Alpha-amylase mutant and application thereof

Abstract

The invention discloses an amylase mutant. The amino acid sequence of the amylase mutant is shown as SEQ ID NO: 2 in a sequence table. The mutant is obtained by carrying out transformation and screening of protein engineering on the high-temperature alpha-amylase from thermococcus eurythermalis, and the high-temperature alpha-amylase is obtained by carrying out transformation and screening of protein engineering on the high-temperature alpha-amylase from thermococcus eurythermalis.

Description

Technical Field

[0001] The invention belongs to the field of gene engineering and genetic engineering, and in particular relates to an amylase mutant and an application thereof. Background Art

[0002] Biomass energy has become a hot area of ​​new energy research due to its renewable nature, clean use, abundant raw materials, and widespread distribution. Biomass energy has the potential to become a renewable energy source that reduces environmental pollution and ultimately replaces fossil fuels. Fuel ethanol is currently the most widely used biomass liquid fuel. The raw materials for fuel ethanol production primarily include starch, sugars, and cellulose. Starch is hydrolyzed into glucose by amylase and saccharifying enzymes, which can then be fermented by Saccharomyces cerevisiae to produce ethanol. Improving amylase activity is key to increasing the yield of starch-based bioethanol.

[0003] Site-directed mutagenesis is one of the two main methods to improve the thermal stability of industrial enzymes. Molecular modification of amylase and screening of amylase mutants that are more suitable for industrial applications will be of great significance for the industrial application of amylase. Summary of the Invention

[0004] The purpose of the present invention is to provide a mutant obtained by point mutation using α-amylase derived from thermophilic archaeon Thermococcus eurythermalis as a parent.

[0005] Another object of the present invention is to provide a gene encoding the above mutant.

[0006] Another object of the present invention is to provide a recombinant vector comprising the mutant gene.

[0007] Another object of the present invention is to provide applications of the mutant.

[0008] Another object of the present invention is to provide a method for preparing α-amylase having high heat resistance.

[0009] According to a specific embodiment of the present invention, the α-amylase mutant is obtained by subjecting the wild-type α-amylase with the amino acid sequence shown in SEQ ID NO: 1 to G54P / P262W / V345N point mutations.

[0010] According to a specific embodiment of the present invention, a recombinant vector comprising the above-mentioned α-amylase mutant gene is also provided, and the starting vector of the recombinant expression vector is specifically pET-22b(+).

[0011] According to a specific embodiment of the present invention, a recombinant strain comprising the above-mentioned α-amylase mutant gene is also provided, and the starting strain of the recombinant bacteria is specifically E. coli BL21 (DE3).

[0012] The method for preparing an α-amylase having high heat resistance according to the present invention comprises the following steps: 1) preparing a recombinant vector containing the mutant gene; 2) transforming a host with the recombinant vector; 3) fermenting and culturing the host and isolating α-amylase.

[0013] The present invention overcomes the shortcomings of the prior art and provides a highly heat-resistant α-amylase mutant suitable for use in the energy, food, and feed sectors. The optimal operating temperature of the mutant enzyme provided by the present invention is increased from 80°C (the wild-type) to 95°C, significantly enhancing thermal stability. When the reaction temperature rises from 80°C to 95°C, the wild-type enzyme activity decreases significantly, while the mutant still exhibits nearly 70% of its peak activity under the same conditions. Even under boiling conditions (100°C), the mutant still maintains over 60% of its activity, while the wild-type retains only 8%. Therefore, the α-amylase mutant provided by the present invention can effectively meet the high-temperature hydrolytic activity requirements of α-amylases used in the energy, food, and feed sectors, and has a very broad application prospect. DETAILED DESCRIPTION

[0014] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. The following specific embodiments further describe the present invention. Example

[0015] 1. Construction of mutant enzymes By analyzing the 3D structure of the wild-type thermostable α-amylase protein, its substrate binding site was identified. PCR-directed saturation mutagenesis was then used to investigate the effects of different amino acid residues at this site on starch hydrolysis, thereby screening for mutant enzymes that hydrolyze starch with high glucose content. The present invention was developed through predictive analysis of numerous sites, followed by mutation studies and comparative screening of multiple candidate sites. The methods described herein merely describe the experimental procedures for obtaining the mutant enzymes of the present invention and do not include descriptions of mutation experiments at other candidate sites.

[0016] PCR site-directed saturation mutagenesis technology, gene expression technology and recombinant protein purification technology, and molecular biology experimental methods not described in detail in this embodiment are all routine experimental methods familiar to molecular biology professionals.

[0017] 2. Gene Expression and Enzyme Preparation The recombinant vector of the mutant enzyme is transformed into an Escherichia coli strain for induced expression, and the target recombinant protein is obtained by nickel column purification. After a single target band is obtained by SDS-PAGE electrophoresis, the enzymatic properties can be analyzed.

[0018] 3. Analysis of the optimal reaction temperature of enzymes At pH 5.5, enzyme activity was measured at 75, 80, 85, 90, 95, and 100°C. The highest activity was taken as 100% and the relative activity at other temperatures was calculated to determine the optimal reaction temperature. The enzyme samples were heat-treated at 75, 80, 85, and 90°C for 60 min. Samples were taken every 10 minutes and the residual enzyme activity was measured using the above-described enzyme activity assay method. The activity of the untreated sample was taken as 100% to determine the thermal stability of the enzyme.

[0019] The activity of the high-temperature α-amylase mutant was measured at 50-100°C at pH 6.0, generating a temperature-dependent activity curve. The mutant's optimal temperature was 100°C, 20°C higher than that of the wild-type. While the activity of the wild-type enzyme decreased significantly when the reaction temperature increased from 80°C to 100°C, the mutant maintained nearly 70% of its peak activity under the same conditions. Even at boiling (100°C), the mutant retained over 60% of its activity, while the wild-type retained only 8%. This indicates that the mutant can effectively hydrolyze starch molecules over a wider temperature range than the wild-type.

[0020] 4. pH stability analysis The enzyme solution was placed in different pH buffers at room temperature for 1 hour and then its residual enzyme activity was measured. The mutant exhibited good stability in all tested pH buffers (pH 4.0 to 8.0), with significantly better stability at pH 3.0 than the wild type (which retained only 30% of its activity after 1 hour at pH 4.0).

[0021] The optimum pH of the amylase mutant was close to that of the wild type, but the specific activity was 4.6 times higher than that of the wild type. The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention can also be implemented in other ways. Any obvious replacement without departing from the concept of the present invention is within the scope of protection of the present invention.

Claims

1. An α-amylase mutant, characterized in that The α-amylase mutant is obtained by subjecting the wild-type α-amylase with the amino acid sequence shown in SEQ ID NO: 1 to G54P / P262W / V345N point mutations.

2. An α-amylase gene, characterized in that Encodes the amylase mutant according to claim 1.

3. A recombinant vector comprising the α-amylase gene according to claim 2. 4 . The recombinant vector of the α-amylase gene according to claim 3 , wherein the starting vector of the recombinant vector is specifically pET-22b(+).

5. A recombinant strain comprising the α-amylase gene according to claim 2.

6. The recombinant strain expressing the α-amylase gene according to claim 3, wherein the starting strain of the recombinant bacteria is specifically E. coli BL21 (DE3).

7. Use of the α-amylase protein encoded by the gene according to claim 1 in hydrolyzing starch.

8. Use of the highly heat-resistant α-amylase mutant according to claim 1 in energy, food and feed.

9. Use of the α-amylase gene according to claim 2 in energy, food and feed.

10. A method for preparing an α-amylase having high heat resistance, comprising the steps of: 1) preparing a recombinant vector comprising the mutant gene according to claim 2; 2) transforming a host with the recombinant vector; 3) fermenting and culturing the host and isolating α-amylase.