Thermostable chitinase mutant and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a heat-stable chitinase mutant and application thereof. According to the specific technical scheme, the chitinase mutant is obtained on the basis of wild chitinase; carrying out any one or more of the following mutations: mutating serine at the 67th site into glycine, mutating lysine at the 177th site into arginine, mutating alanine at the 220th site into valine, mutating asparagine at the 257th site into tyrosine, and mutating asparagine at the 271th site into glutamic acid; the amino acid sequence of the wild chitinase is as shown in SEQ ID NO: 1. Compared with a wild type, the mutant has the advantages that the degradation mode of the mutant on chitin is not obviously changed, and the hydrolysate is still mainly chitobiose; however, the optimal reaction temperature and melting temperature of the mutant are obviously improved, the half-life period at 60 DEG C is obviously prolonged, and the application potential is huge.

Description

Technical Field

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

[0002] Chitin is a polysaccharide composed of N-acetyl-D-glucosamine (GlcNAc) units connected by β-(1,4)-glycosidic bonds. It is the second most abundant biomass resource in nature after cellulose. Due to the high crystallinity and low solubility of chitin, its application is relatively limited. The hydrolysis products of chitin are chitin oligosaccharides (CHOs), which have a variety of biological activities, including antioxidant, antibacterial, immunomodulatory and anti-tumor properties, and can be widely used in food, medicine, agriculture and other industries. Therefore, it is very important to efficiently produce high-quality CHOs from chitin.

[0003] At present, chitin is generally degraded chemically or enzymatically to produce CHOs. However, the chemical hydrolysis of chitin requires the use of a large amount of strong acid and strong base, and has obvious defects such as low conversion rate, serious pollution and high production cost. Moreover, this type of method is non-selective for the degradation of chitin and will produce CHOs with different degrees of polymerization. The enzymatic depolymerization of chitin can be carried out 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 thermostable properties is crucial to improving their potential for industrial applications. Summary of the invention

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

[0007] To achieve the above-mentioned purpose, the technical scheme 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 the serine at position 67 to glycine, mutating the lysine at position 177 to arginine, mutating the alanine at position 220 to valine, mutating the asparagine at position 257 to tyrosine, and mutating the 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, in a strain of Bacillus circulans, the DNA encoding the chitinase mutant is used to replace the 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 invention has the following beneficial effects: the 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°C, 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. Moreover, the mutant Mu5 can be repeatedly used for the degradation of chitin, and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 2Schematic diagram of 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 at different times;

[0023] Figure 5 It is a schematic diagram comparing the melting temperatures of various chitinases;

[0024] Figure 6 This is a schematic diagram comparing the optimal reaction temperatures of various chitinases;

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

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

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

[0028] The 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 edited wild-type chitinase is shown in SEQ ID NO: 2, the amino acid sequence of chitinase mutant Mu5 is shown in SEQ ID NO: 3, and the DNA sequence of edited chitinase mutant Mu5 is shown in SEQ ID NO: 4. Compared with wild-type chitinase, the thermal stability of chitinase mutant Mu5 is significantly improved, which is specifically manifested in that the optimal reaction temperature and dissolution temperature are significantly increased, and the half-life at 60°C is greatly extended.

[0030] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. If not specifically indicated, the technical means used in the embodiment are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least 3 repetitions, and all the repetitions obtained 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 3,5-dinitrosalicylic acid, 18.2 g potassium sodium tartrate, 0.5 g crystalline phenol, 0.5 g sodium sulfite, 2 g sodium hydroxide, heat and mix, then adjust the volume to 100 mL.

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

[0036] 1. Use site-directed mutagenesis primers to perform PCR amplification on the full-length plasmid to obtain the target mutant. The PCR amplification system is: 5× PrimeSTAR Buffer 10μL, dNTP Mixture 4μL, upstream primer (10μM) 1μL, downstream primer (10μM) 1μL, DNA template 1μL, PrimeSTAR HSDNA Polymerase 0.5μL, ddH2O 32.5μL. The PCR amplification conditions are: 98℃ pre-denaturation for 5min; then denaturation at 98℃ for 10s, annealing at 55℃ for 10s, and extension at 72℃ for 7min30s as one cycle, for a total of 30 cycles; 72℃ thorough extension for 5min, and finally insulation at 12℃. The template plasmid in the PCR system was digested with DpnⅠ at 37℃ for 2 hours, and the purified and recovered PCR reaction solution was transformed into E.coli DH5α to screen positive clones and sequence them. The primers for each mutant for single-site mutation of wild-type chitinase are shown in Table 1.

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

[0038] Table 1 Primer comparison table for each mutant

[0039]

[0040] 2. Extract plasmids from the positive clones with correct sequencing obtained in step 1, and transform the plasmids into E. coli BL21 (DE3) for heterologous expression. The specific steps of heterologous expression are: spread the bacterial solution after plasmid transformation on LB solid medium plates containing 60 μg / mL kanamycin, and culture at 37°C until OD 600 0.6~0.8. Then 1mmol of isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium for induction, and the culture was continued at 16℃ for 12h. After the culture was completed, the cells were collected by centrifugation (10000g, 10 minutes, 4℃). The collected cells were resuspended in lysis buffer (50mM Tris-HCl, 500mM NaCl, pH=8.0) and sonicated in ice water to promote cell lysis. After centrifugation (10000g, 30 minutes, 4℃), 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 150mM imidazole. The purified protein samples were analyzed by SDS-PAGE, and the protein concentration was determined using the Bradford method. The extraction and purification methods of wild-type chitinase and each mutant chitinase were the same, all for this purpose.

[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 are wild-type chitinase, N257Y mutant, N271E mutant, K177R mutant, S67G mutant, A220V mutant and Mu5. The results showed that after induced expression and purification, wild-type chitinase and mutant protein samples with high purity were obtained.

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

[0043] 1. Chitinase activity determination method: Using colloidal chitin as a substrate, chitinase activity was determined by a modified 3,5-dinitrosalicylic acid (DNS) method. 10 μL of 3 mg / mL enzyme solution was added to 290 μL of 1% (w / v) colloidal chitin solution. The reaction was incubated at 50°C for 10 minutes, then terminated in a boiling water bath for 5 minutes, and the reducing sugar produced in the reaction was determined by 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) only retained 48.81% of the residual enzyme activity, while the five single-point mutants all retained more than 70% of the enzyme activity and had 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, and then rapidly cooled and centrifuged to remove the precipitate. The enzyme activity in the supernatant of each group was measured.

[0049] The results are as follows Figure 3 As shown. The results showed that the initial enzyme activity of wild-type chitinase was 100%, and the initial enzyme activity of Mu5 was 109%, slightly higher than that of wild-type. After heat treatment at 55℃ for 120min, the wild-type chitinase was almost inactivated, while Mu5 still maintained more than 95% of its enzyme activity; after heat treatment for 300min, Mu5 still maintained nearly 90% of its enzyme activity. This shows that the thermal stability of Mu5 is far superior to that of the wild-type.

[0050] 3. Determination of enzyme activity loss half-life (t 1 / 2 ): 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 activity was measured.

[0051] The results are as follows Figure 4 The results showed that the half-life of wild-type chitinase was 5 minutes, while the half-life 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 T of the enzyme was 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 dispensed into a 96-well PCR plate and subjected to a temperature scan ranging from 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 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 enzyme activity at 50°C, and the enzyme activity of Mu5 was even higher at 55°C.

[0056] In summary, the mutant Mu5 showed better heat resistance than the wild-type chitinase and the chitinase mutant with only single-site mutations. The optimal reaction temperature of Mu5 was higher and the half-life was longer. Compared with the wild-type, the thermal stability of the mutant Mu5 was significantly improved.

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

[0058] 1. Comparison of substrate conversion rate: The purified enzyme (5 mg / mL) obtained in Example 1 was mixed with 1% (w / v) colloidal chitin solution (300 μL) and reacted at 50°C. Samples were collected at specified time intervals and the content of reducing sugar in the hydrolyzate was determined by 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, and only 65% ​​of the substrate was converted within 90 minutes. However, Mu5 achieved a conversion rate of 97% within 90 minutes. This shows that even at the optimal reaction temperature of wild-type chitinase, Mu5 also showed a better ability to degrade chitin.

[0060] 2. Reusability: The purified enzyme (5 mg / mL) obtained in Example 1 was mixed with 1% (w / v) colloidal chitin solution (300 μL), hydrolyzed at 50° C. for 90 min, and the amount of CHOs product was measured. Subsequently, an equal amount of fresh colloidal chitin was directly added to the original reaction system, and the hydrolysis was continued under the same conditions. The substrate conversion was measured for 5 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 show 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 (5 mg / mL) obtained in Example 1 was mixed with 1% (w / v) colloidal chitin solution (300 μL), incubated at 50°C for 90 min, and the reaction was stopped in a boiling water bath for 5 minutes. After centrifugation, the supernatant of the mixture was concentrated 10 times. The degradation products were then analyzed using thin layer chromatography (TLC). The developing agent 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 Fig. 9 The results showed that the main hydrolysis products of the wild type and the Mu5 mutant were chitosan, indicating that the mutation of the Mu5 mutant did not affect the degradation pattern.

[0064] The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design 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 all fall within the protection scope determined by the claims of the present invention.

Claims

1. A chitinase mutant, characterized in that: Based on the wild-type chitinase, any one or more of the following mutations are performed: 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.

2. A chitinase mutant, characterized in that: Based on the wild-type chitinase, the following mutations are performed: 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.

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

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

5. A recombinant plasmid containing nucleotides expressing the amino acids corresponding to the chitinase mutant according to claim 1 or 2.

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

7. A strain of Bacillus circulans, characterized in that: The DNA encoding the chitinase mutant according to claim 1 or 2 is used to replace the DNA encoding the wild-type chitinase.

8. Use of the chitinase according to claim 1 or 2 in the preparation of chitin oligosaccharides.

9. Use of the chitinase according to claim 1 or 2 in enzymatic hydrolysis of chitin.

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

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