CN109486804A

CN109486804A - It is a kind of with the novel chitosan enzyme CsnM of hot recovery characteristics and its application

Info

Publication number: CN109486804A
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: 2019-03-19
Anticipated expiration: 2038-12-26
Also published as: CN109486804B

Abstract

The present invention relates to a kind of with the endo-type chitosan enzyme CsnM of hot recovery characteristics and its application.The chitosan enzyme CsnM amino acid sequence is as shown in SEQ ID NO.1.For chitosan enzyme yield of the present invention up to 680.2U/mL, optimal reactive temperature is 40 DEG C, and there are hot recovery characteristics to be incubated for 30min in ice water after boiling 10min, can restore 89.1% activity.The enzyme effect mode is inscribe, and degradation principal product is chitobiose and chitotriose.Chitosan enzyme yield of the present invention is big, and property is novel, has excellent industrial applications potential quality.

Description

It is a kind of with the novel chitosan enzyme CsnM of hot recovery characteristics and its application

Technical field

The present invention relates to a kind of with the endo-type chitosan enzyme CsnM of hot recovery characteristics and its application, belongs to biotechnology Field.

Background technique

Chitosan oligosaccharide is that the degree of polymerization is less than 20 after hydrolysis for chitosan, and oligosaccharides of the molecular weight less than 3900 is by N- acetyl-D- By β-l, 4- glucosides key connection is formed for Glucosamine (GLcNAc) and D- Glucosamine (GLcN).Due to lower molecule Amount, better dissolubility chitosan oligosaccharide compare chitosan and have more to be widely applied.In addition, chitosan oligosaccharide is also equipped with anti-swell The unique pharmacological function activity such as tumor, blood pressure lowering, strengthen immunity, this makes chitosan oligosaccharide in medicine, health care product, food, cultivation There is bigger application value in equal fields.

Chitosan can be by multiclass enzyme hydrolysis.Including non-specific enzyme: lipase, protease, carbohydrase Deng chitosan oligosaccharide that can be low at the degree of polymerization by chitosan hydrolyzate.But the hydrolysis of non-specific enzyme to a certain extent when, increase enzyme amount Also it is difficult to improve hydrolysis degree, and hydrolysate is more complex, is difficult to separate.And use chitosan enzyme (EC:3.2.1.132) water Solution chitosan come the method for preparing oligosaccharides have many advantages, such as reaction condition mildly, reaction process be easy to control, and product purity compared with Height, the engineering bacteria using genetic engineering building high yield chitosan enzyme are the effective ways of preparation of industrialization chitosan oligosaccharide.Currently, report Chitosan enzyme vigor it is lower, and thermal stability and stable storing row are poor, seriously limit its industrial applications prospect.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a kind of novel chitosan enzyme CsnM and preparation method thereof.This hair The optimal reactive temperature of the bright chitosan enzyme CsnM is 40 DEG C, and optimal reaction pH is 5.9；Its characteristic that there is heat to restore, enzyme It after liquid boils 10min, is placed in ice water (0 DEG C) incubation 30min, its 89.1% activity can be restored, conducive to solving to transport The stability problem of enzyme in journey.The enzyme degrades mode as inscribe, and enzymatic hydrolysis principal product is chitobiose and chitotriose.

On the one hand, the present invention provides a kind of novel chitosan enzyme CsnM, and amino acid sequence is as shown in SEQ ID NO.1.

SEQ ID NO.1:

MKLSCIASFKWHAKVLSLLELGAPVPNLRTRTVAVAVGGLLIASGAIAGTAAQANAVSSLAPAITAVSA ASTGDLSAPAKKEIAMQLVCSAENSSLDWKAQYGYIEDIDDDRGYTGGIIGFTSGTGDMLELVQNYANTKPDNNVLK PFLPVLRKVNGTKSHEGLGQKYVDAWHQAAKDSVFLKEQDKLRDSMYFNPAVSQGKDSNMSNLGQFMYYDAIFMHGP GDSSDSFGGIRKSAMKNAYTAAAQGGDEKTYLQAFATARKKIMKQENAHSDTSRVDDAQLKFLNEGNYDLHTLGKWK VYGDPYEIK

On the other hand, the present invention also provides a kind of corresponding nucleic acid sequences of novel chitosan enzyme CsnM, such as SEQ ID NO.2 It is shown.

SEQ ID NO.2:

ATGAAGTTGTCTTGTATCGCTTCTTTCAAGTGGCACGCTAAGGTTTTGTCTTTGTTGGAATTGGGTGCT CCAGTTCCAAACTTGAGAACTAGAACTGTTGCTGTTGCTGTTGGTGGTTTGTTGATCGCTTCTGGTGCTATCGCTGG TACTGCTGCTCAAGCTAACGCTGTTTCTTCTTTGGCTCCAGCTATCACTGCTGTTTCTGCTGCTTCTACTGGTGACT TGTCTGCTCCAGCTAAGAAGGAAATCGCTATGCAATTGGTTTGTTCTGCTGAAAACTCTTCTTTGGACTGGAAGGCT CAATACGGTTACATCGAAGACATCGACGACGACAGAGGTTACACTGGTGGTATCATCGGTTTCACTTCTGGTACTGG TGACATGTTGGAATTGGTTCAAAACTACGCTAACACTAAGCCAGACAACAACGTTTTGAAGCCATTCTTGCCAGTTT TGAGAAAGGTTAACGGTACTAAGTCTCACGAAGGTTTGGGTCAAAAGTACGTTGACGCTTGGCACCAAGCTGCTAAG GACTCTGTTTTCTTGAAGGAACAAGACAAGTTGAGAGACTCTATGTACTTCAACCCAGCTGTTTCTCAAGGTAAGGA CTCTAACATGTCTAACTTGGGTCAATTCATGTACTACGACGCTATCTTCATGCACGGTCCAGGTGACTCTTCTGACT CTTTCGGTGGTATCAGAAAGTCTGCTATGAAGAACGCTTACACTGCTGCTGCTCAAGGTGGTGACGAAAAGACTTAC TTGCAAGCTTTCGCTACTGCTAGAAAGAAGATCATGAAGCAAGAAAACGCTCACTCTGACACTTCTAGAGTTGACGA CGCTCAATTGAAGTTCTTGAACGAAGGTAACTACGACTTGCACACTTTGGGTAAGTGGAAGGTTTACGGTGACCCAT ACGAAATCAAG

On the other hand, the present invention also provides the preparation and purification methods of chitosan enzyme CsnM a kind of.

On the other hand, the application the present invention also provides the chitosan enzyme CsnM in degradation chitosan.

On the other hand, a method of degradation chitosan, selected chitosan enzyme are CsnM.

It is preferred that: reaction temperature is 0~70 DEG C in the degradation condition.Optimal reactive temperature is 40 DEG C.

It is preferred that: it is 4.9~7.1 that pH is reacted in the degradation condition.Optimal reaction pH is 5.9.

The utility model has the advantages that

1. chitosan enzyme CsnM of the invention be structure and novel functions algin catenase, amino acid sequence with The chitosan enzyme sequence similarity for having property to report is only 63%.

2. the present invention provides a kind of methods for preparing chitosan enzyme CsnM, that is, the technical method of genetic engineering is utilized, it will The gene order heterologous recombination of CsnM is expressed to Escherichia coli, and after fermented, fermentation broth enzyme activity is up to 680.2U/mL, has The potential quality of industrialized production.The enzyme purification method is simple, a step affinity purification is carried out to it using nickel column, the rate of recovery is up to 90.6%, lipidated protein is up to 95%.

3. chitosan enzyme CsnM of the invention has excellent physicochemical property, which is 5.9, has heat The characteristic of recovery, after enzyme solution boils 10min, (0 DEG C) placement 30min, can restore its 89.1% activity in ice water.Utilize this Recombinase has carried out catabolite analysis, which degrades principal product as chitosan oligosaccharide disaccharides and trisaccharide.Chitosan of the present invention Enzyme CsnM has good industrial applications prospect.

Detailed description of the invention

Fig. 1 is chitosan enzyme CsnM protein separation figure (M, Protein standards of the present invention；1, purifying gained shell is poly- Carbohydrase CsnM)；

Fig. 2 is the temperature and pH Adaptability Analysis (A, the optimal reaction of chitosan enzyme CsnM of chitosan enzyme CsnM of the present invention Temperature；B, the temperature stability of chitosan enzyme CsnM；The optimal reaction pH of C, chitosan enzyme CsnM；D, the pH of chitosan enzyme CsnM Stability)；

(A is incubated for 0 He after incubation 1h under different temperatures to the restorative analysis of the heat that Fig. 3 is chitosan enzyme CsnM of the present invention on ice The restorative comparison of heat of CsnM after 30min；B boils the different time influence restorative to enzyme heat；C, different incubation temperatures are to shell The restorative influence of heat of dextranase CsnM；D, heat restorative influence of the different incubation times on chitosan enzyme CsnM)

Fig. 4 is enzymolysis product (M, chitosan oligosaccharide DP=1-4 that thin-layer chromatography (TLC) method detects chitosan enzyme CsnM of the present invention Standard items；0-6 is followed successively by the enzyme degradation chitosan 0min, 1min, 10min, 30min, 120min, 360min, 1440min)；

Fig. 5 is the high-efficient liquid phase chromatogram of the final enzymolysis product of chitosan enzyme of the present invention.

Specific embodiment

The sequence of 1 chitosan enzyme CsnM of embodiment is analyzed and recombinant expression

The producing enzyme gene csnM of chitosan enzyme CsnM of the present invention derives from marine bacteria Pseudoalteromonase Sp.SY39 includes 927 base sequences, encodes 309 amino acid sequences.Utilize National Center for Conserved structure domain analysis Conserved domain (CDD) in Biotechnology Information (NCBI) and multiple Sequence alignment Basic Local Alignment Search Tool (Blast) discovery, which includes one section of polysaccharide hydrolysis The chitosan enzyme conserved region of enzyme GH family.It has been reported that chitosan enzyme in, it is highest with CsnM amino acid sequence similarity to be The chitosan enzyme (Genbank EMF00356) of the 46th family (GH46) of polysaccharide hydrolase, amino acid sequence between the two are similar Spending (Identity) is 63%.Chitosan enzyme CsnM described in the invention belongs to polysaccharide hydrolase (GH46) family.

By the producing enzyme sequence of CsnM using restriction enzyme Nco I and Xho I as restriction enzyme site, design recombination primer is as follows (underscore is restriction endonuclease sites, and italic is restriction enzyme enzyme protection base):

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)

PCR amplification condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, 72 DEG C of extension 1min, Totally 30 circulations；72 DEG C of extension 5min；4 DEG C of stable 15min.It is that Primerstar HS is purchased from that PCR, which reacts archaeal dna polymerase used, Dalian treasured biotech firm.

PCR product carries out double digestion with restriction enzyme NcoI and XhoI, recycles digestion by agarose gel electrophoresis PCR product afterwards.PET22b (+) Plasmid DNA (Invitrogen company, the U.S.) equally uses restriction enzyme Nco I and Xho I carries out double digestion, carries out agarose gel electrophoresis and recycles the product segment after digestion.Enzyme used in digestion and substrate reactions system The description of product operation that (temperature, time, DNA dosage etc.) is provided referring to the precious biology in Dalian.

The PCR product and pET-22b (+) plasmid vector of double digestion processing are referring to DNA ligase (Dalian treasured biotech firm) Specification is attached reaction；Connection product is converted to e.colistraindh5α (Invitrogen company, the U.S.), is coated on (contain 50 μ g/mL ampicillins) on Luria-Bertani (LB) culture medium solid plate, cultivates 12- in 37 DEG C of incubators After 16 hours, picking monoclonal；Monoclonal is forwarded in LB liquid medium (containing 50 μ g/mL ampicillins), revolving speed For overnight incubation in 37 DEG C of shaking tables of 180rpm.Monoclonal is subjected to sequencing, selects positive colony, and be named as pET22b-csnM.Recombinant plasmid transformed will recombinate coliform to e. coli bl21 (DE3) (being purchased from Dalian treasured biotech firm) Strain be named as BL21 (DE3)/pET22b-csnM, be stored in -80 DEG C it is spare.

The preparation of 2 chitosan enzyme CsnM of embodiment and purification process

By recombinant bacterial strain BL21 (DE3)/pET22b-csnM, (50 μ g/mL ammonia benzyls are green in the LB liquid medium of 100mL Mycin), 180rpm shake culture is to OD in 37 DEG C of shaking tables₆₀₀=0.6, the inducer isopropylthio-of final concentration of 0.1mM is added β-D- thiogalactoside (IPTG), in 20 DEG C of induction 20h.Chitosan enzyme activity determination method are as follows: 900 μ are added in 100 μ L enzyme solutions 0.3% chitosan substrate of L (20mM Acetic acid-sodium acetate, pH=5.9) reacts 10min at 40 DEG C, and the DNS examination of 750 μ L is added Agent, reaction 10min develops the color in boiling water, its absorbance is detected at OD520.It is that every min generates 1 μM that enzyme activity, which is defined as 1U, Enzyme amount required for reduced sugar.Through detecting, chitosan enzyme vigor is up to 680.2U/mL in fermentation liquid.

After fermentation stops, 12000rpm is centrifuged 10min, abandons thallus, collects supernatant；Fermented supernatant fluid is splined on 10mL nickel Ion affinity chromatography column, loading flow velocity are 5mL/min, are eluted using 10mM imidazoles, remove foreign protein, recycle the miaow of 150mM Azoles elution, collects elution fraction.By active constituent dialyse removal imidazoles, packing be stored in -20 DEG C it is spare.Pass through one step of nickel ion Affinity purification, protein recovery reach 90.6%.Purifying gained chitosan enzyme carries out polyacrylamide gel electrophoresis (SDS- PAGE), big with the albumen predicted in sequence analysis as shown in Figure 1, the molecular weight of purifying gained chitosan enzyme CsnM is 30kDa It is small consistent.It is found by gel analysis, the purity of protein of purifying gained chitosan enzyme CsnM reaches 95% or more.

The influence of 3 temperature of embodiment and pH to chitosan enzyme CsnM

Purifying gained chitosan enzyme CsnM in embodiment 2 is subjected to enzyme activity determination at different conditions, detects not equality of temperature Degree and influence of the pH to enzyme activity.10min is reacted under different temperatures (0-70 DEG C), detects differential responses temperature to enzyme activity It influences, with highest enzyme activity for 100%, calculates the enzyme activity of CsnM under different temperatures.As shown in Figure 2 A, chitosan enzyme The optimal reactive temperature of CsnM is 40 DEG C.

Purifying gained chitosan enzyme CsnM in embodiment 2 is incubated for 1h under different temperatures (0-70 DEG C), after taking-up, immediately Its enzyme activity is detected under its optimal reactive temperature (40 DEG C), the vigor before being incubated for is as 100%, and as shown in Figure 2 B, shell is poly- Carbohydrase CsnM temperature stability is poor, and 1h is incubated at 40 DEG C, and enzymatic activity only remains 15.8%.

The chitosan that will purifying gained chitosan enzyme CsnM in embodiment 2 and be made into different PH with different buffer solution systems Substrate reactions, the buffer used are respectively sodium acetate buffer (50mM, pH4.49-5.9), phosphate buffer (50mM, ) and Tris-HCl buffer (50mM, pH6.22-7.11) pH5.88-7.12.Activity is detected under optimum temperature, enzymatic activity is most High level is 100%.As shown in Figure 2 C, the optimal reaction pH of chitosan enzyme CsnM is 5.9.

Purifying gained chitosan enzyme CsnM in embodiment 2 is incubated for for 24 hours at condition of different pH (4.7-10.8), is taken out Afterwards, its enzyme activity is detected at its optimal reaction pH (5.9) immediately, the vigor before being incubated for is as 100%, as shown in Figure 2 D.

The restorative measurement of heat of 4 chitosan enzyme CsnM of embodiment

Purifying gained CsnM in embodiment 2 is incubated for 1h under different temperatures (0-70 DEG C), is incubated for 0min on ice respectively And 30min, its enzyme activity is detected in the case where it is 40 DEG C.As shown in Figure 3A, it is incubated for activity measured by 30min on ice, is significantly higher than It is not incubated for the activity directly detected.

Purifying gained chitosan enzyme CsnM in embodiment 2 is boiled into different time (5-40min), then incubates it on ice Its enzyme activity is detected at 40 DEG C after educating 0min and 30min.As shown in Figure 3B, after boiling 5-40min, it is incubated for 30min on ice, Its enzyme activity can partially be restored, wherein boiling time is shorter, and recovery capability is stronger.

10min will be boiled by purifying gained chitosan enzyme in embodiment 2, then take out to be placed on immediately at 0-50 DEG C and be incubated for 30min detects its remaining enzyme activity, not boil the vigor of enzyme solution for 100%；To boil rear directly detection gained enzyme activity masterpiece For negative control；Detect the influence restorative to its heat of different incubation temperatures；As shown in Figure 3 C, chitosan enzyme CsnM is through boiling After 10min, 10 DEG C of incubation 30min are placed on, effect is best, can restore its 92.3% activity.

Purifying gained chitosan enzyme in embodiment 2 is boiled into 10min, is placed on 0 DEG C of incubation different time after taking-up immediately (0-45min) detects the influence restorative to enzyme of different incubation times.As shown in Figure 3D, as incubation time extends, the heat of enzyme Restorative to gradually increase, in 45min, heat is restorative reaches peak, then extends incubation time heat and restorative be not further added by.

5 chitosan enzyme CsnM enzymolysis product thin layer chromatography analysis of embodiment

Will in embodiment 2 the pure enzyme of purifying gained chitosan enzyme CsnM and 0.3% chitosan be incubated for respectively at 30 DEG C 0min, Then 1min, 10min, 30min, 120min, 360min, 1440min are detected at High Performance Thin Layer Chromatography plate (HPTLC).Specifically Are as follows: HPTLC chromatoplate is cut into the sample of the suitable size of 7cm wide, front and back sample point sample at the origin will be incubated for, be placed in exhibition 30min in the exhibition cylinder of agent (n-butanol: glacial acetic acid: water=2:2:1) is opened, chromatoplate is dried up, immerses color developing agent (0.5% ninhydrin Ethanol solution) in 2s, take out drying, 80 DEG C baking, until sample occur.As shown in figure 4, with standard items migration rate it was found that, It is chitobiose (DP2) and chitotriose (DP3) that chitosan enzyme CsnM, which digests principal product,.

As shown in figure 4, enzyme digestion reaction initial stage (0-10min), the oligosaccharides of a large amount of large fragments generates, with enzyme digestion reaction into Row, the oligosaccharides that the oligosaccharides of large fragment gradates as small fragment illustrate that the mode of action of the enzyme is to gather in a manner of inscribe from shell It is cut inside glycan molecule.

6 chitosan enzyme CsnM enzymolysis product Syrups by HPLC of embodiment

The chitosan substrate of purifying gained chitosan enzyme in embodiment 2 and 0.3% is incubated for 24 hours, catabolite 12, 000g is centrifuged 10min, abandons precipitating, by the AMAC solution of the 0.1M of 100 μ L supernatant volumes (5 μ L) (be dissolved in dimethyl sulfoxide and 17:3 (v/v) mixture of glacial acetic acid) and 1M boron Cymag (100 μ L).By mixture in the dark in 90 DEG C of incubation 40min, And stop reaction at -20 DEG C.50% dimethyl sulfoxide of 500 μ L is added and is centrifuged and is splined on the pre-equilibration (carbonic acid of 0.2M Hydrogen ammonium) solvent resistant column (Superdex peptide 10/300).Flow velocity is 0.6mL/min, and mobile phase is the carbonic acid of 0.2M Hydrogen ammonium, detection time 40min, detector are parallax detector, as shown in Figure 5.

Sequence table

<110>University Of Qingdao

<120>a kind of with the novel chitosan enzyme CsnM of hot recovery characteristics and its application

<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 Leu Gly 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 aggttttgtc tttgttggaa 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 kind of novel chitosan enzyme, amino acid sequence is as shown in SEQ ID NO.1.

2. nucleotide sequence corresponding to chitosan enzyme as described in claim 1, the nucleotide sequence such as SEQ ID NO.2 It is shown.

3. the preparation and purification method of chitosan enzyme as described in claim 1.

4. application of the chitosan enzyme as described in claim 1 in degradation chitosan.

5. a kind of method for chitosan of degrading, characterized in that selected chitosan enzyme is chitosan described in claim 1 Enzyme.

6. method as claimed in claim 5, characterized in that reaction temperature is 0~70 DEG C in degradation condition, optimal reactive temperature It is 40 DEG C.

7. method as claimed in claim 5, characterized in that reacting pH in degradation condition is 4.9~7.1, and optimal reaction pH is 5.9。