A high-specific-activity raw starch hydrolase mutant, its encoding gene, expression strain and applications thereof

Abstract

The present invention discloses a high specific activity raw starch hydrolase mutant, its encoding gene, an expression strain thereof and applications thereof. In the present invention, α-amylase Amy with poor stability is used as the starting enzyme, and through site-directed mutagenesis, a mutant Amy:ΔTG / W345A with improved thermal stability is obtained. When corn raw starch is used as the substrate, the specific enzyme activity of the mutant is 85% of that of the starting enzyme, and its thermal stability is greatly improved under the conditions of 30-35 °C. Under the conditions of 35 °C and pH 7.0, the half-life can reach 20 h. While the specific enzyme activity is close to that of the starting enzyme, the thermal stability is greatly improved. This mutant has potential application value in the field of sugar-making industry using corn raw starch as the substrate.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a high-specific-activity raw starch hydrolase mutant, its encoding gene, an expression strain and their applications. Background Art

[0002] Raw starch hydrolase refers to an enzyme that can directly degrade raw starch granules below the gelatinization temperature of starch. Currently, among all the discovered α-amylases, only about 10% of the α-amylases have the ability to degrade raw starch, which are distributed in bacteria, fungi and animals. And these α-amylases can hydrolyze the raw starch of potatoes, wheat, corn, etc. However, their specific activities are generally low. Among the reported raw starch hydrolases, the specific enzyme activity ranges from 4.9 to 2370 U / mg. When using corn as the substrate, the specific enzyme activity of the α-amylase from Bacillus amyloliquefaciens is 44.6 U / mg, the specific enzyme activity of the α-amylase from Streptomyces badius DB-1 is 148.1 U / mg, while the highest specific enzyme activity of the α-amylase from Bacillus acidicola reaches 874.5 U / mg. The low enzyme activity of these α-amylases with raw starch hydrolysis ability limits their wide application in industry.

[0003] In terms of industrial applications, the starch processing industry generally uses a 20-30% (W / V) starch slurry as the raw material. Currently, there are few studies on the direct action of raw starch hydrolase on high-concentration raw starch hydrolysis. Thermal stability is also an important property of starch-degrading enzymes used in starch processing. Many amylases cannot continuously play a hydrolysis role during hydrolysis due to their poor stability. Therefore, using protein engineering technology to obtain a raw starch hydrolase with a relatively high specific enzyme activity and good thermal stability has an important role in the application of hydrolyzing high-concentration corn raw starch. Summary of the Invention

[0004] The present invention provides a high-specific-activity raw starch hydrolase mutant, its encoding gene, an expression strain and their applications. Based on the raw starch hydrolase Amy with poor stability, the present invention obtained a mutant enzyme Amy:ΔTG / W345A with greatly improved thermal stability and retaining the specific activity of the starting enzyme Amy through site-directed mutagenesis. When using corn raw starch as the substrate, the specific enzyme activity of the mutant is 85% of that of the starting enzyme. The kinetic parameter determination results show that the mutant enzyme is close to the starting enzyme. However, its thermal stability is greatly improved. At 35°C and pH 7.0, it is 20 times that of the starting enzyme. In the experiment of hydrolyzing high-concentration corn raw starch, after reacting for 4 h, the hydrolysis rate can reach 43%. This mutant enzyme has potential value in industrial applications based on the hydrolysis of high-concentration corn raw starch.

[0005] The high specific activity raw starch hydrolase mutant of the present invention has an amino acid sequence as shown in SEQ ID No: 1, and the nucleotide sequence encoding the mutant is as shown in SEQ ID No: 2.

[0006] The expression strain of the raw starch hydrolase mutant of the present invention is classified and named Bacillus subtilis WB600 / pBHSs142-AmyZ1(ΔTG / W345A), which has been deposited in the China Center for Type Culture Collection (CCTCC). The deposit number is CCTCC NO: CCTCC M 20221942, the deposit time is December 12, 2022, and the deposit address is Wuhan University, Wuhan, China.

[0007] The construction method of the expression strain of the raw starch hydrolase mutant of the present invention includes the following steps:

[0008] First, taking the α-amylase BLA structure from Bacillus licheniformis as a template, the Swiss-Model was used to perform homologous modeling on the structure of the raw starch hydrolase Amy. Using a semi-rational design strategy, the sequences and structures of Amy and BLA were compared to analyze and determine the target amino acids for mutation.

[0009] According to the gene sequence of the raw starch hydrolase Amy, mutant primers were designed and synthesized. Using the recombinant plasmid containing the raw starch hydrolase Amy gene as a template and the above-synthesized mutant primers as primers, site-directed mutagenesis was carried out based on the overlap extension PCR method to obtain a mutant gene of the raw starch hydrolase with a greatly improved thermal stability.

[0010] Using the unmutated raw starch hydrolase plasmid constructed in E. coli BL21(DE3) as a template, the mutant gene was constructed by the overlap extension PCR method; then, through the POE-PCR method, the mutant gene was ligated with the vector pBHSs142 to obtain a ligation product; the ligation product was transformed into the host bacterium WB600, and positive clones were screened to obtain an engineering strain containing the mutant gene of the present invention.

[0011] The expression plasmid vectors described in the above construction method include pET22b, pBHSs142, etc.

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

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

[0014] Application of the raw starch hydrolase mutant of the present invention is to apply the raw starch hydrolase mutant to hydrolyze high-concentration raw corn starch (concentration 20-30%, W / V). When using raw corn starch as the substrate, at 35°C and pH 7.0, the specific enzyme activity of the mutant enzyme is 85% of that of the wild type. The kinetic parameter determination results show that the catalytic efficiency of the mutant on raw corn starch is not much different from that of the starting enzyme. While maintaining the specific enzyme activity, the stability of the mutant enzyme has been greatly improved. At 30°C and 35°C, the thermal stability is 15 times and 20 times that of the starting enzyme. In the experiment of hydrolyzing high-concentration raw corn starch, after 4 hours of reaction, the hydrolysis rate can reach 43%. This mutant has potential value in industrial applications based on the hydrolysis of high-concentration raw corn starch.

[0015] The present invention measures and compares the specific enzyme activity, optimum temperature, optimum pH, kinetic parameters, stability, etc. of the mutant protein and the original wild-type protein. The measurement results show that when using raw corn starch as the substrate, while the present invention is close to the specific enzyme activity, at 30°C and 35°C, the stability is greatly improved compared with the starting enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 、 2 is the electrophoresis pattern of the PCR amplification product of the present invention: Figure 1 Each lane in is DNA marker, the amplified fragment of PCR; Figure 2 Each lane in is DNA marker, the vector amplified by PCR.

[0017] Figure 3 is the SDS-PAGE pattern of the purified mutant protein and the starting enzyme Amy: Lane 1 is the crude enzyme of the starting enzyme Amy, Lane 2 is the pure enzyme of the starting enzyme Amy, Lane 3 is the crude enzyme of Amy:ΔTG / W345A, Lane 4 is the pure enzyme of Amy:ΔTG / W345A, and M is the protein marker.

[0018] Figure 4 In, a is the measurement result of the optimum temperature, and b is the measurement result of the optimum pH.

[0019] Figure 5 In, a is the stability at 30°C and pH 7.0, and b is the stability at 35°C and pH 7.0. DETAILED DESCRIPTION OF THE INVENTION

[0020] The implementation methods in the following examples are all conventional methods unless otherwise specified.

[0021] (I) Construction of the expression strain containing the raw starch hydrolase mutant gene of the present invention

[0022] 1. Selection of the gene mutation site of the raw starch hydrolase

[0023] Based on sequence alignment, the amino acid sequence identity between raw starch-hydrolyzing enzyme Amy and α-amylase BLA from Bacillus licheniformis is 71%. Using the structure of BLA as a template, the structure of raw starch-hydrolyzing enzyme Amy was homology modeled using Swiss-Model.

[0024] According to the simulated structure and multiple sequence alignment, the sites for site-directed mutagenesis were determined to be threonine T at site 181, glycine at site 182, and tryptophan W at site 345. The mutation direction was to remove threonine T at site 181 and glycine G at site 182, and tryptophan W at site 345 was replaced by alanine A.

[0025] 2. Construction of engineering strains of raw starch-hydrolyzing enzyme mutant genes

[0026] The high specific activity raw starch-hydrolyzing enzyme mutant of the present invention has an amino acid sequence as shown in SEQ ID No: 1, and the nucleotide sequence encoding this mutant is as shown in SEQ ID No: 2.

[0027] Using the recombinant plasmid containing the AmyZ1 gene as the template plasmid, the target fragment was obtained by overlap extension PCR amplification ( Figure 1 ); then pBHS-F and pBHS-R were used to amplify the vector pBHS ( Figure 2 ). The target fragment and the vector were used as templates and primers for POE-PCR ligation. The ligation product was transferred into Bacillus subtilis by chemical transformation, and the transformants with the correct sequence were selected to obtain the engineering strain Bacillus subtilis WB600 / pBHSs142-Amy(ΔTG / W345A) of the mutant gene of the present invention.

[0028] The strain Bacillus subtilis WB600 / pBHSs142-AmyZ1(ΔTG / W345A) of the present invention has been deposited at the China Center for Type Culture Collection (CCTCC), with the deposit number CCTCC NO: CCTCC M 20221942, the deposit date being December 12, 2022, and the deposit address: Wuhan University, Wuhan, China.

[0029] (2) Expression and protein purification of the engineering bacteria containing the raw starch-hydrolyzing enzyme mutant gene of the present invention

[0030] Inoculate the successfully constructed mutant strain into 5 mL of LB medium containing Kana in a small volume, and culture it in a shaker at 37 °C and 200 rpm for 12 h. Use the bacterial solution cultured for 12 h as the seed solution, inoculate 5 mL of the seed solution into 400 mL of TY liquid medium containing 30 μg / mL Kana, and ferment it in a shaker at 30 °C and 200 rpm for 48 - 60 h. Centrifuge the fermentation broth cultured for 48 h at 8000×g and low temperature for 15 min in a large centrifuge. The obtained supernatant is the crude enzyme solution.

[0031] The crude enzyme solution was purified by Ni-NTA column chromatography. The imidazole concentration in the eluent was 200 mM, and 3 column volumes were eluted. The obtained protein was detected to reach the SDS-PAGE purity.

[0032] (III) Detection of the specific enzyme activity of the raw starch hydrolase mutant containing the present invention (DNS method)

[0033] The reaction system was 900 μL. Corn raw starch was dissolved with Na2HPO4-KH2PO4 buffer (50 mM, pH 7.0) to make its final concentration 1%. After incubating at 35 °C for 10 min, the diluted enzyme solution was added and reacted for 10 min. Then 300 μL of DNS was added and placed on ice to terminate the reaction. After the reaction was completed, it was boiled in a boiling water bath for 15 min for color development. The experimental group was set with 3 parallel experiments, and the control group used buffer instead of enzyme solution. Measure the absorbance value at A540 nm, and calculate the amount of reducing sugar according to the maltose standard curve formula. 1 U is the amount of protein required to generate 1 μM of maltose per minute.

[0034] The measurement results showed that when using corn raw starch as the substrate, the specific enzyme activity of the mutant enzyme obtained in the present invention was 85% of the starting enzyme.

[0035] (IV) Detection of the optimal pH and optimal temperature of the raw starch hydrolase mutant containing the present invention

[0036] Select Na2HPO4-KH2PO4 (50 mM, pH 7.0, 1 mM CaCl2) as the buffer, and corn raw starch with a final concentration of 1% as the substrate, and set different temperature gradients. After diluting the mutant enzyme in advance, aliquot it into 2 mL Ep tubes and place them in water baths at different temperatures to measure the enzyme activity of the mutant enzyme at different temperature gradients. Set the highest enzyme activity as 100%, and calculate the relative enzyme activity of the mutant enzyme at other temperatures.

[0037] After obtaining the result of the optimal temperature, under the condition of the optimal temperature, set different pH gradients to measure the enzyme activity of the mutant enzyme under different pH conditions. Set the highest enzyme activity measured at different pHs as 100%, and calculate the relative enzyme activity of the mutant enzyme at the remaining pHs.

[0038] When using corn native starch as the substrate, the optimal temperature of the mutant enzyme is 45 °C and the optimal pH is 6.0 ( Figure 4 ), and it has more than 80% catalytic activity within the range of pH 5.5 - 7.5.

[0039] (V) Determination of kinetic parameters of the native starch hydrolase of the present invention

[0040] The kinetic constants of the mutant enzyme, including K m and V max , were measured using corn native starch as the substrate. The reaction was carried out by incubating the enzyme in Na2HPO4 - KH2PO4 buffer (50 mM, pH 7.0) at 35 °C for 10 min in the presence of different concentrations of corn native starch (1.0 - 30 mg / mL). Then, the kinetic parameters were calculated by fitting the experimental data to the Lineweaver - Burt equation of the Michaelis - Menten model using Origin 8.0.

[0041] The results showed that the affinity of the mutant enzyme for corn native starch decreased slightly, but its K cat was higher than that of the starting enzyme.

[0042]

[0043] (VI) Detection of the stability of the native starch hydrolase of the present invention

[0044] Under the conditions of 30 - 35 °C and pH 7.0, the starting enzyme Amy and the mutant were heat - treated. Samples were taken every half hour or one hour, and taking the initial enzyme activity as 100%, the remaining rate of enzyme activity after heat - treatment for a certain time was calculated.

[0045] The measurement results showed ( Figure 5 ) that at 30 °C, the half - life of the mutant enzyme was 28 h, which was 15 times higher than that of the starting enzyme; at 35 °C, the half - life of the mutant enzyme was 20 h, which was 20 times higher than that of the starting enzyme.

[0046] (VII) Application of the mutant of the native starch hydrolase of the present invention in hydrolyzing high - concentration corn native starch

[0047] The hydrolysis system was Na2HPO4 - KH2PO4 buffer (50 mM, pH 7.0) with the addition of CaCl2 with a final concentration of 1 mM, followed by the addition of 30% (W / V) corn native starch and the mutant of the native starch hydrolase. 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 commercial enzyme BLA with the same enzyme amount was added as a control group.

[0048] In the experiment of hydrolyzing 30% raw corn starch, the results showed that the mutant enzyme basically reached a plateau after 4 h, and its hydrolysis rate for corn could reach 43%. Under the same conditions, it could reach the hydrolysis rate of commercial amylase BLA for high-concentration raw corn starch, showing great application potential.

Claims

1. A high specific activity raw starch hydrolase mutant, characterized in that: The amino acid sequence of the high specific activity raw starch hydrolase mutant is shown in SEQ ID No:

1.

2. A gene encoding the high specific activity raw starch hydrolase mutant according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No:

2.

3. An expression strain of the raw starch hydrolase mutant according to claim 1, characterized in that: The classification name of the expression strain is Bacillus subtilis WB600 / pBHSs142-AmyZ1(ΔTG / W345A), which has been deposited in the China Center for Type Culture Collection (CCTCC), the deposit number is CCTCC NO: CCTCC M20221942, the deposit date is December 12, 2022, and the deposit address: Wuhan University, Wuhan, China.

4. The application of the raw starch hydrolase mutant according to claim 1, characterized in that: Hydrolyzing corn raw starch with the raw starch hydrolase mutant.

5. The application according to claim 4, characterized in that: The concentration of the corn raw starch ≤ 30%.

6. The application according to claim 4, characterized in that: The hydrolysis temperature is 30 - 45 °C, pH 5.5 - 7.5.

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