Chitosanase OUC-CsnA4-E59V-E64G as well as coding gene and application thereof

Abstract

The invention discloses chitosanase OUC-CsnA4-E59V-E64G as well as a coding gene and application of the chitosanase OUC-CsnA4-E59V-E64G, and belongs to the technical field of chitosanase. The amino acid sequence of the chitosanase OUC-CsnA4-E59V-E64G is as shown in SEQ ID NO. 9, and the nucleotide sequence of the coding gene of the chitosanase OUC-CsnA4-E59V-E64G is as shown in SEQ ID NO. 10. According to the application of the chitosanase OUC-CsnA4-E59V-E64G in preparation of chitosan oligosaccharide, the polymerization degree of the chitosan oligosaccharide is 2 to 5. The chitosanase OUC-CsnA4-E59V-E64G disclosed by the invention is obtained through rational design on the basis of the chitosanase OUC-CsnA4-E59V, and compared with the chitosanase OUC-CsnA4-E59V, the chitosanase OUC-CsnA4-E59V has the advantages that the half-life period is prolonged by 4.15 times, and the thermal stability is greatly enhanced.

Description

Technical Field

[0001] The present invention relates to a chitosanase OUC-CsnA4-E59V-E64G, its encoding gene and applications, and belongs to the technical field of chitosanases. Background Art

[0002] Chitosanase is a glycoside hydrolase that catalyzes the degradation of chitosan. Chinese invention patent CN 118165959 A discloses a chitosanase OUC-CsnA4-E59V, its encoding gene and applications. After mutating and modifying chitosanase OUC-CsnA4, chitosanase OUC-CsnA4-E59V is obtained. Degrading chitosan can obtain chitosan oligosaccharides with a degree of polymerization of 2-5, and the enzyme activity is increased by 1.23 times. However, whether it is chitosanase OUC-CsnA4 or chitosanase OUC-CsnA4-E59V, the thermal stability is not ideal enough. Therefore, it is necessary to mutate and modify it in order to obtain a chitosanase with stronger thermal stability. Summary of the Invention

[0003] In view of the above-mentioned prior art, the present invention provides a chitosanase OUC-CsnA4-E59V-E64G, its encoding gene and applications, and belongs to the technical field of chitosanases.

[0004] The present invention is achieved by the following technical solutions: A chitosanase OUC-CsnA4-E59V-E64G, whose amino acid sequence is as shown in SEQ ID NO.9.

[0005] The encoding gene of the chitosanase OUC-CsnA4-E59V-E64G, whose nucleotide sequence is as shown in SEQ ID NO.10.

[0006] The application of the chitosanase OUC-CsnA4-E59V-E64G in the preparation of chitosan oligosaccharides, and the degree of polymerization of the chitosan oligosaccharides is 2-5.

[0007] The chitosanase OUC-CsnA4-E59V-E64G of the present invention is obtained by rational design on the basis of chitosanase OUC-CsnA4-E59V. Compared with chitosanase OUC-CsnA4-E59V, its half-life is increased by 4.15 times, and the thermal stability is greatly enhanced. The present invention provides a potentially highly efficient enzyme for the industrial production of chitosan oligosaccharides, with great application potential and good application prospects.

[0008] All terms and phrases used in the present invention have the general meanings well-known to those skilled in the art. Brief Description of the Drawings

[0009] Figure 1: Pullulan diagram of homology modeling.

[0010] Figure 2 : SDS-PAGE detection results of chitosanase OUC-CsnA4, chitosanase OUC-CsnA4-E59V, and chitosanase OUC-CsnA4-E64G. Here, M represents the standard protein, A4 represents chitosanase OUC-CsnA4, Val represents chitosanase OUC-CsnA4-E59V, and A64 represents chitosanase OUC-CsnA4-E64G.

[0011] Figure 3 : SDS-PAGE detection results of chitosanase OUC-CsnA4-L211F, chitosanase OUC-CsnA4-E59V-E64G, and chitosanase OUC-CsnA4-E59V-L211F. Here, M represents the standard protein, A211 represents chitosanase OUC-CsnA4-L211F, V64 represents chitosanase OUC-CsnA4-E59V-E64G, and V211 represents chitosanase OUC-CsnA4-E59V-L211F.

[0012] Figure 4 : Thermal stability fitting curve of chitosanase OUC-CsnA4. Here, the black broken line represents the relative enzyme activity measured at 60 °C after incubation for different times, and the red curve is the non-linear fitting of the experimental data to determine the half-life of the enzyme.

[0013] Figure 5 : Thermal stability fitting curve of chitosanase OUC-CsnA4-E59V. Here, the black broken line represents the relative enzyme activity measured at 60 °C after incubation for different times, and the red curve is the non-linear fitting of the experimental data to determine the half-life of the enzyme.

[0014] Figure 6 : Thermal stability fitting curve of chitosanase OUC-CsnA4-E64G. Here, the black broken line represents the relative enzyme activity measured at 60 °C after incubation for different times, and the red curve is the non-linear fitting of the experimental data to determine the half-life of the enzyme.

[0015] Figure 7 : Thermal stability fitting curve of chitosanase OUC-CsnA4-E59V-E64G. Here, the black broken line represents the relative enzyme activity measured at 60 °C after incubation for different times, and the red curve is the non-linear fitting of the experimental data to determine the half-life of the enzyme.

[0016] Figure 8: Thermal stability fitting curve of chitosanase OUC-CsnA4-L211F. Among them, the black broken line represents the relative enzyme activity measured after incubation at 60 °C for different times, and the red curve is the non-linear fitting of the experimental data to determine the half-life of the enzyme.

[0017] Figure 9 : Thermal stability fitting curve of chitosanase OUC-CsnA4-E59V-L211F. Among them, the black broken line represents the relative enzyme activity measured after incubation at 60 °C for different times, and the red curve is the non-linear fitting of the experimental data to determine the half-life of the enzyme.

[0018] Figure 10 : Results of relative enzyme activity determination at different temperatures.

[0019] Figure 11 : Results of relative enzyme activity determination at different pH values.

[0020] Figure 12 : Three-dimensional structure model of chitosanase OUC-CsnA4-E59V-E69G. 3.1 in the figure represents the bond length of the hydrogen bond formed by glycine at position 64.

[0021] Figure 13 : Schematic diagram of the interaction forces of the amino acid at position 64 of chitosanase OUC-CsnA4.

[0022] Figure 14 : Schematic diagram of the interaction forces of the amino acid at position 64 of chitosanase OUC-CsnA4-E59V-E69G. Detailed implementation mode

[0023] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0024] The instruments, reagents, and materials involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods and detection methods existing in the prior art.

[0025] Example 1 Improving the thermal stability of chitosanase OUC-CsnA4 based on rational design The amino acid sequence of chitosanase OUC-CsnA4 is shown in SEQ ID NO.1, as follows: HMVTFMPKGDTEYPSNNTEYPSNNTEYPSNNTSNMKNVILQMTSTLENSDTQLHFNYAENLGDERGITFGCIGFCTGTYDGNILIKHYTELNPDNTLAKYIPALDKIDTGPHDAADGDGNPSVEGLSGFIQDVNSCDDPLFKNAQIDKLDELYYNPAMEIADSIGAKNPLTKAFIYDMCVRHGVDQTEDIIKDAGTTPKQGTDENTYLQKLISLRDAKLKQEGIEDVNRNQGYKKLLNSGNVDLKTPFTFVAYGDSFTIDGKLYLGEYQQLE。

[0026] The nucleotide sequence of the coding gene of chitosanase OUC-CsnA4 is shown in SEQ ID NO.2 as follows (direction 5'-3'): CATATGGTGACCTTTATGCCGAAAGGCGATACCGAATATCCGAGCAACAACACCGAATATCCGAGCAACAACACCGAATATCCGAGCAACAACACCAGCAACATGAAAAACGTGATTCTGCAGATGACCAGCACCCTGGAAAACAGCGATACCCAGCTGCATTTTAACTATGCGGAAAACCTGGGCGATGAACGCGGCATTACCTTTGGCTGCATTGGCTTTTGCACCGGCACCTATGATGGCAACATTCTGATTAAACATTACACCGAACTGAACCCGGATAACACCCTGGCGAAATATATTCCGGCGCTGGATAAAATTGATACCGGCCCGCATGATGCGGCGGATGGCGATGGTAATCCTAGCGTGGAAGGCTTAAGCGGTTTTATTCAGGATGTGAACAGCTGCGATGATCCGCTGTTTAAAAACGCGCAGATTGATAAACTGGATGAGCTGTATTATAACCCGGCGATGGAAATTGCGGATAGCATTGGCGCGAAAAACCCGCTGACCAAAGCGTTTATTTATGATATGTGCGTGCGCCACGGCGTGGATCAGACCGAAGATATTATTAAAGATGCGGGCACCACCCCGAAACAGGGCACCGATGAAAACACCTATCTGCAGAAACTGATTAGCCTGCGCGATGCGAAACTGAAACAGGAAGGCATTGAAGATGTGAACCGCAACCAGGGCTATAAAAAGCTGCTGAACAGCGGCAACGTGGATCTGAAAACCCCGTTTACCTTTGTGGCGTATGGCGATAGCTTTACCATTGATGGCAAACTGTATCTGGGCGAATATCAGCAGCTCGAG。

[0027] The amino acid sequence of chitosanase OUC-CsnA4-E59V is shown in SEQ ID NO.3 as follows: HMVTFMPKGDTEYPSNNTEYPSNNTEYPSNNTSNMKNVILQMTSTLENSDTQLHFNYAVNLGDERGITFGCIGFCTGTYDGNILIKHYTELNPDNTLAKYIPALDKIDTGPHDAADGDGNPSVEGLSGFIQDVNSCDDPLFKNAQIDKLDELYYNPAMEIADSIGAKNPLTKAFIYDMCVRHGVDQTEDIIKDAGTTPKQGTDENTYLQKLISLRDAKLKQEGIEDVNRNQGYKKLLNSGNVDLKTGFTFVAYGDSFTIDGKLYLGEYQQLE。

[0028] The nucleotide sequence of the coding gene of chitosanase OUC-CsnA4-E59V is shown in SEQ ID NO.4 as follows (direction 5'-3'): CATATGGTGACCTTTATGCCGAAAGGCGATACCGAATATCCGAGCAACAACACCGAATATCCGAGCAACAACACCGAATATCCGAGCAACAACACCAGCAACATGAAAAACGTGATTCTGCAGATGACCAGCACCCTGGAAAACAGCGATACCCAGCTGCATTTTAACTATGCGGTGAACCTGGGCGATGAACGCGGCATTACCTTTGGCTGCATTGGCTTTTGCACCGGCACCTATGATGGCAACATTCTGATTAAACATTACACCGAACTGAACCCGGATAACACCCTGGCGAAATATATTCCGGCGCTGGATAAAATTGATACCGGCCCGCATGATGCGGCGGATGGCGATGGTAATCCTAGCGTGGAAGGCTTAAGCGGTTTTATTCAGGATGTGAACAGCTGCGATGATCCGCTGTTTAAAAACGCGCAGATTGATAAACTGGATGAGCTGTATTATAACCCGGCGATGGAAATTGCGGATAGCATTGGCGCGAAAAACCCGCTGACCAAAGCGTTTATTTATGATATGTGCGTGCGCCACGGCGTGGATCAGACCGAAGATATTATTAAAGATGCGGGCACCACCCCGAAACAGGGCACCGATGAAAACACCTATCTGCAGAAACTGATTAGCCTGCGCGATGCGAAACTGAAACAGGAAGGCATTGAAGATGTGAACCGCAACCAGGGCTATAAAAAGCTGCTGAACAGCGGCAACGTGGATCTGAAAACCGGCTTTACCTTTGTGGCGTATGGCGATAGCTTTACCATTGATGGCAAACTGTATCTGGGCGAATATCAGCAGCTCGAG。

[0029] In this study, a point mutation strategy based on multiple sequence alignment and computer simulation was used to enhance the thermal stability of chitosanase OUC-CsnA4. The specific process is described below.

[0030] (1)First, perform homology modeling on chitosanase OUC-CsnA4 on the Swiss Model website (https: / / swissmodel.expasy.org / ) and use a Ramachandran plot to evaluate the quality of the model. The Ramachandran plot for homology modeling is as shown in Figure 1 . As can be seen from the figure, more than 95% of the key amino acid residues in this model fall within the allowed region and the most favored region. Therefore, it is considered that this model conforms to stereochemical rules and can be used for computer simulation of thermal stability.

[0031] (2)Then, perform thermal stability prediction on the modeled PDB file using the HotSpot Wizard v3.1 and Consensus Finder online prediction programs to find suitable amino acid sites for site-directed mutagenesis.

[0032] The website of HotSpot Wizard v3.1 is: https: / / loschmidt.chemi.muni.cz / hotspotwizard / .

[0033] The website of Consensus Finder is: http: / / kazlab.umn.edu / .

[0034] The prediction result of HotSpot Wizard v3.1 is: If the amino acid at position 64 is mutated from glutamate (Glu) to glycine (Gly), there is more than an 80% probability of enhancing the thermal stability of the original enzyme.

[0035] The prediction result of Consensus Finder is: The amino acid at position 211 of 92% of the similar proteins is phenylalanine, and the amino acid at position 64 of 90% of the similar proteins is glycine. Maintaining consistency with homologous proteins often improves the stability of proteins.

[0036] Based on the above prediction results, it is preliminarily considered that the following two mutation schemes may enhance the stability of chitosanase: ① Mutate the amino acid at position 64 from glutamate to glycine, that is, mutate the codon for glutamate at position 64 on the coding gene from "GAA" to "GGC"; ② Mutate the amino acid at position 211 from leucine (Leu) to phenylalanine (Phe), that is, mutate the codon for leucine at position 211 on the coding gene from "CTG" to "TTT".

[0037] Correspondingly, the two mutants obtained by performing the above mutations on the basis of chitosanase OUC-CsnA4 are named: chitosanase OUC-CsnA4-E64G and chitosanase OUC-CsnA4-L211F, respectively.

[0038] Two mutants obtained by the above mutations based on chitosanase OUC-CsnA4-E59V were named chitosanase OUC-CsnA4-E59V-E64G and chitosanase OUC-CsnA4-E59V-L211F, respectively.

[0039] Example 2 Preparation of Chitosanase Chitosanase OUC-CsnA4, chitosanase OUC-CsnA4-E59V, and each mutant predicted in Example 1 were expressed and purified by conventional methods as follows: (1) Recombinant plasmids were isolated from Escherichia coli BL21(DE3) / OUC-CsnA4-pET28a and Escherichia coli BL21(DE3) / OUC-CsnA4-E59V-pET28a stored in the laboratory as templates for site-directed mutagenesis. Plasmid extraction was carried out using a plasmid mini-prep kit. Specific primers containing the mutation sites were designed by Snap Gene, and PCR amplification was performed using the extracted recombinant plasmid template to obtain gene fragments containing each mutant; the nucleotide sequences of the specific primers for site-directed mutagenesis are shown below (direction 5'-3'): E64G-T: tgggcgatGGCcgcggcattacctttg, as shown in SEQ ID NO.5; E64G-B: ggtaatgccgcgGCCatcgcccaggtt, as shown in SEQ ID NO.6; L211F-T: cctatctgcagaaaTTTattagcctgcgcgatgcgaaactgaaacagg, as shown in SEQ ID NO.7; L211F-B: cagtttcgcatcgcgcaggctaatAAAtttctgcagataggtgttttc, as shown in SEQ ID NO.8.

[0040] (2) Construction of the recombinant expression vector The gene fragments obtained by the above amplification were transferred into E.coli DH5α competent cells. Positive transformants were screened using LB plates containing kanamycin sulfate. After the clones were verified by colony PCR using T7 universal primers, positive clones were picked for sequencing to obtain recombinant plasmids.

[0041] (3) Construction of the recombinant engineering bacteria The recombinant plasmid with correct sequencing was extracted and transformed into the host E.coli BL21 competent cells, and the constructed engineering bacteria grew on the kanamycin sulfate-resistant plate.

[0042] (4)Expression of chitosanase Pick the recombinant engineering bacteria strains grown on the kanamycin sulfate resistant plate and inoculate them into 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate. Culture at 37 °C and 220 rpm for 12 hours; inoculate into 50 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 1%, and culture at 37 °C and 220 rpm until the OD600 value reaches 0.8; add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.1 mM to induce the expression of chitosanase for 16 hours.

[0043] (5)Purification of chitosanase After the above-mentioned induced expression, take the culture solution, centrifuge at 4 °C and 8000 rpm for 20 minutes, collect the thalli, resuspend them in water, ultrasonically disrupt for 15 minutes, and centrifuge at 4 °C and 8000 rpm for 20 minutes. The supernatant is the crude enzyme solution.

[0044] The crude enzyme solution is purified by affinity chromatography using a Ni - NTA column. Equilibrate the column with 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), then elute the weakly bound miscellaneous proteins with 40 mM imidazole solution (40 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), and elute the target protein with 100 mM imidazole solution (100 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). Collect the eluate of this part. Perform SDS-PAGE detection on the crude enzyme solution, breakthrough solution, and eluate of chitosanase OUC-CsnA4, chitosanase OUC-CsnA4-E59V, and each mutant. Results: The SDS-PAGE detection results of chitosanase OUC-CsnA4, chitosanase OUC-CsnA4-E59V, and chitosanase OUC-CsnA4-E64G are as Figure 2 shown, and the SDS-PAGE detection results of chitosanase OUC-CsnA4-L211F, chitosanase OUC-CsnA4-E59V-E64G, and chitosanase OUC-CsnA4-E59V-L211F are as Figure 3 shown. From Figure 2 , Figure 3 it can be seen that a single band is observed for each mutant, with a molecular weight of approximately 35 KD, which is consistent with the molecular weight of chitosanase OUC-CsnA4. For each eluate, ultrafiltrate with a 10 KDa ultrafiltration membrane at 4 °C and 4000 r / min to obtain the pure enzyme solution.

[0045] Example 3 Primary screening of thermal stability The pure enzyme solutions of chitosanase OUC-CsnA4, chitosanase OUC-CsnA4-E59V, and each mutant obtained in Example 2 were incubated at 60 °C for 0 min, 10 min, 20 min, 30 min, and 40 min, respectively, and then the enzyme activity was measured. The relative enzyme activity was calculated with the enzyme activity at 0 min of incubation as 100%.

[0046] The method for measuring enzyme activity was the dinitrosalicylic acid (DNS) colorimetric method as follows: The reaction system consisted of 10 μL of pure enzyme solution plus 190 μL of chitosan solution, and the reaction was carried out at 55 °C for 15 minutes. After the reaction, it was placed in a boiling water bath for 10 minutes and centrifuged at 12,000 rpm for 2 minutes; 200 μL of the supernatant was taken and reacted with 300 μL of DNS reagent, and then placed in a boiling water bath for 10 minutes for color development; it was centrifuged at 12,000 rpm for 2 minutes, and the absorbance value at 540 nm was measured. The solvent of the chitosan solution was 10 mg / mL acetic acid solution, and the concentration of chitosan was 20 mg / mL. The chitosan was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China), and the degree of deacetylation was ≥95%. One unit (U) of chitosanase activity was defined as: the amount of enzyme required to produce 1 μmol of reducing sugar per minute.

[0047] The incubation time-relative enzyme activity of the enzyme at 60 °C was curve-fitted with Origin 2022 to compare the half-life of each chitosanase at 60 °C (the time required for the enzyme activity to decrease to half when incubated at 60 °C). Results: The thermal stability fitting curve of chitosanase OUC-CsnA4 was as Figure 4 shown, the thermal stability fitting curve of chitosanase OUC-CsnA4-E59V was as Figure 5 shown, the thermal stability fitting curve of chitosanase OUC-CsnA4-E64G was as Figure 6 shown, the thermal stability fitting curve of chitosanase OUC-CsnA4-E59V-E64G was as Figure 7 shown, the thermal stability fitting curve of chitosanase OUC-CsnA4-L211F was as Figure 8 shown, the thermal stability fitting curve of chitosanase OUC-CsnA4-E59V-L211F was as Figure 9As shown. It can be seen that the half-life of chitosanase OUC-CsnA4 is 3.4 min, the half-life of chitosanase OUC-CsnA4-E59V is 3.4 min, the half-life of chitosanase OUC-CsnA4-E64G is 5.9 min, the half-life of chitosanase OUC-CsnA4-E59V-E64G is 14.1 min, the half-life of chitosanase OUC-CsnA4-L211F is 2.4 min, and the half-life of chitosanase OUC-CsnA4-E59V-L211F is 1.4 min. By comparison, it can be known that the half-lives of chitosanase OUC-CsnA4-E64G and chitosanase OUC-CsnA4-E59V-E64G have a relatively obvious increase. Especially for chitosanase OUC-CsnA4-E59V-E64G, which has the longest half-life and is 4.15 times that of chitosanase OUC-CsnA4; while the half-lives of chitosanase OUC-CsnA4-L211F and chitosanase OUC-CsnA4-E59V-L211F have varying degrees of decrease. This indicates that the chitosanase with stronger thermal stability was indeed obtained through the prediction in Example 1, but the prediction result of Example 1 is not completely credible.

[0048] At the same time, it was measured that the specific enzyme activity of chitosanase OUC-CsnA4 is 38.8 U / mg, and the specific enzyme activity of chitosanase OUC-CsnA4-E59V-E69G is 39.8 U / mg. There is no significant difference between the two, which indicates that mutating the amino acid at position 64 from Glu to Gly has a greater improvement in the thermal stability of the enzyme, while having little or negligible effect on the enzyme activity. The reason may be that the intermolecular forces change after the amino acid change.

[0049] The amino acid sequence of chitosanase OUC-CsnA4-E59V-E64G is as shown in SEQ ID NO.9, as follows: HMVTFMPKGDTEYPSNNTEYPSNNTEYPSNNTSNMKNVILQMTSTLENSDTQLHFNYAVNLGDGRGITFGCIGFCTGTYDGNILIKHYTELNPDNTLAKYIPALDKIDTGPHDAADGDGNPSVEGLSGFIQDVNSCDDPLFKNAQIDKLDELYYNPAMEIADSIGAKNPLTKAFIYDMCVRHGVDQTEDIIKDAGTTPKQGTDENTYLQKLISLRDAKLKQEGIEDVNRNQGYKKLLNSGNVDLKTGFTFVAYGDSFTIDGKLYLGEYQQLE.

[0050] The nucleotide sequence of the encoding gene of chitosanase OUC-CsnA4-E59V-E64G is shown in SEQ ID NO.10 as follows (orientation 5'-3'): CATATGGTGACCTTTATGCCGAAAGGCGATACCGAATATCCGAGCAACAACACCGAATATCCGAGCAACAACACCGAATATCCGAGCAACAACACCAGCAACATGAAAAACGTGATTCTGCAGATGACCAGCACCCTGGAAAACAGCGATACCCAGCTGCATTTTAACTATGCGGTGAACCTGGGCGATGGCCGCGGCATTACCTTTGGCTGCATTGGCTTTTGCACCGGCACCTATGATGGCAACATTCTGATTAAACATTACACCGAACTGAACCCGGATAACACCCTGGCGAAATATATTCCGGCGCTGGATAAAATTGATACCGGCCCGCATGATGCGGCGGATGGCGATGGTAATCCTAGCGTGGAAGGCTTAAGCGGTTTTATTCAGGATGTGAACAGCTGCGATGATCCGCTGTTTAAAAACGCGCAGATTGATAAACTGGATGAGCTGTATTATAACCCGGCGATGGAAATTGCGGATAGCATTGGCGCGAAAAACCCGCTGACCAAAGCGTTTATTTATGATATGTGCGTGCGCCACGGCGTGGATCAGACCGAAGATATTATTAAAGATGCGGGCACCACCCCGAAACAGGGCACCGATGAAAACACCTATCTGCAGAAACTGATTAGCCTGCGCGATGCGAAACTGAAACAGGAAGGCATTGAAGATGTGAACCGCAACCAGGGCTATAAAAAGCTGCTGAACAGCGGCAACGTGGATCTGAAAACCGGCTTTACCTTTGTGGCGTATGGCGATAGCTTTACCATTGATGGCAAACTGTATCTGGGCGAATATCAGCAGCTCGAG。

[0051] Example 4 Determination of the Optimal Reaction Conditions of Chitosanase OUC-CsnA4-E59V-E69G Determination of the optimal temperature: In the range of 20 - 90 °C, the enzyme activity of chitosanase OUC-CsnA4-E59V-E69G at different temperatures was determined according to the method for determining enzyme activity in Example 3. Taking the highest enzyme activity as 100%, the relative enzyme activities at different temperatures were calculated. The results of the relative enzyme activity determination at different temperatures are as Figure 10 shown. The optimal reaction temperature is 65 °C, with relatively high activity in the range of 60 - 70 °C, and the relative activity is higher than 80%. It is superior to chitosanase OUC-CsnA4 in terms of heat resistance.

[0052] Determination of the optimal pH: In the range of pH 4.0 - 10.0 (the buffers used are: acetate buffer at pH 4.0 - 6.0, phosphate buffer at pH 6.0 - 8.0, Tris-HCl buffer at pH 8.0 - 9.0, and Gly-NaOH buffer at pH 9.0 - 10.0), the enzyme activity of chitosanase OUC-CsnA4-E59V-E69G at different pH values was determined according to the method for determining enzyme activity in Example 3. Taking the highest enzyme activity as 100%, the relative enzyme activities at different pH values were calculated. The results of the relative enzyme activity determination at different pH values are as Figure 11 shown. The optimal pH is 6.0 (acetate buffer), and there is no significant difference compared with the optimal pH of chitosanase OUC-CsnA4 in phosphate buffer at pH 6.0.

[0053] Example 5 Homology Modeling Quality Assessment and Thermal Stability Mechanism Analysis The homology modeling of chitosanase OUC-CsnA4-E59V-E69G was performed using the Swiss Model online website, and the quality of the model was evaluated using a Ramachandran plot. The model of chitosanase OUC-CsnA4-E59V-E69G was overlapped and compared with the model of A4 in PyMOL to analyze the molecular structure and force changes after the enzymatic properties of chitosanase OUC-CsnA4-E59V-E69G were altered, so as to explore the molecular mechanism of the improved thermal stability.

[0054] Results: The three-dimensional structure model of chitosanase OUC-CsnA4-E59V-E69G is as Figure 12 shown. It can be found that the amino acid at position 64 is near the catalytic pocket of chitosanase OUC-CsnA4. Therefore, it is reasonable to speculate that the structural changes of the amino acid chain near the amino acid at position 64 have a great impact on the activity of chitosanase, and the structural stability of the catalytic pocket directly affects the catalytic activity and stability of the enzyme.

[0055] The schematic diagram of the interaction force of the amino acid at position 64 of chitosanase OUC-CsnA4 is as Figure 13As shown, the schematic diagram of the interaction of the 64th amino acid of chitosanase OUC-CsnA4-E59V-E69G is as Figure 14 As shown, by comparing the differences between chitosanase OUC-CsnA4 and chitosanase OUC-CsnA4-E59V-E69G, it can be found that Glu at the 64th position of chitosanase OUC-CsnA4 has a negatively charged side chain group and there is an obvious steric hindrance with surrounding amino acids; while Gly at the 64th position of chitosanase OUC-CsnA4-E59V-E69G not only has a smaller uncharged side chain group, but also forms a new intramolecular hydrogen bond. Therefore, when Glu at the 64th position is mutated to Gly, the reason for the improved thermal stability of the mutant is most likely attributed to two points: one is that the amino acid residue after mutation has a smaller, non-polar side chain group, resulting in a reduction in steric hindrance; the other is that a new intramolecular hydrogen bond is formed with Asp at the adjacent 63rd position after mutation, which is beneficial to the stability of the catalytic pocket structure, and macroscopically shows an enhancement in thermal stability.

[0056] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A chitosanase OUC-CsnA4-E59V-E64G, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

9.

2. The coding gene of chitosanase OUC-CsnA4-E59V-E64G according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

10.

3. Use of the chitosanase OUC-CsnA4-E59V-E64G according to claim 1 in the preparation of chitosan oligosaccharides, characterized in that: The degree of polymerization of the chitosan oligosaccharide is 2 to 5.

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