A chitinase with antifungal activity, its preparation method and application

Abstract

The present application provides a chitinase with antifungal activity, whose amino acid sequence is SEQ ID NO.3 or SEQ ID NO.7. This chitinase has good activity at room temperature and is insensitive to the presence of iron ions, and is suitable for preparing agricultural preparations for inhibiting fungi such as Fusarium.

Description

Field of the Invention:

[0001] This application belongs to the fields of proteins and pesticides. Specifically, this application provides a chitinase with antifungal activity, its preparation method, and applications. Background Art:

[0002] With the rapid growth of the global population, the demand for food has been increasing year by year. However, fungal infections of crops can cause a decline in food production and quality. But the overuse of chemical fungicides will affect food safety and cause certain damage to the environment. In order not to affect food and environmental safety and follow the path of sustainable agricultural development, the application of biotechnology in agricultural production has been gradually explored and emphasized. Chitinase can degrade the chitin component in the fungal cell wall, causing the fungal cell wall to rupture, thereby achieving the effect of inhibiting fungal growth without causing adverse effects on the plants themselves.

[0003] Chitin, also known as chitosan, chitin, or crustacean, is a macromolecular linear polysaccharide composed of N-acetyl-D-glucosamine (GlcNAc) connected by β-1,4 glycosidic bonds. Chitinase (EC 3.2.1.14) can hydrolyze the β-1,4 glycosidic bonds in chitin to degrade it into chito-oligosaccharides or chito-monosaccharides. The sources of chitinase are very extensive, and there are relevant reports in various organisms, such as bacteria, fungi, plants, and animals. Chitinases are generally classified according to reaction conditions, action modes, sequence conservation, etc. According to different reaction conditions, they can be divided into heat-resistant enzymes, cold-resistant enzymes, acid-resistant enzymes, and alkali-resistant enzymes; according to the action mode, they can be divided into endo-chitinases and exo-chitinases. Among them, endo-enzymes randomly hydrolyze β-1,4 glycosidic bonds inside chitin to form oligosaccharides mainly composed of disaccharides to hexasaccharides, and exo-enzymes can be divided into chitobiase and β-N-acetylglucosaminidase.

[0004] Searching for suitable types of chitinases for controlling fungi in agriculture among such diverse chitinases with various sources and properties has been one of the research directions in this field. Summary of the Invention:

[0005] On the one hand, this application provides a chitinase with antifungal activity, and the amino acid sequence of the chitinase is SEQ ID NO.3 or SEQ ID NO.7.

[0006] On the other hand, this application provides the application of the above chitinase in the preparation of a preparation for inhibiting fungi.

[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 the coding gene of the above chitinase, and the nucleotide sequence of the coding gene is SEQ ID NO.4 or SEQ ID NO.9.

[0011] On the other hand, the present application provides a vector, and the vector contains the above coding gene.

[0012] On the other hand, the present application provides a host cell, and the host cell contains the above 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, including the step of culturing the above host cell.

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

[0016] Those skilled in the art can routinely use the 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, etc. Description of the drawings:

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

[0018] Figure 2 It is the trend diagram of enzyme activity changing with temperature.

[0019] Figure 3 It is the trend diagram of enzyme activity changing with pH in different buffers.

[0020] Figure 4 It is the diagram of enzyme stability at different temperatures.

[0021] Figure 5 It is the example diagram of the antibacterial effect on Fusarium.

[0022] Figure 6 It is the trend diagram of the activity of various variants changing with temperature.

[0023] Figure 7 Showing 25 mM, 50 mM of Fe in the form of ferrous chloride added2 Enzyme activity assay results of ChiCD, ChiCD1, and ChiCD4 Specific implementation methods:

[0024] Main reagents

[0025] Fusarium graminearum, Escherichia coli E.coil TOP10, Escherichia coli E.coil BL21(DE3), and the expression vector pET-28a are all stored in the applicant's laboratory.

[0026] Restriction endonucleases NdeI and XhoI: Beijing TransGen Biotech Co., Ltd.

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

[0028] PDA medium: Qingdao Hope Bio-Technology Co., Ltd.

[0029] Binding buffer (phosphate buffer: 20 mM phosphate, pH 7.2 - 7.4); washing 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 TransGen Biotech Co., Ltd.

[0032] 8 - 14 kD dialysis bag: Shanghai Yuanye Bio-Technology Co., Ltd.

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

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

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

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

[0037] MMKRFAALSALAAAAAGLASPAAQAGAFAPYVDMTLWPTPQIDKLGVNQGIQQFTLAFVVAKGGCAPSW

[0038] GGVLAIPGSGSDQQLSAIRNGINNFRGKGGEVMVSFGGANGTPLQQACTTNASLQAAYQTVLDTYNLSR

[0039] IDFDIEGGAQTDTAANNRNFAVVAALQKNYKAKGKTLHVSLTLPAMPFGLTQDGQRVLASALANGVALD

[0040] TVNIMAMDYGQSNPNMGAAAKQAAQALYSQIDAAYKAHGQTLTDAQLWQKVGVTPMVGLNDTQPETFTV

[0041] DNAKDLYGMANSNRFGLLSMWSISRDKSCPNNGHYVDAQCSGIVQTPYAFSNVFKGFKDHWGSGVTQDP

[0042] NYGGGDNGNGGGPVNGQPWSSGQVYNTGNTVTYSGATWKAQWWTQGDVPGQASVWQQQGGGLQQWSATA

[0043] AYSAKDCALYQGKKYCAKWWTQGNLPAAGDPWVLSN

[0044] Original nucleic acid sequence of ChiCD (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 and cultured overnight, then inoculated into 500 mL of LB liquid medium containing kanamycin at an inoculation amount of 1%, and cultured at 37 °C and 200 rpm until the OD 600 was about 0.8. IPTG was added to a final concentration of 0.1 mmol / L for induction, and induced expression was carried out at 28 °C and 180 rpm for 6 h. The cells were collected by centrifugation at 5000 rpm for 10 min. The cells were washed twice with binding buffer (pH 7.2), and then the cell pellet was resuspended in 60 mL of binding buffer. Ultrasonic disruption was carried out at a power of 300 W with a working / interval time of 3 s / 4 s for 20 min. After centrifugation at 4 °C and 8000 rpm for 10 min, the supernatant was collected to obtain the crude enzyme solution.

[0066] The prepared crude enzyme solution was added to a pre-equilibrated purification column, mixed at 4 °C for 2 - 3 h, and then the waste liquid was discarded. Then, it was rinsed twice with 20 mL of rinsing buffer containing 10 mM imidazole, twice with 20 mL of rinsing buffer containing 20 mM imidazole, and finally twice with 20 mL of rinsing buffer containing 50 mM imidazole. After the rinsing was completed, it was eluted with elution buffer containing 500 mM imidazole, and the purity of the eluate was detected by SDS-PAGE electrophoresis. The eluate was dialyzed overnight using an 8 - 14 kD dialysis bag to remove imidazole and NaCl, and a purified enzyme solution was obtained. The SDS-PAGE electrophoresis after purification is shown in Figure 1 .

[0067] Example 2 Basic properties of Chromobacterium violaceum chitinase

[0068] Enzyme activity assay method:

[0069] The DNS method was used to determine the reducing sugar content. 50 μL of diluted enzyme solution was mixed with 200 μL of 1% colloidal chitin 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 cooled in ice water, and after brief centrifugation, the supernatant was taken to measure the OD 540 value (using the inactivated enzyme solution as a control), and the reducing sugar amount and enzyme activity were calculated according to the glucose standard curve. Definition of enzyme activity unit: Under the above measurement conditions, the amount of enzyme required to hydrolyze the colloidal chitin substrate to produce 1 μmol of reducing sugar per minute is defined as one enzyme activity unit.

[0070] Action temperature:

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

[0072] Action pH:

[0073] The diluted enzyme solution was assayed for enzyme activity using 1% colloidal chitin prepared 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] 50 mM Gly-NaOH buffer, pH 9.0 - 10.0) as the substrate at 60 °C. Taking the maximum enzyme activity as 100%, the relative activity of the enzyme at different pH values was calculated. The results showed that the optimal pH of the recombinant enzyme was 5.0 (acetate buffer), and the enzyme activity was relatively high in the range of pH 4.5 - 6.0, with the enzyme activity remaining above 70%; the enzyme activity decreased rapidly with the increase of pH; under the same pH conditions, acetate buffer was superior to phosphate buffer. The specific results are shown in Figure 3 .

[0075] Temperature stability:

[0076] The enzyme solution was incubated at 50 °C, 55 °C and 60 °C for 60 min, and the enzyme activity was measured every 10 min. The chitinase solution stored at different temperatures for different times was assayed for enzyme activity with 1% colloidal chitin solution prepared with 50 mM acetate buffer (pH 5.0). Taking the enzyme activity of the untreated chitinase as 100%, the relative enzyme activities at different temperatures and storage times were calculated. The results showed that the enzyme had good stability at 50 °C. After being stored at 50 °C for 40 min, the enzyme activity remained above 65%; after being stored at 55 °C and 60 °C for 20 min, the enzyme activity could also be maintained above 50%. The specific results are shown in Figure 4 .

[0077] Inhibitory effect on Fusarium

[0078] Fusarium graminearum was inoculated into 5 mL of PDB medium and cultured at 28 °C and 150 rpm for 24 h. 1 mL of the fermentation broth was aspirated into a 2 mL centrifuge tube and shaken well to prepare a spore suspension. The spore suspension was evenly spread on PDA medium, and the Oxford cup method was used to determine the inhibitory effect of the recombinant chitinase on Fusarium. 200 μL of the recombinant enzyme solution was added to the Oxford cup, and ultrapure water was used as a control. After incubation at 28 °C for 24 h, the size of the inhibition zone was observed. The results showed that the recombinant enzyme had a certain inhibitory ability against Fusarium, and the diameter of the inhibition zone was about 18 mm. The results are shown in Figure 5 .

[0079] Example 3 Obtaining of Chromobacterium violaceum chitinase variants

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

[0081] To overcome the above problems, the applicant used error-prone PCR to perform site-directed mutagenesis on Chromobacterium violaceum chitinase ChiCD:

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

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

[0084] The error-prone PCR product was used to construct a mutant library, which was transformed into the host Escherichia coli TOP10 and spread on a plate containing LA for culture and screening. More than 200 transformants that could be stably passaged were obtained. The enzyme-producing activity was compared at a temperature of 15 °C, and combined with the growth performance of the strains, the mutant ChiCD1 was obtained. After sequencing, its amino acid and nucleotide sequences were SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0085] Amino acid sequence of ChiCD1 (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 introduces G430A, G643C, T862A, and C1134A mutations on the basis of ChiCD, corresponding to E144K, D215H, S288T, and premature termination truncation at position 378.

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

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

[0113] MMKRFAALSALAAAAAGLASPAAQAGAFAPYVDMTLWPTPQIDKLGVNQGIQQFTLAFVVAKGGCAPSW

[0114] GGVLAIPGSGSDQQLSAIRNGINNFRGKGGEVMVSFGGANGTPLQQACTTNASLQAAYQTVLDTYNLSR

[0115] IDFDIKGGAQTDTAANNRNFAVVAALQKNYKAKGKTLHVSLTLPAMPFGLTQDGQRVLASALANGVALD

[0116] TVNIMAMHYGQSNPNMGAAAKQAAQALYSQIDAAYKAHGQTLTDAQLWQKVGVTPMVGLNDTQPETFTV

[0117] DNAKDLYGMANTNRFGLLSMWSISRDKSCPNNGHYVDAQCSGIVQTPYAFSNVFKGFKDHWGSGVTQDP

[0118] NYGGGDNGNGGGPVNGQPWSSGQVYNTGNTVTYSGATWKAQWWTQGDVPGQASVWQQQGGGLQQWSATA

[0119] AYSAKDCALYQGKKYCAKWWTQGNLPAAGDPWVLSN

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

[0121] MMKRFAALSALAAAAAGLASPAAQAGAFAPYVDMTLWPTPQIDKLGVNQGIQQFTLAFVVAKGGCAPSW

[0122] GGVLAIPGSGSDQQLSAIRNGINNFRGKGGEVMVSFGGANGTPLQQACTTNASLQAAYQTVLDTYNLSR

[0123] IDFDIKGGAQTDTAANNRNFAVVAALQKNYKAKGKTLHVSLTLPAMPFGLTQDGQRVLASALANGVALD

[0124] TVNIMAMHYGQSNPNMGAAAKQAAQALYSQIDAAYKAHGQTLTDAQLWQKVGVTPMVGLNDTQPETFTV

[0125] DNAKDLYGMANTNRFGLLSMWSISRDKSCPNNGHYVDAQCSGIVQTPYAFSNVFKGFKDHWGSGVTQDP

[0126] NYGGGDNGNGGGPVNGQPWSSGQVYNTGNTVT

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

[0128] MMKRFAALSALAAAAAGLASPAAQAGAFAPYVDMTLWPTPQIDKLGVNQGIQQFTLAFVVAKGGCAPSW

[0129] GGVLAIPGSGSDQQLSAIRNGINNFRGKGGEVMVSFGGANGTPLQQACTTNASLQAAYQTVLDTYNLSR

[0130] IDFDIKGGAQTDTAANNRNFAVVAALQKNYKAKGKTLHVSLTLPAMPFGLTQDGQRVLASALANGVALD

[0131] TVNIMAMDYGQSNPNMGAAAKQAAQALYSQIDAAYKAHGQTLTDAQLWQKVGVTPMVGLNDTQPETFTV

[0132] DNAKDLYGMANTNRFGLLSMWSISRDKSCPNNGHYVDAQCSGIVQTPYAFSNVFKGFKDHWGSGVTQDP

[0133] NYGGGDNGNGGGPVNGQPWSSGQVYNTGNTVT

[0134] ChiCD 5 amino acid sequence (SEQ ID NO.8)

[0135] MMKRFAALSALAAAAAGLASPAAQAGAFAPYVDMTLWPTPQIDKLGVNQGIQQFTLAFVVAKGGCAPSW

[0136] GGVLAIPGSGSDQQLSAIRNGINNFRGKGGEVMVSFGGANGTPLQQACTTNASLQAAYQTVLDTYNLSR

[0137] IDFDIEGGAQTDTAANNRNFAVVAALQKNYKAKGKTLHVSLTLPAMPFGLTQDGQRVLASALANGVALD

[0138] TVNIMAMHYGQSNPNMGAAAKQAAQALYSQIDAAYKAHGQTLTDAQLWQKVGVTPMVGLNDTQPETFTV

[0139] DNAKDLYGMANTNRFGLLSMWSISRDKSCPNNGHYVDAQCSGIVQTPYAFSNVFKGFKDHWGSGVTQDP

[0140] NYGGGDNGNGGGPVNGQPWSSGQVYNTGNTVT

[0141] ChiCD 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] TCCCGTGATAAATCTTGCCCGAACAACGGCCACTACGTAGATGCTCAGTGTAGCGGTATCGTACAGACT

[0156] CCGTACGCGTTTTCTAACGTGTTCAAGGGTTTCAAAGATCACTGGGGTTCCGGTGTTACTCAAGACCCG

[0157] AACTACGGTGGTGGTGACAACGGTAACGGTGGCGGTCCGGTCAACGGTCAGCCGTGGTCCAGCGGCCAAGTGTACAATACTGGTAACACTGTTACCTAA。

[0158] Properties of Chromobacterium violaceum chitinase variants in Example 4

[0159] Detect the enzyme activities of ChiCD and ChiCD1-5 at different temperatures according to the method of Example 2. The results are as Figure 6 shown. Similar to the trend of the primary screening results, the activity of ChiCD1 is significantly increased near room temperature, reaching about 1.8 times that of the wild type. Moreover, without optimizing the culture and enzyme production conditions, it is preliminarily judged that its yield in Escherichia coli is also at a similar level to that of the wild type.

[0160] The properties of ChiCD2, ChiCD 3, and ChiCD 5 are similar to those of ChiCD, and there is no obvious change in temperature adaptability. ChiCD 4 shows temperature adaptability similar to that of ChiCD 1. It shows that the synergistic effect of E144K in the substrate-binding pocket and S288T at other positions is the key factor for 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 counted as 100%. 25 mM and 50 mM of Fe in the form of ferrous chloride were added to the detection system 2 to measure the enzyme activity. The enzyme activity of ChiCD measured at 60 °C was counted as 100%. 25 mM and 50 mM of Fe in the form of ferrous chloride were added to the detection system 2 to measure the enzyme activity. The results are as Figure 7 shown. ChiCD1 and ChiCD4 provided better enzyme activity retention performance in the presence of iron ions, remaining above 30% even at a concentration of 25 mM, and still having about 10% remaining at a concentration of 50 mM where the enzyme activity of ChiCD had basically disappeared. Such performance provides great convenience for actual field use (inevitably in contact with iron ions in foliar fertilizers or equipment).

[0163] The results of the antibacterial zone experiment carried out according to the method in Example 2 showed that, when using the same 200 μL of enzyme solution (prepared by a method basically the same as that in Example 1), due to better room temperature performance, its antibacterial zone was significantly larger than 18 mm of ChiCD, reaching about 26 mm.

Claims

1. A chitinase having anti-Fusarium graminearum 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 pesticides for inhibiting Fusarium graminearum.

3. 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.

4. A carrier, characterized in that The vector comprises the coding gene according to claim 3.

5. A host cell, characterized in that The host cell comprises the vector according to claim 4.

6. 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 5. The method according to claim 6 , further comprising a purification step.

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