A Stability-Enhanced Starch Hydrolase Mutant and Its Application

Abstract

This invention discloses a mutant of raw starch hydrolase with improved stability and its applications. Using α-amylase AmyZ2 as the starting enzyme, and through computer-aided design to clarify the mutation site, a mutant was heterologously expressed in Bacillus subtilis, resulting in an α-amylase with improved stability. When using raw corn starch as a substrate, the mutant exhibits 1.57 times the specific enzyme activity and 2 times the stability of the starting enzyme. While maintaining the specific enzyme activity, the mutant enzyme shows a significant improvement in thermostability. This mutant has potential application value in the production of starch sugars using raw corn starch as a substrate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a stable mutant of raw starch hydrolase 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 activity is generally low; for example, when using corn as a substrate, the activity from... Bacillus amyloliquefaciens The specific enzyme activity of α-amylase was 44.6 U / mg, derived from... Streptomyces badius DB-1 has a specific enzyme activity of 148.1 U / mg for α-amylase, while the activity derived from... Bacillus acidophilus The highest specific activity of α-amylase reached 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 with improved stability and its applications. Based on the raw starch hydrolase AmyZ2, this invention utilizes computer-aided design to obtain a mutant with significantly improved thermal stability and specific enzyme activity. Using corn raw starch as a substrate, the mutant's specific enzyme activity is 1.57 times that of the original enzyme, and its thermal stability is significantly improved, being twice that of the original enzyme at 35℃ and pH 7.0. In experiments hydrolyzing high-concentration corn raw starch, the hydrolysis rate reached 36.9% after 4 hours of reaction. This mutant enzyme has potential value in industrial applications based on the hydrolysis of high-concentration corn raw starch.

[0005] The present invention relates to a raw starch hydrolase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1. Specifically, the 155th amino acid of the AmyZ2 amino acid sequence, aspartic acid, is mutated to lysine.

[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 is classified and named as follows: Bacillus subtilisWB600 / pBHSSs142-AmyZ2:D155K has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M 20242139, on September 30, 2024, at 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] Firstly, from Bacillus licheniformis Using the α-amylase BLA structure as a template, the structure of the raw starch hydrolase AmyZ2 was homologously modeled using the Swiss-Model. A computer-aided design strategy was adopted, and three energy calculation functions were selected for calculation. The mutation sites for positive screening were obtained by finding the intersection of pairs of mutations. Then, the target amino acids for mutation were obtained by co-evolutionary analysis and sequence conservation analysis.

[0010] Based on the gene sequence of the starch hydrolase AmyZ2, mutant primers were designed and synthesized. Using a recombinant plasmid containing the starch hydrolase AmyZ2 gene as a template and the synthesized mutant primers as primers, site-directed mutagenesis was performed using the overlap extension PCR method, resulting in a mutant gene of starch hydrolase with significantly improved thermostability.

[0011] exist E.coli Using the unmutated raw starch hydrolase plasmid constructed in BL21(DE3) as a template, the mutant gene was constructed by overlapping extension PCR. Then, the mutant gene was ligated to the vector pBHS by POE-PCR to obtain the ligation product. The ligation product was transformed into the host bacterium WB600, and positive clones were screened to obtain the engineered strain containing the mutant gene of this invention.

[0012] The expression plasmid vectors described in the above construction method include pET22b, pBHS, etc.

[0013] The host bacteria mentioned in the above construction method include E.coli BL21(DE3) and WB600, etc.

[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] Specifically, the described raw starch hydrolase mutant can be applied to hydrolyze corn raw starch emulsions with a concentration ≤30%. Using corn raw starch as a substrate, at 35℃ and pH 7.0, the specific enzyme activity of the mutant enzyme is 1.57 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 twice that of the original enzyme. In experiments hydrolyzing high-concentration corn raw starch, the hydrolysis rate reached 36.9% after 4 hours of reaction. This mutant has potential value in industrial applications based on the hydrolysis of high-concentration corn raw starch.

[0017] This invention measured and compared the specific enzyme activity, optimal temperature, optimal pH, and stability of the mutant protein and the original starting enzyme. The results showed that, using corn starch as a substrate, this invention maintained high specific enzyme activity while exhibiting twice the stability of the starting enzyme at 35°C. Attached Figure Description

[0018] Figure 1 This is the electrophoretic pattern of the PCR amplification product of the present invention.

[0019] Figure 2 SDS-PAGE images of the purified mutant protein and the starting enzyme AmyZ2. Where: M is the marker; 1 is the extracellular supernatant of AmyZ2:D155K; 2 is the 200 mM imidazole elution buffer of AmyZ2:D155K; 3 is the extracellular supernatant of AmyZ2; 4 is the 200 mM imidazole elution buffer of AmyZ2.

[0020] Figure 3 The results represent the optimal temperature measurement.

[0021] Figure 4 The results represent the optimal pH measurement.

[0022] Figure 5 Stability at 35℃ and pH 7.0.

[0023] Figure 6 The hydrolysis rate of high-concentration corn starch was determined by the mutant protein and the starting enzyme AmyZ2. Detailed Implementation

[0024] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0025] (i) Construction of expression strains containing the mutant gene of the starch hydrolase of the present invention

[0026] 1. Selection of mutation sites in raw starch hydrolase gene

[0027] Based on sequence alignment, the starch hydrolase AmyZ2 and its derivatives from... Bacillus licheniformisThe amino acid sequence identity of the α-amylase BLA was 71%. Using the structure of BLA as a template, the structure of the raw starch hydrolase AmyZ2 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.).

[0028] Based on the simulated structure and multiple sequence alignment, the site of the site-directed mutation was determined, and aspartic acid D at position 155 was replaced by lysine K.

[0029] 2. Construction of mutant strains of raw starch hydrolase

[0030] Based on the gene sequence of the starch hydrolase AmyZ2 and the selected mutation site 155D, a recombinant plasmid containing the AmyZ2 gene was used as a template plasmid for overlap extension PCR amplification to obtain the target fragment. The vector pBHS was then amplified using pBHS-F and pBHS-R. The target fragment and the vector were used as templates and primers for POE-PCR ligation. The ligation product was transformed into Bacillus subtilis via chemical transformation, and transformants with the correct sequence were selected to obtain the engineered strain containing the mutant gene of this invention. Bacillus subtilis WB600 / pBHS-AmyZ2:D155K.

[0031] The expression strain of the raw starch hydrolase mutant of this invention is classified and named as follows: Bacillus subtilis WB600 / pBHSSs142-AmyZ2:D155K has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO: M 20242139, on September 30, 2024, at Wuhan University, Wuhan, China.

[0032] (II) Expression and protein purification of genetically engineered bacteria containing the mutant starch hydrolase of the present invention

[0033] The successfully constructed mutant strain was inoculated into a small volume of 5 mL LB medium containing kanamycin and cultured for 12 h at 37°C and 200 rpm in a shaker. The 12-h culture was used as a seed culture, and 4 mL of the seed culture was inoculated into 400 mL of YT liquid medium containing 30 mg / mL kanamycin. Fermentation was carried out for 48-60 h at 30°C and 200 rpm in a shaker. The fermentation broth after 48 h of culture was centrifuged at 8000 g for 15 min in a large centrifuge. The resulting supernatant was the crude enzyme solution.

[0034] The crude enzyme solution was purified by Ni-NTA column chromatography. The imidazole concentration in the eluent was 200 mM, and elution was performed for 3 column volumes. The obtained protein was tested and found to meet SDS-PAGE purity.

[0035] When using raw corn starch as a substrate, the optimal temperature for the mutant enzyme is 45℃, the optimal pH is 7.0, and it exhibits over 80% catalytic activity within the pH range of 5.5-7.5.

[0036] (III) Detection of specific enzyme activity of the raw starch hydrolase mutant of the present invention (DNS method)

[0037] 1. Definition of enzyme activity

[0038] 1 U is the amount of protein required to generate 1 µM of maltose per minute.

[0039] 2. Enzyme activity assay

[0040] The reaction system consisted of 600 μL. 270 μL of 50 mM Na₂HPO₄-KH₂PO₄ buffer was transferred to a 2 mL EP tube. 300 μL of a 1% corn starch solution was added, and the mixture was incubated at 45°C for 10 min. 30 μL of enzyme solution was then transferred to an EP tube. 30 μL of buffer was added to the control group, 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.

[0041] 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 1.57 times that of the starting enzyme.

[0042] (iv) Detection of the stability of the raw starch hydrolytic enzyme of the present invention

[0043] Under conditions of 35℃ and pH 7.0, the starting enzyme AmyZ2 and the mutant enzyme were heat-treated. Samples were taken every 3 hours. With the initial enzyme activity as 100%, the enzyme activity remaining rate after a certain period of heat treatment was calculated. The formula is as follows: Enzyme activity remaining rate = Enzyme activity after heat treatment / Enzyme activity before heat treatment × 100%.

[0044] The results showed that at 35°C, the half-life of the mutant enzyme was 18 h, which was twice that of the starting enzyme.

[0045] (v) Application of the raw starch hydrolytic enzyme mutant of the present invention in the hydrolysis of high-concentration corn raw starch

[0046] The hydrolysis system consisted of Na₂HPO₄-KH₂PO₄ buffer (50 mM, pH 7.0) with 1 mM CaCl₂ added, followed by raw starch and the raw starch hydrolase mutant. The hydrolysis reaction was carried out in a shaker at 35°C 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 AmyZ₂ was added as a control group.

[0047] In the experiment of hydrolyzing 30% corn starch, the results showed that the mutant enzyme reached a plateau in hydrolysis after 4 hours, with a hydrolysis rate of 36.9% for corn. Under the same conditions, the starting enzyme had a hydrolysis rate of only 33.2% for high-concentration corn starch, indicating that the mutant enzyme has great potential for application in the hydrolysis of high-concentration corn starch to produce starch sugars.

Claims

1. A mutant of raw starch hydrolase with improved stability, the amino acid sequence of which is shown in SEQ ID NO:

1.

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

2.

3. The expression strain of the starch hydrolase mutant according to claim 1, characterized in that: The strains expressed are classified as follows: Bacillus subtilis WB600 / pBHSSs142-AmyZ2:D155K has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20242139, on September 30, 2024, 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: Raw starch concentration ≤30%.

6. The application according to claim 4, characterized in that: The hydrolysis system was maintained at a temperature of 35℃ and a pH value of 6.5-7.5.

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