Raw starch hydrolase mutant Amyh1: G207H with improved stability and application of raw starch hydrolase mutant Amyh1: G207H
By performing amino acid mutations on the raw starch hydrolase Amyh1, a mutant Amyh1:G207H with improved thermal stability and specific enzyme activity was constructed, which solved the problem of insufficient stability of the existing enzyme, achieved efficient hydrolysis of algae raw starch and extended the shelf life, and is suitable for industrial applications of algae biomass.
Patent Information
- Application Number
- CN202511259911.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-04
- Publication Date
- 2025-10-17
AI Technical Summary
Existing raw starch hydrolyzing enzymes have poor thermal stability, resulting in a short shelf life and an inability to function continuously during the hydrolysis process, especially low efficiency in hydrolyzing algal raw starch.
Through computer-aided design, the amino acid glycine at position 207 of the raw starch hydrolase Amyh1 was mutated to histidine, constructing a mutant Amyh1:G207H with improved thermal stability and specific enzyme activity. Specific methods included homology modeling, co-evolution analysis, and overlap extension PCR, and the expression strain Escherichia coli BL21(DE3)/pET28a(+)-Amyh1:G207H was constructed.
The mutant enzyme's thermal stability at 35°C was increased by 3.5 times, and its specific enzyme activity was maintained or improved. The efficiency of hydrolyzing raw algae starch reached 33%, extending the shelf life, making it suitable for industrial applications using algae biomass as raw materials.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biotechnology, and particularly relates to a raw starch hydrolyzing enzyme mutant Amyh1:G207H with improved stability and application thereof. BACKGROUND
[0002] Raw starch hydrolyzing enzyme refers to an enzyme that can directly degrade raw starch granules below the gelatinization temperature of starch. At present, among all the discovered alpha-amylases, only about 10% of the alpha-amylases have the ability to degrade raw starch, which are distributed in bacteria, fungi and animals. These alpha-amylases can hydrolyze raw starch of potato, wheat, corn, rice and the like. However, the specific activity is generally low, for example, the specific enzyme activity of the alpha-amylase from Bacillus amyloliquefaciens is 44.6 U / mg, the specific enzyme activity of the alpha-amylase from Streptomyces badius DB-1 is 148.1 U / mg, and the highest specific enzyme activity of the alpha-amylase from Bacillus acidicola reaches 874.5 U / mg. There are only a few reports about raw starch hydrolyzing alpha-amylase hydrolyzing raw starch of algae, and the specific enzyme activity of the alpha-amylase from Clostridium acetobutylicum to raw starch of microalgae is only 303.2 U / mg. At present, algae are a potential new source of starch, so microalgae that can accumulate high starch content are being paid more and more attention as a suitable substitute for food crops in the production of biofuels, and there is an urgent need for a raw starch hydrolyzing enzyme with high hydrolysis capacity to raw starch of algae.
[0003] Thermal stability is one of the important properties required for enzymes used in starch processing. Many starch amylases have a short shelf life due to their poor stability, and cannot continuously exert hydrolysis during the hydrolysis process. Therefore, using protein engineering technology to obtain raw starch hydrolyzing enzymes with high specific activity and good thermal stability plays an important role in hydrolyzing raw starch of algae, which is a potential new source of starch. SUMMARY
[0004] The present application provides a raw starch hydrolytic enzyme mutant Amyh1:G207H with improved stability and its application to solve the above problems of the prior art. The present application is based on raw starch hydrolytic enzyme Amyh1, and a mutant with greatly improved thermal stability is obtained through computer-aided design. The specific enzyme activity of the mutant is 8311 U / mg, which is 1.1 times that of the starting enzyme when corn raw starch is used as the substrate. When algal raw starch is used as the substrate, the specific enzyme activity of the mutant reaches 2622 U / mg, which is 1.02 times that of the starting enzyme. Moreover, the thermal stability of the mutant is greatly improved, and the thermal stability is 3.5 times that of the starting enzyme under the conditions of 35℃ and pH 7.0. In the algal starch hydrolysis experiment, 367.5 mg / g DW (dry weight of biomass) of reducing sugar is produced after 3 h of reaction, and the hydrolysis rate can reach 33%. The mutant enzyme has potential value in industrial applications using algal biomass as raw material.
[0005] The raw starch hydrolytic enzyme mutant Amyh1:G207H of the present application has an amino acid sequence as shown in SEQ ID NO: 1. Specifically, the 207th amino acid glycine in the Amyh1 amino acid sequence is mutated to histidine.
[0006] The coding gene of the raw starch hydrolytic enzyme mutant of the present application has a nucleotide sequence as shown in SEQ ID NO: 2.
[0007] The expression strain of the raw starch hydrolytic enzyme mutant of the present application is classified and named as Escherichia coli BL21(DE3) / pET28a(+)-Amyh1:G207H, which has been preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M 2025726, a preservation time of April 8, 2025, and a preservation address of Wuhan, China.
[0008] The construction method of the expression strain of the raw starch hydrolytic enzyme mutant of the present application includes the following steps:
[0009] First, the structure of alpha-amylase BLA from Escherichia coli is used as a template to perform homology modeling of the structure of raw starch hydrolytic enzyme Amyh1. Three energy calculation functions are selected for calculation using computer-aided design strategy, and the intersection of each two is obtained to obtain the mutation sites of positive selection. Then, co-evolution analysis and sequence conservation analysis are performed to obtain the target amino acid of mutation.
[0010] According to the gene sequence of the raw starch hydrolyzing enzyme Amyh1, a mutant primer is designed and synthesized, and a recombinant plasmid containing the raw starch hydrolyzing enzyme Amyh1 gene is used as a template, the mutant primer is used as a primer, and a site-directed mutation is carried out based on the overlap extension PCR method, so that the mutant gene of the raw starch hydrolyzing enzyme with greatly improved thermal stability is obtained.
[0011] The unmutated raw starch hydrolyzing enzyme plasmid constructed in E.coli BL21(DE3) is used as a template, and the mutant gene is constructed by using the overlap extension PCR method; then, the mutant gene is cut by double enzyme digestion, and the mutant gene is connected after being cut by the vector pET28a, so that a connection product is obtained; the connection product is transformed into the host bacteria BL21(DE3), and a positive clone is screened, so that the engineering strain containing the mutant gene of the application is obtained.
[0012] The expression plasmid vector in the construction method includes pET28a and the like.
[0013] The host bacteria in the construction method include E.coli BL21(DE3) and the like.
[0014] The raw starch hydrolyzing enzyme mutant of the application can be obtained by fermentation of the expression strain.
[0015] The raw starch hydrolyzing enzyme mutant of the application is applied to hydrolysis of raw starch.
[0016] The temperature of the hydrolysis system is 40 DEG C, and the pH value is 7.0.
[0017] The addition amount of the raw starch hydrolyzing enzyme mutant in the hydrolysis system is 8000 U / g, based on the mass of the raw starch in the hydrolysis system.
[0018] The raw starch hydrolyzing enzyme mutant is applied to hydrolysis of 5% (w / v) algal raw starch emulsion. When corn and algal raw starch are used as substrates, the specific enzyme activity of the mutant enzyme is 8311 U / mg and 2622 U / mg under the condition of 40 DEG C and pH 7.0, which is 1.1 and 1.02 times of the wild type, respectively. In the case that the specific enzyme activity does not decrease obviously, even increases, the stability of the mutant enzyme is greatly improved. The thermal stability is 3.5 times of the starting enzyme under the condition of 35 DEG C. In the experiment of hydrolyzing raw starch of chlorella, the hydrolysis rate can reach 33% after 3 h of reaction. The mutant maintains the same hydrolysis capacity, and the stability is greatly improved, so that the shelf life is prolonged, and the mutant has potential value in industrial application using algal biomass as raw material.
[0019] The application determines and compares specific enzyme activity, optimum temperature, optimum pH, stability and the like of the mutant protein and the original starting enzyme. The determination result shows that when corn raw starch is used as the substrate, the application keeps high specific enzyme activity, and the stability is 3.5 times of the starting enzyme under the condition of 35℃. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 It is an electrophoretogram of the PCR amplification product of the application.
[0021] Figure 2 It is an SDS-PAGE spectrum of the purified mutant protein and the starting enzyme Amyh1. Wherein: Amyh1 from left to right is: M is Marker; S is the cell crushing supernatant of Amyh1; P is the cell crushing precipitate of Amyh1; 2 is the 200 mM imidazole eluent of Amyh1. Amyh1:G207H from left to right is: M is Marker; S is the cell crushing supernatant of Amyh1:G207H; P is the cell crushing precipitate of Amyh1:G207H; 1 is the 200 mM imidazole eluent of Amyh1:G207H.
[0022] Figure 3 It is the determination result of the optimum temperature of the starting enzyme Amyh1 and the mutant enzyme.
[0023] Figure 4 Fig. a and Fig. b are respectively the determination result of the optimum pH of the starting enzyme Amyh1 and the mutant enzyme.
[0024] Figure 5 It is the stability of the starting enzyme Amyh1 and the mutant enzyme under the condition of 35℃ and pH 7.0.
[0025] Figure 6 It is the hydrolysis rate of the starting enzyme Amyh1 and the mutant enzyme in hydrolyzing raw starch of algae. DETAILED DESCRIPTION
[0026] The implementation methods in the following examples are all conventional methods unless otherwise specified.
[0027] (I) Construction of expression strain containing raw starch hydrolytic enzyme mutant gene of the application
[0028] 1. Selection of raw starch hydrolytic enzyme gene mutation site
[0029] Based on sequence alignment, the starch-hydrolyzing enzyme Amyh1 has 48.22% amino acid sequence identity with the alpha-amylase BLA from Escherichia coli. Using the structure of BLA as a template, the structure of the starch-hydrolyzing enzyme Amyh1 was homologously modeled using Swiss-Model (http: / / swissmodel.expasy.org / ; Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T. The SWISS-MODEL Repository and associated resources. Nucleic Acids Research. 2009, 37, D387-392.).
[0030] According to the simulated structure and multiple sequence alignment, the site of site-directed mutation was determined, and the glycine G at the 207th site was replaced with histidine H.
[0031] 2. Construction of a starch-hydrolyzing enzyme mutant gene engineering strain
[0032] According to the gene sequence of the starch-hydrolyzing enzyme Amyh1 and the selected mutation site 207, a recombinant plasmid containing the Amyh1 gene was used as a template plasmid, and the target fragment was obtained by overlap extension PCR amplification; the vector pET28a was double-digested with Nde I and Xho I; the target fragment and the vector were ligated by T4 DNA ligase. The ligation product was transformed into E. coli by chemical transformation, and the transformant with correct sequence was selected to obtain the mutant gene engineering strain Escherichia coli BL21 (DE3) / pET28a (+) -Amyh1: G207H of the application.
[0033] The expression strain of the starch-hydrolyzing enzyme mutant of the application is classified and named as Escherichia coli BL21 (DE3) / pET28a (+) -Amyh1: G207H, which has been preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M 2025726, a preservation time of April 8, 2025, and a preservation address of Wuhan, China.
[0034] (II) Expression and protein purification of the starch-hydrolyzing enzyme mutant gene engineering strain of the application
[0035] The mutant strain with successful construction is inoculated into 5 mL LB medium containing kanamycin in a small volume, and cultured in a 37℃, 200rpm shaker for 12h. The bacterial liquid cultured for 12h is used as seed liquid, 4mL seed liquid is inoculated into 400mL LB medium containing kanamycin, and is fermented in a 16℃, 200rpm shaker for 12-16h. The fermentation liquid cultured for 16h is centrifuged at 8000xg in a large centrifuge for 15min, the supernatant is discarded, the bacterial cells at the bottom of the centrifuge bottle are collected, and the buffer is added to resuspend the bacterial cells; the cell disrupter is used at a frequency of 350W, and the cells are ultrasonically broken for 50min in an ice bath. When the bacterial liquid changes from milky turbidity to clear and translucent, it is centrifuged at 8000xg for 30min, and the supernatant is collected to obtain the crude enzyme liquid.
[0036] The crude enzyme liquid is purified by Ni-NTA column chromatography, the imidazole concentration in the eluate is 200mM, and one column volume is eluted. The obtained protein is detected to reach SDS-PAGE purity.
[0037] When corn raw starch is used as the substrate, the optimal temperature of the mutant enzyme is 45℃, the optimal pH is 7.0, and the catalytic activity is more than 80% in the pH range of 7.0-8.0.
[0038] (Three) detection of specific enzyme activity of the raw starch hydrolyzing enzyme mutant
[0039] 1. Enzyme activity definition
[0040] 1 U is the amount of enzyme required to produce 1 µM maltose per minute.
[0041] 2. Specific enzyme activity determination
[0042] The reaction system is 600 µL, 270 µL of 50 mM Tris-HCl buffer is taken into a 2 mL EP tube, 300 µL of corn starch solution with a substrate concentration of 2% is added, the mixture is uniformly placed in a 40℃ water bath for 10 min, 30 µL of enzyme liquid is taken into the EP tube, 30 µL of buffer is added to the control group, and the substrate is reacted for 10 min, 300 µL of DNS is added and boiled in boiling water for 15 min to terminate the reaction, the sample to be tested is placed on ice to cool to room temperature, 12,000xg centrifugation for 2 min, 200 µL of supernatant is taken into a 96-well plate, and the absorbance value is read at A540 nm, the content of reducing sugar is calculated according to the DNS standard curve, and the enzyme activity is calculated according to the enzyme activity definition.
[0043] The determination results show that the specific enzyme activity of the mutant enzyme obtained by the application is 8311 U / mg when corn raw starch is used as a substrate, which is 1.1 times of the starting enzyme; the specific enzyme activity of the mutant enzyme obtained by the application is 2622 U / mg when Chlorella raw starch is used as a substrate, which is 1.02 times of the starting enzyme.
[0044] (Four) Detection of the stability of the raw starch hydrolyzing enzyme
[0045] Under the condition of 35 DEG C and pH 7.0, the starting enzyme Amyh1 and the mutant enzyme are subjected to heat treatment, and samples are taken every 3 hours, taking the initial enzyme activity as 100%, and the residual rate of the enzyme activity after heat treatment for a certain time is calculated, and the formula is as follows: residual rate of enzyme activity = enzyme activity after heat treatment / enzyme activity before heat treatment x 100%.
[0046] The determination results show that the half-life of the mutant enzyme is 113 h under the condition of 35 DEG C, which is 3.5 times of the starting enzyme.
[0047] (Five) Application of the mutant raw starch hydrolyzing enzyme in hydrolysis of algal raw starch
[0048] The hydrolysis system is 5% (w / v) Chlorella starch treated by 4% dilute sulfuric acid at 121 DEG C for 30 min, and 8000 U / g raw starch hydrolyzing enzyme mutant (based on the mass of Chlorella starch), and the hydrolysis reaction is carried out at 40 DEG C with 180 rpm water bath shaking. Samples are taken at appropriate intervals, and the DNS method is used to determine the content of reducing sugar in the hydrolysis system, and the same amount of enzyme is added to the starting enzyme Amyh1 to set up a control group.
[0049] In the experiment of hydrolyzing 5% Chlorella raw starch, the results show that the hydrolysis of the mutant enzyme basically reaches a plateau after 3 h, and the hydrolysis rate of the mutant enzyme to algae can reach 33%. Under the same conditions, the hydrolysis rate of the starting enzyme to algal raw starch is 31%, and the mutant enzyme greatly prolongs the shelf life on the basis of keeping the hydrolysis ability unchanged, and has great application potential in the industrial production of microalgae biomass.
Claims
1. A raw starch hydrolase mutant Amyh1:G207H with improved stability, characterized in that: The amino acid sequence of the raw starch hydrolase mutant Amyh1:G207H is shown in SEQ ID NO:
1.
2. The gene encoding the raw starch hydrolyzing enzyme mutant according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO:
2.
3. The expression strain of the raw starch hydrolyzing enzyme mutant according to claim 1, characterized in that: The strain is classified and named Escherichia coli BL21(DE3) / pET28a(+)-Amyh1:G207H, and has been sent to the China Center for Type Culture Collection (CCTCC) for preservation. The preservation number is CCTCC NO: M 2025726, the preservation date is April 8, 2025, and the preservation address is: Wuhan University, Wuhan, China.
4. Use of the raw starch hydrolase mutant according to claim 1 in hydrolyzing raw starch.
5. The use according to claim 4, characterized in that: The raw starch includes one or more of corn raw starch and algae raw starch.
6. The use according to claim 4, characterized in that: The temperature of the hydrolysis system was 40°C and the pH value was 7.
0.
7. The use according to claim 4, characterized in that: The addition amount of the raw starch hydrolyzing enzyme mutant in the hydrolysis system is 8000 U / g, based on the mass of the raw starch in the hydrolysis system.