys Ile Met Pro Trp Asp Gly Gly Phe Asn

180 185 190

Gly Gly Ser Ser Gln Val Ser Ala Leu Glu Ala Phe His Gly Leu Leu

195 200 205

Met Ser His Met Gly Trp Asp Ser Ala Thr Ala Tyr Ala His Glu Cys

210 215 220

Phe Ser Gly Met Asn Gly Lys Ser Asp Ala Ala Glu Met Phe Tyr Thr

225 230 235 240

Ser Asp Phe Gln Thr Val Tyr Asp Tyr Ala Thr Ser His Gly Leu Gly

245 250 255

Arg Phe Thr Phe Phe Ser Val Asn Arg Asp Arg Ala Cys Val Gly Thr

260 265 270

Thr Asp Asn Gly Val Cys Ser Asn Val Pro Gln Asn Asp Trp Asp Phe

275 280 285

Thr Lys Phe Ser Val Arg Phe Ala Gly Ala Cys Pro Pro Gln Thr Thr

290 295 300

Pro Pro Val Thr Thr Thr Pro Thr Thr Pro Gly Asn Gly Ser Cys Thr

305 310 315 320

Ala Ala Glu Trp Asp Arg Thr Lys Val Tyr Val Lys Asp Asn Val Val

325 330 335

Ser His Asn Ser His Lys Trp Thr Ala Lys Trp Trp Thr Gln Gly Glu

340 345 350

Glu Pro Gly Thr Thr Gly Glu Trp Gly Val Trp Gln Asp Asn Gly Ala

355 360 365

Cys

<210> 3

<211> 4836

<212> DNA

<213> Gene sequence information of expression vector pGAPGA-pdi (Gene sequence information of expression vector pGAPGA-pdi)

<400> 3

agatcttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca tctctgaaat 60

atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc cggggtaaaa 120

cttaaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct tccaccgccc 180

gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc cccttgcagc 240

aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg acctagcagc 300

ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg catgtcatga 360

gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca attttggttt 420

ctcctgaccc aaagacttta aatttaattt atttgtccct atttcaatca attgaacaac 480

tatttcgaaa cgaggaattc atgcaattca actggaatat taaaactgtg gcaagtattt 540

tgtccgctct cacactagca caagcaagtg atcaggaggc tattgctcca gaggactctc 600

atgtcgtcaa attgactgaa gccacttttg agtctttcat caccagtaat cctcacgttt 660

tggcagagtt ttttgcccct tggtgtggtc actgtaagaa gttgggccct gaacttgttt 720

ctgctgccga gatcttaaag gacaatgagc aggttaagat tgctcaaatt gattgtacgg 780

aggagaagga attatgtcaa ggctacgaaa ttaaagggta tcctactttg aaggtgttcc 840

atggtgaggt tgaggtccca agtgactatc aaggtcaaag acagagccaa agcattgtca 900

gctatatgct aaagcagagt ttaccccctg tcagtgaaat caatgcaacc aaagatttag 960

acgacacaat cgccgaggca aaagagcccg tgattgtgca agtactaccg gaagatgcat 1020

ccaacttgga atctaacacc acattttacg gagttgccgg tactctcaga gagaaattca 1080

cttttgtctc cactaagtct actgattatg ccaaaaaata cactagcgac tcgactcctg 1140

cctatttgct tgtcagacct ggcgaggaac ctagtgttta ctctggtgag gagttagatg 1200

agactcattt ggtgcactgg attgatattg agtccaaacc tctatttgga gacattgacg 1260

gatccacctt caaatcatat gctgaagcta acatcccttt agcctactat ttctatgaga 1320

acgaagaaca acgtgctgct gctgccgata ttattaaacc ttttgctaaa gagcaacgtg 1380

gcaaaattaa ctttgttggc ttagatgccg ttaaattcgg taagcatgcc aagaacttaa 1440

acatggatga agagaaactc cctctatttg tcattcatga tttggtgagc aacaagaagt 1500

ttggagttcc tcaagaccaa gaattgacga acaaagatgt gaccgagctg attgagaaat 1560

tcatcgcagg agaggcagaa ccaattgtga aatcagagcc aattccagaa attcaagaag 1620

agaaagtctt caagctagtc ggaaaggccc acgatgaagt tgtcttcgat gaatctaaag 1680

atgttctagt caagtactac gccccttggt gtggtcactg taagagaatg gctcctgctt 1740

atgaggaatt ggctactctt tacgccaatg atgaggatgc ctcttcaaag gttgtgattg 1800

caaaacttga tcacactttg aacgatgtcg acaacgttga tattcaaggt tatcctactt 1860

tgatccttta tccagctggt gataaatcca atcctcaact gtatgatgga tctcgtgacc 1920

tagaatcatt ggctgagttt gtaaaggaga gaggaaccca caaagtggat gccctagcac 1980

tcagaccagt cgaggaagaa aaggaagctg aagaagaagc tgaaagtgag gcagacgctc 2040

acgacgagct ttaagcggcc gccagcttgg gcccgaacaa aaactcatct cagaagagga 2100

tctgaatagc gccgtcgacc atcatcatca tcatcattga gttttagcct tagacatgac 2160

tgttcctcag ttcaagttgg gcacttacga gaagaccggt cttgctagat tctaatcaag 2220

aggatgtcag aatgccattt gcctgagaga tgcaggcttc atttttgata cttttttatt 2280

tgtaacctat atagtatagg attttttttg tcattttgtt tcttctcgta cgagcttgct 2340

cctgatcagc ctatctcgca gctgatgaat atcttgtggt aggggtttgg gaaaatcatt 2400

cgagtttgat gtttttcttg gtatttccca ctcctcttca gagtacagaa gattaagtga 2460

gaccttcgtt tgtgcggatc ccccacacac catagcttca aaatgtttct actccttttt 2520

tactcttcca gattttctcg gactccgcgc atcgccgtac cacttcaaaa cacccaagca 2580

cagcatacta aattttccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag 2640

gtttggaaaa gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa 2700

aatttttatc acgtttcttt ttcttgaaat tttttttttt agtttttttc tctttcagtg 2760

acctccattg atatttaagt taataaacgg tcttcaattt ctcaagtttc agtttcattt 2820

ttcttgttct attacaactt tttttacttc ttgttcatta gaaagaaagc atagcaatct 2880

aatctaaggg cggtgttgac aattaatcat cggcatagta tatcggcata gtataatacg 2940

acaaggtgag gaactaaacc atgagccata ttcaacggga aacgtcttgc tcgaggccgc 3000

gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg 3060

ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc 3120

tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact 3180

ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg 3240

catggttact caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc 3300

ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga 3360

ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat 3420

cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc 3480

ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg gattcagtcg 3540

tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt 3600

gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga 3660

actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg 3720

ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaacacg 3780

tccgacggcg gcccacgggt cccaggcctc ggagatccgt cccccttttc ctttgtcgat 3840

atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc 3900

gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg 3960

ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg 4020

tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct 4080

ttaatttgca agctggagac caacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 4140

aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 4200

aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 4260

ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 4320

tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc 4380

agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 4440

gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 4500

tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 4560

acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 4620

tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 4680

caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 4740

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 4800

aactcacgtt aagggatttt ggtcatgcat gagatc 4836

<210> 4

<211> 31

<212> DNA

<213> Y7C-fw

<400> 4

tgtctaacaa ctggtgcgct gctgctccat a 31

<210> 5

<211> 31

<212> DNA

<213> Y7C-rev

<400> 5

tatggagcag cagcgcacca gttgttagac a 31

<210> 6

<211> 32

<212> DNA

<213> T299C-fw

<400> 6

attcgctggt gcttgtccac cacaaactac tc 32

<210> 7

<211> 32

<212> DNA

<213> T299C-rev

<400> 7

gagtagtttg tggtggacaa gcaccagcga at 32

<210> 8

<211> 34

<212> DNA

<213> A10C-fw

<400> 8

aactggtacg ctgcttgtcc atacttgatg ccac 34

<210> 9

<211> 34

<212> DNA

<213> A10C-rev

<400> 9

gtggcatcaa gtatggacaa gcagcgtacc agtt 34

<210> 10

<211> 33

<212> DNA

<213> A34C-fw

<400> 10

gctactggtt tgaagtgttt ccaattggct ttc 33

<210> 11

<211> 33

<212> DNA

<213> A34C-rev

<400> 11

gaaagccaat tggaaacact tcaaaccagt agc 33

<210> 12

<211> 33

<212> DNA

<213> L41C-fw

<400> 12

aattggcttt catctgtgct ccaaacggtg gtg 33

<210> 13

<211> 33

<212> DNA

<213> L41C-rev

<400> 13

caccaccgtt tggagcacag atgaaagcca att 33

<210> 14

<211> 33

<212> DNA

<213> G54C-fw

<400> 14

caacttggga ctgtacttct gctgtttctt ctg 33

<210> 15

<211> 33

<212> DNA

<213> G54C-rev

<400> 15

tcagaagaaa cagcagaagt acagtcccaa gtt 33

<210> 16

<211> 36

<212> DNA

<213> W52C-S56C-fw

<400> 16

gttctccaac ttgtgacggt acttgtgctg tttctt 36

<210> 17

<211> 36

<212> DNA

<213> W52C-S56C-rev

<400> 17

aagaaacagc acaagtaccg tcacaagttg gagaac 36

<210> 18

<211> 38

<212> DNA

<213> I71C-G76C-fw

<400> 18

tctctagatg tagaggtgct ggttgtgacg tttctgtt 38

<210> 19

<211> 38

<212> DNA

<213> I71C-G76C-rev

<400> 19

aacagaaacg tcacaaccag cacctctaca tctagaga 38

<210> 20

<211> 31

<212> DNA

<213> Y125C-fw

<400> 20

gaagaaccag aatgtgaaaa cactgctgct a 31

<210> 21

<211> 31

<212> DNA

<213> Y125C-rev

<400> 21

tagcagcagt gttttcacat tctggttctt c 31

<210> 22

<211> 32

<212> DNA

<213> L168C-fw

<400> 22

ttcggtactc aatgtatcga ccaagctaag tc 32

<210> 23

<211> 32

<212> DNA

<213> L168C-rev

<400> 23

gacttagctt ggtcgataca ttgagtaccg aa 32

<210> 24

<211> 32

<212> DNA

<213> S183C-fw

<400> 24

ccaaacaact tctgtatcat gccattcgac gg 32

<210> 25

<211> 32

<212> DNA

<213> S183C-rev

<400> 25

ccgtcgaatg gcatgataca gaagttgttt gg 32

<210> 26

<211> 33

<212> DNA

<213> G224C-fw

<400> 26

cttacgctca cgaatgtttc tctggtatga acg 33

<210> 27

<211> 33

<212> DNA

<213> G224C-rev

<400> 27

cgttcatacc agagaaacat tcgtgagcgt aag 33

<210> 28

<211> 32

<212> DNA

<213> G230C-fw

<400> 28

tctctggtat gaactgtaag tctgacgctg ct 32

<210> 29

<211> 32

<212> DNA

<213> G230C-rev

<400> 29

agcagcgtca gacttacagt tcataccaga ga 32

<210> 30

<211> 35

<212> DNA

<213> V281C-fw

<400> 30

gtgtttgttc taactgtcca caaaacgact gggac 35

<210> 31

<211> 35

<212> DNA

<213> V281C-rev

<400> 31

gtcccagtcg ttttgtggac agttagaaca aacac 35

<210> 32

<211> 36

<212> DNA

<213> Q283C-D287C-fw

<400> 32

tctaacgttc catgtaacga ctggtgtttc actaag 36

<210> 33

<211> 36

<212> DNA

<213> Q283C-D287C-rev

<400> 33

cttagtgaaa caccagtcgt tacatggaac gttaga 36

<210> 34

<211> 21

<212> DNA

<213> Sequence information of primer 5'AOX-fw (Sequence information of primer 5' AOX-fw)

<400> 34

gactggttcc aattgacaag c 21

<210> 35

<211> 22

<212> DNA

<213> Sequence information of primer 3'AOX-rev (Sequence information of primer 3' AOX-rev)

<400> 35

ggcacctggc attctgacat cc 22

<210> 36

<211> 25

<212> DNA

<213> gap-fw

<400> 36

acaaggactg gagaggtggt agaac 25

<210> 37

<211> 25

<212> DNA

<213> gap-rev

<400> 37

gagacaacgg catcttcagt gtaac 25

<210> 38

<211> 18

<212> DNA

<213> chimss-fw

<400> 38

ctgctatcgc taacgaat 18

<210> 39

<211> 19

<212> DNA

<213> chimss-rev

<400> 39

gaaagtgaat ctacccaaa 19

Claims (8)

1. The chitinase mutant ChiM-SS is characterized in that the amino acid sequence of the chitinase mutant ChiM-SS is shown as SEQ ID NO. 2.

2. The chitinase mutant ChiM-SS according to claim 1, wherein the sequence encoding said amino acids is a polynucleotide sequence as shown in SEQ ID No. 1.

3. A recombinant expression vector pPICZ α a-chimss comprising the polynucleotide sequence of claim 2.

4. A recombinant bacterium comprising the recombinant expression vector pPICZ α A-chimss according to claim 3.

5. The recombinant strain as claimed in claim 4, further comprising a chaperonin expression vector pGAPGA-pdi for expressing the chitinase mutant ChiM-SS, wherein the gene sequence information of the expression vector pGAPGA-pdi is shown as SEQ ID No. 3.

6. The recombinant strain of claim 5, wherein the recombinant strain takes pichia pastoris engineering bacteria as a host, and the pichia pastoris engineering bacteria comprise pichia pastoris X33.

7. The use of the chitinase mutant ChiM-SS of claim 1 in the preparation of chitooligosaccharides.

8. The use of claim 7, wherein the chitooligosaccharide is prepared by hydrolyzing pretreated shrimp and crab shells by chitinase mutant ChiM-SS.

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