CN110144340A - A kind of novel chitosan enzyme CsnQ and its application - Google Patents

A kind of novel chitosan enzyme CsnQ and its application Download PDF

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CN110144340A
CN110144340A CN201910454263.2A CN201910454263A CN110144340A CN 110144340 A CN110144340 A CN 110144340A CN 201910454263 A CN201910454263 A CN 201910454263A CN 110144340 A CN110144340 A CN 110144340A
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csnq
enzyme
chitosan enzyme
chitosan
gly
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CN110144340B (en
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杨文钰
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Zaozhuang Quanding Biotechnology Co., Ltd
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Shandong Haoyue Pharmaceutical Technology Co Ltd
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2442—Chitinase (3.2.1.14)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01014—Chitinase (3.2.1.14)

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Abstract

The present invention relates to a kind of novel chitosan enzyme CsnQ and its applications.The chitosan enzyme CsnQ amino acid sequence is as shown in SEQ ID NO.1.The present invention provides the preparation method of chitosan enzyme CsnQ a kind of, and yield is up to 1058.2 U/mL.Chitosan enzyme CsnQ property of the present invention is stablized, and optimal reactive temperature is 60 DEG C, keeps stablizing between pH 4.0-6.0, and 40 DEG C are incubated for the activity that 1 h still maintains 61.2%.Degradation mode is inscribe, degrade principal product chitobiose and chitotriose.Chitosan enzyme CsnQ yield of the present invention is high, stability is good, has good industrial applications potential quality.

Description

A kind of novel chitosan enzyme CsnQ and its application
Technical field
The present invention relates to a kind of novel chitosan enzyme CsnQ and its applications, belong to field of biotechnology.
Background technique
Chitosan oligosaccharide has the important biomolecules such as antibacterial, antitumor, anti-inflammatory activity, is known as " the hexa-atomic element of life ".Shell is few Sugar is by 2-acetylamino-2-deoxy-D-glucose (GLcNAc) and D- Glucosamine (GLcN) by β-l, 4- glucosides key connection and At for molecular weight less than 3900, the degree of polymerization is highly soluble in water less than 20, in fields such as medicine, health care product, food, cultivation than big point Seed chitosan has bigger application value.
Chitosan oligosaccharide is formed by the degradation of chitosan of macromolecular.Usual biodegrading process is divided into physical method, chemical method and biology Enzymic degradation three types.Wherein biological enzymatic degradation has reaction condition is mild, catabolite is single, reaction process is easy to control The advantages such as system, environmental-friendly.Chitosan oligosaccharide preparation process is increasingly becoming using chitosan enzyme biological enzyme degradation macromolecular chitosan In main stream approach.But current market sales of chitosan enzyme is expensive, vigor is lower, and stability is poor, seriously Limit the application prospect of chitosan enzyme.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of novel chitosan enzyme CsnQ and preparation method thereof.This hair The bright chitosan enzyme CsnQ yield is up to 1058.2 U/mL, and optimal reactive temperature is 60 DEG C, protects between pH 4.0-6.0 It is fixed to keep steady, and 40 DEG C are incubated for the activity that 1 h still maintains 61.2%, and the enzyme activity of various metals ion pair chitosan enzyme CsnQ, which has, to be promoted Into effect.Degradation mode is inscribe, and degradation principal product is chitobiose and chitotriose.Chitosan enzyme CsnQ yield of the present invention Height, stability are good, have good industrial applications potential quality.
On the one hand, the present invention provides a kind of novel chitosan enzyme CsnQ, and amino acid sequence is as shown in SEQ ID NO. 1.
SEQ ID NO.1:
MKYLLPTAAAGLLLLAAQPAMAMGVGGTGATHAGAAGSGVNLTDPHKKEIAMELVSSAENSSLDWKAQYKYIE DIGDGRGYTGGIIGFCSGTGDMLELVQHYTDLEPGNILAKYLPALKKVNGSASHSGLGTPFTKDWATAAKDTVFQQA QNDERDRVYFDPAVSQAKADGLRALGQFAYYDAIVMHGPGNDPTSFGGIRKTAMKKARTPAQGGDETTYLNAFLDAR KAAMLTEAAHDDTSRVDTEQRVFLKAGNLDLNPPLHWKTYGDSYSINSLEHTTTPLRSGC
On the other hand, the present invention also provides a kind of corresponding nucleic acid sequences of chitosan enzyme CsnQ, as shown in SEQ ID NO.2.
SEQ ID NO.2:
ATGAAATACTTATTACCAACAGCAGCAGCAGGTTTATTATTATTAGCAGCACAACCAGCAATGGCAATGGGTG TAGGTGGTACAGGTGCAACACATGCAGGTGCAGCAGGTTCTGGTGTAAACTTAACAGATCCACATAAAAAAGAAATT GCAATGGAATTAGTATCTTCTGCAGAAAACTCTTCTTTAGATTGGAAAGCACAATACAAATACATTGAAGATATTGG TGATGGTCGTGGTTACACAGGTGGTATTATTGGTTTCTGTTCTGGTACAGGTGATATGTTAGAATTAGTACAACATT ACACAGATTTAGAACCAGGTAACATTTTAGCAAAATACTTACCAGCATTAAAAAAAGTAAACGGTTCTGCATCTCAT TCTGGTTTAGGTACACCATTCACAAAAGATTGGGCAACAGCAGCAAAAGATACAGTATTCCAACAAGCACAAAACGA TGAACGTGATCGTGTATACTTCGATCCAGCAGTATCTCAAGCAAAAGCAGATGGTTTACGTGCATTAGGTCAATTCG CATACTACGATGCAATTGTAATGCATGGTCCAGGTAACGATCCAACATCTTTCGGTGGTATTCGTAAAACAGCAATG AAAAAAGCACGTACACCAGCACAAGGTGGTGATGAAACAACATACTTAAACGCATTCTTAGATGCACGTAAAGCAGC AATGTTAACAGAAGCAGCACATGATGATACATCTCGTGTAGATACAGAACAACGTGTATTCTTAAAAGCAGGTAACT TAGATTTAAACCCACCATTACATTGGAAAACATACGGTGATTCTTACTCTATTAACTCTTTAGAACATACAACAACA CCATTACGTTCTGGTTGT
On the other hand, the present invention also provides the preparation and purification methods of chitosan enzyme CsnQ a kind of.
On the other hand, the application the present invention also provides the chitosan enzyme CsnQ in degradation chitosan.
On the other hand, a method of degradation chitosan, selected chitosan enzyme are CsnQ.
It is preferred that: reaction temperature is 0 ~ 80 DEG C in the degradation condition.Optimal reactive temperature is 60 DEG C.
It is preferred that: it is 4.0 ~ 9.6 that pH is reacted in the degradation condition.Optimal reaction pH is 5.0.
The utility model has the advantages that
1. chitosan enzyme CsnQ of the invention is novel chitosan enzyme, the chitosan enzyme of conserved region and existing structure report (PDB:1CHK) amino acid sequence similarity is 96.63%, almost the same, so that it maintains good catalytic activity (Rate activity Reach 690.5 U/mg)；Chitosan enzyme (PDB:1CHK) sequence of chitosan enzyme CsnQ of the present invention and existing structure report Difference is mainly shown as: chitosan enzyme CsnQ includes 287 amino acid sequences, and chitosan enzyme (PDB:1CHK) only includes 238 amino acid sequences.The N-terminal of chitosan enzyme CsnQ of the present invention includes one section of 39 amino acid (Met1-Gly39) composition N-trunk structure, C sections include 9 amino acid C-tail structure (His278-Cys287).The two includes that there are many rigidity Amino acid increases the rigidity of the three-dimensional structure of chitosan enzyme, is its structure basis with better stability.
2. the present invention provides a kind of methods for preparing chitosan enzyme CsnQ, that is, the technical method of genetic engineering is utilized, it will The gene order heterologous recombination of chitosan enzyme CsnQ is expressed to Escherichia coli, and after fermented, fermentation broth enzyme activity is up to 1058.2 U/mL, the potential quality with industrialized production.The enzyme purification method is simple, and a step affinity purification is carried out to it using nickel column, recycling Rate is up to 79.6%, and lipidated protein is up to 95%.
3. chitosan enzyme CsnQ of the invention has excellent physicochemical property, optimal pH 5.0, in pH 4.0-6.0 range Interior holding is stablized, and 40 DEG C are incubated for the activity that 1 h still maintains 61.2%, the enzyme activity tool of various metals ion pair chitosan enzyme CsnQ There is facilitation.Degradation mode is inscribe, degrade principal product chitobiose and chitotriose.Chitosan enzyme CsnQ yield of the present invention Height, stability are good, have good industrial applications potential quality.
Detailed description of the invention
Evolutionary relationship result of the Fig. 1 between chitosan enzyme CsnQ of the present invention and known chitosan enzyme sequence；
Fig. 2 is protein separation figure (M, the Protein standards of chitosan enzyme CsnQ of the present invention；1, sample before purification, 2, Purifying gained chitosan enzyme CsnQ)；
Fig. 3 is the temperature and pH Adaptability Analysis (A, the optimal reactive temperature of chitosan enzyme CsnQ of chitosan enzyme CsnQ of the present invention； B, the temperature stability of chitosan enzyme CsnQ；The optimal reaction pH of C, chitosan enzyme CsnQ；The pH of D, chitosan enzyme CsnQ stablize Property)；
Fig. 4 is the degradation mode and enzymolysis product (M, chitosan oligosaccharide that thin-layer chromatography (TLC) method detects chitosan enzyme CsnQ of the present invention DP1-4, chitosan oligosaccharide 1-4 saccharide)；
Fig. 5 is the catabolite that anion first mass spectrometric method detects chitosan enzyme CsnQ of the present invention.
Specific embodiment
The sequence of 1 chitosan enzyme CsnQ of embodiment is analyzed
The producing enzyme gene csnQ of chitosan enzyme CsnQ of the present invention is artificial synthesized sequence.In early-stage study, it has been found that sea Foreign bacterium Vibrio sp. Q108 chitosan enzyme activity with higher, when carrying out full genome sequencing to it, it is found that it contains one The chitosan enzyme sequence of a prediction.In the case where amino acid sequence is constant, we are by the gene order according to host's (large intestine bar Bacterium) codon preference, carried out base sequence optimization, carried out high efficient expression in Escherichia coli conducive to it.Sequence is closed It is carried out in Huada gene company.Chitosan enzyme CsnQ of the present invention includes 861 base sequences, encodes 287 amino Acid sequence.Utilize the conserved domain in National Center for Biotechnology Information (NCBI) Analyze Conserved domain (CDD) and Multiple sequence alignments Basic Local Alignment Search Tool (Blast) it finds, which includes the conserved region of the 46th family (GH-46) of a polysaccharide hydrolase.Chitosan of the invention Enzyme CsnQ guards chitosan enzyme (PDB:1CHK) sequence similarity that region amino acid sequence is reported with existing crystal structure 96.63%, it is consistent substantially, so that it maintains good catalytic activity (Rate activity reaches 690.5 U/mg)；With the present invention Chitosan enzyme (PDB:1CHK) sequence difference that the chitosan enzyme CsnQ is reported with existing crystal structure is mainly shown as: shell is poly- Carbohydrase CsnQ includes 287 amino acid sequences, and chitosan enzyme (PDB:1CHK) only includes 238 amino acid sequences.Shell The N-terminal of dextranase CsnQ includes one section of 39 amino acid (Met1-Gly39) composition N-trunk structure, C section include 9 ammonia One section of C-tail structure (His of base acid278-Cys287), the two includes to increase the three of chitosan enzyme there are many rigid amino acid The rigidity for tieing up structure is the structure basis that chitosan enzyme CsnQ of the present invention has better stability.
Chitosan enzyme CsnQ of the present invention carries out Blast points with the amino acid sequence for belonging to same family (GH46) Analysis carries out Multiple sequence alignments using ClustalX software, and carries out phylogenetic analysis using 7.1 software of Mega.Such as Fig. 1 institute Show, chitosan enzyme CsnQ and other it has been reported that chitosan enzyme have farther away affiliation, be a novel chitosan Enzyme.
By the base sequence of chitosan enzyme CsnQ using restriction enzyme Nco I and Xho I as restriction enzyme site, design recombination Primer is following (underscore is restriction endonuclease sites, and italic is restriction enzyme enzyme protection base):
Forward primer: SEQ ID NO.3:PcsnQ-F:
5’- CATGCCATGG ATGAAATACTTATTACCAAC-3’ (Nco I)
Reverse primer: SEQ ID NO.4:PcsnQ-R:
5’- CCGCTCGAGACAACCAGAACGTAATGGT-3’ (Xho I)
PCR amplification condition are as follows: 94 DEG C of 3 min of initial denaturation；94 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, 72 DEG C of 1 min of extension, altogether 30 circulations；72 DEG C of 5 min of extension；4 DEG C are stablized 15 min.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 Nco I and Xho I, recycles enzyme by agarose gel electrophoresis PCR product after cutting.PET22b (+) Plasmid DNA (Invitrogen company, the U.S.), equally with restriction enzyme Nco I and Xho I carries out double digestion, carries out agarose gel electrophoresis and recycles the product segment after digestion.Enzyme and substrate reactions used in digestion The description of product operation that system (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, is cultivated in 37 DEG C of incubators After 12-16 hours, picking monoclonal；Monoclonal is forwarded in LB liquid medium (containing 50 μ g/mL ampicillins), Revolving speed is overnight incubation in 37 DEG C of shaking tables of 180 rpm.Monoclonal is subjected to sequencing, selects positive colony, and ordered Entitled pET22b-csnQ.Recombinant plasmid transformed is big by recombination to e. coli bl21 (DE3) (being purchased from Dalian treasured biotech firm) Intestines Strain Designation be BL21 (DE3)/pET22b-csnQ, be stored in -80 DEG C it is spare.
The preparation of 2 chitosan enzyme CsnQ of embodiment and purification process
By recombinant bacterial strain BL21 (DE3)/pET22b-csnQ in the LB liquid medium of 100 mL (50 μ g/mL ammonia benzyl moulds Element), 180 rpm shake cultures are to OD in 37 DEG C of shaking tables600=0.6, the inducer isopropylthio-β-of final concentration of 0.1 mM is added D- thiogalactoside (IPTG), in 20 DEG C of 20 h of induction.The active standard determination method of chitosan enzyme CsnQ are as follows: 100 μ L enzymes 900 μ L, 0.3% chitosan substrate (20 mM Acetic acid-sodium acetates, pH=5.9) are added in liquid, and 10 min are reacted at 60 DEG C, are added The DNS reagent of 750 μ L reacts 10 min in boiling water and develops the color, its absorbance is detected at OD520.Enzyme activity is defined as 1 U is that every min generates enzyme amount required for 1 μM of reduced sugar.Through detecting, chitosan enzyme vigor is up to 1058.2 U/ in fermentation liquid mL。
After fermentation stops, 12000 rpm are centrifuged 10 min, abandon thallus, collect supernatant；Fermented supernatant fluid is splined on 10 ML nickel ion affinity chromatograph column, loading flow velocity are 5 mL/min, are eluted using 10 mM imidazoles, remove foreign protein, recycle 150 The imidazoles of mM elutes, and collects elution fraction.By active constituent dialyse removal imidazoles, packing be stored in -20 DEG C it is spare.By nickel from A sub step affinity purification, protein recovery reach 79.6%.Purifying gained chitosan enzyme CsnQ carries out polyacrylamide gel electrophoresis (SDS-PAGE), as shown in Fig. 2, the molecular weight of chitosan enzyme CsnQ is 27 kDa, with the albumen size predicted in sequence analysis Unanimously.It is found by gel analysis, the purity of protein of purifying gained chitosan enzyme CsnQ reaches 95% or more.
The influence of 3 temperature of embodiment and pH to chitosan enzyme CsnQ
To carry out at different conditions enzyme activity determination by purifying gained chitosan enzyme CsnQ in embodiment 2, detection different temperatures and Influence of the pH to enzyme activity.10 min, shadow of the detection differential responses temperature to enzyme activity are reacted under different temperatures (0-80 DEG C) It rings, with highest enzyme activity for 100%, calculates the enzyme activity of chitosan enzyme CsnQ under different temperatures.As shown in Figure 3A, shell is poly- The optimal reactive temperature of carbohydrase CsnQ is 60 DEG C.
Purifying gained chitosan enzyme CsnQ in embodiment 2 is incubated for 1 h under different temperatures (0-80 DEG C), after taking-up, Its enzyme activity is detected under its optimal reactive temperature (60 DEG C), using the vigor before being incubated for as 100%, as shown in Figure 3B, chitosan enzyme CsnQ temperature stability is preferable, and 1 h is incubated at 40 DEG C, and enzymatic activity can reach 61.2% before being incubated for.
In three kinds of different buffer (50 mM, Sodium acetate buffer, Phosphate of different pH Buffer, Gly-NaOH buffer) under detect the activity of purifying gained chitosan enzyme CsnQ in embodiment 2, enzymatic activity peak It is 100%.As shown in Figure 3 C, the optimal reaction pH of chitosan enzyme CsnQ is 5.0.
Purifying gained chitosan enzyme CsnQ in embodiment 2 is incubated for 24 h under condition of different pH, after taking-up, immediately at it Optimal reaction pH(5.0) under detect its enzyme activity, using be incubated for before vigor as 100%, as shown in Figure 3D, chitosan enzyme CsnQ Vigor can keep 80% or more in the range of pH 4.0-6.0.
Influence of 4 different metal ions of embodiment to chitosan enzyme CsnQ
Purifying gained chitosan enzyme CsnQ in embodiment 2 is added to the different metal ions of 5 mM during enzyme activity determination, It is incubated for for 24 hours, under the conditions of the standard of chitosan enzyme CsnQ surveys living, detects different metal ions to chitosan enzyme CsnQ enzyme activity It influences, not add the enzyme solution of metal ion as compareing, as shown in Table 1, various metals ion pair chitosan enzyme CsnQ enzyme activity Power has certain facilitation.
Influence of 1 different metal ions of table to chitosan enzyme CsnQ enzyme activity of the present invention
Ion Concentration (mM) Enzyme activity (%)
None -- 100
Aluminium ion 5 48±10.1
Ferric ion 5 88±2.6
Potassium ion 5 101±4.3
Sodium ion 5 104±3.9
Magnesium ion 5 106±0.7
Lithium ion 5 106±3.3
Ammonium ion 5 107±3.1
Ferrous ion 5 108±2.6
Copper ion 5 108±3.3
Barium ions 5 109±4.2
Cobalt ions 5 110±2.4
Calcium ion 5 112±3.4
Zinc ion 5 114±3.8
Manganese ion 5 121±3.1
5 chitosan enzyme CsnQ enzymolysis product thin layer chromatography analysis of embodiment
Purifying gained chitosan enzyme CsnQ and 0.3% chitosan in embodiment 2 are incubated for different time at 60 DEG C respectively, then It is detected at High Performance Thin Layer Chromatography plate (HPTLC).Specifically: HPTLC chromatoplate is cut into the sample of the suitable size of 7 cm wide, it will It is incubated for front and back sample point sample at the origin, is placed in 30 min in the exhibition cylinder of solvent (n-butanol: glacial acetic acid: water=2:2:1), Chromatoplate is dried up, 2s in color developing agent (0.5% ethanol solution of ninhydrin) is immersed, takes out drying, 80 DEG C of bakings, until sample occurs.Such as Shown in Fig. 4, it was found that, it is chitobiose (DP2) and chitotriose that chitosan enzyme CsnQ, which digests principal product, with standard items migration rate (DP3).
As shown in figure 4, enzyme digestion reaction initial stage (0-10 min), the oligosaccharides of a large amount of large fragments is generated, with enzyme digestion reaction It carries out, the oligosaccharides that the oligosaccharides of large fragment gradates as small fragment illustrates that the mode of action of the enzyme is in a manner of inscribe, from shell It is cut inside glycan molecule.
6 chitosan enzyme Csn Q enzymolysis product mass spectrometric determination of embodiment
The chitosan substrate of purifying gained chitosan enzyme CsnQ and 0.3% in embodiment 2 is incubated for 24 hours, catabolite 12, 000 g is centrifuged 10 minutes, abandons precipitating.Gained sample is simultaneously mixed with acetonitrile in equal volume, carries out the detection of anion first mass spectrometric, Determine the molecular weight of enzymolysis product.Ion mode first mass spectrometric is the result shows that (Fig. 5), degradation principal product are chitobiose and shell three Sugar, it is consistent with TLC testing result in embodiment 5.
Sequence table
<110>Shandong Hao Yue Pharmaceutical Technology Co., Ltd
<120>a kind of novel chitosan enzyme CsnQ and its application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 287
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Met Gly Val Gly Gly Thr Gly Ala Thr His
20 25 30
Ala Gly Ala Ala Gly Ser Gly Val Asn Leu Thr Asp Pro His Lys Lys
35 40 45
Glu Ile Ala Met Glu Leu Val Ser Ser Ala Glu Asn Ser Ser Leu Asp
50 55 60
Trp Lys Ala Gln Tyr Lys Tyr Ile Glu Asp Ile Gly Asp Gly Arg Gly
65 70 75 80
Tyr Thr Gly Gly Ile Ile Gly Phe Cys Ser Gly Thr Gly Asp Met Leu
85 90 95
Glu Leu Val Gln His Tyr Thr Asp Leu Glu Pro Gly Asn Ile Leu Ala
100 105 110
Lys Tyr Leu Pro Ala Leu Lys Lys Val Asn Gly Ser Ala Ser His Ser
115 120 125
Gly Leu Gly Thr Pro Phe Thr Lys Asp Trp Ala Thr Ala Ala Lys Asp
130 135 140
Thr Val Phe Gln Gln Ala Gln Asn Asp Glu Arg Asp Arg Val Tyr Phe
145 150 155 160
Asp Pro Ala Val Ser Gln Ala Lys Ala Asp Gly Leu Arg Ala Leu Gly
165 170 175
Gln Phe Ala Tyr Tyr Asp Ala Ile Val Met His Gly Pro Gly Asn Asp
180 185 190
Pro Thr Ser Phe Gly Gly Ile Arg Lys Thr Ala Met Lys Lys Ala Arg
195 200 205
Thr Pro Ala Gln Gly Gly Asp Glu Thr Thr Tyr Leu Asn Ala Phe Leu
210 215 220
Asp Ala Arg Lys Ala Ala Met Leu Thr Glu Ala Ala His Asp Asp Thr
225 230 235 240
Ser Arg Val Asp Thr Glu Gln Arg Val Phe Leu Lys Ala Gly Asn Leu
245 250 255
Asp Leu Asn Pro Pro Leu His Trp Lys Thr Tyr Gly Asp Ser Tyr Ser
260 265 270
Ile Asn Ser Leu Glu His Thr Thr Thr Pro Leu Arg Ser Gly Cys
275 280 285
<210> 2
<211> 861
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgaaatact tattaccaac agcagcagca ggtttattat tattagcagc acaaccagca 60
atggcaatgg gtgtaggtgg tacaggtgca acacatgcag gtgcagcagg ttctggtgta 120
aacttaacag atccacataa aaaagaaatt gcaatggaat tagtatcttc tgcagaaaac 180
tcttctttag attggaaagc acaatacaaa tacattgaag atattggtga tggtcgtggt 240
tacacaggtg gtattattgg tttctgttct ggtacaggtg atatgttaga attagtacaa 300
cattacacag atttagaacc aggtaacatt ttagcaaaat acttaccagc attaaaaaaa 360
gtaaacggtt ctgcatctca ttctggttta ggtacaccat tcacaaaaga ttgggcaaca 420
gcagcaaaag atacagtatt ccaacaagca caaaacgatg aacgtgatcg tgtatacttc 480
gatccagcag tatctcaagc aaaagcagat ggtttacgtg cattaggtca attcgcatac 540
tacgatgcaa ttgtaatgca tggtccaggt aacgatccaa catctttcgg tggtattcgt 600
aaaacagcaa tgaaaaaagc acgtacacca gcacaaggtg gtgatgaaac aacatactta 660
aacgcattct tagatgcacg taaagcagca atgttaacag aagcagcaca tgatgataca 720
tctcgtgtag atacagaaca acgtgtattc ttaaaagcag gtaacttaga tttaaaccca 780
ccattacatt ggaaaacata cggtgattct tactctatta actctttaga acatacaaca 840
acaccattac gttctggttg t 861
<210> 3
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
catgccatgg atgaaatact tattaccaac 30
<210> 4
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ccgctcgaga caaccagaac gtaatggt 28

Claims (7)

1. a kind of novel chitosan enzyme CsnQ, amino acid sequence is as shown in SEQ ID NO.1.
2. nucleotide sequence corresponding to chitosan enzyme CsnQ as described in claim 1, the nucleotide sequence such as SEQ ID Shown in NO.2.
3. the preparation and purification method of chitosan enzyme CsnQ as described in claim 1.
4. application of the chitosan enzyme CsnQ 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 enzyme described in claim 1 CsnQ。
6. method as claimed in claim 5, characterized in that reaction temperature is 0 ~ 80 DEG C in degradation condition, optimal reactive temperature It is 60 DEG C.
7. method as claimed in claim 5, characterized in that reacting pH in degradation condition is 4.0 ~ 9.6, and optimal reaction pH is 5.0。
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CN113493781B (en) * 2021-06-25 2023-07-18 青岛大学 Chitosan enzyme and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012100345A1 (en) * 2011-01-28 2012-08-02 Socpra Sciences Et Genie S.E.C. Thermostable chitosanase
CN108330119A (en) * 2018-04-23 2018-07-27 中国海洋大学 A kind of chitosan enzyme and its application in chitosan oligosaccharide preparation
CN109486804A (en) * 2018-12-26 2019-03-19 青岛大学 It is a kind of with the novel chitosan enzyme CsnM of hot recovery characteristics and its application
CN109640649A (en) * 2016-03-16 2019-04-16 斯波根生物技术公司 Promote the method for plant health using resolvase and the microorganism for being overexpressed enzyme
CN109652437A (en) * 2019-02-21 2019-04-19 中国农业大学 It is a kind of produce chitosan enzyme recombinant bacterium construction method and its application

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012100345A1 (en) * 2011-01-28 2012-08-02 Socpra Sciences Et Genie S.E.C. Thermostable chitosanase
CN109640649A (en) * 2016-03-16 2019-04-16 斯波根生物技术公司 Promote the method for plant health using resolvase and the microorganism for being overexpressed enzyme
CN108330119A (en) * 2018-04-23 2018-07-27 中国海洋大学 A kind of chitosan enzyme and its application in chitosan oligosaccharide preparation
CN109486804A (en) * 2018-12-26 2019-03-19 青岛大学 It is a kind of with the novel chitosan enzyme CsnM of hot recovery characteristics and its application
CN109652437A (en) * 2019-02-21 2019-04-19 中国农业大学 It is a kind of produce chitosan enzyme recombinant bacterium construction method and its application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAN Y等: "N-Terminal seven-amino-acid extension simultaneously improves the pH stability, optimal temperature,thermostability and catalytic efficiency of chitosanase CsnA", 《BIOTECHNOL LETT》 *

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