CN116590260A - Chitosan mutant for producing chitetrasaccharide and application thereof - Google Patents
Chitosan mutant for producing chitetrasaccharide and application thereof Download PDFInfo
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- 229920001661 Chitosan Polymers 0.000 title claims abstract description 67
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- 102000004190 Enzymes Human genes 0.000 claims abstract description 33
- 108010089807 chitosanase Proteins 0.000 claims abstract description 24
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- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 6
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 5
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- 150000004044 tetrasaccharides Chemical class 0.000 claims description 2
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- 238000003259 recombinant expression Methods 0.000 claims 2
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- 150000001413 amino acids Chemical group 0.000 abstract description 12
- 102220625480 RING finger protein 24_T50E_mutation Human genes 0.000 abstract description 10
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- 101100007330 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS2 gene Proteins 0.000 abstract description 2
- 101100007331 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS3 gene Proteins 0.000 abstract description 2
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- 230000002378 acidificating effect Effects 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract description 2
- 238000003032 molecular docking Methods 0.000 abstract description 2
- 238000002741 site-directed mutagenesis Methods 0.000 abstract description 2
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- 239000013612 plasmid Substances 0.000 description 7
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- 241000588724 Escherichia coli Species 0.000 description 3
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 101100007332 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) COS4 gene Proteins 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
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- 238000012257 pre-denaturation Methods 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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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
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Abstract
The invention belongs to the technical field of enzyme engineering, and particularly relates to a chitosanase mutant for producing chitetraose. The chitosan enzyme is derived from 46 family glycoside hydrolase chitosan enzyme (Bsn 46A) of bacillus subtilis, and has an amino acid sequence shown in SEQ ID NO:1, and performing computer-aided simulation analysis on a chitosan enzyme substrate channel, and simultaneously performing molecular docking on the Bsn 46A and a substrate to determine amino acid sites which possibly have influence on the polymerization degree of a chitosan enzyme hydrolysate, and performing site-directed mutagenesis on the amino acid sites to respectively mutate into an acidic amino acid E (glutamic acid) and a basic amino acid K (lysine). The invention obtains two kinds of chitosan enzyme mutants T50E and E203K, and compared with the original enzyme, the mutant enzyme hydrolyzes chitosan to produce COS2 and COS3, and also produces chitosan tetraose (COS 4).
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a chitosanase mutant for producing chitetraose and application thereof.
Background
Chitosan is the only basic polysaccharide in nature, is formed by connecting beta-1, 4-glycosidic bond with glucosamine to form a linear structure, and is mainly formed by deacetylating chitin in shrimp shells and crab shells through alkali treatment. Chitosan is very abundant in nature, next to cellulose. The research shows that chitosan has excellent biological activity of resisting bacteria, resisting tumor, raising immunity, etc. and thus has wide application foreground in food, medicine, agriculture, cosmetics, etc. However, since the absorption of human body to biological macromolecules is very limited, chitosan is difficult to be effectively utilized, and there are reports on how to obtain chitosan with low polymerization degree, which has more remarkable antibacterial effect and is easy to be absorbed by human body after the chitosan is degraded into chitosan oligosaccharide, has become a research hot spot in recent years.
The production methods of chitosan oligosaccharide mainly comprise three methods: chemical, physical and biological enzymatic methods. Chemical and physical methods are common industrial technical means, but in consideration of product purity, production cost, environmental pollution and subsequent purification, an enzymatic method is generally adopted to degrade chitosan, and compared with the former two methods, the enzymatic method has the advantages of strong specificity of chitosan hydrolysis, mild reaction conditions, easy preparation and safer and more mature production mode.
Chitosanase is a glycoside hydrolase specially used for degrading chitosan to be chitosan oligosaccharide, beta-1, 4-glycosidic bond in chitosan molecule is generally broken in an inscribed way, and the chitosanase is discovered for the first time in 1973 Shimosaka until the moment, and reported chitosanase can be divided into 7 families according to the amino acid sequence, namely GH3, GH5, GH7, GH8, GH46, GH75 and GH80 families, although various glycoside hydrolases can hydrolyze chitosan to produce chitosan oligosaccharide at present, and the catalytic activity is far higher than that of glycoside hydrolases of other families, so that the research on the chitosanase of the GH46 family is most widely conducted at present. The GH46 family chitosanase is mostly produced by bacterial fermentation, and the products of hydrolyzing the chitosanase are mainly chitosan and chitosan trisaccharide. Research reports show that the chitosan oligosaccharide can exert the antibacterial property to the greatest extent only when the polymerization degree is 4-8. Current research reports on chitosanase are mainly focused on how to improve the catalytic activity and stability of the enzyme to adapt the enzyme to industrial application, while the research reports on preparing chitosan oligosaccharide with specific polymerization degree are less common. In order to improve the application value of chitosan oligosaccharide produced by the hydrolysis of chitosan enzyme, the invention aims to obtain a chitosan enzyme mutant which can degrade chitosan to obtain chitosan oligosaccharide with the polymerization degree of more than 4.
Disclosure of Invention
The invention aims to provide a chitosanase mutant for producing chitetraose and application thereof.
The bacillus subtilis (Bacillus subtilis) chitosan enzyme is a wild chitosan enzyme (Bsn 46A) obtained by laboratory early cloning, and the amino acid sequence of the bacillus subtilis (Bacillus subtilis) chitosan enzyme is SEQ ID NO:1, the gene sequence is SEQ ID NO:2.
the invention selects sites with potential influence value on the generation of products through substrate channel simulation and molecular butt joint screening to mutate the 50 th threonine in the amino acid sequence of the chitosan enzyme Bsn 46A into glutamic acid (T50E), the 203 th glutamic acid into lysine (E203K), and the functional expression is carried out in E.coli BL21 (DE 3).
The amino acid sequence of the chitosan enzyme mutant T50E is SEQ ID NO:3, the coded nucleotide sequence is SEQ ID NO:4, a step of; the amino acid sequence of the chitosan enzyme mutant E203K is SEQ ID NO:5, the coded nucleotide sequence is SEQ ID NO:6.
a recombinant vector carrying the gene encoding the chitosanase mutant.
A recombinant bacterium comprising the recombinant vector.
The invention further provides application of the chitosan enzyme mutant in degrading chitosan, and in particular relates to application in degrading chitosan to generate chitosan disaccharide (COS 2) and chitosan trisaccharide (COS 3) exo-chitosan tetrasaccharide (COS 4).
Compared with wild chitosan enzyme, the chitosan enzyme mutant provided by the invention can generate chitosan tetraose COS4 in addition to chitosan COS2 and chitosan trisaccharide COS3 in the process of hydrolyzing chitosan by enzyme.
Drawings
FIG. 1 is a thin-layer chromatogram of chitosan degradation products of wild-type chitosanase and mutants thereof in an embodiment of the present invention.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
Example 1
The invention provides Bsn 46A gene sequence SEQ ID NO:2 into plasmid pET-28a to construct recombinant plasmid pET-Bsn 46A, upstream primer CGGGATCCGCGGGACTGAATAAAGATC, downstream primer CCCAAGCTTTTAAGGGATTACAAAATTACC; simulation is carried out on chitosanase (BsCsn 46A) by using Swiss-Model online software to obtain a space structure of the chitosanase; by simulating a substrate channel and finding a site which is possibly related to the polymerization degree of an enzyme hydrolysate by molecular docking, the invention respectively mutates 50 and 203 sites into acidic amino acid E or basic amino acid K, and a chitosan enzyme mutant T50E, T50K, E K is intended to be obtained.
And designing a site-directed mutagenesis primer, obtaining a mutant chitosan enzyme gene through a PCR technology, cloning the amplified target gene into an expression vector pET-28a, and constructing a recombinant plasmid.
The primer sequences were as follows:
| primer name | Primer use | Primer (5 '-3') |
| bscsnF | BsCsn46A | CGGTAATTTTGTAATCCCTTAAAAGCTTGCGGCC |
| bscsnR | BsCsn46A | GGCCGCAAGCTTTTAAGGGATTACAAAATTACCG |
| T50EF | T50E | CAGGCTTTACAACGGCTGAAGGGGATGCATTGGAAG |
| T50ER | T50E | CTTCCAATGCATCCCCTTCAGCCGTTGTAAAGCCTG |
| T50KF | T50K | CAGGCTTTACAACGGCTAAAGGGGATGCATTGGAAG |
| T50KR | T50K | CTTCCAATGCATCCCCTTTAGCCGTTGTAAAGCCTG |
| E203KF | E203K | CATGACACCCGTGACAAATGGAGAGAATCAG |
| E203KR | E203K | CTGATTCTCTCCATTTGTCACGGGTGTCATG |
PCR system:
| reagent name | Volume (mu L) |
| Template | 1 |
| PCR Buffer | 5 |
| dNTP | 1 |
| Upstream and downstream primers | 1 |
| PfuDNA polymerase | 1.2 |
| ddH 2 O | 39.8 |
| Total volume of | 50 |
The templates are shown in SEQ ID NO:2. SEQ ID NO: 4. SEQ ID NO:6. SEQ ID NO: shown at 8. PCR amplification conditions: pre-denaturation at 95℃for 3 min; denaturation at 95℃for 30 s, annealing at 64℃for 1 min, elongation at 68℃for 10 min,15 cycles, incubation at 4 ℃.
The obtained recombinant plasmid was digested with DpnI restriction enzyme to obtain plasmid template. 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.
And (3) transforming the mutant plasmid with correct sequencing into E.coli BL21 (DE 3) for induction culture, centrifuging, collecting thalli, ultrasonically crushing cells, and purifying protein by using a Ni-NTA affinity chromatographic column to obtain the mutant chitosanase.
The specific procedures for protein purification were as follows: the bacterial liquid activated by 1mL is transferred into a conical flask filled with 50 mL of LB liquid medium, 25 mu L of 50 mg/L kanamycin (Kana) is added into the medium, and the medium is placed into a constant temperature shaking table at 37 ℃ and 160 r/min for 3 hours. After 3 hours, inducer (IPTG) with a final concentration of 1 mu mol/L was added to the bacterial liquid to induce bacteria to produce protein, the bacteria were cultured overnight in a constant temperature shaking table at 16℃and 160 r/min, the bacteria after the expansion culture were poured into a 50 mL centrifuge tube, placed in a refrigerated centrifuge, centrifuged at 8000 rpm for 5 min, the supernatant was removed, the bacterial cells precipitated at the bottom of the centrifuge tube were collected, 5 mL M0 (0.02M Tris-HCl, pH 8.0,0.5M NaCl,10% glycerol) was added, the bacteria originally split into two tubes were combined into one tube, centrifuged at 8000 rpm for 5 min, the supernatant was removed again, and 5 mL M0 was added for resuspension. The crude enzyme solution obtained by sonicating cells was applied to a Ni-NTA affinity column, eluted with 0.02M imidazole eluent (0.02M Tris-HCl, pH 8.0,0.5M NaCl,0.02M imidazole and 10% glycerol), eluted with 0.08M imidazole eluent (0.02M Tris-HCl, pH 8.0,0.5M NaCl,0.08M imidazole and 10% glycerol), the eluent containing the activity of the chitosanase was collected, imidazole was removed by dialysis, and the obtained mutant enzyme was stored at-20 ℃.
Example 2
The chitosan enzyme mutant T50E, T50K, E K was used to hydrolyze chitosan.
Weighing 1 g chitosan powder, dissolving the chitosan powder in 0.5% (v/v) hydrochloric acid solution, and adjusting the pH of the chitosan solution to 5.5 with NaOH to obtain 1% colloidal chitosan solution.
In a 1mL reaction systemAdding 450. Mu.L of 1% colloidal chitosan solution, 50. Mu.L of purified enzyme solution, 500. Mu.L of pH buffer and 18. Mu.L of Mn of 100 mM 2+ After 24 h reaction in a shaker at 28 ℃, the reaction was stopped by taking out and boiling water bath for 10 min.
The purified enzyme solutions are wild-type chitosanase Bsn 46A and chitosanase mutant T50E, T50K, E K enzyme solutions respectively.
Product analysis: sample application was performed at a position about 1. 1 cm from the bottom of the thin-layer plate, using glucosamine (DP 1), chitosan (DP 2), chitosan (DP 3), chitosan (DP 4) and chitosan (DP 5) as mixed standard samples, and the thin-layer plate thus sampled was placed in a chromatography cylinder with a developing agent so that the lower end of the chromatography plate was immersed in the developing agent. And after the developing agent fully wets the top end of the thin layer plate, taking out the thin layer plate, drying the thin layer plate by using a blower until ammonia water smell does not exist, spraying the color-developing agent, uniformly spraying, putting the thin layer plate into a baking oven at 120 ℃ which is prepared in advance, drying for 10 min, enabling the thin layer plate to show colored spots, comparing the positions of standard samples, and analyzing the experimental results. The thin-layer spot plate results show that the hydrolysate of the wild-type chitosanase Bsn 46A and the chitosanase mutant T50K is chitosan disaccharide and chitosan trisaccharide; the hydrolysis products of the chitosanase mutant T50E, E K are chitosan, chitosan and chitosan tetraose.
As can be seen from FIG. 1, the two mutants T50E, E203K provided by the invention have the advantages that compared with the original enzyme, the hydrolysate identification result has the new generation of chitosan oligosaccharide COS4, and the industrial application potential.
Claims (5)
1. The chitosanase mutant for producing chitetrasaccharide is characterized in that threonine at the 50 th position on the amino acid sequence of encoding bacillus subtilis chitosanase Bsn 46A is mutated into glutamic acid, and the amino acid sequence is shown as SEQ ID NO:3 is shown in the figure; or the 203 th glutamic acid is mutated into lysine, and the amino acid sequence of the lysine is shown as SEQ ID NO:5 is shown in the figure; the amino acid sequence of the coded bacillus subtilis chitosan enzyme Bsn 46A is shown as SEQ ID NO: 1.
2. A nucleotide sequence encoding a chitetrasaccharide-producing chitosanase mutant according to claim 1, characterized in that it encodes the sequence of SEQ ID NO:3 is shown in SEQ ID NO:4 is shown in the figure; encoding SEQ ID NO:5 has a nucleotide sequence shown in SEQ ID NO: shown at 6.
3. A recombinant expression vector comprising one of the nucleotide sequences of claim 2.
4. A recombinant bacterium comprising the recombinant expression vector of claim 3.
5. The use of a chitosanase mutant of claim 1 for degrading chitosan to chitosan, chitosan trisaccharide and chitosan tetrasaccharide.
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