Chitinase mutant as well as preparation method and application thereof

Abstract

The invention discloses a chitinase mutant obtained based on random mutation as well as a preparation method and application of the chitinase mutant. On the basis of an error-prone PCR (Polymerase Chain Reaction) technology, the mutant ChiA-7 (T165I / C253Y / A391T / N480S / T508S / K547T / I550V / N607T) of the chitinase ChiA of the bacillus thuringiensis is obtained by the invention. Compared with ChiA, the ChiA-7 has better stability, and the enzyme activity of the ChiA-7 is about 90% of the initial enzyme activity after the ChiA-7 is subjected to heat preservation for 3 hours at 40 DEG C in a phosphate buffer solution with the optimal catalysis condition of 40 DEG C and the pH value of 4.0, while the enzyme activity of the ChiA-7 is attenuated to 75% of the initial enzyme activity. The chiA-7 is connected with a carrier pET20b (+) and is transferred into escherichia coli Escherichia coli BL21 (DE3), and the extracellular enzyme activity of a recombinant bacterium reaches 10.23 U / mL, which is improved by 13.41% compared with the enzyme activity of a control bacterium for expressing chiA. The chitinase mutant obtained by the preparation method disclosed by the invention has good thermal stability and enzymatic activity, and can be applied to hydrolysis of chitin and inhibition of mould.

Description

Technical Field

[0001] The invention relates to a chitinase mutant modified based on random mutation and application thereof, belonging to the technical field of protein engineering and enzyme engineering. Background Art

[0002] Chitin is one of the most abundant biological resources on Earth and the second most readily available polysaccharide in nature. It primarily originates from the exoskeletons of arthropods, such as shrimp, crab, and other crustaceans, which have high chitin content in their shells. Chitin is also present in specialized tissue structures of certain mollusks, most notably the cartilage of squid. Fungal cell walls are also a significant source of chitin, with chitin being the primary component of the cell walls of most molds.

[0003] Chitinase (ChiA) from Bacillus thuringiensis is an exo-chitinase that hydrolyzes chitin into soluble oligomeric products, ultimately producing monomeric N-acetylglucosamine and diacetylchitobiose. In addition to hydrolyzing chitin, chitinase also exhibits significant antifungal activity, effectively protecting against fungal infection by degrading chitin in fungal cell walls.

[0004] The extracellularly expressed protein is directly secreted into the culture medium and can be collected directly from the culture medium, avoiding cell disruption and complex intracellular extraction steps, significantly simplifying the purification process and reducing production costs. Extracellular expression can also reduce the potential interference of the protein's large-scale accumulation in the cell with the host cell's internal environment. Directed evolution belongs to irrational design, which refers to simulating Darwin's natural evolution process in the laboratory, targeting the gene of a certain protein enzyme, inducing gene-level mutations and constructing a library, and then screening to obtain excellent mutants. The present invention constructed a recombinant Escherichia coli (Escherichia coli BL21 (DE3)) that secretes and expresses ChiA from Bacillus thuringiensis by fusion expression with the signal peptide PelB, and obtained the ChiA mutant ChiA-7 (T165I / C253Y / A391T / N480S / T508S / K547T / I550V /

[0005] N607T). Compared with ChiA, ChiA-7 has better stability and higher extracellular enzyme activity when expressed in culture. Summary of the Invention

[0006] The present invention aims to provide a chitinase mutant, a preparation method, and an application thereof. The chitinase mutant is based on chitinase (ChiA) from Bacillus thuringiensis with an amino acid sequence as shown in SEQ ID NO. 1, wherein the threonine at position 165 of the parent chitinase is mutated to isoleucine, the cysteine at position 253 is mutated to tyrosine, the alanine at position 391 is mutated to threonine, the aspartic acid at position 480 is mutated to serine, the threonine at position 508 is mutated to serine, the lysine at position 547 is mutated to threonine, the isoleucine at position 550 is mutated to valine, and the aspartic acid at position 607 is mutated to threonine.

[0007] A ChiA mutant library was constructed by error-prone PCR of the parental ChiA gene. This library was then ligated into the pET20b(+) vector using overlap extension PCR and transformed into Escherichia coli BL21(DE3) for gene expression. Library screening and sequencing analysis yielded the ChiA-7 mutant. Compared to ChiA, ChiA-7 exhibits superior stability. Under optimal catalytic conditions, at 40°C and pH 4.0 phosphate buffer, after 3 hours of incubation, ChiA-7 maintained approximately 90% of its initial activity, while ChiA decreased to 75% of its initial activity.

[0008] E. coli BL21 / pET20b(+)-chiA-7 carries the pET20b(+) plasmid linked to the DNA fragment encoding SEQ ID NO. 2, which can be used for extracellular expression of ChiA-7. The recombinant bacteria were inoculated into fermentation medium (1-2% Triton X-100, 11.8 g / L peptone, 23.6 g / L yeast extract, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, 5 g / L glycerol) and cultured at 25°C, 220 rpm for 2 hours. 0.5 mM IPTG was added and the culture was continued for 12-60 hours. The supernatant was collected to obtain the chitinase mutant.

[0009] The beneficial effects of the present invention are:

[0010] The present invention provides a chitinase mutant, a preparation method and an application thereof. Based on the error-prone PCR technology, the present invention obtains a mutant ChiA-7 of the chitinase ChiA of Bacillus thuringiensis. Compared with ChiA, ChiA-7 has better thermal stability. The extracellular secretion expression of the recombinant protein can avoid the steps of cell fragmentation and removal of intracellular foreign proteins, greatly reducing costs. ChiA-7 was connected to the vector pET20b(+) and transferred into Escherichia coli BL21(DE3). The extracellular enzyme activity of the recombinant bacteria reached 10.23U / mL, which was 13.41% higher than the enzyme activity of the control bacteria expressing ChiA. The present invention provides a useful method for chitinase production research and provides a valuable solution for promoting the comprehensive utilization of chitin. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Recombinant Escherichia coli extracellular chitinase activity;

[0012] Figure 2 Analysis of enzymatic properties of chitinase mutant ChiA-7;

[0013] Figure 3 Alignment of amino acid sequences of chitinase mutant ChiA-7 with its parent;

[0014] Figure 4 Structural modeling analysis of the chitinase mutant ChiA-7 protein;

[0015] Figure 5 The chitinase mutant ChiA-7 hydrolyzes chitin to produce N-acetylglucosamine;

[0016] Figure 6 Inhibition test of fungal spore germination by chitinase mutant ChiA-7. DETAILED DESCRIPTION

[0017] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0018] Example 1: Construction and screening of chitinase mutant library

[0019] Increase the change of Mn 2+A low-fidelity DNA polymerase (0.2 mM) and a low-fidelity DNA polymerase can increase the frequency of base mutations in the amplified product, allowing incorrect bases to be randomly incorporated into the amplified gene at a certain frequency, increasing the diversity of the mutation spectrum. This results in a DNA sequence with random mutations, allowing the selection of proteins that meet the needs. Using the Bacillus thuringiensis genome as a template, the chitinase gene chiA was amplified using primers 1-chiA-pelB(Nco I)+:CCAGCCGGCGATGGC CATGGCTATGAGGTCTCAAAAATTCAC and 1-chiA(Xho I)-:GTGGTGGTGGTG GTGCTCGAGGTTTTCGCTAATGACGGCATTTA. The plasmid pET-20b(+)-pelB-chiA was constructed by one-step cloning and ligation into pET-20b(+). Error-prone PCR was performed using pET-20b(+)-pelB-chiA as a template using primers mutation-F: TTGTTTAACTTTAAGAAGGAG ATATACATATG and mutation-R: GGTGGTGGTGGTGCTCGAG, as shown in Table 1. The resulting product was digested with Dpn I inhibitory endonuclease to remove the plasmid template. The reaction was performed for 1 hour and the product was recovered by gel recovery. The resulting product was the mutant library gene.

[0020] Overlap extension PCR was used to connect the mutant library gene to the vector. The pET-20b(+) plasmid was first linearized using primers pET-20b(+)-F: CATATGTATATC TCCTTCTTAAAGTTAAACAA and pET-20b(+)-R: CTCGAGCACCACCACCACC. The PCR amplification system is shown in Table 2, and the reaction conditions are the same as in Table 1. The mutant product and the linearized plasmid were then used as templates for overlap extension PCR to generate the mutant recombinant plasmid. The overlap extension PCR amplification system and reaction conditions are the same as in Table 2 and Table 1, respectively. The overlap extension PCR products were transferred into E. coli BL21 (DE3) and screened by colony PCR to obtain a mutant library. The recombinant bacteria were inoculated into fermentation medium (1-2% Triton X-100, peptone, yeast powder), cultured at 25°C and 220 rpm for 2 hours, 0.5 mM IPTG was added, and the culture was continued for 12-60 hours. The supernatant was collected to obtain the chitinase mutant.

[0021] Table 1 Parameters of error-prone PCR amplification system

[0022]

[0023] Table 2 PCR amplification system

[0024]

[0025] Chitinase activity assay method: Take 15g of white chitin powder and place it in a beaker. Add 250mL of concentrated hydrochloric acid and stir for 3-4h until no obvious granular material remains. Place it in a 4℃ refrigerator and let it stand for 24h. Add 600mL of 50% ethanol solution and stir for 1.5h. Centrifuge at 8000rpm for 10min. Collect the precipitate and wash it repeatedly with ultrapure water until it is neutral. Weigh 30g of the above precipitate and dilute it to 1L with PBS buffer of a specific pH. Mix it thoroughly and it can be used to obtain colloidal chitin (3%, w / v) solution. Take a 1.5mL centrifuge tube and add 200μL of colloidal chitin and 200μL of crude enzyme solution. Mix them by oscillation with phosphate buffer of appropriate pH and react at 37℃ for 1h. After the reaction, centrifuge at 12000rpm for 2min. Take 75μL of the supernatant and add 75μL of DNS reagent. Mix them by oscillation. Heat at 100℃ for 10min. After cooling, measure the OD value. 540 The light absorption value was measured three times. Chitinase activity unit (U) is defined as the amount of enzyme required to release 1 μmol NAG per hour under appropriate conditions.

[0026] The mutant ChiA-7 (1-2% Triton X-100, 11.8 g / L peptone, 23.6 g / L yeast extract, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, 5 g / L glycerol) with the highest chitinase activity was screened out, with an enzyme activity of 10.23 U / mL, which was 13.41% higher than that of the control group. Figure 1 .

[0027] Example 2: Analysis of enzymatic properties of mutant ChiA-7

[0028] In order to further understand the enzymatic properties of chitinase, the optimal temperature, optimal pH and thermal stability of the enzyme before and after mutation were analyzed and compared. The enzymatic activity of chitinase was measured at different temperatures (30-70°C). The temperature corresponding to the peak of the enzyme activity was determined as the optimal reaction temperature of the recombinant enzyme. The enzymatic activity of chitinase was measured in 10mM buffer diluted with 3.0-10.0 pH and at the optimal temperature, and the pH corresponding to the peak of the enzyme activity was determined as the optimal reaction pH of the enzyme. The recombinant enzyme was placed in a 40°C environment and after different periods of heat treatment, its enzymatic activity was measured. The relative residual enzyme activity of the recombinant enzyme after different periods of heat treatment was compared to understand the thermal stability of the enzyme. The results are shown in Figure 2The enzyme activity of BL21 / pET-20b(+)-chiA-7 was higher than that of other recombinant strains, maintaining a certain level of activity between 30 and 50°C, with peak activity at 37-50°C and reaching its peak at 40°C. Therefore, its optimal reaction temperature was determined to be 40°C, while activity decreased significantly above 40°C. The enzyme exhibited excellent thermostability, with a residual activity exceeding 96% after 60 minutes at 40°C, approximately 93% after 120 minutes, and approximately 89% after 180 minutes. In contrast, the enzyme activity and stability of BL21 / pET-20b(+)-pelB-chiA were lower, with a residual activity of approximately 75% after 180 minutes. The activity of recombinant ChiA was higher within the pH range of 3.0-5.0. The highest activity of BL21 / pET-20b(+)-chiA-7 was 10.22 U / mL at pH 3.0, while the activity of BL21 / pET-20b(+)-pelB-chiA was 8.61 U / mL at pH 4.0.

[0029] Example 3: Comparison of amino acid sequences of mutant ChiA-7 and partial structural changes at mutation sites

[0030] The inserted sequence of plasmid pET-20b(+)-chiA-7 was analyzed by Sanger sequencing to obtain the DNA sequence of mutant chiA-7, as shown in SEQ ID NO. 2:

[0031]

[0032] The protein sequence of ChiA-7 was derived from the DNA sequence, as shown in SEQ ID NO.1:

[0033] MAMRSQKFTLLLLSLLLFLPLFLTNFITPNLALADSPKQSQKIVGYFPSWG

[0034] VYGRNYQVA

[0035] DIDASKLTHLNYAFADICWNGKHGNPSTHPDNPNKQTWNCKESGVPLQNKEVPNGTLVLGEPWADVTKSYPGSGTTWEDCDKYARCGNFGELKRLKAKYPHLKTIISVGGWTWSNRFSDMAADEKTRKVFAESTVAFLRAYGFDGVDLDWE YPGVETIPGGSYRPEDKQNFTLLLQDVRNALNKAGAEDGKQYLLTIASGASQRYADHTELKKISQILDWINIMTYDFHGGWEATSNHNAALYKDPNDPAANTNFYVDGAINVYTNEGVPVDKLVLGVPFYGRGWKSCGKENNGQYQPCKPGSDGKLASKGTWDDYSTGDTGVYDYGDLATNYVNKNGFVRYWNDTAKVPYLYNATTGTFISYDDNESMKYKTDYIKTKGLSGAMFWELSGDCRTSPKYSCSGPKLLDTLVKELLGGPISQKDTEPPTNVKNIVVTNKNSNSVQLNWSASTDNVGVTEYEITAGEEKWSTTTNSITIKNLKPNTEYTFSVIAKDAAGNKSQPTALTVKTDEANTTPPDGNGTATFSVTSNWGSGYNFSIIIKNNGTTPIKNWKLEFDYSGNLTQVWDSKISSKTNNHYVITNAGWNGEIPPGGSITIGGAGTGNPAELLNAVISENLEHHHHHH(SEQ ID NO.1)

[0036] Multiple sequence alignments were performed between the mutant sequences and the wild-type gene to identify the base and corresponding ChiA-7 amino acid change sites (T165I / C253Y / A391T / N480S / T508S / K547T / I550V / N607T). To analyze the effects of the mutations on protein structure, a three-dimensional structural model of the mutants was constructed using a deep learning algorithm. A molecular visualization system was then used to analyze the topological differences between the mutants and the wild-type protein, focusing on the molecular conformational changes caused by the mutations and their effects on the spatial arrangement of key functional domains (such as the active center or ligand binding site). The most important mutations were located in the TIM-barrel core structure. The T165I mutation replaced threonine with isoleucine, enhancing local stability through hydrophobic interactions; the C253Y mutation introduced tyrosine, whose aromatic ring and hydroxyl groups can form π-π interactions and hydrogen bonds, optimizing protein folding; and the N480S mutation replaced asparagine with serine, whose shorter hydroxyl side chain may participate in proton transfer or stabilize the transition state, thereby improving catalytic rate. Results are shown in Figure 3. Figure 3 and Figure 4 .

[0037] Example 4: Application of ChiA-7 in hydrolyzing chitin to prepare N-acetylglucosamine

[0038] ChiA-7 can efficiently cut the β-1,4-glycosidic bonds in chitin and is used to hydrolyze chitin to produce N-acetylglucosamine. When 1.00g / L colloidal chitin is used as the substrate, the effects of different enzyme activity levels and reaction time combinations on the yield of N-acetylglucosamine show significant differences. Among them, the yield of N-acetylglucosamine reached a maximum value of 0.15g / L under the reaction conditions of 2U enzyme activity and 10h reaction. The results are shown in Figure 5 .

[0039] Example 5: Chitinase inhibition test on fungal spore germination

[0040] Take out the slant solid culture medium containing Aspergillus niger and Aspergillus kawachii from the 4°C refrigerator, dip the inoculating needle into the spores and streak them onto the PDA plate without antibiotics. Place the plate in a constant temperature and humidity incubator at 28°C. When a large single colony grows and produces spores, pour sterile ultrapure water into the plate, scrape the spores with a spreader and count them with a hemocytometer to prepare a concentration of 3×10 6 CFU / mL spore suspension. Take 200μL of spore suspension and evenly spread it on a PDA plate without antibiotics, and place Oxford cups in different areas. Add 200μL of ddH2O and crude enzyme solution respectively. After 48h of incubation, observe and record the antibacterial effect, take photos and keep them, and measure the diameter of the inhibition zone (unit: cm) from three different angles. The diameter of the inhibition zone of the control group is recorded as the diameter of the Oxford cup (0.6cm). The results are shown in Figure 6(A: Aspergillus kawachii; B: Aspergillus niger (I: dd H2O, II: ChiA)) and Table 3. Figure 6 As shown in the figure, the diameter of the inhibition zone was measured after 48 hours of incubation. The results showed that the chitinase ChiA had a significant inhibitory effect on both A. niger and A. kawachii, with a particularly strong inhibitory effect on A. niger. This phenomenon is closely related to the difference in chitin content in the cell walls of the two fungi. A. niger has a relatively high chitin content in its cell wall, accounting for 15-20% of the dry weight of the cell wall, while A. kawachii only has 10-15%. Therefore, the stronger inhibitory effect of ChiA on A. niger is consistent with the difference in chitin content in the cell wall.

[0041] Table 3 Data analysis of the inhibitory effect of ChiA on fungal spore germination

[0042]

[0043] Note: Data are presented as mean ± standard deviation

[0044] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A chitinase mutant, characterized in that: The amino acid sequence of the chitinase mutant ChiA-7 is shown in SEQ ID NO.

1.

2. The chitinase mutant according to claim 1, characterized in that Chitinase (ChiA) from Bacillus thuringiensis was used as the parent and obtained by error-prone PCR. The mutants were characterized by mutations at position 165: threonine to isoleucine, position 253: cysteine to tyrosine, position 391: alanine to threonine, position 480: aspartic acid to serine, position 508: threonine to serine, position 547: lysine to threonine, position 550: isoleucine to valine, and position 607: aspartic acid to threonine.

3. The gene fragment of the mutant according to claim 1, characterized in that The nucleotide sequence of the gene fragment is shown in SEQ ID NO:

2.

4. A recombinant expression vector containing the coding gene according to claim 3.

5. A recombinant host cell containing the recombinant expression vector according to claim 3.

6. The method for preparing the chitinase mutant according to claim 1, characterized in that The following steps are involved: (1) Recombinant Escherichia coli BL21 (DE3) containing the pET20b(+) plasmid linked to the DNA fragment described in SEQ ID NO: 2, named BL21 / pET20b(+)-chiA-7, can be used for extracellular expression of ChiA-7. (2) The recombinant bacteria BL21 / pET20b(+)-chiA-7 were inoculated into a fermentation medium (1-2% Triton X-100, 11.8 g / L peptone, 23.6 g / L yeast powder, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, 5 g / L glycerol), cultured at 25°C and 220 rpm for 2 h, 0.5 mM IPTG was added, and the culture was continued for 12-60 h. The supernatant was collected to obtain the chitinase mutant.

7. The use of the chitinase mutant according to claim 1, characterized in that: The application is to utilize the chitinase mutant to hydrolyze chitin to obtain N-acetylglucosamine and inhibit the germination of fungal spores.

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