Raw starch hydrolase mutant Amyh1: C142Q with improved stability and application of raw starch hydrolase mutant Amyh1: C142Q

Abstract

The invention discloses a raw starch hydrolase mutant Amyh1: C142Q with improved stability and an application of the raw starch hydrolase mutant Amyh1: C142Q. According to the invention, alpha-amylase Amyh1 is taken as a starting enzyme, and on the basis, computer-aided design is carried out, mutation sites are clear, heterologous expression is carried out in escherichia coli, and alpha-amylase with improved stability is obtained. When raw corn starch is used as a substrate, the specific enzyme activity of the mutant is 1.05 times that of a starting enzyme, and the stability of the mutant is 3.25 times that of the starting enzyme. Compared with the starting enzyme, the thermal stability of the mutant enzyme is greatly improved while the specific enzyme activity is maintained. The mutant has potential application value in starch sugar production with corn raw starch as a substrate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a stable raw starch hydrolase mutant Amyh1:C142Q and its applications. Background Technology

[0002] Starch hydrolases are enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. Currently, of all discovered α-amylases, only about 10% are capable of degrading raw starch, and these are found in bacteria, fungi, and animals. These α-amylases can hydrolyze raw starch from potatoes, wheat, corn, rice, etc. However, their specific activities are generally low. For example, when using corn as a substrate, the specific activity of α-amylase from *Bacillus amyloliquefaciens* is 44.6 U / mg, the specific activity of α-amylase from *Streptomyces badius* DB-1 is 148.1 U / mg, while the highest specific activity of α-amylase from *Bacillus acidicola* reaches 874.5 U / mg.

[0003] Thermal stability is one of the important characteristics required for enzymes used in starch processing. Many amylases cannot sustain their hydrolytic activity during the process due to their poor stability. Therefore, obtaining raw starch hydrolases with high specific activity and good thermal stability using protein engineering technology is of great importance for the application of hydrolyzing high-concentration corn raw starch. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing a mutant of raw starch hydrolase, Amyh1: C142Q, with improved stability, and its applications. Based on the raw starch hydrolase Amyh1, this invention utilizes computer-aided design to obtain a mutant with significantly improved thermostability and a slightly increased specific enzyme activity. Using corn raw starch as a substrate, the mutant's specific enzyme activity (7956 U / mg) is 1.05 times that of the original enzyme, and its thermostability is significantly improved, reaching 3.25 times that of the original enzyme at 40 °C and pH 7.0. In experiments hydrolyzing high-concentration corn raw starch, the hydrolysis rate reached 37.83% after 8 hours of reaction. By maintaining the hydrolysis rate and specific enzyme activity without decreasing, the enzyme stability of this mutant is greatly extended. Therefore, this mutant enzyme has application value in industrial applications based on the hydrolysis of high-concentration corn raw starch.

[0005] The present invention relates to a starch hydrolase mutant, Amyh1: C142Q, whose amino acid sequence is shown in SEQ ID NO: 1. Specifically, the 142nd amino acid in the Amyh1 amino acid sequence is mutated from cysteine ​​to glutamine.

[0006] The coding gene of the raw starch hydrolase mutant has the nucleotide sequence shown in SEQ ID NO: 2.

[0007] The expression strain of the raw starch hydrolase mutant of this invention, classified and named Escherichia coli BL21(DE3) / pET28a(+)-Amyh1: C142Q, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025725, deposit date April 8, 2025, and deposit address: Wuhan University, Wuhan, China.

[0008] The method for constructing a raw starch hydrolase mutant expression strain of the present invention includes the following steps:

[0009] First, using the α-amylase BLA structure from Bacillus licheniformis as a template, the structure of the raw starch hydrolase Amyh1 was homologously modeled using Swiss-Model. A computer-aided design strategy was adopted, and three energy calculation functions were selected for calculation. Mutation sites for positive screening were obtained by finding the intersection of pairs of mutations. Then, negative screening was performed by co-evolutionary analysis and sequence conservation analysis to obtain the target amino acids for mutation.

[0010] Based on the gene sequence of the starch hydrolase Amyh1, mutant primers were designed and synthesized. Using a recombinant plasmid containing the starch hydrolase Amyh1 gene as a template, site-directed mutagenesis was performed using the synthesized mutant primers and the overlapping extension PCR method to obtain the mutant gene of the starch hydrolase.

[0011] The mutant gene was then ligated to the digestion product of the vector pET28a using a double enzyme digestion method; the ligation product was transformed into the host bacterium E. coli BL21(DE3), and positive clones were screened to obtain an engineered strain containing the mutant gene of this invention.

[0012] The expression plasmid vector described in the above construction method includes pET28a.

[0013] The host bacterium mentioned in the above construction method includes E. coli BL21(DE3).

[0014] The raw starch hydrolase mutant of the present invention can be obtained by fermentation of the expressed strain.

[0015] The application of the raw starch hydrolase mutant of the present invention in the hydrolysis of raw starch.

[0016] The hydrolysis system was maintained at a temperature of 35-45℃ and a pH of 7.0.

[0017] Specifically, the mutant raw starch hydrolase can be applied to the hydrolysis of 30% corn raw starch emulsion. Using corn raw starch as a substrate, at 40℃ and pH 7.0, the specific enzyme activity of the mutant enzyme (7956 U / mg) is 1.05 times that of the wild type. While the specific enzyme activity did not decrease significantly, and even increased somewhat, the stability of the mutant enzyme was significantly improved. At 35℃, its thermostability is 3.25 times that of the original enzyme. In experiments hydrolyzing high-concentration corn raw starch, the hydrolysis rate reached 37.83% after 8 hours of reaction. This mutant enzyme has application value in the industrial application of hydrolyzing high-concentration corn raw starch.

[0018] This invention measured and compared the specific enzyme activity, optimal temperature, optimal pH, and stability of the mutant enzyme and the original starting enzyme. The results showed that, using corn starch as a substrate, this invention maintained high specific enzyme activity while exhibiting 3.25 times the stability of the starting enzyme at 35°C. Attached Figure Description

[0019] Figure 1 This is the electrophoretic pattern of the PCR amplification product of the present invention.

[0020] Figure 2 SDS-PAGE images of the purified mutant protein and the starting enzyme Amyh1. Wherein: M is the marker; 1 is the cell lysis supernatant of Amyh1; 2 is the cell lysis pellet of Amyh1; 3 is the 200 mM imidazole elution buffer of Amyh1; 4 is the cell lysis supernatant of Amyh1:C142Q; 5 is the cell lysis pellet of Amyh1:C142Q; 6 is the 200 mM imidazole elution buffer of Amyh1:C142Q.

[0021] Figure 3 The results show the optimal temperature determination of the mutant enzyme and the starting enzyme Amyh1.

[0022] Figure 4 The results show the optimal pH values ​​for the mutant enzyme and the starting enzyme Amyh1.

[0023] Figure 5 The stability of the mutant enzyme and the starting enzyme Amyh1 at 35°C and pH 7.0 was measured.

[0024] Figure 6 The hydrolysis rate of high-concentration corn starch was determined by the mutant enzyme and the starting enzyme Amyh1. Detailed Implementation

[0025] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0026] (i) Construction of expression strains containing the mutant gene of the starch hydrolase of the present invention

[0027] 1. Selection of mutation sites in raw starch hydrolase gene

[0028] Based on sequence alignment, the amino acid sequence identity of the raw starch hydrolase Amyh1 is 48.22% with that of the α-amylase BLA from Bacillus licheniformis. Using the structure of BLA as a template, the structure of the raw starch hydrolase 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.).

[0029] Based on the simulated structure and multiple sequence alignment, the site of the mutation was determined, and the cysteine ​​at position 142 was replaced by glutamine.

[0030] 2. Construction of mutant strains of raw starch hydrolase

[0031] Based on the gene sequence of the starch hydrolase Amyh1 and the selected mutation site 142C, a recombinant plasmid containing the Amyh1 gene was used as a template plasmid, and overlap extension PCR was used to amplify the target fragment. The vector pET28a was then amplified using Nde I and Xho I. The target fragment and the vector were ligated using T4 NDA ligase. The ligation product was transformed into *E. coli* via chemical transformation, and transformants with the correct sequence were selected to obtain the engineered strain *Escherichiacoli* BL21(DE3) / pET28a(+)-Amyh1: C142Q, which contains the mutant gene of this invention.

[0032] The expression strain of the raw starch hydrolase mutant of this invention, classified and named Escherichia coli BL21(DE3) / pET28a(+)-Amyh1: C142Q, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025725, deposit date April 8, 2025, and deposit address: Wuhan University, Wuhan, China.

[0033] (II) Expression and protein purification of genetically engineered bacteria containing the mutant starch hydrolase of the present invention

[0034] The successfully constructed mutant strain was inoculated into a small volume of 5 mL LB medium containing kanamycin and cultured for 12 h in a shaker at 37°C and 200 rpm. The 12-h culture was then used as a seed culture, and 4 mL of the seed culture was inoculated into 400 mL of LB liquid medium containing 30 μg / mL kanamycin. When the bacterial culture reached the OD... 600 When the absorbance is around 0.6, add IPTG to a final concentration of 0.15 mM to induce the expression of the target protein, and incubate overnight at 16°C for 12-16 h. Centrifuge the cultured fermentation broth at 8000×g, 4°C for 15 min, collect the cells, resuspend the cells in buffer, and sonicate them in an ultrasonic homogenizer at 350W for 1 h. After homogenization, centrifuge at 8000×g, 4°C for 30 min, and the supernatant obtained is the crude enzyme solution.

[0035] The crude enzyme solution was purified by Ni-NTA column chromatography. The elution buffer contained 200 mM imidazole, and eluted for one column volume. The purity of the obtained protein was determined by SDS-PAGE.

[0036] When using raw corn starch as a substrate, the optimal temperature for the mutant enzyme is 45 °C, the optimal pH is 7.0, and it exhibits catalytic activity of over 85% within a pH range of 6.5-7.5.

[0037] (III) Detection of specific enzyme activity of the raw starch hydrolase mutant of the present invention (DNS method)

[0038] 1. Definition of enzyme activity

[0039] 1 U is the amount of enzyme required to produce 1 µM maltose per minute.

[0040] 2. Enzyme activity assay

[0041] The reaction system consisted of 600 μL. 270 μL of 50 mM Tris-HCl buffer was transferred to a 2 mL EP tube, and 300 μL of 2% corn starch solution was added. After mixing thoroughly, the mixture was incubated in a 45 ℃ water bath for 10 min. 30 μL of enzyme solution was then transferred to an EP tube. For the control group, 30 μL of buffer was added, and the mixture was reacted with the substrate for 10 min. 300 μL of DNS was added, and the reaction was terminated by boiling in water for 15 min. The sample was cooled to room temperature on ice, centrifuged at 12,000 g for 2 min, and 200 μL of the supernatant was transferred to a 96-well plate. The absorbance was read at A540 nm. The reducing sugar content was calculated based on the DNS standard curve, and the enzyme activity was calculated according to the definition of enzyme activity.

[0042] The test results showed that, when using corn starch as a substrate, the specific enzyme activity of the mutant enzyme obtained in this invention was 7956 U / mg, which was 1.05 times that of the starting enzyme.

[0043] (iv) Detection of the stability of the raw starch hydrolytic enzyme of the present invention

[0044] Under conditions of 35 ℃ and pH 7.0, the starting enzyme Amyh1 and the mutant enzyme were heat-treated. With the initial enzyme activity as 100%, the enzyme activity remaining rate after a certain period of heat treatment was calculated using the following formula: Enzyme activity remaining rate = Enzyme activity after heat treatment / Enzyme activity before heat treatment × 100%.

[0045] The results showed that at 35 °C, the half-life of the mutant enzyme was 104 h, which was 3.25 times that of the original enzyme.

[0046] (v) Application of the raw starch hydrolytic enzyme mutant of the present invention in the hydrolysis of high-concentration corn raw starch

[0047] The hydrolysis system consisted of Tris-HCl buffer (50 mM, pH 7.0) with 1 mM CaCl2 added, followed by raw starch. The raw starch hydrolase mutant was added at a concentration of 20 U / mg corn raw starch to the reaction system, and the hydrolysis reaction was carried out in a shaking water bath at 40 ℃ and 200 rpm. Samples were taken at appropriate time intervals, and the reducing sugar content in the hydrolysis system was determined using the DNS method. Simultaneously, the same amount of the starting enzyme Amyh1 was added as a control group.

[0048] In the experiment of hydrolyzing 30% corn starch, the results showed that the mutant enzyme reached a plateau in hydrolysis after 8 hours, with a hydrolysis rate of 37.83% for corn. Under the same conditions, the starting enzyme had a hydrolysis rate of 36.56% for high-concentration corn starch. The mutant enzyme, without reducing the hydrolysis rate, showed greater stability than the wild type in the hydrolysis of high-concentration corn starch to produce starch sugars, indicating significant application potential.

Claims

1. A starch hydrolase mutant with improved stability, Amyh1:C142Q, characterized in that: The amino acid sequence of the raw starch hydrolase mutant Amyh1:C142Q is shown in SEQ ID NO:

1.

2. The encoding gene of the starch hydrolase mutant according to claim 1, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

2.

3. The expression strain of the starch hydrolase mutant according to claim 1, characterized in that: The strain is classified as Escherichia coli BL21(DE3) / pET28a(+)-Amyh1: C142Q and has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025725, deposited on April 8, 2025, at Wuhan University, Wuhan, China.

4. The application of the raw starch hydrolase mutant of claim 1 in the hydrolysis of raw starch.

5. The application according to claim 4, characterized in that: The hydrolysis system was maintained at a temperature of 35-45℃ and a pH of 7.0.

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