CN116949015A - Chitosan mutant with improved catalytic activity and application thereof - Google Patents
Chitosan mutant with improved catalytic activity and application thereof Download PDFInfo
- Publication number
- CN116949015A CN116949015A CN202310871726.1A CN202310871726A CN116949015A CN 116949015 A CN116949015 A CN 116949015A CN 202310871726 A CN202310871726 A CN 202310871726A CN 116949015 A CN116949015 A CN 116949015A
- Authority
- CN
- China
- Prior art keywords
- chitosan
- mutant
- enzyme
- amino acid
- catalytic activity
- 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.)
- Pending
Links
- 229920001661 Chitosan Polymers 0.000 title claims abstract description 58
- 230000003197 catalytic effect Effects 0.000 title abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 54
- 102000004190 Enzymes Human genes 0.000 claims abstract description 53
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 12
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 11
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 4
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims abstract description 4
- 235000004279 alanine Nutrition 0.000 claims abstract description 4
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 3
- 239000004220 glutamic acid Substances 0.000 claims abstract description 3
- 108010089807 chitosanase Proteins 0.000 claims description 14
- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 claims description 11
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 4
- 125000000539 amino acid group Chemical group 0.000 claims description 3
- 239000000758 substrate Substances 0.000 abstract description 4
- 150000001413 amino acids Chemical group 0.000 abstract description 3
- 108010031186 Glycoside Hydrolases Proteins 0.000 abstract description 2
- 102000005744 Glycoside Hydrolases Human genes 0.000 abstract description 2
- 235000001014 amino acid Nutrition 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 14
- 238000000034 method Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 229920001542 oligosaccharide Polymers 0.000 description 3
- 150000002482 oligosaccharides Chemical class 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 229920002101 Chitin Polymers 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000002144 chemical decomposition reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 238000007852 inverse PCR Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- MSWZFWKMSRAUBD-QZABAPFNSA-N beta-D-glucosamine Chemical compound N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-QZABAPFNSA-N 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000000850 deacetylating effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000012038 nucleophile Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 239000002341 toxic gas Substances 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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)
-
- 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
- C12P19/14—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
-
- 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)
- C12Y302/01132—Chitosanase (3.2.1.132)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a chitosan enzyme mutant with improved enzyme catalytic activity and application thereof, belonging to the technical field of enzyme engineering. The chitosan enzyme is derived from 46 family glycoside hydrolase (Bsn 46A) of bacillus subtilis, the mutant of the chitosan enzyme is formed by mutating the 203 th glutamic acid of the amino acid sequence of Bsn 46A into small-volume side chain amino acid alanine, the mutant is E203A, and the catalytic activity of the mutant is obviously improved compared with that of a wild type by taking chitosan as a substrate.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a chitosan enzyme mutant with improved catalytic activity and application thereof.
Background
Chitosanase is a glycoside hydrolase with higher catalytic activity on chitosan, and can convert high molecular weight chitosan into low molecular weight functional chitosan oligosaccharide. Chitosanase is mainly distributed in GH8, GH46, GH75 and GH80 families, with the research of chitosanase in GH46 family being the most intensive. The whole structure of the peptide consists of an N-terminal small structural domain and a C-terminal large structural domain, wherein 2 structural domains are connected through a long and bent alpha spiral and are in an asymmetric dumbbell shape, an active center is provided with two catalytic residues Asp and Glu, one amino acid residue is used as a nucleophile, and the other amino acid residue is used as generalized acid/alkali.
Chitosan is a macromolecule which is obtained by highly deacetylating chitin and is called chitosan, the main constituent unit of chitosan is D-glucosamine (Glc N), and the chitosan is an alkaline polysaccharide which is formed by connecting beta-1.4-glycosidic bonds. The chitosan has good performance in bacteriostasis, biocompatibility, film forming property and biodegradability, and is mainly used for chemical applications such as food packaging, active antibacterial agents, water treatment and the like. Chitosan is widely used in chitin animals such as shrimps and crabs, large fungi such as algae plants and mushrooms, has wide sources and rich resources, and is a second type of polymer compound next to cellulose. Chitosan has limited applications due to its large relative molecular mass and poor solubility. And chitosan can be hydrolyzed to produce chitosan oligosaccharide (polymerization degree 2-10) or glucosamine. Chitosan oligosaccharides are the only basic, positively charged oligosaccharides of the known oligosaccharides. The research reports show that the chitosan oligosaccharide has all the effects of the chitosan oligosaccharide, has a plurality of advantages of easy absorption, good water solubility and the like, has the functions of resisting tumor, resisting inflammation, resisting bacteria, improving the immunity of organisms, promoting the growth of lactic acid bacteria and the like, and has wider application prospect in the fields of medicines, foods, agriculture, cosmetics and the like.
The production method of chitosan oligosaccharide has physical degradation method, chemical degradation method and enzymolysis method. The physical method mainly comprises ultrasonic treatment, microwave degradation, ultraviolet irradiation and the like. The yield is low, and the large-scale production of the chitosan oligosaccharide is limited. The chemical degradation method has the defects of severe chemical reaction, poor product selectivity, difficult separation and purification, toxic gas generation and the like. The enzymolysis method has the characteristics of high selectivity, high activity, high efficiency and the like, and has the advantages of mild reaction, high yield of chitosan oligosaccharide, obvious cost advantage and little environmental pollution. Therefore, protein engineering of chitosan enzymes, optimizing enzyme catalytic efficiency is a current research hotspot. At present, the catalytic activity of the chitosan enzyme is low, so that the obtained chitosan enzyme with high catalytic activity has important value in the industrial production of chitosan oligosaccharide.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a chitosan enzyme mutant with improved catalytic activity and application thereof.
According to the invention, through modifying the bacillus subtilis (Bacillus subtilis) chitosan enzyme Bsn 46A molecules, through analyzing substrate channel sites, site-directed mutation is carried out on the 203 th site, and the sites are respectively mutated into tryptophan W with a large volume of R groups or mutated into small volume of R group alanine A, so that the chitosan enzyme mutant with improved catalytic efficiency is obtained.
The amino acid sequence of the bacillus subtilis chitosan enzyme Bsn 46A is shown as SEQ ID NO. 1, and the coded nucleotide sequence is shown as SEQ ID NO. 2.
The invention relates to a chitosan enzyme mutant, which is characterized in that site-directed mutagenesis is carried out on the 203 th site of the amino acid sequence of bacillus subtilis chitosan enzyme Bsn 46A, glutamic acid is mutated into alanine with a small-volume R group, and the mutant is marked as E203A.
The amino acid sequence of the chitosan enzyme mutant E203A is SEQ ID NO. 3, and the nucleotide sequence of the chitosan enzyme mutant is SEQ ID NO. 4.
The invention also provides a recombinant vector carrying the gene for encoding the chitosan enzyme mutant and recombinant bacteria for transforming/transfecting the recombinant vector; the host bacteria of the recombinant bacteria are escherichia coli E.coli BL21 (DE 3).
The invention further provides application of the chitosan enzyme mutant in catalyzing and hydrolyzing chitosan to produce chitosan oligosaccharide, and particularly, the catalytic activity of the mutant is obviously improved in the process of catalyzing and hydrolyzing chitosan.
Furthermore, in the chitosan reaction catalysis of the chitosan mutant, the optimal temperature is 60 ℃, and the optimal pH is 6.2.
Compared with the wild chitosan Bsn 46A, the catalytic activity of the chitosan mutant provided by the invention is obviously improved, and the application of the chitosan mutant in the hydrolysis of chitosan oligosaccharide production has a wide application prospect.
Drawings
FIG. 1 shows the enzymatic activities of Wild-type and mutant, wherein Wild-type is B.subtilis (Bacillus subtilis) chitosanase Bsn 46A;
FIG. 2 is an enzyme activity pH stability;
FIG. 3 is the enzyme activity temperature stability;
FIG. 4 is a protein structure diagram of the chitosan mutant E203A.
Description of the embodiments
The invention is further described in detail below in connection with the examples: these examples are provided only to illustrate the invention and are not intended to limit the scope of the invention.
1. Determination of mutation sites
Submitting the Bsn 46A amino acid sequence shown in SEQ ID NO. 1 to SWISS-MODEL to construct the three-dimensional structure of the enzyme; analyzing the three-dimensional structure of the obtained chitosan enzyme by using a substrate channel analysis technology to obtain an amino acid site capable of affecting the catalytic activity of the enzyme, and determining the 203 th site positioned in a substrate channel as a potential site affecting the catalytic activity; site 203 was mutated to W for the bulky R group or to small R group A, respectively. Designing a primer, and obtaining the mutant chitosan enzyme gene by a PCR technology.
Wherein the amino acid sequence of the wild type chitosan enzyme Bsn 46A is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2;
the amino acid sequence of the chitosan enzyme mutant E203A is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 4;
the amino acid sequence of the chitosan enzyme mutant E203W is shown as SEQ ID NO. 5, and the nucleotide sequence is shown as SEQ ID NO. 6.
TABLE 1 primer sequences
| Primer name | Primer use | Primer (5 '-3') |
| bscsnF | BsCsn46A | CGGTAATTTTGTAATCCCTTAAAAGCTTGCGGCC |
| bscsnF | BsCsn46A | GGCCGCAAGCTTTTAAGGGATTACAAAATTACCG |
| E203WF | E203W | CAATCATGACACCCGTGACTGGTGGAGAGAATCAGTTGCC |
| E203WR | E203W | GGCAACTGATTCTCTCCACCAGTCACGGGTGTCATGATTG |
| E203AF | E203A | CATGACACCCGTGACGCTTGGAGAGAATCAGTTG |
| E203AR | E203A | CAACTGATTCTCTCCAAGCGTCACGGGTGTCATG |
TABLE 2 inverse PCR System:
| reagent name | Volume (mu L) |
| Template | 2 |
| PCR Buffer | 5 |
| dNTPs(10 mM) | 1 |
| Upstream/downstream primer (100 mM) | Each 0.3 |
| PfuDNA polymerase | 1.5 |
| ddH 2 O | 40 |
| Total volume of | 50 |
Inverse PCR amplification conditions: pre-denaturation at 95℃for 5min, denaturation at 95℃for 50 s, annealing at 65℃for 30s, elongation at 68℃for 12.5min,12 cycles; preserving heat at 4 ℃.
2. Construction of expression vectors
Cloning the target gene amplified by PCR into an expression vector pET-28a to construct a recombinant plasmid. mu.L of the PCR reaction mixture was taken, 1. Mu.L of DpnI restriction enzyme was directly added thereto, and the mixture was reacted in a water bath at 37℃to digest the template plasmid. 10 mu L of the enzyme digestion product is directly transformed into E.coli DH5 alpha competent cells. Recombinant cells carrying the mutant plasmid were sent to Shanghai Bioengineering Co.Ltd for sequencing. The mutant plasmid sequenced correctly was transformed to e.coli BL21.
3. Protein purification
After the induced expression mutant is cultured by a shaking table, the thalli are collected and crushed by ultrasonic waves. The supernatant was purified by Ni-IDA affinity chromatography. Loading the supernatant onto Ni-IDA column, eluting the unbound protein with a buffer solution, eluting the protein with an eluent (50 mM Tris-HCl,0.5 mM NaCl,0.1M imidazole, pH 8.0), collecting the eluted protein sample, and preserving at-20deg.C. The protein content of the enzyme solution was determined by the Broadford method.
4. Enzyme activity assay
Enzyme activity is determined by using a DNS method, 1475 [ mu ] L of pH 6.2 phosphate buffer solution, 500 [ mu ] L of 1% colloidal chitosan solution and 18 [ mu ] L of Mn of 100 mM are added into a colorimetric tube 2+ Finally adding 25 mu L of purified chitosanase (the protein content is 25 mu g/mL), uniformly mixing, immediately adding 1.5 mL of DNS reagent into the mixture for uniform mixing in a water bath at 55 ℃ for 5min after the water bath is finished, and stopping the reaction, wherein a sample without enzyme liquid is used as a blank control; boiling water bath for 5min, adding water to 25 mL, and measuring absorbance at 520, 520 nm. Under this condition, the amount of enzyme catalyzing the production of 1. Mu.M reducing sugar per minute is defined as one enzyme activity unit (U).
5. Enzymatic Properties
(1) Optimum pH
Enzyme activity of chitosanase was determined at different pH (ph=6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2) phosphate buffers at a temperature of 50 ℃. The highest point of the enzyme activity is taken as 100 percent.
(2) pH stability
Under the condition of 4 ℃, the chitosan enzyme is placed in phosphate buffer solution with pH of 6.2 to be preserved for 2 hours, the enzyme activity of the chitosan enzyme measured at 0 hour is taken as 100%, and the residual enzyme activity of the chitosan enzyme is measured after 2 h.
(3) Optimum temperature
Under the condition of the optimal pH, the reaction system is respectively placed at 40-75 ℃ (40, 45, 50, 55, 60, 65, 70 and 75) for reaction for 5min, and the enzyme activity of the chitosanase is measured. The highest enzyme activity point is taken as 100%, and the temperature is the optimal temperature.
(4) Temperature stability
The enzyme activity of the chitosan enzyme measured when the chitosan enzyme was stored at 55℃for 2 hours and 0 hour was taken as 100%. 2h, the residual enzyme activity of the chitosanase was determined.
The enzymatic properties of the bacillus subtilis (Bacillus subtilis) chitosanase BsCsn46A (WT) and its mutant (E203W, E203A) are shown in table 3. The E203A mutant has significantly higher catalytic activity than the wild-type bacillus subtilis (Bacillus subtilis) chitosanase Bsn 46A.
TABLE 3 enzymatic Properties
From the above results, it can be seen that the catalytic activity of the chitosanase mutant E203A was improved by 1.17 times as compared with the wild type.
Simple variations or equivalent alternatives of the technical solution, which are obvious to the person skilled in the art, fall within the scope of the present invention, within the technical scope of the present disclosure.
Claims (7)
1. A chitosan enzyme mutant, which is characterized in that the chitosan enzyme mutant is mutant E203A in which the 203 rd glutamic acid of the amino acid sequence of the chitosan enzyme Bsn 46A from bacillus subtilis is mutated into alanine and the amino acid residues at other positions are kept unchanged; the amino acid sequence of the chitosan enzyme mutant is shown as SEQ ID NO. 3.
2. A gene encoding the chitosanase mutant of claim 1.
3. The gene according to claim 2, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 4.
4. A recombinant vector comprising the mutant of claim 1 or the gene of claim 2.
5. A recombinant bacterium comprising the recombinant vector of claim 4.
6. The use of a chitosanase mutant as defined in claim 1, wherein said chitosanase mutant is used for catalyzing chitosan to produce chitosan oligosaccharide.
7. The use of a chitosan enzyme mutant according to claim 6, wherein the chitosan enzyme mutant has an optimum temperature of 60 ℃ and an optimum pH of 6.2 in catalyzing the chitosan reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310871726.1A CN116949015A (en) | 2023-07-17 | 2023-07-17 | Chitosan mutant with improved catalytic activity and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310871726.1A CN116949015A (en) | 2023-07-17 | 2023-07-17 | Chitosan mutant with improved catalytic activity and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116949015A true CN116949015A (en) | 2023-10-27 |
Family
ID=88443881
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310871726.1A Pending CN116949015A (en) | 2023-07-17 | 2023-07-17 | Chitosan mutant with improved catalytic activity and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116949015A (en) |
-
2023
- 2023-07-17 CN CN202310871726.1A patent/CN116949015A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109295043B (en) | Alginate lyase, and preparation method and application thereof | |
| CN113755471B (en) | Chitosan mutant and construction method and application thereof | |
| CN112708609B (en) | Chitosanase OUC-CsnPa and application thereof | |
| CN114410611B (en) | Kunmu polysaccharide degrading enzyme OUC-BsLam26 and application thereof | |
| CN109810966A (en) | Chitinase CmChi6 gene and cloning expression and application thereof | |
| CN101633931A (en) | Hyaluronidase expression vector and application thereof | |
| CN114015675B (en) | Lambda-carrageenase OUC-LuV and application thereof | |
| CN109777817A (en) | A kind of algin catenase and its gene and application | |
| CN102676557B (en) | Encoding gene of type I pullulanase as well as recombinant expression and application thereof | |
| CN113980937A (en) | Lambda-carrageenase OUC-G150-L7 and application thereof | |
| CN114480350B (en) | Application of carrageenase in degrading kappa-carrageenan and furcellaran | |
| CN109652395A (en) | One Bacillus species chitinase and its application | |
| CN109182303A (en) | A kind of balun Pueraria lobota hereby series bacillus β-N-acetylglucosamine glycosides enzyme and its encoding gene and application | |
| CN111676206A (en) | Truncated mutant of alpha-L-rhamnosidase and application thereof | |
| CN116640744B (en) | Chitosanase OUC-CsnA4-S49I, application thereof and method for preparing chitosan oligosaccharide | |
| CN114457057B (en) | Chitosan mutant and application thereof | |
| CN116640747B (en) | Chitosanase OUC-CsnA4-S49P and application thereof | |
| CN111334488B (en) | Laminarin enzyme OUC-L1, and coding gene and application thereof | |
| CN110029097B (en) | Glycoside hydrolase CmNAGase and clone expression and application thereof | |
| CN110511918B (en) | Alginate lyase system and application thereof | |
| CN112795556A (en) | beta-N-acetylglucosaminidase 159 and cloning expression and application thereof | |
| CN114752584B (en) | High-temperature-stability mutant chitosan enzyme | |
| CN107603967A (en) | A kind of chitosan enzyme CSN4 and its encoding gene and application | |
| CN116064484B (en) | Mutant chitinase SsChi18A-2 and application thereof | |
| CN116949015A (en) | Chitosan mutant with improved catalytic activity 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 |