Chitinase with antifungal activity as well as preparation method and application of chitinase

Abstract

The invention provides chitinase with antifungal activity, and the amino acid sequence of the chitinase is SEQ ID NO.3 or SEQ ID NO.7. The chitinase has good activity at room temperature, is insensitive to the existence of iron ions, and is suitable for preparing agricultural preparations for inhibiting fungi such as fusarium.

Description

Field of the Invention

[0001] The present application belongs to the field of proteins and pesticides. Specifically, the present application provides a chitinase with antifungal activity and a preparation method and application thereof. Background Art

[0002] With the rapid growth of the global population, people's demand for food is also rising year by year, and fungal infection of crops will cause a decrease in food production and quality. However, excessive use of chemical antibacterial agents will affect food safety and cause certain damage to the environment. In order not to affect food and environmental safety and take the path of sustainable agricultural development, the application of biotechnology in agricultural production has been gradually explored and valued. Chitinase can degrade the chitin component in the fungal cell wall and rupture the fungal cell wall, thereby achieving the effect of inhibiting fungal growth without causing adverse effects on the plant itself.

[0003] Chitin, also known as chitosan, chitosan or chitosan, is a macromolecular linear polysaccharide composed of N-acetyl-D-glucosamine (GlcNAc) connected by β-1,4 glycosidic bonds. Chitinase (EC3.2.1.14) can hydrolyze the β-1,4 glycosidic bonds in chitin and degrade it into chito-oligosaccharides or chito-monosaccharides. The sources of chitinase are very wide, and there are related reports in various organisms, such as bacteria, fungi, plants and animals. Chitinase is generally classified according to reaction conditions, mode of action and sequence conservation. According to the different reaction conditions, they can be divided into high-temperature resistant enzymes, low-temperature resistant enzymes, acid-resistant enzymes and alkali-resistant enzymes; according to the mode of action, they can be divided into endo-chitinases and exo-chitinases. Endo-chitinases randomly hydrolyze β-1,4 glycosidic bonds from the inside of chitin to form oligosaccharides mainly composed of disaccharides to hexasaccharides, and exo-chitinases can be divided into chitobiose exo-chitobiose enzymes and β-N-acetylglucosaminidase.

[0004] Finding chitinases that are suitable for controlling fungi in agriculture among chitinases with diverse sources and properties has always been one of the research directions in this field. Summary of the invention

[0005] In one aspect, the present application provides a chitinase having antifungal activity, wherein the amino acid sequence of the chitinase is SEQ ID NO.3 or SEQ ID NO.7.

[0006] On the other hand, the present application provides the use of the chitinase in preparing a fungus-inhibiting preparation.

[0007] Furthermore, the fungus is Fusarium sp.

[0008] Furthermore, the fungus is Fusarium graminearum.

[0009] Furthermore, the preparation is a pesticide.

[0010] On the other hand, the present application provides a gene encoding the chitinase, wherein the nucleotide sequence of the gene encoding is SEQ ID NO.4 or SEQ ID NO.9.

[0011] On the other hand, the present application provides a vector comprising the above encoding gene.

[0012] In another aspect, the present application provides a host cell, which comprises the above-mentioned vector.

[0013] The host cell is preferably Escherichia coli, and other hosts, such as Bacillus subtilis and Streptomyces, can also be used. Those skilled in the art can select the vector and adjust the gene according to the codon preference of the host.

[0014] On the other hand, the present application provides a method for preparing the above chitinase, comprising the step of culturing the above host cell.

[0015] Furthermore, the method also includes a purification step.

[0016] Those skilled in the art can routinely use protein purification methods known in the art, including but not limited to centrifugation, ammonium sulfate precipitation, molecular sieve chromatography, anion chromatography, cation chromatography, affinity chromatography, renaturation, dialysis, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the SDS-PAGE electrophoresis of purified ChiCD;

[0018] Figure 2 This is a diagram showing the enzyme activity changing with temperature.

[0019] Figure 3 The figure shows the trend of enzyme activity changing with pH in different buffers.

[0020] Figure 4 This is a diagram showing the stability of the enzyme at different temperatures.

[0021] Figure 5 This is an example of the antibacterial effect on Fusarium.

[0022] Figure 6 The figure shows the trend of activity of various variants changing with temperature.

[0023] Figure 7 Shown is the addition of 25mM and 50mM Fe in the form of ferrous chloride 2The enzyme activity assay results of ChiCD, ChiCD1 and ChiCD4 were shown. DETAILED DESCRIPTION

[0024] Main reagents

[0025] Fusarium graminearum, E.coil TOP10, E.coilBL21 (DE3), and expression vector pET-28a were all stored in the applicant's laboratory.

[0026] Restriction endonucleases NdeI and XhoI: Beijing Quanshijin Biotechnology Co., Ltd.).

[0027] LB medium (containing 1% peptone, 0.5% yeast extract, 1% NaCl and optionally 50 μg / mL kanamycin per liter); PDB medium: Golden Clone (Beijing) Biotechnology Co., Ltd.

[0028] PDA culture medium: Qingdao Haibo Biotechnology Co., Ltd.

[0029] Binding buffer (phosphate buffer: 20 mM phosphate, pH 7.2-7.4); rinsing buffer (500 mM NaCl, 10-50 mM imidazole, 20 mM phosphate, pH 7.4); elution buffer (500 mM NaCl, 500 mM imidazole, 20 mM phosphate, pH 7.4); 3,5-dinitrosalicylic acid: Sinopharm Chemical Reagent Co., Ltd.

[0030] Kanamycin: Sangon Biotech (Shanghai) Co., Ltd.

[0031] Ni-NTA Resin: Beijing Quanshijin Biotechnology Co., Ltd.).

[0032] 8~14kD dialysis bag: Shanghai Yuanye Biotechnology Co., Ltd.

[0033] Error-prone PCR kit: Biolabs, BTN101005-100T.

[0034] Example 1 Amplification and expression of chitinase from Chromobacterium violaceum

[0035] The chitinase ChiCD gene sequence was obtained by analyzing the genome sequencing data of Chromobacterium violaceum ATCC 12472, and the gene sequence was optimized to make it suitable for expression in Escherichia coli. The optimized chitinase ChiCD gene sequence (amino acid and nucleotide sequences are SEQ ID NO.1 and SEQ ID NO.2, respectively) was obtained by artificial synthesis, and restriction sites NdeI and XhoI were added.

[0036] ChiCD original amino acid sequence (SEQ ID NO.1)

[0037] MMKRFAALSALAAAAAAGLASPAAQAGAFAPYVDMTLWPTPQIDKLGVNQGIQQFTLAFVVAKGGCAPSW

[0038] GGVLAIPGSGSDQQLSAIRNGINNFRGKGGEVMVSFGGANGTPLQQACTTNASLQAAYQTVLDTYNLSR

[0039] IDFDIEGGAQTDTAANNRNFAVVAALQKNYKAKGKTLHVSLTLPAMPFGLTQDGQRVLASALANGVALD

[0040] TVNIMAMDYGQSNPNMGAAAKQAAQALYSQIDAAYKAHGQTLTDAQLWQKVGVTPMVGLNDTQPETFTV

[0041] DNAKDLYGMANSNRFGLLSMWSISRDKSCPNNGHYVDAQCSGIVQTPYAFSNVFKGFKDHWGSGVTQDP

[0042] NYGGGDNGNGGGPVNGQPWSSGQVYNTGNTVTYSGATWKAQWWTQGDVPGQASVWQQQGGGLQQWSATA

[0043] AYSAKDCALYQGKKYCAKWWTQGNLPAAGDPWVLSN

[0044] ChiCD original nucleic acid sequence (SEQ ID NO.2)

[0045] ATGATGAAACGTTTTGCCGCTCTGAGCGCCCTGGCAGCAGCTGCAGCTGGCCTGGCATCTCCGGCTGCG

[0046] CAAGCAGGTGCATTCGCTCCGTATGTTGACATGACCCTGTGGCCGACCCCGCAAATCGACAAGCTGGGT

[0047] GTTAACCAGGGTATCCAGCAGTTCACTCTGGCCTTCGTTGTTGCGAAGGGCGGCTGTGCCCCGTCTTGG

[0048] GGTGGCGTTCTGGCTATCCCGGGTTCCGGCAGCGACCAGCAGCTGTCCGCTATCCGTAACGGCATCAAC

[0049] AACTTCCGTGGCAAGGGCGGTGAAGTGATGGTAAGCTTTGGCGGCGCGAACGGTACGCCGCTGCAGCAA

[0050] GCCTGTACCACCAATGCTAGCCTGCAAGCCGCTTATCAGACCGTACTGGATACCTATAACCTGAGCCGC

[0051] ATCGACTTCGATATCGAAGGTGGCGCGCAAACTGACACTGCAGCTAACAACCGCAACTTTGCGGTTGTG

[0052] GCTGCACTGCAGAAAAACTACAAAGCGAAAGGTAAAACCCTGCACGTATCCCTGACCCTGCCAGCCATG

[0053] CCATTTGGCCTGACTCAGGATGGCCAGCGTGTACTGGCAAGCGCGCTGGCTAACGGTGTGGCCCTGGAC

[0054] ACTGTAAACATTATGGCCATGGACTATGGCCAGAGCAACCCAAACATGGGTGCCGCTGCAAAACAGGCG

[0055] GCTCAGGCACTGTACAGCCAGATTGATGCAGCGTACAAAGCACACGGTCAGACTCTGACTGACGCTCAG

[0056] CTGTGGCAAAAAGTAGGCGTTACTCCAATGGTAGGTCTGAACGATACCCAGCCGGAAACTTTCACCGTT

[0057] GATAATGCGAAAGACCTGTACGGTATGGCGAACTCTAACCGTTTCGGCCTGCTGAGCATGTGGTCCATC

[0058] TCCCGTGATAAATCTTGCCCGAACAACGGCCACTACGTAGATGCTCAGTGTAGCGGTATCGTACAGACT

[0059] CCGTACGCGTTTTCTAACGTGTTCAAGGGTTTCAAAGATCACTGGGGTTCCGGTGTTACTCAAGACCCG

[0060] AACTACGGTGGTGGTGACAACGGTAACGGTGGCGGTCCGGTCAACGGTCAGCCGTGGTCCAGCGGCCAA

[0061] GTGTACAATACTGGTAACACTGTTACCTACTCTGGCGCGACTTGGAAAGCCCAGTGGTGGACTCAAGGT

[0062] GATGTACCGGGTCAGGCTTCTGTCTGGCAACAACAGGGCGGTGGCCTGCAGCAGTGGTCTGCTACCGCG

[0063] GCGTATAGCGCAAAGGACTGCGCGCTGTATCAGGGCAAGAAATACTGTGCAAAATGGTGGACCCAGGGT

[0064] AATCTGCCTGCAGCGGGCGATCCATGGGTGCTGTCTAACTGA

[0065] The recombinant plasmid containing the chitinase gene was transformed into E. coli BL21 (DE3) to obtain a recombinant strain containing the chitinase gene. The recombinant strain was inoculated into 5 mL of LB liquid medium containing 50 μg / ml kanamycin for overnight culture, and then inoculated into 500 mL of LB liquid medium containing kanamycin at a 1% inoculum, and cultured at 37°C and 200 rpm until OD 600 The pH value was about 0.8, IPTG was added to make the final concentration 0.1mmol / L for induction, and the expression was induced for 6h at 28℃ and 180rpm, and the cells were collected by centrifugation at 5000rpm for 10min. The cells were washed twice with binding buffer (pH7.2), and then resuspended in 60mL binding buffer, ultrasonically disrupted for 20min at a power of 300W and a working / interval time of 3s / 4s, and centrifuged at 4℃ and 8000rpm for 10min, and the supernatant was collected to obtain the crude enzyme solution.

[0066] Add the prepared crude enzyme solution to the pre-equilibrated purification column, mix at 4°C for 2-3 hours, and then discard the waste liquid. Rinse twice with 20 mL of rinsing buffer containing 10 mM imidazole, rinse twice with 20 mL of rinsing buffer containing 20 mM imidazole, and finally rinse twice with 20 mL of rinsing buffer containing 50 mM imidazole. After rinsing, elute with elution buffer containing 500 mM imidazole, and perform SDS-PAGE electrophoresis on the eluate to detect its purity. Use 8-14 kD dialysis bags to dialyze the eluate overnight to remove imidazole and NaCl to obtain the purified enzyme solution. SDS-PAGE electrophoresis after purification shows Figure 1 .

[0067] Example 2 Basic properties of Chromobacterium violaceum chitinase

[0068] Enzyme activity assay method:

[0069] The reducing sugar content was determined by DNS method. 50 μL of diluted enzyme solution and 200 μL of 1% colloidal chitin were mixed and reacted at 60°C for 50 min. After the reaction was completed, 500 μL of DNS reagent was added, boiled for 5 min, immediately placed in ice water for cooling, centrifuged briefly, and the supernatant was taken to determine the OD 540 The value (with inactivated enzyme solution as control) was used to calculate the amount of reducing sugar and enzyme activity according to the glucose standard curve. Definition of enzyme activity unit: Under the above determination conditions, the amount of enzyme required to hydrolyze the colloidal chitin substrate to produce 1 μmol of reducing sugar per minute was defined as one enzyme activity unit.

[0070] Working temperature:

[0071] The enzyme activity of the diluted enzyme solution was measured at 25-100°C with 1% colloidal chitin prepared in 50mM acetate buffer (pH 5.0) as the substrate. The relative enzyme activity at different temperatures was calculated with the maximum enzyme activity as 100%. The results showed that the optimal reaction temperature of the recombinant enzyme was 60°C, and the enzyme activity was higher in the range of 50-70°C, and its enzyme activity was maintained above 75%; in the range of 40-75°C, its enzyme activity could also be maintained above 45%. For specific results, see Figure 2 .

[0072] pH:

[0073] The diluted enzyme solution was heated at 60°C with different buffers (50 mM acetate buffer, pH 4.0-6.0; 50 mM phosphate buffer, pH 6.0-8.0; 50 mM Tris-HCl buffer, pH 7.0-9.0;

[0074] 1% colloidal chitin prepared with 50mM Gly-NaOH buffer (pH 9.0-10.0) was used as a substrate for enzyme activity determination. The maximum enzyme activity was taken as 100%, and the relative activity of the enzyme at different pH was calculated. The results showed that the optimal pH of the recombinant enzyme was 5.0 (acetate buffer), and the enzyme activity was higher in the pH range of 4.5-6.0, and its enzyme activity was maintained above 70%; the enzyme activity of the enzyme also decreased rapidly with the increase of pH; under the same pH conditions, acetate buffer was better than phosphate buffer. For specific results, see Figure 3 .

[0075] Temperature stability:

[0076] The enzyme solution was kept at 50°C, 55°C and 60°C for 60 minutes, and the enzyme activity was measured every 10 minutes. The chitinase solution placed at different temperatures for different times was tested for enzyme activity with a 1% colloidal chitin solution prepared with 50mM acetate buffer (pH 5.0). The activity of the untreated chitinase was taken as 100%, and the relative enzyme activity at different temperatures and storage times was calculated. The results showed that the enzyme had good stability at 50°C. After being stored at 50°C for 40 minutes, its enzyme activity remained above 65%; after being stored at 55°C and 60°C for 20 minutes, the enzyme activity could also be maintained above 50%. For specific results, see Figure 4 .

[0077] Inhibitory effect on Fusarium

[0078] Inoculate Fusarium graminearum into 5 mL PDB medium and culture at 28°C and 150 rpm for 24 hours. Pipette 1 mL of fermentation solution into a 2 mL centrifuge tube and shake it thoroughly to prepare a spore suspension. Spread the spore suspension evenly on the PDA medium. Use the Oxford cup method to determine the inhibitory effect of recombinant chitinase on Fusarium. Add 200 μL of recombinant enzyme solution to the Oxford cup. Use ultrapure water as a control. Incubate at 28°C for 24 hours and observe the size of the inhibition zone. The results show that the recombinant enzyme has a certain inhibitory ability against Fusarium, and the diameter of the inhibition zone is about 18 mm. See the results. Figure 5 .

[0079] Example 3 Obtaining Chitinase Variants of Chromobacterium violaceum

[0080] The results of Example 2 show that the chitinase ChiCD of Chromobacterium violaceum can meet the requirements of agricultural preparation preparation in terms of stability and pH adaptability. However, its enzyme activity decreases rapidly at normal room temperature or field temperature of about 20°C, and is only one-third of that at the optimal temperature (about 60°C). Moreover, according to previous studies, the enzyme is sensitive to iron ions commonly found in fertilizers and pesticides. 25mM Fe 2+ The presence of can reduce its vitality to less than one tenth.

[0081] To overcome the above problems, the applicant used error-prone PCR to carry out directed mutagenesis of the chitinase ChiCD of Chromobacterium violaceum:

[0082] The 30 μL error-prone PCR reaction system is: 10x PCR Mix (provided by the kit) 3 μL, 10x dNTP (provided by the kit) 3 μL, 2.5 mM MnCl 2 3μL, 10ng / μL template (ChiCD gene PCR product) 1μL, 10μm primers 10pmol each, Taq enzyme (provided by the kit) 5U.

[0083] After denaturation at 94°C for 3 min, the cycle was started: 94°C for 1 min, 45°C for 1 min, and 72°C for 1 min for 10 cycles (the expected number of mutations is about 5).

[0084] The mutant library was constructed with error-prone PCR products, transformed into the host Escherichia coli TOP10, and plated on a plate containing LA for culture and screening, obtaining more than 200 transformants that can be stably propagated. The enzyme activity was compared at a temperature of 15°C, and combined with the growth performance of the strain, the mutant ChiCD1 was obtained. The amino acid and nucleotide sequences were sequenced as SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0085] ChiCD1 amino acid sequence (SEQ ID NO.3)

[0086] MMKRFAALSALAAAAAGLASPAAQAGAFAPYVDMTLWPTPQIDKLGVNQGIQQFTLAFVVAKGGCAPSW

[0087] GGVLAIPGSGSDQQLSAIRNGINNFRGKGGEVMVSFGGANGTPLQQACTTNASLQAAYQTVLDTYNLSR

[0088] IDFDIKGGAQTDTAANNRNFAVVAALQKNYKAKGKTLHVSLTLPAMPFGLTQDGQRVLASALANGVALD

[0089] TVNIMAMHYGQSNPNMGAAAKQAAQALYSQIDAAYKAHGQTLTDAQLWQKVGVTPMVGLNDTQPETFTV

[0090] DNAKDLYGMANTNRFGLLSMWSISRDKSCPNNGHYVDAQCSGIVQTPYAFSNVFKGFKDHWGSGVTQDP

[0091] NYGGGDNGNGGGPVNGQPWSSGQVYNTGNTVT

[0092] Nucleotide sequence of ChiCD1 (SEQ ID NO.4)

[0093] ATGATGAAACGTTTTGCCGCTCTGAGCGCCCTGGCAGCAGCTGCAGCTGGCCTGGCATCTCCGGCTGCG

[0094] CAAGCAGGTGCATTCGCTCCGTATGTTGACATGACCCTGTGGCCGACCCCGCAAATCGACAAGCTGGGT

[0095] GTTAACCAGGGTATCCAGCAGTTCACTCTGGCCTTCGTTGTTGCGAAGGGCGGCTGTGCCCCGTCTTGG

[0096] GGTGGCGTTCTGGCTATCCCGGGTTCCGGCAGCGACCAGCAGCTGTCCGCTATCCGTAACGGCATCAAC

[0097] AACTTCCGTGGCAAGGGCGGTGAAGTGATGGTAAGCTTTGGCGGCGCGAACGGTACGCCGCTGCAGCAA

[0098] GCCTGTACCACCAATGCTAGCCTGCAAGCCGCTTATCAGACCGTACTGGATACCTATAACCTGAGCCGC

[0099] ATCGACTTCGATATCAAAGGTGGCGCGCAAACTGACACTGCAGCTAACAACCGCAACTTTGCGGTTGTG

[0100] GCTGCACTGCAGAAAAACTACAAAGCGAAAGGTAAAACCCTGCACGTATCCCTGACCCTGCCAGCCATG

[0101] CCATTTGGCCTGACTCAGGATGGCCAGCGTGTACTGGCAAGCGCGCTGGCTAACGGTGTGGCCCTGGAC

[0102] ACTGTAAACATTATGGCCATGCACTATGGCCAGAGCAACCCAAACATGGGTGCCGCTGCAAAACAGGCG

[0103] GCTCAGGCACTGTACAGCCAGATTGATGCAGCGTACAAAGCACACGGTCAGACTCTGACTGACGCTCAG

[0104] CTGTGGCAAAAAGTAGGCGTTACTCCAATGGTAGGTCTGAACGATACCCAGCCGGAAACTTTCACCGTT

[0105] GATAATGCGAAAGACCTGTACGGTATGGCGAACACTAACCGTTTCGGCCTGCTGAGCATGTGGTCCATC

[0106] TCCCGTGATAAATCTTGCCCGAACAACGGCCACTACGTAGATGCTCAGTGTAGCGGTATCGTACAGACT

[0107] CCGTACGCGTTTTCTAACGTGTTCAAGGGTTTCAAAGATCACTGGGGTTCCGGTGTTACTCAAGACCCG

[0108] AACTACGGTGGTGGTGACAACGGTAACGGTGGCGGTCCGGTCAACGGTCAGCCGTGGTCCAGCGGCCAA

[0109] GTGTACAATACTGGTAACACTGTTACCTAA

[0110] Comparison shows that ChiCD1 introduced G430A, G643C, T862A, and C1134A mutations based on ChiCD, corresponding to E144K, D215H, S288T, and premature termination truncation at position 378.

[0111] According to the interaction simulation results with chitin, E144K and D215H are mutations located in the substrate binding pocket. Based on this, the full-length variant ChiCD 2 containing E144K and D215T, the truncated variant ChiCD 3 containing E144K and D215T, the truncated variant ChiCD4 containing E144K and S288T, and the truncated variant ChiCD5 containing D215H and S288T were further constructed by a method similar to Example 1.

[0112] ChiCD 2 amino acid sequence (SEQ ID NO.5)

[0113] MMKRFAALSALAAAAAGLASPAAQAGAFAPYVDMTLWPTPQIDKLGVNQGIQQFTLAFVVAKGGCAPSW

[0114] GGVLAIPGSGSDQQLSAIRNGINNFRGKGGEVMVSFGGANGTPLQQACTTNASLQAAYQTVLDTYNLSR

[0115] IDFDIKGGAQTDTAANNRNFAVVAALQKNYKAKGKTLHVSLTLPAMPFGLTQDGQRVLASALANGVALD

[0116] TVNIMAMHYGQSNPNMGAAAKQAAQALYSQIDAAYKAHGQTLTDAQLWQKVGVTPMVGLNDTQPETFTV

[0117] DNAKDLYGMANTNRFGLLSMWSISRDKSCPNNGHYVDAQCSGIVQTPYAFSNVFKGFKDHWGSGVTQDP

[0118] NYGGGDNGNGGGPVNGQPWSSGQVYNTGNTVTYSGATWKAQWWTQGDVPGQASVWQQQGGGLQQWSATA

[0119] AYSAKDCALYQGKKYCAKWWTQGNLPAAGDPWVLSN

[0120] ChiCD 3 amino acid sequence (SEQ ID NO.6)

[0121] MMKRFAALSALAAAAAGLASPAAQAGAFAPYVDMTLWPTPQIDKLGVNQGIQQFTLAFVVAKGGCAPSW

[0122] GGVLAIPGSGSDQQLSAIRNGINNFRGKGGEVMVSFGGANGTPLQQACTTNASLQAAYQTVLDTYNLSR

[0123] IDFDIKGGAQTDTAANNRNFAVVAALQKNYKAKGKTLHVSLTLPAMPFGLTQDGQRVLASALANGVALD

[0124] TVNIMAMHYGQSNPNMGAAAKQAAQALYSQIDAAYKAHGQTLTDAQLWQKVGVTPMVGLNDTQPETFTV

[0125] DNAKDLYGMANTNRFGLLSMWSISRDKSCPNNGHYVDAQCSGIVQTPYAFSNVFKGFKDHWGSGVTQDP

[0126] NYGGGDNGNGGGPVNGQPWSSGQVYNTGNTVT

[0127] ChiCD 4 amino acid sequence (SEQ ID NO.7)

[0128] MMKRFAALSALAAAAAGLASPAAQAGAFAPYVDMTLWPTPQIDKLGVNQGIQQFTLAFVVAKGGCAPSW

[0129] GGVLAIPGSGSDQQLSAIRNGINNFRGKGGEVMVSFGGANGTPLQQACTTNASLQAAYQTVLDTYNLSR

[0130] IDFDIKGGAQTDTAANNRNFAVVAALQKNYKAKGKTLHVSLTLPAMPFGLTQDGQRVLASALANGVALD

[0131] TVNIMAMDYGQSNPNMGAAAKQAAQALYSQIDAAYKAHGQTLTDAQLWQKVGVTPMVGLNDTQPETFTV

[0132] DNAKDLYGMANTNRFGLLSMWSISRDKSCPNNGHYVDAQCSGIVQTPYAFSNVFKGFKDHWGSGVTQDP

[0133] NYGGGDNGNGGGPVNGQPWSSGQVYNTGNTVT

[0134] Amino acid sequence of ChiCD (SEQ ID NO.8)

[0135] MMKRFAALSALAAAAAGLASPAAQAGAFAPYVDMTLWPTPQIDKLGVNQGIQQFTLAFVVAKGGCAPSW

[0136] GGVLAIPGSGSDQQLSAIRNGINNFRGKGGEVMVSFGGANGTPLQQACTTNASLQAAYQTVLDTYNLSR

[0137] IDFDIEGGAQTDTAANNRNFAVVAALQKNYKAKGKTLHVSLTLPAMPFGLTQDGQRVLASALANGVALD

[0138] TVNIMAMHYGQSNPNMGAAAKQAAQALYSQIDAAYKAHGQTLTDAQLWQKVGVTPMVGLNDTQPETFTV

[0139] DNAKDLYGMANTNRFGLLSMWSISRDKSCPNNGHYVDAQCSGIVQTPYAFSNVFKGFKDHWGSGVTQDP

[0140] NYGGGDNGNGGGPVNGQPWSSGQVYNTGNTVT

[0141] ChiCD 4 nucleotide sequence (SEQ ID NO.9)

[0142] ATGATGAAACGTTTTGCCGCTCTGAGCGCCCTGGCAGCAGCTGCAGCTGGCCTGGCATCTCCGGCTGCG

[0143] CAAGCAGGTGCATTCGCTCCGTATGTTGACATGACCCTGTGGCCGACCCCGCAAATCGACAAGCTGGGT

[0144] GTTAACCAGGGTATCCAGCAGTTCACTCTGGCCTTCGTTGTTGCGAAGGGCGGCTGTGCCCCGTCTTGG

[0145] GGTGGCGTTCTGGCTATCCCGGGTTCCGGCAGCGACCAGCAGCTGTCCGCTATCCGTAACGGCATCAAC

[0146] AACTTCCGTGGCAAGGGCGGTGAAGTGATGGTAAGCTTTGGCGGCGCGAACGGTACGCCGCTGCAGCAA

[0147] GCCTGTACCACCAATGCTAGCCTGCAAGCCGCTTATCAGACCGTACTGGATACCTATAACCTGAGCCGC

[0148] ATCGACTTCGATATCAAAGGTGGCGCGCAAACTGACACTGCAGCTAACAACCGCAACTTTGCGGTTGTG

[0149] GCTGCACTGCAGAAAAACTACAAAGCGAAAGGTAAAACCCTGCACGTATCCCTGACCCTGCCAGCCATG

[0150] CCATTTGGCCTGACTCAGGATGGCCAGCGTGTACTGGCAAGCGCGCTGGCTAACGGTGTGGCCCTGGAC

[0151] ACTGTAAACATTATGGCCATGGACTATGGCCAGAGCAACCCAAACATGGGTGCCGCTGCAAAACAGGCG

[0152] GCTCAGGCACTGTACAGCCAGATTGATGCAGCGTACAAAGCACACGGTCAGACTCTGACTGACGCTCAG

[0153] CTGTGGCAAAAAGTAGGCGTTACTCCAATGGTAGGTCTGAACGATACCCAGCCGGAAACTTTCACCGTT

[0154] GATAATGCGAAAGACCTGTACGGTATGGCGAACACTAACCGTTTCGGCCTGCTGAGCATGTGGTCCATC

[0155] TCCCGTGATAAAATCTTGCCCGAACAACGGCCACTACGTAGATGCTCAGTGTAGCGGTATCGTACAGACT

[0156] CCGTACGCGTTTTCTAACGTGTTCAAGGGTTTCAAAGATCACTGGGGTTCCGGTGTTACTCAAGACCCG

[0157] AACTACGGTGGTGGTGACAACGGTAACGGTGGCGGTCCGGTCAACGGTCAGCCGTGGTCCAGCGGCCAAGTGTACAATACTGGTAACACTGTTACCTAA.

[0158] Example 4 Properties of Chromobacterium violaceum chitinase variants

[0159] The enzyme activities of ChiCD and ChiCD1-5 at different temperatures were detected according to the method of Example 2. The results are as follows: Figure 6 As shown in the figure, similar to the trend of the initial screening results, the activity of ChiCD1 at room temperature was significantly improved, reaching about 1.8 times that of the wild type. Moreover, without optimizing the culture and enzyme production conditions, it was preliminarily judged that its production in E. coli was also at a similar level to that of the wild type.

[0160] ChiCD2, ChiCD 3, and ChiCD 5 have similar properties to ChiCD, with no significant changes in temperature adaptability, while ChiCD 4 exhibits temperature adaptability similar to that of ChiCD 1. This indicates that the synergistic effect of E144K in the substrate binding pocket and S288T at other positions is the key factor in the change in temperature adaptability.

[0161] ChiCD1 and ChiCD4 showed similar effects to ChiCD in terms of pH and temperature stability.

[0162] The enzyme activities of ChiCD1 and ChiCD4 measured at 50°C were calculated as 100%. 25 mM and 50 mM Fe in the form of ferrous chloride were added to the detection system. 2 The ChiCD enzyme activity measured at 60°C was counted as 100%. 25 mM and 50 mM Fe in the form of ferrous chloride were added to the detection system. 2 , and the enzyme activity was determined. Figure 7 As shown, ChiCD1 and ChiCD4 provide better enzyme activity retention performance in the presence of iron ions, and can still maintain more than 30% at a concentration of 25mM. At a concentration of 50mM where the ChiCD enzyme activity basically disappears, there is still about 10% remaining; such performance provides great convenience for actual field use (which inevitably involves contact with iron ions in foliar fertilizers or equipment).

[0163] The results of the inhibition zone experiment performed with reference to the method in Example 2 showed that when using the same 200 μL enzyme solution (obtained by basically the same preparation method as in Example 1), due to better room temperature performance, its inhibition zone also significantly exceeded ChiCD's 18 mm and reached about 26 mm.

Claims

1. A chitinase having antifungal activity, characterized in that: The amino acid sequence of the chitinase is SEQ ID NO.3 or SEQ ID NO.

7.

2. Use of the chitinase according to claim 1 in the preparation of a fungus-inhibiting preparation.

3. The use according to claim 2, wherein the fungus is Fusarium sp.

4. The use according to claim 2, wherein the fungus is Fusarium graminearum.

5. The use according to any one of claims 2 to 4, wherein the preparation is a pesticide.

6. The chitinase encoding gene according to claim 1, characterized in that: The nucleotide sequence of the coding gene is SEQ ID NO.4 or SEQ ID NO.

9.

7. A carrier, characterized in that The vector comprises the coding gene according to claim 6.

8. A host cell, characterized in that The host cell comprises the vector according to claim 7.

9. A method for preparing the chitinase according to claim 1, characterized in that: The method comprises the step of culturing the host cell according to claim 8.

10. The method according to claim 9, further comprising a purification step.

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