CN109486804B

CN109486804B - Novel chitosanase CsnM with heat recovery characteristic and application thereof

Info

Publication number: CN109486804B
Application number: CN201811596664.3A
Authority: CN
Inventors: 李尚勇; 韩彦弢; 周宇
Original assignee: Qingdao University
Current assignee: Qingdao University
Priority date: 2018-12-26
Filing date: 2018-12-26
Publication date: 2020-09-01
Anticipated expiration: 2038-12-26
Also published as: CN109486804A

Abstract

The invention relates to endo-chitosanase CsnM with heat recovery characteristic and application thereof. The amino acid sequence of the chitosanase CsnM is shown in SEQ ID NO. 1. The chitosanase of the invention has the yield of 680.2U/mL, the optimal reaction temperature of 40 ℃, the heat recovery characteristic, and the 89.1% activity can be recovered after being boiled for 10min and incubated in ice water for 30 min. The enzyme has an action mode of endo-cutting, and the main degradation products are chitobiose and chitotriose. The chitosanase of the invention has high yield, novel property and excellent industrial application potential.

Description

Novel chitosanase CsnM with heat recovery characteristic and application thereof

Technical Field

The invention relates to an endo-chitosanase CsnM with heat recovery characteristic and application thereof, belonging to the field of biotechnology.

Background

The chitosan oligosaccharide is oligosaccharide with the polymerization degree of less than 20 and the molecular weight of less than 3900 after chitosan is hydrolyzed, and is formed by connecting N-acetyl-D-glucosamine (GLcNAc) and D-glucosamine (GLcN) through beta-l, 4-glycosidic bonds. Due to lower molecular weight and better solubility, the chitosan oligosaccharide has wider application compared with chitosan. In addition, the chitosan oligosaccharide also has unique pharmacological functional activities of resisting tumors, reducing blood pressure, enhancing immunity and the like, so that the chitosan oligosaccharide has greater application value in the fields of medicines, health-care products, foods, cultivation and the like.

Chitosan can be hydrolyzed by various types of enzymes. Including non-specific enzymes: lipases, proteases, carbohydrases, etc., which can hydrolyze chitosan into chitosan oligosaccharides having a low degree of polymerization. However, when the non-specific enzymes are hydrolyzed to a certain degree, the hydrolysis degree is difficult to improve by increasing the enzyme amount, and the hydrolysate is complex and difficult to separate. The method for preparing the oligosaccharide by hydrolyzing chitosan with chitosan enzyme (EC: 3.2.1.132) has the advantages of mild reaction conditions, easy control of the reaction process and the like, and the product purity is higher, and the construction of the engineering bacteria for high yield of chitosan enzyme by using genetic engineering is an effective way for industrially preparing the chitosan oligosaccharide. At present, the reported chitosan enzyme has low activity, poor thermal stability and storage stability, and the industrial application prospect of the chitosan enzyme is severely limited.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides novel chitosanase CsnM and a preparation method thereof. The optimum reaction temperature of the chitosanase CsnM is 40 ℃, and the optimum reaction pH is 5.9; the enzyme solution has the characteristic of heat recovery, after being boiled for 10min, the enzyme solution is placed in ice water (0 ℃) for incubation for 30min, and 89.1% of activity of the enzyme solution can be recovered, so that the problem of stability of the enzyme in the transportation process can be solved. The enzyme degradation mode is endo-cutting, and the main products of enzymolysis are chitobiose and chitotriose.

On one hand, the invention provides novel chitosanase CsnM, the amino acid sequence of which is shown in SEQ ID NO. 1.

SEQ ID NO.1：

MKLSCIASFKWHAKVLSLLELGAPVPNLRTRTVAVAVGGLLIASGAIAGTAAQANAVSSLAPAITAVSAASTGDLSAPAKKEIAMQLVCSAENSSLDWKAQYGYIEDIDDDRGYTGGIIGFTSGTGDMLELVQNYANTKPDNNVLKPFLPVLRKVNGTKSHEGLGQKYVDAWHQAAKDSVFLKEQDKLRDSMYFNPAVSQGKDSNMSNLGQFMYYDAIFMHGPGDSSDSFGGIRKSAMKNAYTAAAQGGDEKTYLQAFATARKKIMKQENAHSDTSRVDDAQLKFLNEGNYDLHTLGKWKVYGDPYEIK

On the other hand, the invention also provides a nucleic acid sequence corresponding to the novel chitosanase CsnM, which is shown in SEQ ID NO. 2.

SEQ ID NO.2：

ATGAAGTTGTCTTGTATCGCTTCTTTCAAGTGGCACGCTAAGGTTTTGTCTTTGTTGGAATTGGGTGCTCCAGTTCCAAACTTGAGAACTAGAACTGTTGCTGTTGCTGTTGGTGGTTTGTTGATCGCTTCTGGTGCTATCGCTGGTACTGCTGCTCAAGCTAACGCTGTTTCTTCTTTGGCTCCAGCTATCACTGCTGTTTCTGCTGCTTCTACTGGTGACTTGTCTGCTCCAGCTAAGAAGGAAATCGCTATGCAATTGGTTTGTTCTGCTGAAAACTCTTCTTTGGACTGGAAGGCTCAATACGGTTACATCGAAGACATCGACGACGACAGAGGTTACACTGGTGGTATCATCGGTTTCACTTCTGGTACTGGTGACATGTTGGAATTGGTTCAAAACTACGCTAACACTAAGCCAGACAACAACGTTTTGAAGCCATTCTTGCCAGTTTTGAGAAAGGTTAACGGTACTAAGTCTCACGAAGGTTTGGGTCAAAAGTACGTTGACGCTTGGCACCAAGCTGCTAAGGACTCTGTTTTCTTGAAGGAACAAGACAAGTTGAGAGACTCTATGTACTTCAACCCAGCTGTTTCTCAAGGTAAGGACTCTAACATGTCTAACTTGGGTCAATTCATGTACTACGACGCTATCTTCATGCACGGTCCAGGTGACTCTTCTGACTCTTTCGGTGGTATCAGAAAGTCTGCTATGAAGAACGCTTACACTGCTGCTGCTCAAGGTGGTGACGAAAAGACTTACTTGCAAGCTTTCGCTACTGCTAGAAAGAAGATCATGAAGCAAGAAAACGCTCACTCTGACACTTCTAGAGTTGACGACGCTCAATTGAAGTTCTTGAACGAAGGTAACTACGACTTGCACACTTTGGGTAAGTGGAAGGTTTACGGTGACCCATACGAAATCAAG

On the other hand, the invention also provides a preparation and purification method of the chitosanase CsnM.

On the other hand, the invention also provides application of the chitosanase CsnM in degrading chitosan.

On the other hand, the chitosan degrading method adopts CsnM chitosan enzyme.

Preferably: the reaction temperature in the degradation condition is 0-70 ℃. The optimum reaction temperature is 40 ℃.

Preferably: the reaction pH value in the degradation condition is 4.9-7.1. The optimum reaction pH was 5.9.

Has the advantages that:

1. the chitosanase CsnM is alginate lyase with novel structure and function, and the similarity of the amino acid sequence and the chitosanase sequence reported by the existing properties is only 63%.

2. The invention provides a method for preparing chitosanase CsnM, namely, a gene engineering technical method is utilized to carry out heterologous recombination expression on a gene sequence of CsnM to escherichia coli, after fermentation, the enzyme activity of a fermentation liquid is up to 680.2U/mL, and the method has the potential of industrial production. The enzyme purification method is simple, and the recovery rate is up to 90.6% and the protein purity is up to 95% by one-step affinity purification by using a nickel column.

3. The chitosanase CsnM has excellent physicochemical property, the optimum reaction pH of the chitosanase is 5.9, the chitosanase CsnM has the characteristic of heat recovery, and the chitosanase CsnM can recover 89.1% of the activity after the enzyme solution is boiled for 10min and placed in ice water (0 ℃) for 30 min. The recombinant enzyme is used for analyzing degradation products, and main degradation products of the enzyme are chitosan oligosaccharide disaccharide and trisaccharide. The chitosanase CsnM has good industrial application prospect.

Drawings

FIG. 1 shows the separation and purification of chitosanase CsnM protein (M, protein standard; 1, purified chitosanase CsnM);

FIG. 2 shows the temperature and pH adaptation analysis of the chitosanase CsnM of the present invention (A, the optimal reaction temperature of the chitosanase CsnM; B, the temperature stability of the chitosanase CsnM; C, the optimal reaction pH of the chitosanase CsnM; D, the pH stability of the chitosanase CsnM);

FIG. 3 shows the results of the thermal recovery analysis of chitosanase CsnM of the invention (A, comparison of the thermal recovery of CsnM after incubation for 1h at different temperatures and 0min on ice and 30 min; B, effect of boiling for different times on the thermal recovery of the enzyme; C, effect of different incubation temperatures on the thermal recovery of chitosanase CsnM; D, effect of different incubation times on the thermal recovery of chitosanase CsnM)

FIG. 4 is a Thin Layer Chromatography (TLC) method for detecting the enzymolysis products of the chitosanase CsnM (M, chitooligosaccharide DP ═ 1-4 standard substance; 0-6 is the enzyme degradation chitosan 0min, 1min, 10min, 30min, 120min, 360min, 1440min in sequence);

FIG. 5 is a high performance liquid chromatogram of the final enzymatic hydrolysate of chitosanase of the present invention.

Detailed Description

Example 1 sequence analysis and recombinant expression of chitosanase CsnM

The enzyme-producing gene csnM of the chitosanase CsnM is derived from marine bacterium Pseudoalteromonas sp.SY39, and comprises 927 base sequences and 309 coded amino acid sequences. Conserved domain analysis (CDD) and multiplex Alignment of Basic Local Alignment Search Tool (Blast) using Conserved domains in the National Center for Biotechnology Information (NCBI) revealed that this sequence contains a Conserved region of the chitosanase of the polysaccharide hydrolase GH family. Among chitosanases reported, chitosanase (Genbank EMF00356) of family 46 (GH46) of polysaccharide hydrolases with the highest amino acid sequence similarity to CsnM had an amino acid sequence similarity (Identity) of 63%. The chitosanase CsnM disclosed by the invention belongs to polysaccharide hydrolase (GH46) family.

Taking restriction enzymes Nco I and Xho I as enzyme cutting sites of the enzyme-producing sequence of CsnM, designing recombinant primers as follows (restriction enzyme sites are underlined, restriction enzyme protecting bases are italicized):

a forward primer: SEQ ID NO. 3: PcsnM-F:

5’-CATGCCATGGAAGTTGTCTTGTATCGCT-3’(Nco I)

reverse primer: SEQ ID NO. 4: PcsnM-R:

5’-CCGCTCGAGCTTGATTTCGTATGGGTCA-3’(Xho I)

the PCR amplification conditions were: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 1min for 30 cycles; extending for 5min at 72 ℃; stabilizing at 4 deg.C for 15 min. The DNA polymerase used for the PCR reaction was Primerstar HS, available from Dalibao Bio.

The PCR product was double-digested with restriction enzymes NcoI and XhoI, and the digested PCR product was recovered by agarose gel electrophoresis. pET22b (+) plasmid DNA (Invitrogen, USA) was also double-digested with restriction enzymes Nco I and XhoI, subjected to agarose gel electrophoresis, and the product fragment after the digestion was recovered. The enzyme and substrate reaction system (temperature, time, DNA dosage and the like) used in the enzyme digestion are operated according to the product instruction provided by the Dalianbao organism.

Performing ligation reaction on the PCR product subjected to double digestion treatment and a pET-22b (+) plasmid vector according to the instruction of DNA ligase (Dalibao biological Co., Ltd.); the ligation product was transformed into E.coli DH5 alpha strain (Invitrogen, USA), spread on Luria-Bertani (LB) medium solid plate (containing 50. mu.g/mL ampicillin), cultured in an incubator at 37 ℃ for 12-16 hours, and then single clone was picked; the single clones were transferred to LB liquid medium (containing 50. mu.g/mL ampicillin) and cultured overnight in a shaker at 37 ℃ at 180 rpm. The single clone was sequenced, and a positive clone was selected and named pET22 b-csnM. The recombinant plasmid was transformed into E.coli BL21(DE3) (purchased from Dalibao Bio Inc.), and the recombinant Escherichia coli strain was named BL21(DE3)/pET22b-csnM and stored at-80 ℃ for further use.

Example 2 preparation and purification methods of chitosanase CsnM

The recombinant strain BL21(DE3)/pET22b-csnM was cultured in 100mL LB liquid medium (50. mu.g/mL ampicillin) in a shaker at 37 ℃ with shaking at 180rpm to OD₆₀₀Adding 0.6 mM of inducer isopropyl- β -D-thiogalactoside (IPTG) with the final concentration of 0.1mM, and inducing for 20h at 20 ℃, wherein the activity of the chitosan enzyme is determined by adding 900 mul of 0.3% chitosan substrate (20mM acetic acid-sodium acetate, pH 5.9) into 100 mul of enzyme solution, reacting for 10min at 40 ℃, adding 750 mul of DNS reagent, reacting for 10min in boiling water for color development, detecting the absorbance at OD520, wherein the enzyme activity is defined as 1U and the enzyme amount required for generating 1 mul of reducing sugar per min, and the enzyme activity in the fermentation liquor can reach 680.2U/mL.

After fermentation is stopped, centrifuging at 12000rpm for 10min, discarding thalli, and collecting supernatant; and (3) loading the fermentation supernatant into a 10mL nickel ion affinity chromatography column at the loading flow rate of 5mL/min, eluting by using 10mM imidazole to remove impure proteins, eluting by using 150mM imidazole, and collecting the eluted components. Dialyzing the active ingredient to remove imidazole, packaging and storing at-20 deg.C for use. Through one-step affinity purification of nickel ions, the recovery rate of protein reaches 90.6%. The purified chitosanase was subjected to polyacrylamide gel electrophoresis (SDS-PAGE), and as shown in FIG. 1, the molecular weight of CsnM of the purified chitosanase was 30kDa, which is consistent with the protein size predicted in sequence analysis. The protein purity of the purified chitosanase CsnM reaches more than 95% by gel analysis.

Example 3 Effect of temperature and pH on chitosanase CsnM

And (3) carrying out enzyme activity determination on the purified chitosanase CsnM in the example 2 under different conditions, and detecting the influence of different temperatures and pH values on the enzyme activity. Reacting for 10min at different temperatures (0-70 ℃), detecting the influence of different reaction temperatures on the enzyme activity, and calculating the relative enzyme activity of CsnM at different temperatures by taking the highest enzyme activity as 100%. As shown in FIG. 2A, the optimum reaction temperature for chitosanase CsnM was 40 ℃.

The chitosanase CsnM purified in example 2 was incubated at different temperatures (0-70 ℃) for 1h, and after being taken out, the enzymatic activity was immediately detected at the optimum reaction temperature (40 ℃) and the activity before incubation was taken as 100%, as shown in FIG. 2B, the chitosanase CsnM is poor in temperature stability, and after incubation at 40 ℃ for 1h, the enzymatic activity was only 15.8%.

The chitosan enzyme CsnM purified in example 2 was reacted with chitosan substrates formulated to different pH with different buffer systems, which were sodium acetate buffer (50mM, pH4.49-5.9), phosphate buffer (50mM, pH5.88-7.12) and Tris-HCl buffer (50mM, pH6.22-7.11), respectively. The activity is detected at the optimum temperature, and the highest value of the enzyme activity is 100 percent. As shown in FIG. 2C, the optimum reaction pH for chitosanase CsnM is 5.9.

The chitosanase CsnM purified in example 2 was incubated for 24h under different pH conditions (4.7-10.8), and immediately after being taken out, its enzymatic activity was measured at its optimum reaction pH (5.9), taking the activity before incubation as 100%, as shown in FIG. 2D.

Example 4 Heat recovery assay for chitosanase CsnM

The CsnM purified in example 2 was incubated at different temperatures (0-70 ℃) for 1h, respectively for 0min and 30min on ice, and its enzymatic activity was measured at 40 ℃. As shown in fig. 3A, the activity measured by incubation on ice for 30min was significantly higher than that measured directly without incubation.

The chitosanase CsnM purified in example 2 was boiled for various times (5-40min), and then incubated on ice for 0min and 30min before measuring its enzymatic activity at 40 ℃. As shown in FIG. 3B, after boiling for 5-40min, the enzyme activity was partially recovered by incubating on ice for 30min, wherein the shorter the boiling time, the stronger the recovery.

Boiling the purified chitosanase of example 2 for 10min, taking out, immediately placing at 0-50 deg.C, incubating for 30min, and detecting the residual enzyme activity, with the activity of enzyme solution without boiling being 100%; enzyme activity obtained by direct detection after boiling is used as negative control; detecting the influence of different incubation temperatures on the thermal recovery of the incubation temperatures; as shown in FIG. 3C, the chitosanase CsnM was boiled for 10min, incubated at 10 ℃ for 30min, with the best effect, and 92.3% of its activity was recovered.

The chitosanase purified in example 2 was boiled for 10min, taken out and immediately incubated at 0 ℃ for various times (0-45min), and the effect of the various incubation times on enzyme recovery was examined. As shown in FIG. 3D, the thermal recovery of the enzyme was gradually increased with the increase of the incubation time, and the thermal recovery reached the highest value at 45min and did not increase any more with the increase of the incubation time.

Example 5 thin layer chromatography analysis of chitosanase CsnM enzymatic hydrolysate

The purified chitosanase CsnM pure enzyme obtained in example 2 and 0.3% chitosan were incubated at 30 deg.C for 0min, 1min, 10min, 30min, 120min, 360min, 1440min, respectively, and then detected on High Performance Thin Layer Chromatography (HPTLC). The method specifically comprises the following steps: cutting the HPTLC chromatographic plate into samples with the width of 7cm and the proper size, spotting the samples before and after incubation at the origin, placing the samples in a developing tank with a developing agent (n-butyl alcohol: glacial acetic acid: water: 2:1) for 30min, drying the chromatographic plate, immersing the plate in a color developing agent (0.5% ninhydrin ethanol solution) for 2s, taking out the plate, drying the plate, and baking the plate at 80 ℃ until the samples appear. As shown in figure 4, compared with the standard product migration rate, the chitosanase CsnM enzymolysis main product is chitobiose (DP2) and chitotriose (DP 3).

As shown in FIG. 4, in the initial stage of the enzymatic hydrolysis (0-10min), a large amount of large-fragment oligosaccharides are produced, and as the enzymatic hydrolysis proceeds, the large-fragment oligosaccharides are gradually converted into small-fragment oligosaccharides, which indicates that the mode of action of the enzyme is to cut from the interior of chitosan molecules in an endo-mode.

Example 6 high Performance liquid chromatography assay of chitosanase CsnM enzymatic hydrolysate

The chitosanase purified in example 2 was incubated with 0.3% chitosan substrate for 24h, the degradation product was centrifuged at 12,000g for 10min, the precipitate was discarded, and 100. mu.L of the supernatant and a volume (5. mu.L) of 0.1M AMAC solution (17: 3(v/v) in DMSO and glacial acetic acid) and 1M sodium borocyanide (100. mu.L) were added. The mixture was incubated in the dark at 90 ℃ for 40min and stopped at-20 ℃. 500 μ L of 50% dimethyl sulfoxide was added and centrifuged and loaded onto a pre-equilibrated (0.2M ammonium bicarbonate) gel filtration column (Superdex peptide 10/300). The flow rate was 0.6mL/min, the mobile phase was 0.2M ammonium bicarbonate, the detection time was 40min, and the detector was a parallax detector, as shown in FIG. 5.

Sequence listing

<110> Qingdao university

<120> novel chitosanase CsnM with heat recovery characteristic and application thereof

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>309

<212>PRT

<213> Unknown (Unknown)

<400>1

Met Lys Leu Ser Cys Ile Ala Ser Phe Lys Trp His Ala Lys Val Leu

1 5 10 15

Ser Leu Leu Glu LeuGly Ala Pro Val Pro Asn Leu Arg Thr Arg Thr

20 25 30

Val Ala Val Ala Val Gly Gly Leu Leu Ile Ala Ser Gly Ala Ile Ala

35 40 45

Gly Thr Ala Ala Gln Ala Asn Ala Val Ser Ser Leu Ala Pro Ala Ile

50 55 60

Thr Ala Val Ser Ala Ala Ser Thr Gly Asp Leu Ser Ala Pro Ala Lys

65 70 75 80

Lys Glu Ile Ala Met Gln Leu Val Cys Ser Ala Glu Asn Ser Ser Leu

85 90 95

Asp Trp Lys Ala Gln Tyr Gly Tyr Ile Glu Asp Ile Asp Asp Asp Arg

100 105 110

Gly Tyr Thr Gly Gly Ile Ile Gly Phe Thr Ser Gly Thr Gly Asp Met

115 120 125

Leu Glu Leu Val Gln Asn Tyr Ala Asn Thr Lys Pro Asp Asn Asn Val

130 135 140

Leu Lys Pro Phe Leu Pro Val Leu Arg Lys Val Asn Gly Thr Lys Ser

145 150 155 160

His Glu Gly Leu Gly Gln Lys Tyr Val Asp Ala Trp His Gln Ala Ala

165 170 175

Lys Asp Ser Val Phe Leu Lys Glu Gln Asp Lys Leu Arg Asp Ser Met

180 185 190

Tyr Phe Asn Pro Ala Val Ser Gln Gly Lys Asp Ser Asn Met Ser Asn

195 200 205

Leu Gly Gln Phe Met Tyr Tyr Asp Ala Ile Phe Met His Gly Pro Gly

210 215 220

Asp Ser Ser Asp Ser Phe Gly Gly Ile Arg Lys Ser Ala Met Lys Asn

225 230 235 240

Ala Tyr Thr Ala Ala Ala Gln Gly Gly Asp Glu Lys Thr Tyr Leu Gln

245 250 255

Ala Phe Ala Thr Ala Arg Lys Lys Ile Met Lys Gln Glu Asn Ala His

260 265 270

Ser Asp Thr Ser Arg Val Asp Asp Ala Gln Leu Lys Phe Leu Asn Glu

275 280 285

Gly Asn Tyr Asp Leu His Thr Leu Gly Lys Trp Lys Val Tyr Gly Asp

290 295 300

Pro Tyr Glu Ile Lys

305

<210>2

<211>927

<212>DNA

<213> Unknown (Unknown)

<400>2

atgaagttgt cttgtatcgc ttctttcaag tggcacgcta aggttttgtctttgttggaa 60

ttgggtgctc cagttccaaa cttgagaact agaactgttg ctgttgctgt tggtggtttg 120

ttgatcgctt ctggtgctat cgctggtact gctgctcaag ctaacgctgt ttcttctttg 180

gctccagcta tcactgctgt ttctgctgct tctactggtg acttgtctgc tccagctaag 240

aaggaaatcg ctatgcaatt ggtttgttct gctgaaaact cttctttgga ctggaaggct 300

caatacggtt acatcgaaga catcgacgac gacagaggtt acactggtgg tatcatcggt 360

ttcacttctg gtactggtga catgttggaa ttggttcaaa actacgctaa cactaagcca 420

gacaacaacg ttttgaagcc attcttgcca gttttgagaa aggttaacgg tactaagtct 480

cacgaaggtt tgggtcaaaa gtacgttgac gcttggcacc aagctgctaa ggactctgtt 540

ttcttgaagg aacaagacaa gttgagagac tctatgtact tcaacccagc tgtttctcaa 600

ggtaaggact ctaacatgtc taacttgggt caattcatgt actacgacgc tatcttcatg 660

cacggtccag gtgactcttc tgactctttc ggtggtatca gaaagtctgc tatgaagaac 720

gcttacactg ctgctgctca aggtggtgac gaaaagactt acttgcaagc tttcgctact 780

gctagaaaga agatcatgaa gcaagaaaac gctcactctg acacttctag agttgacgac 840

gctcaattga agttcttgaa cgaaggtaac tacgacttgc acactttggg taagtggaag 900

gtttacggtg acccatacga aatcaag 927

<210>3

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

catgccatgg aagttgtctt gtatcgct 28

<210>4

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ccgctcgagc ttgatttcgt atgggtca 28

Claims

1. A novel chitosanase CsnM, its amino acid sequence is shown in SEQ ID NO. 1.

2. The novel chitosanase CsnM of claim 1, having the nucleotide sequence shown in SEQ ID No. 2.

3. The method for preparing the novel chitosanase CsnM according to claim 1, comprising the following steps:

taking the enzyme-producing sequence of CsnM and restriction enzymes Nco I and Xho I as enzyme cutting sites, designing recombinant primers as follows: a forward primer: SEQ ID NO.3, reverse primer: SEQ ID NO. 4; the PCR amplification conditions were: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1min for 30 cycles, extension at 72 ℃ for 5min, and stabilization at 4 ℃ for 15 min;

carrying out double enzyme digestion on the PCR product by using restriction enzymes NcoI and XhoI, recovering the PCR product after enzyme digestion through agarose gel electrophoresis, carrying out double enzyme digestion on pET22b (+) plasmid DNA by using the restriction enzymes NcoI and XhoI, carrying out agarose gel electrophoresis, and recovering a product fragment after enzyme digestion, wherein enzyme and a substrate reaction system for enzyme digestion refer to product description operations provided by the Dalibao organism;

performing ligation reaction on the PCR product subjected to double enzyme digestion and a pET-22b (+) plasmid vector by referring to a DNA ligase specification, transforming the ligation product into an Escherichia coli DH5 alpha strain, coating the Escherichia coli DH5 alpha strain on a Luria-Bertani (LB) culture medium solid plate containing 50 mu g/mL ampicillin, culturing the Escherichia coli DH5 alpha strain in a 37 ℃ incubator for 12-16 hours, picking out a monoclonal, transferring the monoclonal into an LB liquid culture medium, culturing the LB liquid culture medium containing 50 mu g/mL ampicillin in a shaker at the rotation speed of 180rpm for overnight at 37 ℃, performing sequence determination on the monoclonal, selecting a positive clone, naming the positive clone as pET22b-csnM, transforming a recombinant plasmid into Escherichia coli BL21(DE3), naming the recombinant Escherichia coli strain as BL21(DE3)/pET22b-csnM, and storing the recombinant Escherichia coli strain at-80 ℃ for later use;

the enzyme producing gene csnM of the chitosanase CsnM is from marine bacteriaPseudoalteromonasesp .SY39；

Recombinant strain BL21(DE3)/pET22b-csnM in 100mL LB liquid medium, containing 50 ug/mL ampicillin, in 37 ℃ shaking table 180rpm shake culture until OD600 ═ 0.6, adding 0.1mM final concentration of inducer isopropyl-beta-D-thiogalactoside, at 20 ℃ for 20h induction;

after fermentation is stopped, centrifuging at 12000rpm for 10min, discarding thalli, and collecting supernatant;

loading the fermentation supernatant into a 10mL nickel ion affinity chromatography column at a flow rate of 5mL/min, eluting with 10mM imidazole to remove impure proteins, eluting with 150mM imidazole, and collecting the eluted components;

dialyzing the active ingredient to remove imidazole, subpackaging and storing at-20 deg.C for use;

one-step affinity purification by nickel ions.

4. Use of the chitosanase of claim 1 for degrading chitosan.

5. A method for degrading chitosan is characterized by comprising the following steps: adding 900 μ L of 0.3% chitosan substrate and 20mM acetic acid-sodium acetate into 100 μ L of chitosan solution, and reacting, wherein the selected chitosan enzyme is the chitosan enzyme in claim 1.

6. The method according to claim 5, wherein the degradation conditions are carried out at a reaction temperature of 0 to 70 ℃.

7. The method according to claim 5, wherein the degradation conditions are such that the reaction pH is from 4.9 to 7.1.