Chitin deacetylase mutant and its encoding gene and application

Abstract

The present invention discloses a chitin deacetylase mutant and its encoding gene and application. Marine Cyclobacterium ) were studied by directed evolution of chitin deacetylase from wild-type chitin deacetylase, and the parent chitin deacetylase was screened for thermal stability using advanced computer-aided rational design technology, and finally a chitin deacetylase mutant A58C / 133C with significantly improved thermal stability was obtained. The half-life of the mutant A58C / 133C at 50°C is 1.3 times that of the wild-type chitin deacetylase, and the yield of glucosamine is increased by 16%. At the same time, with the help of computer simulation technology, the principle of improved thermal stability is explained at the molecular level. The mutant obtained by the present invention is more suitable for the industrial application of glucosamine than the wild-type chitin deacetylase, and has good market application prospects and industrial value.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and specifically relates to a chitin deacetylase mutant and a coding gene and application thereof. Background Art

[0002] Glucosamine (GlcN), 2-amino-2-deoxy-D-glucose, is a monosaccharide derivative generated by replacing the 2nd hydroxyl group in the glucose molecule with an amino group. It is the first amino sugar discovered. GlcN is widely present in nature, usually in the form of N-acetyl (chitin) or N-acetyl-3-O-lactic acid ether (muramic acid) or N-sulfate in microorganisms. Fungal cell walls and the exoskeletons of shrimps and crabs are rich in chitin and chitosan. Chitin is not only a component of the cell wall, but also can protect organisms from exogenous physical or biological damage. Glucosamine is an important precursor substance for the biochemical synthesis of glycoproteins or glycolipids in animals and humans. In humans and animals, GlcN is an important nutrient for the formation of chondrocytes and plays an important role in the formation of cartilage tissue. Studies have found that this type of substance is also involved in the formation of human eye lenses. Studies have shown that GlcN has the effects of preventing and treating arthritis, anti-oxidation, and anti-tumor, and is widely used in food health care and medicine.

[0003] Chitin is a linear polysaccharide composed of N-acetyl-D-glucosamine linked by β-(1, 4)-glycosidic bonds. Chitin deacetylase (abbreviated as CDA) is an enzyme that catalyzes the hydrolysis of acetyl groups in chitin or chitin oligosaccharides to produce chitosan. CDA is widely present in bacteria, fungi and insects, and plays an important role in the metabolism of chitin. CDA plays an important role in the interaction between plants and pathogens. For example, pathogens protect themselves from degradation by chitinase in plant hosts by expressing CDA, while allowing hyphae to penetrate plant cell walls smoothly.

[0004] However, most natural proteins are not heat-resistant. In order to achieve the purpose of industrial application, the stability of chitin deacetylase at room temperature must be improved. Summary of the invention

[0005] Technical problems solved: In view of the above technical problems, the present invention provides a chitin deacetylase mutant and its encoding gene and application, which can effectively solve the shortcomings of the above chitin deacetylase such as poor thermal stability.

[0006] Technical solution: In a first aspect, the present invention provides a chitin deacetylase mutant, wherein the alanine at position 58 is mutated to cysteine ​​and the phenylalanine at position 133 is mutated to cysteine, and the amino acid sequence of the mutant is shown in SEQ ID NO: 3.

[0007] In a second aspect, the present invention provides a coding gene for the chitin deacetylase mutant described in the first aspect, wherein the coding gene comprises a mutation of G at position 172 to T, a mutation of C at position 173 to G, and a mutation of T at position 398 to G, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.4.

[0008] In a third aspect, the present invention provides a recombinant expression vector comprising the gene encoding the chitin deacetylase mutant according to the second aspect.

[0009] In a fourth aspect, the present invention provides a genetically engineered bacterium comprising the recombinant expression vector described in the third aspect.

[0010] In a fifth aspect, the present invention provides a method for preparing the chitin deacetylase mutant according to the first aspect, comprising the following steps:

[0011] S1. Determination of mutation sites: Alanine at position 58 and phenylalanine at position 133 of chitin deacetylase of marine bacillus are used as disulfide bond mutation sites, and the amino acid sequence of chitin deacetylase of marine bacillus is shown in SEQ ID NO.1;

[0012] S2. Construction of chitin deacetylase mutants: Using the recombinant plasmid carrying the chitin deacetylase encoding gene from Bacillus marineus as a template, a whole-plasmid PCR reaction was performed, a point mutation primer pair was designed, a site-directed mutant was constructed, and the mutant was transformed into Escherichia coli to obtain a mutant.

[0013] Preferably, the nucleotide sequence of the point mutation primer pair in step S2 is shown as SEQ ID NO.9 to SEQ ID NO.12.

[0014] In a sixth aspect, the present invention provides use of the chitin deacetylase mutant described in the first aspect in preparing glucosamine, and use of the prepared glucosamine in preparing health products.

[0015] In a seventh aspect, the present invention provides use of the chitin deacetylase mutant described in the first aspect in the preparation of a plant fungus control product.

[0016] Beneficial effect: The present invention adopts a rationally designed method to treat the marine bacillus ( Cyclobacterium marinum) is used for predictive analysis of chitin deacetylase structure, screening obtains potential sites that affect enzyme stability, and through site-directed mutagenesis, obtains the mutant of chitin deacetylase, and the half-life and glucosamine output of mutant A58C-F133C are greatly improved. Chitin deacetylase and its catalytic product are widely used in multiple fields at present, such as glucosamine in food health care and medicine field, and chitin deacetylase can also be used for plant disease prevention and treatment. The chitin deacetylase mutant with improved stability of the present invention can be better applied in the above field. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A map predicting potential disulfide bond mutation sites for chitin deacetylase;

[0018] Figure 2 This is the gel electrophoresis of the whole plasmid PCR product;

[0019] Figure 3 This is the electrophoresis diagram of the purified protein expressed by the mutant;

[0020] Figure 4 The figure is the TLC result of mutant and wild type activity detection;

[0021] Figure 5 The enzymatic properties of mutant A58C / F133C under different conditions are shown in the figure;

[0022] Figure 6 The diagram is a mechanism diagram for the improvement of mutant stability by molecular dynamics simulation;

[0023] Figure 7 This figure shows the comparison of the yield of glucosamine produced by acetylglucosamine catalyzed by mutant and wild-type chitin deacetylase. DETAILED DESCRIPTION

[0024] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments:

[0025] Main materials and reagents:

[0026] Escherichia coli BL21 and plasmid pET30a(+) were purchased from Invitrogen;

[0027] Plasmid extraction kit, fragment purification and recovery kit, and restriction endonuclease were purchased from Takara Biotechnology (Dalian) Co., Ltd.;

[0028] Kanamycin, ampicillin, and IPTG were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.;

[0029] Protein Marker: Blue Plus II Protein Marker (14-120 kDa) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

[0030] LB liquid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L;

[0031] LB solid medium: Add 1.0%-2.0% (m / v) agar to the basic formula of LB liquid medium.

[0032] Example 1: Disulfide bond mutation site prediction and site-directed mutagenesis

[0033] Computer-aided design prediction of disulfide bonds: by finding proteins with high homology to chitin deacetylase CmCBDA in the PDB database, and then using them as templates for homology modeling; the amino acid sequence of chitin deacetylase CmCBDA is shown in SEQ ID NO.1:

[0034] SEQ ID NO.1:

[0035] MNAAQKLGFTESTKLLIIHADDAGLAHAENRATIQSLQKGIVNSYSIMVPCPWFYEMAIFAKNNNQYDNGVHLTLTCEWENYRFGPVLPISEVPSLVDENGYFFKKRDKLAQNAKAEHVEKELTAQIERALKFGIKPTHIDSHMY SVGAKPEFLNVYRRIAKKYKLPLVLNQQLFEMVGLEMDLSDFKDELLIDNVFMGEFKYFEKGELANFYATALDKMEGGLNLILIHPAFDDDEMKGITINHPNFCGSEWRQIDFDFFTSEEAQSKLKEQNIQLITWDEIREKIYKD

[0036] By analyzing its protein structure, the mutation sites that can form disulfide bonds in the chitin deacetylase protein are found, and the sites that affect the enzyme activity are eliminated. The specific method is: by searching the protein database RCSB database, after blast comparison, the protein 2E67 (PDB number, 31% homology) with high homology to the chitin deacetylase is found, and the spatial structure of CmCBDA is simulated using the software Swiss-model. The constructed model is evaluated using the online software SAVES, and the evaluated model is optimized using the online software Chiron, and finally the optimized model is re-evaluated using the SAVES software. At the same time, the online software AI Lab is used to predict its structure. The PDB file of the CmCBDA crystal structure is used as the parent, and the PDBfile is imported into Pymol to obtain its three-dimensional protein structure. Using the disulfide bond prediction software Disulfide by Design and BridgeD, the chitin deacetylase is screened for disulfide bonds using 2E67 as a template. The prediction finally determined three pairs of disulfide bond sites, namely, serine at position 44 and aspartic acid at position 68, threonine at position 138 and tryptophan at position 279, and alanine at position 58 and phenylalanine at position 133. The specific prediction results are shown in Figure 1 .

[0037] Site-directed mutagenesis: Six sites were mutated by whole plasmid PCR. Primers were designed based on the nucleic acid sequence of chitin deacetylase (SEQ ID NO.2).

[0038] SEQ ID NO.2:

[0039] atgaatgcagcacaaaaattaggttttacagaatctactaagctattaattatccatgcagatgatgcaggacttgctcatgctgaaaacagagcaaccattcaatccttacaaaaagggattgtaaattcgtacagtattatggtgccttgtccatggttttatgaaatggctatttttgcaaagaacaacaatcaatatgacaatggggtacacctgaccttaacctgtgaatgggaaaattatcggtttggaccggttctgcctatttcagaagttccaagtttagtggatgaaaatggctatttcttcaaaaaaagggataagttagcccaaaatgcaaaggctgaacacgttgaaaaagaacttaccgcacaaatagaaagagccttgaaatttggaattaaacctacccatatagattcccatatgtatagtgttggtgcaaagcctgaatttctaaatgtctatcgaaggatagcaaaaaaatacaaactgcctctggtgcttaatcaacagttatttgaaatggtaggtttagaaatggatctttccgattttaaagacgagttattgatcgataatgtatttatgggagagtttaagtattttgaaaaaggagaattagcaaatttttatgctactgccttggataaaatggagggagggttaaacttgattttaattcaccctgcttttgatgatgatgagatgaaaggaataactataaatcaccctaattttggttcagaatggaggcagattgactttgatttttttacttctgaggaggcccaatcaaaactcaaagaacaaaatattcaattgattacctgggatgaaattagggaaaaaatatataaagactaa

[0040] The PCR primers are shown in Table 1 below:

[0041] Table 1 Mutation primer design table based on multiple sequence alignment results

[0042]

[0043] Note: The mutation sites are bold and underlined.

[0044] The recombinant plasmid with site-directed mutation was subjected to whole plasmid PCR using the corresponding primers. The gel electrophoresis of the whole plasmid PCR product is shown in Figure 2 As shown in the figure, it can be seen that the PCR was successful. It was digested with DpnⅠ and transformed into E. coli BL21. The biological company sequenced the mutant site to identify whether it was successfully mutated. The amino acid sequence of the mutant A58C / F133C is shown in SEQ ID NO.3.

[0045] SEQ ID NO.3:

[0046] MNAAQKLGFTESTKLLIIHADDAGLAHAENRATIQSLQKGIVNSYSIMVPCPWFYEMCIFAKNNNQYDNGVHLTLTCEWENYRFGPVLPISEVPSLVDENGYFFKKRDKLAQNAKAEHVEKELTAQIERALKCGIKPTHIDSHMY SVGAKPEFLNVYRRIAKKYKLPLVLNQQLFEMVGLEMDLSDFKDELLIDNVFMGEFKYFEKGELANFYATALDKMEGGLNLILIHPAFDDDEMKGITINHPNFCGSEWRQIDFDFFTSEEAQSKLKEQNIQLITWDEIREKIYKD

[0047] The nucleotide sequence of mutant A58C / F133C is shown in SEQ ID NO.4.

[0048] SEQ ID NO.4:

[0049] .

[0050] Example 2: Expression, purification and enzyme activity identification of mutants

[0051] 2.1 Expression and purification of mutant proteins

[0052] The recombinant mutant obtained in Example 1 was activated overnight. The overnight culture was inoculated into 400 mL of fresh LB liquid medium without antibiotics, and then continued to be cultured at 37°C and 200 rpm. After two hours, the cell concentration A600 of the culture was measured using a Thermo Fisher microplate reader. When OD A600 When the p-value was between 0.5 and 0.8, 400 μL of 1 M IPTG was added to the remaining bacterial solution to induce protein expression, and then the expression was carried out at 18°C ​​and 200 rpm for 24 hours.

[0053] After the expression is completed, centrifuge at 4°C, 4500 rpm for 10 minutes, discard the supernatant, and collect the cells. Add 10 mL of lysis buffer to the cell pellet and pipette several times to suspend the cells. The lysis buffer contains 50 mMTris-HCl, 100 mM NaCl, 1% (volume ratio) Triton X-100, and the pH is adjusted to 8.0;

[0054] The cells were broken and centrifuged at 4°C, 12,000 rpm for 20 minutes, and the supernatant was collected. First, the column was balanced with 5 column volumes of Washing Buffer (50 mM Tris-HCl, 50 mM NaCl, pH 8.0). The supernatant after centrifugation was loaded, and the target protein was now bound to the nickel column. The impurities that were not bound to the nickel column were washed with Washing Buffer until the protein A280nm of the effluent solution was less than 0.1. The target protein was eluted with elution buffer (50 mM Tris-HCl, 500 mM imidazole, 50 mM NaCl, pH 8.0) and the target protein was collected; the specific expression of the purification results is as follows: Figure 3 As shown in the figure, it can be seen that the three mutants are over-expressed, and the mutant S44C / D68C forms a large number of inclusion bodies during the expression process, which exist in the form of precipitation.

[0055] 2.2 Activity detection of mutants

[0056] Enzyme activity determination method: In a 50 μL reaction system, 500 mM N-acetylglucosamine is used as the substrate, and the enzyme solution of wild-type chitin deacetylase or its mutants A58C / F133C, S44C / D68C or T138C / W279C is added at a final concentration of 20 μM, and the reaction is shaken at 37°C for half an hour. The concentration of the product glucosamine is detected by a microplate reader and silica gel thin layer chromatography (TLC). Enzyme activity calculation method: The amount of enzyme required to generate 1 μmol of glucosamine per unit time;

[0057] The microplate reader detected that only the enzyme activity of the A58C / F133C mutant was higher than that of the wild type, while no enzyme activity was detected for the other two mutants. At the same time, the results of TLC detection of enzyme activity verified the results of the microplate reader. The specific TLC detection results are as follows: Figure 4 shown.

[0058] Example 3: Thermal stability and enzymatic properties of mutant A58C / F133C

[0059] To analyze whether the mutant had improved stability, the half-life (T 1 / 2 ) and T The half-life was determined by incubating the wild-type and mutant enzyme solutions at 50°C for different time periods (0 min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h) and then measuring their half-lives. It refers to the temperature at which the enzyme activity remains at 50% after incubation at different temperatures for 30 minutes. The specific test results are shown in Table 2 below:

[0060] Table 2 Half-life and T30 50 of mutant and wild-type chitin deacetylase

[0061]

[0062] From Table 2, we can see that the half-life of mutant A58C / F133C is greatly improved. It also increased by 6.5℃;

[0063] The enzymatic properties of the mutants were analyzed using an ELISA method, including the optimal reaction temperature, pH, and storage stability at 4°C. Figure 5 As can be seen from the figure, the optimal reaction pH and reaction temperature of the mutant did not change, but its stability range became wider. The storage time of the mutant at 4°C was also increased. After 30 days, the activity of the mutant still remained about 42%, while the activity of the wild-type enzyme was only about 20%.

[0064] Example 4: Conformational changes of mutant A58C / F133C at the molecular level

[0065] The newly formed disulfide bond A58C / F133C is located on the surface of the protein, away from the catalytic center of the enzyme, which increases the thermal stability of the enzyme. In this mutant, the random coil and α-helix structures are restricted by the newly formed disulfide bond cross-linking, which reduces the conformational entropy of the unfolded state, thereby improving the thermal stability of the protein.

[0066] Discovery studio 4.0 was used to simulate the protein structure changes before and after the introduction of disulfide bond mutations. Molecular dynamics simulations were performed at 330K with a simulation time of 10 ns. Figure 6 As shown in (A), the total molecular root mean square deviation (RMSD) of the mutant A58C / F133C is higher than that before the mutation. This indicates that during the heat treatment, the structure becomes looser than the wild-type structure, and therefore the introduction of the A58C / F133C8 disulfide bond improves the overall rigidity of the protein structure. Figure 6 As shown in (B), the total molecular root mean square fluctuation (RMSF) also shows that the overall structure of A58C / F133C is more rigid than that of the wild type, thereby enhancing the thermal stability of the enzyme.

[0067] Example 5: Application of mutant A58C / F133C in the preparation of glucosamine

[0068] The mutant A58C / F133C with improved stability in Example 4 was applied to the preparation of glucosamine, a food supplement. In the example, a 1 mL reaction system was prepared, in which the substrate concentration was 110 mg / mL, the enzyme concentration was maintained at 20 μM, and the reaction was carried out in a phosphate buffer at pH 8.0 and a concentration of 200 mM at different time points (0 min, 10 min, 30 min, 1 h, 2 h, 6 h, 12 h, 24 h, 48 h). At the same time, wild-type chitin deacetylase was used as a positive control, and the reaction system without enzyme solution was used as a negative control. The specific reaction results are shown in Figure 7 .from Figure 7 It can be seen that with the extension of time, the efficiency of mutant A58C / F133C in producing glucosamine is gradually higher than that of the wild-type enzyme. Moreover, at 48 h, the concentration of glucosamine in the mutant reaction solution can reach 55.6 mg / mL, while the output of wild-type chitin deacetylase is 45.1 mg / mL, an increase of 22.3%.

[0069] In summary, the mutant obtained by the present invention not only maintains the original enzyme specificity, but also has significantly improved thermal stability, and has good application value.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A chitin deacetylase mutant, characterized in that: The mutant comprises a mutation of alanine at position 58 to cysteine ​​and a mutation of phenylalanine at position 133 to cysteine, and the amino acid sequence of the mutant is as follows: MNAAQKLGFTESTKLLIIHADDAGLAHAENRATIQSLQKGIVNSYSIMVPCPWFYEMCIFAKNNNQYDNGVHLTLTCEWENYRFGPVLPISEVPSLVDENGYFFKKRDKLAQNAKAEHVEKELTAQIERALKCGIKPTHIDSHMY SVGAKPEFLNVYRRIAKKYKLPLVLNQQLFEMVGLEMDLSDFKDELLIDNVFMGEFKYFEKGELANFYATALDKMEGGLNLILIHPAFDDDEMKGITINHPNFGSEWRQIDFDFFTSEEAQSKLKEQNIQLITWDEIREKIYKD.

2. A gene encoding a chitin deacetylase mutant according to claim 1, characterized in that: The coding gene comprises a mutation of G to T at position 172, a mutation of C to G at position 173, and a mutation of T to G at position 398. The nucleotide sequence of the coding gene is as follows: .

3. A recombinant expression vector comprising the chitin deacetylase mutant encoding gene according to claim 2.

4. A genetically engineered bacterium comprising the recombinant expression vector according to claim 3.

5. Use of the chitin deacetylase mutant according to claim 1 in the preparation of glucosamine.

6. Use of the chitin deacetylase mutant according to claim 1 in the preparation of a plant control fungus product.

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