Raw starch hydrolase mutant with improved stability and application thereof

Abstract

The invention discloses a raw starch hydrolase mutant with improved stability and application thereof. According to the invention, alpha-amylase AmyZ2 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 bacillus subtilis, 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 1.57 times that of a starting enzyme, and the stability of the mutant is 2 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] The invention belongs to the field of biotechnology, and particularly relates to a raw starch hydrolase mutant with improved stability and application thereof. Background Art

[0002] Raw starch hydrolyzing enzymes refer to enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. At present, among all the α-amylases discovered, only about 10% have the ability to degrade raw starch, which are distributed in bacteria, fungi and animals. These α-amylases can hydrolyze raw starch from potatoes, wheat, corn, rice, etc. However, the specific activity is generally low. For example, when corn is used as the substrate, Bacillus amyloliquefaciens The specific enzyme activity of α-amylase from Streptomyces badius The specific enzyme activity of α-amylase from DB-1 was 148.1 U / mg, while that from Bacillus acidophilus The highest specific activity of α-amylase reached 874.5 U / mg.

[0003] Thermal stability is one of the important properties required for starch processing enzymes. Many amylases cannot continue to play a hydrolytic role during the hydrolysis process due to their poor stability. Therefore, using protein engineering technology to obtain raw starch hydrolyzing enzymes with higher specific activity and better thermal stability is important for the application of hydrolyzing high-concentration corn raw starch. Summary of the invention

[0004] In view of the deficiencies of the above-mentioned prior art, the present invention provides a raw starch hydrolase mutant with improved stability and its application. Based on the raw starch hydrolase AmyZ2, the present invention obtains a mutant with significantly improved thermal stability through computer-aided design, and its specific enzyme activity is also improved. When corn raw starch is used as a substrate, the specific enzyme activity of the mutant is 1.57 times that of the starting enzyme, and its thermal stability is greatly improved. Under the conditions of 35°C and pH 7.0, it is 2 times that of the starting enzyme. In the experiment of hydrolyzing high-concentration corn raw starch, the hydrolysis rate can reach 36.9% after 4 hours of reaction. The mutant enzyme has potential value in industrial applications based on the hydrolysis of high-concentration corn raw starch.

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

[0006] The nucleotide sequence of the gene encoding the raw starch hydrolyzing enzyme mutant is shown in SEQ ID NO:2.

[0007] The expression strain of the raw starch hydrolyzing enzyme mutant of the present invention is classified and named Bacillus subtilisWB600 / pBHSSs142-AmyZ2:D155K has been sent to the China Center for Type Culture Collection (CCTCC) for preservation, with the deposit number CCTCCNO: M 20242139. The deposit date is September 30, 2024, and the deposit address is Wuhan University, Wuhan, China.

[0008] The method for constructing a raw starch hydrolyzing enzyme mutant expression strain of the present invention comprises the following steps: First, from Bacillus licheniformis The α-amylase BLA structure was used as a template, and Swiss-Model was used to perform homology modeling on the structure of raw starch hydrolase AmyZ2. 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 pairwise intersection, and then co-evolution analysis and sequence conservation analysis were used for negative screening to obtain the target amino acids for mutation.

[0009] According to the gene sequence of raw starch hydrolase AmyZ2, mutant primers were designed and synthesized. Using the recombinant plasmid containing the raw starch hydrolase AmyZ2 gene as a template and the above-mentioned synthetic mutant primers as primers, site-directed mutagenesis was performed based on the overlapping extension PCR method, and a mutant gene of the raw starch hydrolase with significantly improved thermal stability was obtained.

[0010] exist E.coli The unmutated raw starch hydrolase plasmid constructed in BL21 (DE3) is used as a template, and the mutant gene is constructed by overlapping extension PCR; then the mutant gene is connected to the vector pBHS by the POE-PCR method to obtain a connection product; the connection product is transformed into the host bacteria WB600, and positive clones are screened to obtain an engineered strain containing the mutant gene of the present invention.

[0011] The expression plasmid vectors described in the above construction method include pET22b, pBHS and the like.

[0012] The host bacteria in the above construction method include E.coli BL21(DE3) and WB600, etc.

[0013] The raw starch hydrolyzing enzyme mutant of the present invention can be obtained by fermenting the expression strain.

[0014] Application of the raw starch hydrolyzing enzyme mutant of the present invention in hydrolyzing raw starch.

[0015] Specifically, the raw starch hydrolase mutant can be applied to hydrolyze corn raw starch emulsion with a hydrolysis concentration of ≤30%. When corn raw starch is used as a substrate, the specific enzyme activity of the mutant enzyme is 1.57 times that of the wild type at 35°C and pH 7.0. The stability of the mutant enzyme is greatly improved when the specific enzyme activity does not decrease significantly or even increases. At 35°C, the thermal stability is twice that of the starting enzyme. In the experiment of hydrolyzing high-concentration corn raw starch, the hydrolysis rate can reach 36.9% after 4 hours of reaction. The mutant has potential value in industrial applications based on the hydrolysis of high-concentration corn raw starch.

[0016] The present invention measured and compared the specific enzyme activity, optimal temperature, optimal pH, stability, etc. of the mutant protein and the original starting enzyme. The measurement results show that when corn starch is used as a substrate, the present invention maintains a high specific enzyme activity and is twice as stable as the original enzyme at 35°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The electrophoresis pattern of the PCR amplification product of the present invention is shown in FIG.

[0018] Figure 2 The SDS-PAGE profiles of the purified mutant protein and the starting enzyme AmyZ2. Wherein: M is a marker; 1 is the extracellular supernatant of AmyZ2:D155K; 2 is the 200 mM imidazole eluate of AmyZ2:D155K; 3 is the extracellular supernatant of AmyZ2; 4 is the 200 mM imidazole eluate of AmyZ2.

[0019] Figure 3 The optimum temperature measurement result.

[0020] Figure 4 The optimum pH value was determined.

[0021] Figure 5 The stability is at 35°C and pH 7.0.

[0022] Figure 6 The hydrolysis rates of high-concentration raw corn starch by the mutant protein and the starting enzyme AmyZ2. DETAILED DESCRIPTION

[0023] The implementation methods in the following embodiments are all conventional methods unless otherwise specified.

[0024] (I) Construction of an expression strain containing the mutant gene of the raw starch hydrolase of the present invention 1. Selection of mutation sites of raw starch hydrolase genes Based on sequence alignment, raw starch hydrolase AmyZ2 is similar to Bacillus licheniformisThe amino acid sequence identity of the α-amylase BLA of the raw starch hydrolase AmyZ2 was 71%. Using the structure of BLA as a template, 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.) was used to perform homology modeling on the structure of raw starch hydrolase AmyZ2.

[0025] Based on the simulated structure and multiple sequence alignment, the site of site-directed mutation was determined, and aspartic acid D at position 155 was replaced by lysine K.

[0026] 2. Construction of genetic engineering strains producing raw starch hydrolase mutations According to the gene sequence of raw starch hydrolase AmyZ2 and the selected mutation site 155D, the recombinant plasmid containing the AmyZ2 gene is used as a template plasmid, and overlap extension PCR amplification is used to obtain the target fragment; pBHS-F and pBHS-R are then used to amplify the vector pBHS. The target fragment and the vector are used as templates and primers for POE-PCR connection. The connection product is transferred into Bacillus subtilis by chemical transformation, and the transformant with the correct sequence is selected to obtain the engineered strain of the mutant gene of the present invention. Bacillus subtilis WB600 / pBHS-AmyZ2:D155K.

[0027] The expression strain of the raw starch hydrolyzing enzyme mutant of the present invention is classified and named Bacillus subtilis WB600 / pBHSSs142-AmyZ2:D155K has been sent to the China Center for Type Culture Collection (CCTCC) for preservation, with the deposit number CCTCCNO: M 20242139. The deposit date is September 30, 2024, and the deposit address is Wuhan University, Wuhan, China.

[0028] (II) Expression and protein purification of genetically engineered bacteria containing the raw starch hydrolyzing enzyme mutation of the present invention The successfully constructed mutant strain was inoculated into a small volume of 5 mL LB medium containing kanamycin, and cultured in a shaker at 37°C and 200 rpm for 12 hours. The bacterial liquid cultured for 12 hours was used as the seed liquid, and 4 mL of the seed liquid was inoculated into 400 mL of YT liquid medium containing 30 mg / mL Kana, and fermented in a shaker at 30°C and 200 rpm for 48-60 hours. The fermented liquid cultured for 48 hours was centrifuged at 8000g in a large centrifuge for 15 minutes. The supernatant obtained is the crude enzyme liquid.

[0029] The crude enzyme solution was purified by Ni-NTA column chromatography. The imidazole concentration in the eluent was 200 mM and eluted for 3 column volumes. The obtained protein was tested to reach SDS-PAGE purity.

[0030] When corn starch is used as substrate, the optimum temperature of the mutant enzyme is 45°C and the optimum pH is 7.0. It has more than 80% catalytic activity in the pH range of 5.5-7.5.

[0031] (III) Detection of specific enzyme activity of the raw starch hydrolyzing enzyme mutant of the present invention (DNS method) 1. Definition of enzyme activity 1 U is the amount of protein required to generate 1 µM maltose per minute.

[0032] 2. Enzyme activity determination The reaction system is 600 uL, 270 uL of 50 mM Na2HPO4-KH2PO4 buffer solution is pipetted into a 2 mL EP tube, 300 uL of corn starch solution with a substrate concentration of 1% is added, the two are mixed evenly and placed in a 45°C water bath for 10 minutes, 30 uL of enzyme solution is pipetted into the EP tube, 30 uL of buffer solution is added to the control group, reacted with the substrate for 10 minutes, 300 uL of DNS is added and boiled in boiling water for 15 minutes to terminate the reaction, the sample to be tested is placed on ice to cool to room temperature, centrifuged at 12,000g for 2 minutes, 200 uL of supernatant is pipetted into a 96-well plate, the absorbance is read at A540 nm, the reducing sugar content is calculated according to the DNS standard curve, and then the enzyme activity is calculated according to the enzyme activity definition.

[0033] The test results show that when corn starch is used as a substrate, the specific enzyme activity of the mutant enzyme obtained by the present invention is 1.57 times that of the original enzyme.

[0034] (IV) Detection of the stability of the raw starch hydrolyzing enzyme of the present invention The starting enzyme AmyZ2 and the mutant enzyme were heat-treated at 35°C and pH 7.0. Samples were taken every 3 hours. The initial enzyme activity was taken as 100% to calculate the residual enzyme activity after a certain period of heat treatment. The formula is as follows: residual enzyme activity = enzyme after heat treatment / enzyme activity before heat treatment × 100%.

[0035] The measurement results showed that at 35°C, the half-life of the mutant enzyme was 18 h, which was twice that of the original enzyme.

[0036] (V) Application of the raw starch hydrolyzing enzyme mutant of the present invention in hydrolyzing high-concentration raw corn starch The hydrolysis system was a Na2HPO4-KH2PO4 buffer (50 mM, pH 7.0) buffer in which CaCl2 with a final concentration of 1 mM was added, followed by the addition of raw starch and the raw starch hydrolase mutant, and the hydrolysis reaction was carried out in a shaking water bath at 35°C and 200 rpm. Samples were taken at appropriate intervals, and the reducing sugar content in the hydrolysis system was determined by the DNS method. At the same time, the starting enzyme AmyZ2 with the same enzyme amount was added as a control group.

[0037] In the experiment of hydrolyzing 30% corn starch, the results showed that the mutant enzyme reached a plateau after 4 hours, and its hydrolysis rate for corn can reach 36.9%. Under the same conditions, the hydrolysis rate of the starting enzyme for high-concentration corn starch was only 33.2%. The mutant enzyme has great application potential in hydrolyzing high-concentration corn starch to produce starch sugar.

Claims

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

1.

2. The gene encoding the raw starch hydrolyzing enzyme mutant according to claim 1, wherein the nucleotide sequence thereof 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 expression strains were classified as Bacillus subtilis WB600 / pBHSSs142-AmyZ2:D155K has been sent to the China Center for Type Culture Collection (CCTCC) for preservation, with the deposit number being CCTCC NO: M 20242139, the deposit date being September 30, 2024, and the deposit address being Wuhan University, Wuhan, China.

4. Use of the raw starch hydrolyzing enzyme mutant according to claim 1 in hydrolyzing raw starch.

5. The use according to claim 4, characterized in that: Raw starch concentration ≤30%.

6. The use according to claim 4, characterized in that: The temperature of the hydrolysis system is 35°C and the pH value is 6.5-7.5.

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