CN114752584A - Mutant chitosanase with high temperature stability - Google Patents

Mutant chitosanase with high temperature stability Download PDF

Info

Publication number
CN114752584A
CN114752584A CN202210409302.9A CN202210409302A CN114752584A CN 114752584 A CN114752584 A CN 114752584A CN 202210409302 A CN202210409302 A CN 202210409302A CN 114752584 A CN114752584 A CN 114752584A
Authority
CN
China
Prior art keywords
chitosanase
mutant
gly
ala
temperature stability
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202210409302.9A
Other languages
Chinese (zh)
Other versions
CN114752584B (en
Inventor
郭静
高文君
潘文鑫
满在伟
蔡志强
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Changzhou University
Original Assignee
Changzhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Changzhou University filed Critical Changzhou University
Priority to CN202210409302.9A priority Critical patent/CN114752584B/en
Publication of CN114752584A publication Critical patent/CN114752584A/en
Application granted granted Critical
Publication of CN114752584B publication Critical patent/CN114752584B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The present invention relates to gene cloning, site-directedMutation and gene recombination technology, and specifically discloses a mutant of a novel 46-family glycoside hydrolase chitosanase (Sacsn46A) cloned from streptomyces avermitilis, wherein the mutant consists of 271 amino acids and comprises 34 amino acid signal peptides. By analyzing the high unfolding free energy site on the structure of the enzyme protein, the mutation site is selected for mutation, and the chitosanase mutant with improved temperature stability is screened out. The 237 th glycine of the zymogen SaCsn46A is mutated into alanine (G237A), and the mutant is expressed in Escherichia coli Rosetta. By comparison with the wild-type chitosanase, the temperature stability of the mutant enzyme was found to be increased by 4.8-fold compared to the temperature stability of the wild-type strain. The thin layer chromatography results show that the enzymatic hydrolysate is mainly glucosamine GlcN and (GlcN)2。

Description

Mutant chitosanase with high temperature stability
Technical Field
The invention relates to gene cloning, site-directed mutagenesis and gene recombination technology, and particularly relates to mutant chitosanase with high temperature stability.
Background
Chitosan is a polysaccharide linked by glucosamine (GlcN) via β -1, 4-glycosidic linkages. Chitosan is a deacetylated chitin product, widely exists in shells and cartilage of lower plant fungi, algae cells, shellfish and mollusks (such as salmon and squid), is the second largest natural high molecular compound next to cellulose on earth, and is a renewable biological resource.
Research reports show that the chitosan has excellent functions and excellent biological activities of resisting bacteria and tumors, improving immunity and the like, so the chitosan has wide application prospects in the fields of food, medicine, agriculture, cosmetics and the like. However, research reports indicate that many functional properties of chitosan are only expressed when the chitosan is degraded to a certain degree, and most of the degradation products are chitosan oligosaccharide, which not only has all the properties of chitosan, but also is easily soluble in water, has hygroscopicity and moisture retention, and can be well applied to the food, medicine, agriculture and cosmetics industries.
There are three main methods for producing chitosan oligosaccharide: physical, chemical, enzymatic hydrolysis. From the viewpoints of product safety, degradation efficiency and environmental protection, the enzymatic hydrolysis method is generally adopted for industrial degradation of chitosan. In addition, the enzymatic hydrolysis method has become a research hotspot in recent years due to strong specificity, mild reaction conditions and easy preparation.
Chitosanase is a hydrolase specially used for degrading chitosan into chitosan oligosaccharide, and is prepared according to carbohydrate activity enzyme database (www.cazy.org) Chitosanases are known to be classified as Glycoside Hydrolases (GH) families 46, 75, 80 and 8. GH46, GH75 and GH80 currently contain only chitosanases, while the GH8 family contains some other glycoside hydrolases. It is understood that chitosanases of fungal origin are mainly distributed in the GH75 family, whereas those of bacterial origin mainly belong to the GH46 family, and a few to GH 80. The streptomyces avermitilis is a gram-positive filamentous bacterium, and is a main production strain of avermectin and ivermectin. Heggset has reported that a chitosanase of Streptomyces avermitilis GH75 family mainly produces chitooligosaccharide with DP more than or equal to 2. However, at present, there are few reports about modification of streptomyces avermitilis GH46 family chitosanase enzyme molecules, a new chitosanase SaCsn46A is cloned by using a PCR technology at the early stage of the research, and the research shows that the chitosanase has a wide substrate spectrum, but the temperature stability needs to be improved.
Disclosure of Invention
The invention aims to provide a mutant chitosanase with high temperature stability by carrying out molecular modification on chitosanase SaCsn46A, so that an enzymatic hydrolysis method can be better applied to industrial production of chitosan oligosaccharide.
In order to realize the purpose of the invention, the adopted technical scheme is as follows:
the high-temperature stable mutant chitosanase is obtained by mutating the 237 th glycine of the amino acid sequence of the chitosanase Sacsn46A into alanine (G237A), wherein the amino acid sequence of the chitosanase Sacsn46A is SEQ NO. 1, and the amino acid sequence of the mutant chitosanase is SEQ ID NO. 3.
Specifically, the invention screens out the sites which possibly influence the temperature stability of the Sacsn46A by using PoPMuSiC software, selects 1 site to mutate, and performs functional expression in Escherichia coli Rosetta (DE 3). The study of its biochemical properties showed that the temperature stability of the mutant enzyme was improved compared to the original enzyme.
The invention provides a mutant recombinant chitosanase modified by site-directed mutagenesis, and the mutant recombinant chitosanase is expressed in Escherichia coli Rosetta. The site of the mutated amino acid is that the 237 th glycine of chitosanase (SaCsn46A) is mutated into alanine (G237A).
The mutant method comprises the following steps:
1. Designing two primers, amplifying a chitosanase Sacsn46A gene, removing a signal peptide, wherein the gene sequence of the chitosanase Sacsn46A is SEQ NO.2, cloning an amplified target gene into an expression vector pET-28a, and constructing a recombinant plasmid pET-Sacsn46 a;
the two primers are as follows: upstream primer CGGGATCCGCACCCGTCGGCCTGGACGAC downstream primer CCCAAGCTTTCAGCCGATGTGGTAGCTGTC。
2. The chitosanase Sacsn46A was simulated by Swiss-Model online software to obtain the spatial structure of chitosanase (Sacsn 46A).
3. And (3) submitting the spatial structure of the SacSn46A obtained in the step (2) to PoPMuSiC prediction software, calculating the unfolding free energy change of each mutant amino acid of the SacSn46A by using the PoPMuSiC prediction software, determining a key amino acid site related to the stability of the SacSn46A, and taking the key amino acid site as a mutation site.
4. Designing site-directed mutagenesis primer, upstream primer CGGGATCCGCACCCGTCGGCCTGGACGAC downstream primer CCCAAGCTTTCAGGCGATGTGGTAGCTGTC are provided. Obtaining mutant chitosanase gene by PCR technology, cloning the amplified target gene into expression vector pET-28a, constructing recombinant plasmid pET-G237A, the nucleotide sequence of chitosanase is SEQ NO. 4.
5. And (3) transferring the recombinant vector obtained in the step (4) into escherichia coli Rosetta (DE3) for induction culture, centrifuging, collecting thalli, ultrasonically breaking cells, and purifying protein by using a Ni-NTA affinity chromatography column to obtain the mutant chitosanase with improved temperature stability.
Compared with the prior art, the invention has the following technical advantages: the chitosanase Sacsn46A used in the invention has a wider substrate spectrum, the temperature stability of the provided mutant is obviously improved, and the temperature stability is improved without influencing other characteristics of the chitosanase. Under proper conditions, the mutant chitosanase still can polymerize the shellHydrolysis of sugars to GlcN and (GlcN)2Therefore, the mutant chitosanase has good application prospect in industrial production.
Drawings
FIG. 1 shows the temperature stability of wild-type chitosanase and its mutants in examples of the present invention.
Detailed Description
The present invention is not limited to the following embodiments, and those skilled in the art can implement the present invention in other embodiments according to the disclosure of the present invention, or make simple changes or modifications on the design structure and idea of the present invention, and fall into the protection scope of the present invention. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention is described in more detail below with reference to the following examples:
1. identification of chitosanase mutation sites
A protein three-dimensional structure simulation is carried out on chitosanase Sacsn46A by using a Swiss-Model and Streptomyces N174(Streptomyces sp.N174) chitosanase (1CHK _ A) as a template to obtain a space structure of the chitosanase Sacsn46A, the unfolding free energy change (delta G) of each mutant amino acid of the Sacsn46A is calculated by PoPMuSiC prediction software to assist in designing and improving the stability of the chitosanase, a key amino acid site related to the stability of the Sacsn46A is determined, and as the unfolding free energy change of the 237 th amino acid of the chitosanase is relatively large, an upper primer and a lower primer are designed and artificially synthesized to carry out PCR to obtain a corresponding mutant gene.
The gene sequence of the chitosanase Sacsn46A is shown in SEQ NO. 2.
The amino acid sequence of the upstream primer is CGGGATCCGCACCCGTCGGCCTGGACGAC, the downstream primer has the amino acid sequence of CCCAAGCTTTCAGCCGATGTGGTAGCTGTC。
The restriction sites are BamHI and HindIII.
And (3) PCR system:
Figure BDA0003603485350000041
Figure BDA0003603485350000051
the template is pET-Sacsn46 a.
PCR amplification conditions: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 64 ℃ for 1min, extension at 68 ℃ for 10min, 15 cycles, and heat preservation at 4 ℃.
DpnI digestion template plasmid
20 μ of the LPCR product was taken and 1 μ of LDpnI restriction enzyme was directly added to the PCR product.
3. Transferred into Escherichia coli
10. mu.L of the enzyme-cleaved product was directly transformed into E.coli DH 5. alpha. competent cells. The recombinant cells carrying the mutated plasmids were sent to Shanghai bioengineering, Inc. for sequencing. Coli BL21 was transformed with the correctly sequenced mutant plasmid.
4. Protein purification
Loading the crude enzyme solution obtained by ultrasonic cell disruption to Ni-NTA affinity chromatography column, eluting with 0.02M imidazole eluent (0.02M Tris-HCl, pH 8.0, 0.5M NaCl, 0.02M imidazole and 10% glycerol), eluting with 0.08M imidazole eluent (0.02M Tris-HCl, pH 8.0, 0.5M NaCl, 0.08M imidazole and 10% glycerol), collecting the eluate containing chitosanase activity, removing imidazole by dialysis to obtain mutant chitosanase (mutant G237A), and storing at-20 deg.C.
5. Detection of enzymatic Properties
Measuring enzyme activity by using a DNS method, adding 1475 mu L of pH buffer solution and 500 mu L of colloidal chitosan solution into a 2mL reaction system, adding 25 mu L of mutant chitosanase obtained in the step 4, fully mixing enzyme solution uniformly, immediately reacting in a 40 ℃ water bath kettle for 10min, taking out, adding 1.5mL of DNS solution to terminate the reaction, boiling with boiling water for 5min, finally using distilled water to 25mL, cooling to room temperature, mixing uniformly, transferring into a 50mL centrifuge tube, setting 8000rpm, centrifuging for 5min, taking supernatant to measure the OD value under 520nm, adding 25 mu L ddH, fixing the volume to 25mL, adding2Zero-setting O solution as blank control group。
Definition of enzyme activity: the amount of enzyme required to catalytically produce a reducing sugar corresponding to 1. mu. mol of glucosamine in 1 minute at 40 ℃ was 1 enzyme activity unit (U).
(1) Optimum pH
The enzymatic activities of chitosanase were determined in different pH buffers (pH 3.0-8.0) at a temperature of 40 ℃. The highest point of enzyme activity is taken as 100 percent.
(2) pH stability
The chitosanase is stored in phosphate buffer solution with pH 6.4 for 2h at 0 deg.C, and the enzyme activity of the chitosanase is 100%.
(3) Optimum temperature
Under the condition of the most suitable pH, the reaction systems are respectively placed at 25-80 ℃ for reaction, and the enzyme activity of the chitosanase is measured by taking a temperature gradient every 5 ℃. The highest point of enzyme activity is taken as 100 percent.
(4) Temperature stability
The chitosanase is stored for 2 hours under the conditions of 60 ℃ and pH 6.4, and the enzyme activity of the chitosanase is determined as 100% at the beginning.
6. Wild type chitosanase (the wild type chitosanase in the invention is chitosanase Sacsn46A) and its mutant enzymological properties
The enzymology properties of the wild-type chitosanase and the mutant are shown in figure 1 and table 1, and the temperature stability of the mutant G237A is obviously higher than that of the wild-type chitosanase (the temperature stability is improved by 4.8 times), but the specific enzyme activity is reduced.
TABLE 1 enzymological Properties of wild-type chitosanases and their mutants
Figure BDA0003603485350000071
The enzyme cutting mode of the mutant chitosanase is characterized by showing an endo-type, and being capable of hydrolyzing a plurality of polysaccharides connected by beta-1, 4-glycosidic bonds but incapable of hydrolyzing polysaccharides connected by alpha-1, 4-glycosidic bonds. Hydrolysis products of the mutant chitosanase are glucosamine GlcN and chitobiose (GlcN) 2。
SEQ NO.1
Avermitilis chitosanase protein sequence
APVGLDDPAKKEIAMKLVSSAENSSLDWKAQYKYIEDIGDGRGYTAGIIGFCSGTGDMLDLVELYTQRKPGNVLATYLPALRNVNGGDSHQGLDPGFPGDWRRAAQDSAFQQAQNDERDRVYFDPAVRQGKADGIGVLGQFTYYDAIVMHGDGGDSTSFSSIRGRALAKAEPPAQGGNEVTYLNAFLDARVWAMRQEEAHSDTSRVDTAQRVFLTKGNLNLDPPLDWKVYGDSYHIG
SEQ NO.2
Avermitilis chitosanase gene sequence
gcacccgt cggcctggac gacccggcga agaaagagat cgccatgaag ctcgtgtcca gcgcggagaa ctcctcgctggactggaagg cccagtacaa gtacatcgag gacatcggcg acggccgcgg ctacaccgccgggatcatcg gcttctgctc cggcaccggc gacatgctcg acctcgtcga gctctacacc cagcgcaagc cggggaacgt cctggccacg tatctgcccg ccctgcgcaa cgtcaacggcggcgactcgc accagggcct ggacccgggc ttccccggcg actggcgccg cgcggcccag gactcggcgt tccagcaggc ccagaacgac gaacgcgacc gcgtctactt cgacccggcc gtccggcagg ggaaggcgga cggtatcggc gtactcggac agttcacgta ctacgacgccatcgtcatgc acggggacgg cggtgacagc accagcttca gcagcatccg cgggcgcgccctggccaagg ccgagccgcc ggcgcagggc ggcaacgagg tgacgtacct gaacgccttcctcgacgccc gggtctgggc gatgcggcag gaggaggccc actcggacac cagccgggtc gacaccgccc agcgggtctt cctgacgaag ggcaacctga acctggatcc gccactggac tggaaggtgt acggggacag ctaccacatc ggctga
SEQ ID NO.3
Chitosanase mutant amino acid sequence
APVGLDDPAKKEIAMKLVSSAENSSLDWKAQYKYIEDIGDGRGYTAGIIGFCSGTGDMLDLVELYTQRKPGNVLATYLPALRNVNGGDSHQGLDPGFPGDWRRAAQDSAFQQAQNDERDRVYFDPAVRQGKADGIGVLGQFTYYDAIVMHGDGGDSTSFSSIRGRALAKAEPPAQGGNEVTYLNAFLDARVWAMRQEEAHSDTSRVDTAQRVFLTKGNLNLDPPLDWKVYGDSYHIA
SEQ NO.4
Chitosanase mutant gene sequence
gcacccgt cggcctggac gacccggcga agaaagagat cgccatgaag ctcgtgtcca gcgcggagaa ctcctcgctggactggaagg cccagtacaa gtacatcgag gacatcggcg acggccgcgg ctacaccgccgggatcatcg gcttctgctc cggcaccggc gacatgctcg acctcgtcga gctctacacc cagcgcaagc cggggaacgt cctggccacg tatctgcccg ccctgcgcaa cgtcaacggcggcgactcgc accagggcct ggacccgggc ttccccggcg actggcgccg cgcggcccag gactcggcgt tccagcaggc ccagaacgac gaacgcgacc gcgtctactt cgacccggcc gtccggcagg ggaaggcgga cggtatcggc gtactcggac agttcacgta ctacgacgccatcgtcatgc acggggacgg cggtgacagc accagcttca gcagcatccg cgggcgcgccctggccaagg ccgagccgcc ggcgcagggc ggcaacgagg tgacgtacct gaacgccttcctcgacgccc gggtctgggc gatgcggcag gaggaggccc actcggacac cagccgggtc gacaccgccc agcgggtctt cctgacgaag ggcaacctga acctggatcc gccactggac tggaaggtgt acggggacag ctaccacatc gcctga
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be equivalent or changed within the technical scope of the present invention.
Sequence listing
<110> university of Changzhou
<120> high temperature stability mutant chitosanase
<141> 2022-04-19
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
Cys Gly Gly Gly Ala Thr Cys Cys Gly Cys Ala Cys Cys Cys Gly Thr
1 5 10 15
Cys Gly Gly Cys Cys Thr Gly Gly Ala Cys Gly Ala Cys
20 25
<210> 2
<211> 30
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
Cys Cys Cys Ala Ala Gly Cys Thr Thr Thr Cys Ala Gly Cys Cys Gly
1 5 10 15
Ala Thr Gly Thr Gly Gly Thr Ala Gly Cys Thr Gly Thr Cys
20 25 30
<210> 3
<211> 29
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
Cys Gly Gly Gly Ala Thr Cys Cys Gly Cys Ala Cys Cys Cys Gly Thr
1 5 10 15
Cys Gly Gly Cys Cys Thr Gly Gly Ala Cys Gly Ala Cys
20 25
<210> 4
<211> 30
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
Cys Cys Cys Ala Ala Gly Cys Thr Thr Thr Cys Ala Gly Gly Cys Gly
1 5 10 15
Ala Thr Gly Thr Gly Gly Thr Ala Gly Cys Thr Gly Thr Cys
20 25 30
<210> 5
<211> 237
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 5
Ala Pro Val Gly Leu Asp Asp Pro Ala Lys Lys Glu Ile Ala Met Lys
1 5 10 15
Leu Val Ser Ser Ala Glu Asn Ser Ser Leu Asp Trp Lys Ala Gln Tyr
20 25 30
Lys Tyr Ile Glu Asp Ile Gly Asp Gly Arg Gly Tyr Thr Ala Gly Ile
35 40 45
Ile Gly Phe Cys Ser Gly Thr Gly Asp Met Leu Asp Leu Val Glu Leu
50 55 60
Tyr Thr Gln Arg Lys Pro Gly Asn Val Leu Ala Thr Tyr Leu Pro Ala
65 70 75 80
Leu Arg Asn Val Asn Gly Gly Asp Ser His Gln Gly Leu Asp Pro Gly
85 90 95
Phe Pro Gly Asp Trp Arg Arg Ala Ala Gln Asp Ser Ala Phe Gln Gln
100 105 110
Ala Gln Asn Asp Glu Arg Asp Arg Val Tyr Phe Asp Pro Ala Val Arg
115 120 125
Gln Gly Lys Ala Asp Gly Ile Gly Val Leu Gly Gln Phe Thr Tyr Tyr
130 135 140
Asp Ala Ile Val Met His Gly Asp Gly Gly Asp Ser Thr Ser Phe Ser
145 150 155 160
Ser Ile Arg Gly Arg Ala Leu Ala Lys Ala Glu Pro Pro Ala Gln Gly
165 170 175
Gly Asn Glu Val Thr Tyr Leu Asn Ala Phe Leu Asp Ala Arg Val Trp
180 185 190
Ala Met Arg Gln Glu Glu Ala His Ser Asp Thr Ser Arg Val Asp Thr
195 200 205
Ala Gln Arg Val Phe Leu Thr Lys Gly Asn Leu Asn Leu Asp Pro Pro
210 215 220
Leu Asp Trp Lys Val Tyr Gly Asp Ser Tyr His Ile Gly
225 230 235
<210> 6
<211> 714
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
gcacccgtcg gcctggacga cccggcgaag aaagagatcg ccatgaagct cgtgtccagc 60
gcggagaact cctcgctgga ctggaaggcc cagtacaagt acatcgagga catcggcgac 120
ggccgcggct acaccgccgg gatcatcggc ttctgctccg gcaccggcga catgctcgac 180
ctcgtcgagc tctacaccca gcgcaagccg gggaacgtcc tggccacgta tctgcccgcc 240
ctgcgcaacg tcaacggcgg cgactcgcac cagggcctgg acccgggctt ccccggcgac 300
tggcgccgcg cggcccagga ctcggcgttc cagcaggccc agaacgacga acgcgaccgc 360
gtctacttcg acccggccgt ccggcagggg aaggcggacg gtatcggcgt actcggacag 420
ttcacgtact acgacgccat cgtcatgcac ggggacggcg gtgacagcac cagcttcagc 480
agcatccgcg ggcgcgccct ggccaaggcc gagccgccgg cgcagggcgg caacgaggtg 540
acgtacctga acgccttcct cgacgcccgg gtctgggcga tgcggcagga ggaggcccac 600
tcggacacca gccgggtcga caccgcccag cgggtcttcc tgacgaaggg caacctgaac 660
ctggatccgc cactggactg gaaggtgtac ggggacagct accacatcgg ctga 714
<210> 7
<211> 237
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 7
Ala Pro Val Gly Leu Asp Asp Pro Ala Lys Lys Glu Ile Ala Met Lys
1 5 10 15
Leu Val Ser Ser Ala Glu Asn Ser Ser Leu Asp Trp Lys Ala Gln Tyr
20 25 30
Lys Tyr Ile Glu Asp Ile Gly Asp Gly Arg Gly Tyr Thr Ala Gly Ile
35 40 45
Ile Gly Phe Cys Ser Gly Thr Gly Asp Met Leu Asp Leu Val Glu Leu
50 55 60
Tyr Thr Gln Arg Lys Pro Gly Asn Val Leu Ala Thr Tyr Leu Pro Ala
65 70 75 80
Leu Arg Asn Val Asn Gly Gly Asp Ser His Gln Gly Leu Asp Pro Gly
85 90 95
Phe Pro Gly Asp Trp Arg Arg Ala Ala Gln Asp Ser Ala Phe Gln Gln
100 105 110
Ala Gln Asn Asp Glu Arg Asp Arg Val Tyr Phe Asp Pro Ala Val Arg
115 120 125
Gln Gly Lys Ala Asp Gly Ile Gly Val Leu Gly Gln Phe Thr Tyr Tyr
130 135 140
Asp Ala Ile Val Met His Gly Asp Gly Gly Asp Ser Thr Ser Phe Ser
145 150 155 160
Ser Ile Arg Gly Arg Ala Leu Ala Lys Ala Glu Pro Pro Ala Gln Gly
165 170 175
Gly Asn Glu Val Thr Tyr Leu Asn Ala Phe Leu Asp Ala Arg Val Trp
180 185 190
Ala Met Arg Gln Glu Glu Ala His Ser Asp Thr Ser Arg Val Asp Thr
195 200 205
Ala Gln Arg Val Phe Leu Thr Lys Gly Asn Leu Asn Leu Asp Pro Pro
210 215 220
Leu Asp Trp Lys Val Tyr Gly Asp Ser Tyr His Ile Ala
225 230 235
<210> 8
<211> 714
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 8
gcacccgtcg gcctggacga cccggcgaag aaagagatcg ccatgaagct cgtgtccagc 60
gcggagaact cctcgctgga ctggaaggcc cagtacaagt acatcgagga catcggcgac 120
ggccgcggct acaccgccgg gatcatcggc ttctgctccg gcaccggcga catgctcgac 180
ctcgtcgagc tctacaccca gcgcaagccg gggaacgtcc tggccacgta tctgcccgcc 240
ctgcgcaacg tcaacggcgg cgactcgcac cagggcctgg acccgggctt ccccggcgac 300
tggcgccgcg cggcccagga ctcggcgttc cagcaggccc agaacgacga acgcgaccgc 360
gtctacttcg acccggccgt ccggcagggg aaggcggacg gtatcggcgt actcggacag 420
ttcacgtact acgacgccat cgtcatgcac ggggacggcg gtgacagcac cagcttcagc 480
agcatccgcg ggcgcgccct ggccaaggcc gagccgccgg cgcagggcgg caacgaggtg 540
acgtacctga acgccttcct cgacgcccgg gtctgggcga tgcggcagga ggaggcccac 600
tcggacacca gccgggtcga caccgcccag cgggtcttcc tgacgaaggg caacctgaac 660
ctggatccgc cactggactg gaaggtgtac ggggacagct accacatcgc ctga 714

Claims (5)

1. A mutant chitosanase stable at high temperature, comprising: the chitosanase is obtained by mutating the 237 th glycine of the amino acid sequence of the chitosanase Sacsn46A into alanine (G237A), and the amino acid sequence of the mutated chitosanase is SEQ ID NO. 3.
2. A gene, characterized by: the gene codes the mutant chitosanase of claim 1, and the nucleotide sequence of the gene is SEQ NO. 4.
3. A recombinant plasmid, characterized in that: comprising the gene of claim 2.
4. A host cell, characterized in that: comprising the gene of claim 2 or comprising the recombinant plasmid of claim 3.
5. The use of the mutant chitosanase of claim 1, wherein said mutant chitosanase is used to degrade chitosan into chitosan oligosaccharides and glucosamine.
CN202210409302.9A 2022-04-19 2022-04-19 High-temperature-stability mutant chitosan enzyme Active CN114752584B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210409302.9A CN114752584B (en) 2022-04-19 2022-04-19 High-temperature-stability mutant chitosan enzyme

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210409302.9A CN114752584B (en) 2022-04-19 2022-04-19 High-temperature-stability mutant chitosan enzyme

Publications (2)

Publication Number Publication Date
CN114752584A true CN114752584A (en) 2022-07-15
CN114752584B CN114752584B (en) 2024-06-21

Family

ID=82330250

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210409302.9A Active CN114752584B (en) 2022-04-19 2022-04-19 High-temperature-stability mutant chitosan enzyme

Country Status (1)

Country Link
CN (1) CN114752584B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116486903A (en) * 2023-04-17 2023-07-25 深圳新锐基因科技有限公司 Method and device for improving protein stability based on combination of homologous protein sequence evolution direction and free energy change

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004000131A (en) * 2001-12-11 2004-01-08 Sankyo Lifetech Co Ltd Chitosanase and its use
CN113755471A (en) * 2021-08-30 2021-12-07 常州大学 Chitosanase mutant and construction method and application thereof
CN113862241A (en) * 2021-12-02 2021-12-31 深圳润康生态环境股份有限公司 Chitosanase Csncv, mutant CsnB thereof and application of mutant CsnB

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004000131A (en) * 2001-12-11 2004-01-08 Sankyo Lifetech Co Ltd Chitosanase and its use
CN113755471A (en) * 2021-08-30 2021-12-07 常州大学 Chitosanase mutant and construction method and application thereof
CN113862241A (en) * 2021-12-02 2021-12-31 深圳润康生态环境股份有限公司 Chitosanase Csncv, mutant CsnB thereof and application of mutant CsnB

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116486903A (en) * 2023-04-17 2023-07-25 深圳新锐基因科技有限公司 Method and device for improving protein stability based on combination of homologous protein sequence evolution direction and free energy change
CN116486903B (en) * 2023-04-17 2023-12-29 深圳新锐基因科技有限公司 Method and device for improving protein stability based on combination of homologous protein sequence evolution direction and free energy change

Also Published As

Publication number Publication date
CN114752584B (en) 2024-06-21

Similar Documents

Publication Publication Date Title
Dong et al. A unique β-agarase, AgaA, from a marine bacterium, Vibrio sp. strain PO-303
CN114410611B (en) Kunmu polysaccharide degrading enzyme OUC-BsLam26 and application thereof
CN113755471B (en) Chitosan mutant and construction method and application thereof
Lee et al. Molecular Cloning, Overexpression, and Enzymatic Characterization of Glycosyl Hydrolase Family 16${\beta} $-Agarase from Marine Bacterium Saccharophagus sp. AG21 in Escherichia coli
CN112708609B (en) Chitosanase OUC-CsnPa and application thereof
CN110452919B (en) Truncated alginate lyase Aly7B-CDII gene and application thereof
WO2021208519A1 (en) Endo-beta-mannanohydrolase man01929, method for mutating same into glycosyltransferase, and use thereof
CN109929859B (en) Kappa-carrageenan enzyme coding gene and preparation and application thereof
CN114480350B (en) Application of carrageenase in degrading kappa-carrageenan and furcellaran
Sasaki et al. Family 19 chitinase from rice (Oryza sativa L.): substrate-binding subsites demonstrated by kinetic and molecular modeling studies
CN114752584B (en) High-temperature-stability mutant chitosan enzyme
CN114015708B (en) Deep sea bacteria-derived alpha-glucosidase QsGH13 and encoding gene and application thereof
CN113717963B (en) Chitinase and AfChi18 gene and expression method and application thereof
CN114457057B (en) Chitosan mutant and application thereof
CN109486804A (en) It is a kind of with the novel chitosan enzyme CsnM of hot recovery characteristics and its application
Tajana et al. Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus
CN110036108A (en) A kind of bacterium beta-1,3-glucanase and its encoding gene and application
JP5967725B2 (en) Complex sugar chain hydrolase
CN114736889B (en) Chitosan mutant with improved stability of N-terminal mutant enzyme and application thereof
CN112626051B (en) 1,3/1, 4-xylanase MLX1034 and coding gene and application thereof
CN114317495A (en) Glucanase mutant with improved heat stability and application thereof
CN113046376A (en) Mannase gene VbMan26A, recombinant plasmid, recombinant strain, mannase and application thereof
CN109762798A (en) The preparation method and application of a kind of balun Pueraria lobota hereby series bacillus chitosan enzyme
KR102300386B1 (en) Use of alpha-L-fucosidase having dual enzymatic activity for cleaving alpha- and beta-1,4-glycosidic linkages
CN103602646A (en) Optimal reaction temperature increased beta-glucosidase mutant and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant