A thermostable chitinase mutant and its application

Abstract

The present invention belongs to the field of enzyme engineering technology and specifically relates to a thermostable chitinase mutant and its application. The specific technical solution includes: a chitinase mutant, based on a wild-type chitinase, undergoing any one or more of the following mutations: mutation of serine at position 67 to glycine, lysine at position 177 to arginine, alanine at position 220 to valine, asparagine at position 257 to tyrosine, and asparagine at position 271 to glutamate; the amino acid sequence of the wild-type chitinase is shown in SEQ ID NO: 1. Compared with the wild-type chitinase, the mutant exhibits no significant change in its chitin degradation pattern, with chitobiose still being the primary hydrolysis product; however, the mutant exhibits significantly higher optimal reaction and melting temperatures, and a significantly prolonged half-life at 60°C, suggesting significant application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and particularly relates to a thermostable chitinase mutant and application thereof. Background Art

[0002] Chitin is a polysaccharide composed of N-acetyl-D-glucosamine (GlcNAc) units linked by β-(1,4)-glycosidic bonds. It is the second most abundant biomass resource in nature, second only to cellulose. Chitin's high crystallinity and low solubility limit its applications. Chitin oligosaccharides (CHOs), the hydrolysis products of chitin, exhibit a variety of biological activities, including antioxidant, antimicrobial, immunomodulatory, and anti-tumor properties, and are widely used in the food, pharmaceutical, and agricultural industries. Therefore, the efficient production of high-quality CHOs from chitin is crucial.

[0003] Currently, CHOs are typically produced by chemical or enzymatic degradation of chitin. However, chemical hydrolysis of chitin requires the use of large amounts of strong acids and bases, resulting in significant drawbacks such as low conversion rates, severe pollution, and high production costs. Furthermore, these methods are non-selective for chitin degradation, producing CHOs with varying degrees of polymerization. Enzymatic depolymerization of chitin, on the other hand, can be performed under relatively mild conditions and produce specific CHOs in a controlled manner.

[0004] Chitinase plays a vital role in the degradation of chitin. However, due to the poor stability of the enzyme itself, especially poor thermal stability, its enzymatic hydrolysis effect is not satisfactory.

[0005] Therefore, providing new chitinases with ideal thermostability is crucial to improving their potential for industrial applications. Summary of the Invention

[0006] The purpose of the present invention is to provide a thermostable chitinase mutant and application thereof.

[0007] To achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is: a chitinase mutant, based on the wild-type chitinase, undergoes any one or more of the following mutations: mutating serine at position 67 to glycine, mutating lysine at position 177 to arginine, mutating alanine at position 220 to valine, mutating asparagine at position 257 to tyrosine, and mutating asparagine at position 271 to glutamate; the amino acid sequence of the wild-type chitinase is shown in SEQ ID NO: 1.

[0008] Correspondingly, a chitinase mutant is prepared by subjecting the wild-type chitinase to the following mutations: the serine at position 67 is mutated to glycine, the lysine at position 177 is mutated to arginine, the alanine at position 220 is mutated to valine, the asparagine at position 257 is mutated to tyrosine, and the asparagine at position 271 is mutated to glutamate; the amino acid sequence of the wild-type chitinase is shown in SEQ ID NO: 1.

[0009] Accordingly, the DNA encoding the chitinase mutant.

[0010] Correspondingly, an expression plasmid containing said DNA.

[0011] Correspondingly, a recombinant plasmid containing nucleotides expressing the corresponding amino acids of the chitinase mutant.

[0012] Correspondingly, a host microorganism containing the expression plasmid or the recombinant plasmid.

[0013] Accordingly, a strain of Bacillus circulans is transformed with DNA encoding the chitinase mutant to replace DNA encoding the wild-type chitinase.

[0014] Correspondingly, the chitinase is used in the preparation of chitin oligosaccharides.

[0015] Correspondingly, the chitinase is used in enzymatic hydrolysis of chitin.

[0016] Preferably, the application temperature is 30-60°C, more preferably 40-60°C.

[0017] The present invention has the following beneficial effects: the present invention provides a new chitinase mutant Mu5, which can significantly improve the thermal stability of chitinase (BcChiA) produced by Bacillus circulans WL-12.

[0018] Compared with the wild type, the degradation pattern of chitin by mutant Mu5 did not change significantly, and its hydrolysis products were still mainly chitobiose; however, the optimal reaction temperature of mutant Mu5 increased by 5℃, and the melting temperature (T m ) increased by 10°C, and the half-life at 60°C was extended from 5 minutes to 295 minutes. Furthermore, the mutant Mu5 can be reused for chitin degradation, showing great potential for application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 SDS-PAGE electrophoresis detection of each chitinase;

[0020] Figure 2Schematic diagram of the enzyme activity of each chitinase after incubation at 55°C for 15 min;

[0021] Figure 3 Schematic diagram of the enzyme activity of each chitinase after incubation at 55°C for different time periods;

[0022] Figure 4 Schematic diagram of the changes in enzyme activity of each chitinase at 60°C for different time periods;

[0023] Figure 5 Schematic diagram comparing the melting temperatures of various chitinases;

[0024] Figure 6 Schematic diagram for comparing the optimal reaction temperatures of various chitinases;

[0025] Figure 7 Schematic diagram comparing the substrate conversion rates of various chitinases;

[0026] Figure 8 This is a schematic diagram of the reuse of chitinase Mu5;

[0027] Figure 9 Schematic diagram of the hydrolysis products of various chitinases. DETAILED DESCRIPTION

[0028] The present invention provides a chitinase mutant Mu5, which is obtained by mutating the serine (S) at position 67 to glycine (G), the lysine (K) at position 177 to arginine (R), the alanine (A) at position 220 to valine (V), the asparagine (N) at position 257 to tyrosine (Y), and the asparagine (N) at position 271 to glutamic acid (E) on the basis of a wild-type chitinase.

[0029] The amino acid sequence of wild-type chitinase is shown in SEQ ID NO: 1, the DNA sequence of the edited wild-type chitinase is shown in SEQ ID NO: 2, the amino acid sequence of the chitinase mutant Mu5 is shown in SEQ ID NO: 3, and the DNA sequence of the edited chitinase mutant Mu5 is shown in SEQ ID NO: 4. Compared to wild-type chitinase, the chitinase mutant Mu5 exhibits significantly improved thermal stability, as demonstrated by significantly higher optimal reaction and dissolution temperatures and a significantly prolonged half-life at 60°C.

[0030] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, rather than all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least three repetitions, and all the data obtained in each repetition are valid data.

[0031] The culture medium and reagents used in the examples are as follows:

[0032] Equilibration buffer: 50 mM Tris-HCl, pH = 8.0, 20 mM imidazole, 500 mM sodium chloride;

[0033] Elution buffer: 50 mM Tris-HCl, pH 8.0, 150 mM imidazole, 500 mM sodium chloride;

[0034] DNS solution: 0.65 g of 3,5-dinitrosalicylic acid, 18.2 g of potassium sodium tartrate, 0.5 g of crystalline phenol, 0.5 g of sodium sulfite, and 2 g of sodium hydroxide. Heat and mix thoroughly, then adjust the volume to 100 mL.

[0035] Example 1: Construction and acquisition of chitinase mutants

[0036] 1. Target mutants were obtained by PCR amplification of the full-length plasmid using site-directed mutagenesis primers. The PCR amplification system consisted of: 10 μL of 5× PrimeSTAR Buffer, 4 μL of dNTP Mixture, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 1 μL of DNA template, 0.5 μL of PrimeSTAR HSDNA Polymerase, and 32.5 μL of ddH2O. PCR amplification conditions were: initial denaturation at 98°C for 5 min; 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C for 7 min 30 s; a final extension at 72°C for 5 min, followed by a final incubation at 12°C. The template plasmid in the PCR system was digested with Dpn I at 37°C for 2 hours. The purified PCR reaction was transformed into E. coli DH5α, and positive clones were screened and sequenced. The primers for each mutant for single-site mutation of wild-type chitinase are shown in Table 1.

[0037] Chitinase mutants S67G, K177R, A220V, N257Y, N271E and Mu5 (S67G / K177R / A220V / N257Y / N271E) were constructed respectively, that is, chitinase mutants with only one site mutated and chitinase mutants with all five sites mutated (Mu5) were constructed respectively.

[0038] Table 1 Comparison table of primers for each mutant

[0039]

[0040] 2. Extract the plasmid from the positive clones obtained in step 1 and transform the plasmid into E. coli BL21 (DE3) for heterologous expression. The specific steps of heterologous expression are as follows: spread the transformed bacterial solution on LB solid medium plates containing 60 μg / mL kanamycin and culture at 37°C until the OD 600 The expression of β-D-thiogalactopyranoside (IPTG) was 0.6–0.8. Induction was then performed by adding 1 mmol of isopropyl-β-D-thiogalactopyranoside (IPTG) to the culture medium, and the cells were cultured at 16°C for 12 h. After completion of the culture, the cells were harvested by centrifugation (10,000 g, 10 min, 4°C). The harvested cells were resuspended in lysis buffer (50 mM Tris-HCl, 500 mM NaCl, pH = 8.0) and sonicated in ice water to promote cell lysis. After centrifugation (10,000 g, 30 min, 4°C), the supernatant was transferred to a Ni-NTA affinity chromatography column treated with equilibration buffer, and the target protein was collected using an elution buffer containing 150 mM imidazole. The purified protein samples were analyzed by SDS-PAGE, and the protein concentration was determined using the Bradford method. The wild-type chitinase and the various mutant chitinases were extracted and purified using the same methods.

[0041] The results were detected by SDS-PAGE electrophoresis. Figure 1 shown. Figure 1 In the figure, M is a protein marker, and 1-7 represent wild-type chitinase, N257Y mutant, N271E mutant, K177R mutant, S67G mutant, A220V mutant, and Mu5, respectively. The results showed that after induced expression and purification, highly pure wild-type chitinase and mutant protein samples were obtained.

[0042] Example 2: Performance demonstration of wild-type chitinase and chitinase mutants

[0043] 1. Chitinase Activity Assay: Chitinase activity was determined using a modified 3,5-dinitrosalicylic acid (DNS) method using colloidal chitin as the substrate. 10 μL of a 3 mg / mL enzyme solution was added to 290 μL of a 1% (w / v) colloidal chitin solution. The reaction was incubated at 50°C for 10 minutes, then terminated by boiling in a water bath for 5 minutes. The reducing sugars produced were then determined using the DNS method.

[0044] Definition of chitinase activity (U): The amount of enzyme required to produce 1 μmol of reducing sugar per minute is one unit of enzyme activity.

[0045] 2. Effect of heat treatment on enzyme activity:

[0046] (1) The wild-type chitinase and the five single-point mutants obtained in Example 1 were incubated at 55°C for 15 min, then rapidly cooled and centrifuged to remove the precipitate. The enzyme activity in the supernatant of each group was measured.

[0047] The initial enzyme activity (gray) and enzyme activity after heat treatment (red) of each chitinase are shown in Figure 2. Figure 2 The results showed that after heat treatment, the wild type (WT) retained only 48.81% of the residual enzyme activity, while the five single-point mutation mutants retained more than 70% of the enzyme activity, showing higher thermal stability.

[0048] (2) The wild-type chitinase and the Mu5 mutant obtained in Example 1 were incubated at 55°C for 15, 30, 45, 60, 120, 180, 240, 270, and 300 min, respectively, followed by rapid cooling and centrifugation to remove the precipitate. The enzyme activity in the supernatant of each group was measured.

[0049] The results are as follows Figure 3 The results showed that, while the initial enzymatic activity of wild-type chitinase was 100%, that of Mu5 was 109%, slightly higher than that of wild-type chitinase. After heat treatment at 55°C for 120 minutes, wild-type chitinase was nearly inactivated, while Mu5 still retained over 95% of its activity. Even after heat treatment for 300 minutes, Mu5 still retained nearly 90% of its activity, demonstrating that Mu5's thermal stability is far superior to that of the wild-type.

[0050] 3. Determination of enzyme activity loss half-life (t 1 / 2 ) is defined as the time required for the enzyme to lose 50% activity at 60° C. Wild-type chitinase and Mu5 were treated at 60° C. for different time periods, and the enzyme activities were measured.

[0051] The results are as follows Figure 4 The results showed that the half-life of wild-type chitinase was 5 minutes, while that of Mu5 was as long as 295 minutes, which was 59 times that of the wild-type.

[0052] 4. Protein melting temperature (T m ): This is an important indicator for measuring the thermal stability of enzymes, reflecting the conformational changes of enzymes under high temperature conditions. The enzyme T is determined by differential scanning fluorimetry (DSF). mvalue. The specific operation is as follows: It was carried out on a CFX96 real-time PCR instrument (Bio Rad, Hercules CA, USA) with an excitation wavelength of 490 nm and an emission wavelength of 575 nm. The purified enzyme was diluted to 10 mg / mL and mixed with 10×SYPRO dye in a 20 μL reaction system (50 mM Tris-HCl, pH 8.0). The mixture was divided into 96-well PCR plates and subjected to a temperature scan in the range of 20°C to 95°C at a heating rate of 1°C per minute. During the entire temperature scan, the changes in the fluorescence signal were monitored in real time to determine the melting point of the protein. All measurements were repeated three times.

[0053] The results are as follows Figure 5 As shown. Wild-type T m is 50±0.3℃, T of Mu5 m It is 60±0.4℃.

[0054] 5. Optimum reaction temperature: The enzyme activities of wild-type chitinase and mutant Mu5 were measured at temperatures of 20, 30, 40, 45, 50, 55, 60 and 70°C, respectively.

[0055] The results are as follows Figure 6 The results showed that the wild-type chitinase had the highest activity at 50°C, and the Mu5 had even higher activity at 55°C.

[0056] In summary, the Mu5 mutant exhibited improved heat resistance compared to wild-type chitinase and chitinase mutants with single-site mutations. Mu5 had a higher optimal reaction temperature and a longer half-life. Compared to the wild-type, the Mu5 mutant exhibited significantly improved thermal stability.

[0057] Example 3: Hydrolysis characteristics of wild-type chitinase and chitinase mutant products

[0058] 1. Comparison of Substrate Conversion Rates: The purified enzyme obtained in Example 1 (5 mg / mL) was mixed with a 1% (w / v) colloidal chitin solution (300 μL) and reacted at 50°C. Samples were collected at designated time intervals, and the reducing sugar content in the hydrolyzate was determined using the DNS method. The substrate conversion rate was calculated based on the molecular weight of the disaccharide.

[0059] The results are as follows Figure 7 The results showed that the reaction rate of wild-type chitinase dropped rapidly after 30 minutes, with only 65% ​​of the substrate converted within 90 minutes. Mu5, on the other hand, achieved a 97% conversion rate within 90 minutes. This suggests that even at the optimal reaction temperature of wild-type chitinase, Mu5 exhibited superior chitin degradation.

[0060] 2. Reusability: The purified enzyme obtained in Example 1 (5 mg / mL) was mixed with a 1% (w / v) colloidal chitin solution (300 μL) and hydrolyzed at 50°C for 90 min. The CHOs product was measured. An equal amount of fresh colloidal chitin was then added directly to the original reaction system, and hydrolysis was continued under the same conditions. Substrate conversion was measured for five cycles in the same reaction system.

[0061] The results are as follows Figure 8 As shown. The results showed that after five rounds of reaction, the cumulative amount of total product of WT was only 23.6mM. In contrast, Mu5 exhibited excellent catalytic performance. After five rounds of reaction, the concentration of monosaccharide (CHO) released by Mu5 was 86.14mM, which was 3.65 times that of WT. In addition, in the fifth round of reaction, the conversion rate of Mu5 to the substrate remained above 80%, indicating that Mu5 has good reusability. These results indicate that Mu5 exhibits higher catalytic efficiency and better stability in the continuous hydrolysis of colloidal chitin.

[0062] 3. Comparison of Hydrolysis Products: The purified enzyme obtained in Example 1 (5 mg / mL) was mixed with a 1% (w / v) colloidal chitin solution (300 μL) and incubated at 50°C for 90 min. The reaction was stopped in a boiling water bath for 5 minutes. After centrifugation, the supernatant of the mixture was concentrated 10-fold. Degradation products were then analyzed using thin-layer chromatography (TLC). The developing solvent consisted of n-butanol, methanol, 28% ammonia water, and water (10:8:4:2, v / v). After development, the plate was treated with aniline-diphenylamine stain (4 mL aniline, 4 g diphenylamine, 200 mL acetone, and 30 mL 85% phosphoric acid) and then baked at 120°C until spots appeared.

[0063] The results are as follows Figure 9 The results showed that the major hydrolysis product of both the wild type and the Mu5 mutant was chitobiose, indicating that the mutation of the Mu5 mutant did not affect the degradation pattern.

[0064] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A chitinase mutant, characterized in that: Based on the wild-type chitinase, the following mutations were simultaneously performed: serine at position 67 was mutated to glycine, lysine at position 177 was mutated to arginine, alanine at position 220 was mutated to valine, asparagine at position 257 was mutated to tyrosine, and asparagine at position 271 was mutated to glutamate; the amino acid sequence of the wild-type chitinase is shown in SEQ ID NO:

1.

2. A DNA encoding the chitinase mutant according to claim 1.

3. An expression plasmid containing the DNA according to claim 2.

4. A recombinant plasmid containing nucleotides that express the amino acids corresponding to the chitinase mutant according to claim 1.

5. A host microorganism containing the expression plasmid according to claim 3 or the recombinant plasmid according to claim 4.

6. A strain of Bacillus circulans characterized by: The DNA encoding the chitinase mutant according to claim 1 is used to replace the DNA encoding the wild-type chitinase.

7. Use of the chitinase mutant according to claim 1 in the preparation of chitin oligosaccharides.

8. Use of the chitinase mutant according to claim 1 in enzymatic hydrolysis of chitin.

9. The use according to claim 7 or 8, characterized in that: The application temperature is 30-60°C.

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