Thermostable alpha-amylase mutants with high specific activity, genes and uses thereof

Abstract

The present application relates to the field of agricultural biotechnology, and in particular, the present application relates to a heat-resistant alpha-amylase mutant with high specific activity, and genes and applications thereof. The present application obtains the mutant of the alpha-amylase by performing single-point mutation of A262D on the mutant alpha-amylase K152H / A166C / E168H (M1) with the amino acid sequence shown in SEQ ID NO. 1. The alpha-amylase mutant of the present application has greatly improved specific activity compared with the alpha-amylase M1, and the optimal temperature remains 95 DEG C.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a heat-resistant α-amylase mutant with high specific activity, its gene, and its applications. Background Technology

[0002] Enzymes are among the most important biological products necessary for sustaining life on Earth. Alpha-amylase is one of the most widely used enzyme preparations in industry. Alpha-amylase is a starch hydrolase that randomly cleaves the α-1,4-D-glucosidic bonds in starch molecules, releasing glucose, short-chain oligosaccharides, limit dextrins, maltotriose, and maltose. Starch is composed of two polymers: amylose (composed of α-1,4-glycosidic bonds) and amylopectin (containing both α-1,4-glycosidic and α-1,6-glycosidic bonds). Using inexpensive starch as a raw material to produce high-value-added products is an economically viable approach.

[0003] Most industrial applications of α-amylases are derived from microorganisms. The main advantages of microbially derived α-amylases for industrial production are their greater stability, cost-effectiveness, and ease of manipulation at both the genetic and protein levels. α-Amylases have a wide range of applications, such as in sugar liquefaction and starch gelatinization, paper desizing in the textile industry, biofuel production, pharmaceuticals, and analytical chemistry. However, wild-type α-amylases require modification to withstand harsh industrial production environments. Further improving their catalytic efficiency after achieving sufficient stability remains a significant challenge. Therefore, protein engineering is used to molecularly modify thermostable α-amylases to enhance their catalytic efficiency, resulting in mutants that improve production efficiency and reduce production costs. Summary of the Invention

[0004] One object of the present invention is to provide a mutant obtained by point mutation of the α-amylase mutant K152H / A166C / E168H(M1) derived from the thermophilic archaea Thermococcus eurythermalis.

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

[0006] Another object of the present invention is to provide the amino acid sequence of the above-mentioned mutant.

[0007] Another object of the present invention is to provide a recombinant vector containing the above-mentioned mutant gene.

[0008] Another object of the present invention is to provide a recombinant strain containing the above-mentioned mutant gene.

[0009] According to a specific embodiment of the present invention, site-directed mutagenesis was performed on the α-amylase mutant K152H / A166C / E168H(M1) with an amino acid sequence as shown in SEQ ID NO:1.

[0010] SEQ ID NO:1

[0011] AKYLELEEGGVIMQAFYWDVPSGGIWDTIRQKIPEWYDAGISAIWIPPASKGMGG

[0012] AYSMGYDPYDFFDLGEYDQKGTVETRFGSKQELVNMINTAHAYGIKVIADIVINH

[0013] RHRAGGDLEWNPFVNDYTWTDFSKVASGKYTANYLDFHPNEVHCCDEGTFGGFP

[0014] DICHHKSWDQYWLWASNESYAAYLRSIGVDAWRFDYVKGYGAWVVKDWLDW

[0015] WGGWAVGEYWDTNVDALLNWAYSSDAKVFDFPLYYKMDAAFDNKNIPALVEAL

[0016] KNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNK

[0017] DRLKNLIWIHDHLAGGSTDIVYYDNDELIFVRNGYGDKPGLITYINLGSSKAGRW

[0018] VYVPKFAGACIHEYTGNLGGWVDKWVDSSGWVYLEAPAHDPANGYYGYSVWSYCGVG.

[0019] According to a specific embodiment of the present invention, the wild-type α-amylase with the amino acid sequence shown in SEQ ID NO.1 is mutated at position 262, changing alanine to aspartic acid, thereby obtaining an α-amylase mutant.

[0020] The α-amylase mutant with high heat resistance according to the present invention has an amino acid sequence as shown in SEQ ID NO:2, consisting of 435 amino acids.

[0021] SEQ ID NO:2:

[0022] AKYLELEEGGVIMQAFYWDVPSGGIWDTIRQKIPEWYDAGISAIWIPPASKGMGG

[0023] AYSMGYDPYDFFDLGEYDQKGTVETRFGSKQELVNMINTAHAYGIKVIADIVINH

[0024] RHRAGGDLEWNPFVNDYTWTDFSKVASGKYTANYLDFHPNEVHCCDEGTFGGFP

[0025] DICHHKSWDQYWLWASNESYAAYLRSIGVDAWRFDYVKGYGAWVVKDWLDW

[0026] WGGWAVGEYWDTNVDALLNWAYSSDAKVFDFPLYYKMDAAFDNKNIPDLVEAL

[0027] KNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNK

[0028] DRLKNLIWIHDHLAGGSTDIVYYDNDELIFVRNGYGDKPGLITYINLGSSKAGRW

[0029] VYVPKFAGACIHEYTGNLGGWVDKWVDSSGWVYLEAPAHDPANGYYGYSVWSYCGVG.

[0030] According to a specific embodiment of the present invention, a gene encoding the above-mentioned α-amylase mutant with high heat resistance is also provided, the nucleotide sequence of which is shown in SEQ ID NO: 3, and is 1305 bp in total.

[0031] SEQ ID NO: 3:

[0032] GCTAAATACCTGGAACTGGAAGAAGGTGGTGTTATCATGCAGGCTTTCTACTGG

[0033] GACGTTCCGTCTGGTGGTATCTGGTGGGACACCATCCGTCAGAAAATCCCGGAA

[0034] TGGTACGACGCTGGTATCTCTGCTATCTGGATCCCGCCGGCTTCTAAAGGTATGG

[0035] GTGGTGCTTACTCTATGGGTTACGACCCGTACGACTTCTTCGACCTGGGTGAAT

[0036] ACGACCAGAAAGGTACCGTTGAAACCCGTTTCGGTTCTAAACAGGAACTGGTT

[0037] AACATGATCAACACCGCTCACGCTTACGGTATCAAAGTTATCGCTGACATCGTTA

[0038] TCAACCACCGTGCTGGTGGTGACCTGGAATGGAACCCGTTCGTTAACGACTAC

[0039] ACCTGGACCGACTTCTCTAAAGTTGCTTCTGGTAAATACACCGCTAACTACCTG

[0040] GACTTCCACCCGAACGAAGTTCATTGCTGCGACGAAGGTACCTTCGGTGGTTT

[0041] CCCGGACATCTGTCACCATAAATCTTGGGACCAGTACTGGCTGTGGGCTTCTAA

[0042] CGAATCTTACGCTGCTTACCTGCGTTCTATCGGTGTTGACGCTTGGCGTTTCGAC

[0043] TACGTTAAAGGTTACGGTGCTTGGGTTGTTAAAGACTGGCTGGACTGGTGGGG

[0044] TGGTTGGGCTGTTGGTGAATACTGGGACACCAACGTTGACGCTCTGCTGAACT

[0045] GGGCTTACTCTTCTGACGCTAAAGTTTTCGACTTCCCGCTGTACTACAAAATGG

[0046] ACGCTGCTTTCGACAACAAAAACATCCCGGATCTGGTTGAAGCTCTGAAAAAC

[0047] GGTGGTACCGTTGTTTCTCGTGACCCGTTCAAAGCTGTTACCTTCGTTGCTAAC

[0048] CACGACACCGACATCATCTGGAACAAATACCCGGCTTACGCTTTCATCCTGACC

[0049] TACGAAGGTCAGCCGACCATCTTCTACCGTGACTACGAAGAATGGCTGAACAA

[0050] AGACCGTCTGAAAAACCTGATCTGGATCCACGACCACCTGGCTGGTGGTTCTA

[0051] CCGACATCGTTTACTACGACAACGACGAACTGATCTTCGTTCGTAACGGTTACG

[0052] GTGACAAACCGGGTCTGATCACCTACATCAACCTGGGTTTCTTCTAAAGCTGGTC

[0053] GTTGGGTTTACGTTCCGAAATTCGCTGGTGCTTGCATCCACGAATACACCGGTA

[0054] ACCTGGGTGGTTGGGTTGACAAATGGGTTGACTCTTCTGGTTGGGTTTACCTGG

[0055] AAGCTCCGGCTCACGACCCGGCTAACGGTTACTACGGTTACTCTGTTTGGTCTTACTGCGGTGTTGGT.

[0056] According to a specific embodiment of the present invention, a recombinant vector containing the above-mentioned α-amylase mutant gene is also provided, wherein the starting vector of the recombinant expression vector is specifically pET-22b(+).

[0057] According to a specific embodiment of the present invention, a recombinant strain containing the above-mentioned α-amylase mutant gene is also provided, wherein the starting strain of the recombinant strain is E. coli BL21(DE3).

[0058] The recombinant expression vector is specifically pET-22b(+); the recombinant strain is specifically E. coli BL21(DE3).

[0059] The method for preparing α-amylase with high heat resistance according to the present invention comprises the following steps:

[0060] 1) Prepare a recombinant vector containing the above mutant gene;

[0061] 2) Transform the host using the recombinant vector;

[0062] 3) Ferment the host and isolate α-amylase.

[0063] Compared with α-amylase M1, the α-amylase mutant of the present invention maintains the same optimal temperature for α-amylase hydrolysis, but its specific activity is improved. The specific activity at 95°C is 18594.79 U / mg, while that of M1 is 12648.07 U / mg. The mutant is 47% more active than the parent.

[0064] This invention provides applications of the above-mentioned α-amylase mutant with high specific activity, specifically in the fields of energy, food, and feed.

[0065] This invention overcomes the shortcomings of existing technologies and provides an α-amylase mutant with high heat resistance and high specific activity, suitable for applications in the energy, food, and feed industries. The mutant enzyme provided by this invention has an optimal operating temperature of 95℃ and an optimal pH of 5.5℃, and its specific activity at 95℃ is 47% higher than that of the parent M1. Therefore, the α-amylase mutant provided by this invention can well meet the requirements for high-temperature α-amylase hydrolytic activity in applications in the energy, food, and feed industries, and has a very broad application prospect. Attached Figure Description

[0066] Figure 1 The optimal temperature results for the purified α-amylase parent and mutant are shown;

[0067] Figure 2 The optimal pH results for the purified α-amylase parent and mutant are shown;

[0068] Figure 3 The results show the kinetics of the purified α-amylase parent and mutant. Detailed Implementation

[0069] Experimental materials and reagents

[0070] 1. Strains and vectors: Expression host E. coli BL21(DE3), expression plasmid vector pET-22b(+).

[0071] 2. Enzymes and other biochemical reagents: endonucleases, ligases, substrate soluble starch;

[0072] 3. Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, natural pH).

[0073] Note: Molecular biology experimental methods not specifically described in the following examples were performed in accordance with the specific methods listed in J. Sambrook's "Molecular Cloning: A Laboratory Manual" (3rd Edition), or according to the kit and product instructions.

[0074] Example 1: Preparation of recombinant strain BL21 (pET-22b(+)-teamy)

[0075] 1. Construct the recombinant strain BL21(pET-22b(+)-teamy)

[0076] The previously obtained mutant K152H / A166C / E168H(M1) recombinant Escherichia coli strain BL21 (pET-22b(+)-teamy-K152H / A166C / E168H) was plated on LB (containing 100 μg / mL Amp) for activation. The results were then verified by nucleic acid gel electrophoresis.

[0077] Example 2: Preparation of recombinant strain BL21 (pET-22b(+)-m1-A262D)

[0078] 1. Construction of recombinant plasmid pET-22b(+)-m1-A262D

[0079] The optimized mutation site design involved mutating alanine to aspartic acid at position 262. The mutation site was introduced using a point mutagenesis kit and verified by sequencing, ultimately yielding the α-amylase mutant plasmid pET-22b(+)-m1-A262D. The primers used are shown in Table 1.

[0080] Table 1. Mutant α-amylase-specific primers

[0081]

[0082] 2. Construct the recombinant strain BL21(pET-22b(+)-m1-A262D)

[0083] The correctly sequenced single clones were inoculated into 50 mL LB medium and cultured overnight in a shaker (37°C). Plasmids were extracted using a plasmid mini-prep kit. The plasmids were transformed into the expression host *E. coli* BL21(DE3) and plated on LB medium (containing 100 μg / mL Amp) for selection. The recombinant *E. coli* strain BL21 (pET-22b(+)-m1-A262D) was obtained.

[0084] Example 3: Obtaining the α-amylase protein parent M1 and mutant A262D

[0085] 1. Inducible expression of proteins M1 and A262D

[0086] The obtained recombinant expression strains BL21(pET-22b(+)-teamy-m1) and BL21(pET-22b(+)-m1-A262D) were inoculated into 50 ml LB medium for seed culture. After culturing at 200 rpm and 37 ℃ for 16 h, they were transferred to 400 ml LB medium at a 1% inoculation rate and cultured at 200 rpm and 37 ℃ for 2-4 h. The cell concentration was measured by reading the absorbance at 600 nm using a microplate reader. When the absorbance reached 0.6-0.8, IPTG was added to a final concentration of 1 Mm, and expression was induced at 200 rpm and 16 ℃.

[0087] 2. Purification of proteins M1 and A262D

[0088] The induced bacterial culture was centrifuged at 12000 rpm for 10 min to collect the cells. The cells were then resuspended in 10 mM Tris-HCl solution (pH 7.6), followed by sonication and centrifugation to collect the supernatant. The protein was purified by nickel affinity chromatography using a solution of 1 M imidazole, 20 mM Tris-HCl, and 0.5 M NaCl. The eluent was collected, and the purified protein was then desalted.

[0089] Example 4: Determination of the optimal operating temperature of α-amylase M1 and A262D

[0090] After induction of expression, M1 and A262D were purified and their enzyme activity was measured.

[0091] Enzyme activity assay (DNS (3,5-dinitrosalicylic acid) method): A 2% soluble starch solution was diluted to a final concentration of 1% with a pH 5.5 buffer solution (0.1 M HAc-NaAc). The measurement system consisted of 900 μL of substrate and 100 μL of appropriately diluted enzyme solution. The enzyme activity was measured at concentrations of 60, 70, 80, 85, 90, and 95 μL.

[0092] The reaction was carried out in a 100℃ water bath for 30 min. After stopping the reaction by adding 1.5 mL of DNS reagent, the mixture was placed in a boiling water bath for 5 min. After rapid cooling to room temperature, 250 μL of the mixture was taken and the absorbance was read at 540 nm using a microplate reader. One blank control and three replicates were set up for each reaction. The results are as follows: Figure 1 As shown, the optimal temperature for the parent M1 is 95℃, and the optimal temperature for the mutant A262D is also 95℃.

[0093] Enzyme activity unit (U) definition: Under optimal conditions, the amount of enzyme required to hydrolyze soluble starch to produce 1 μmol of glucose per minute is defined as one enzyme activity unit.

[0094] Example 5: Determination of the optimal pH for α-amylase M1 and A262D

[0095] The prepared 2% soluble starch was diluted to a final concentration of 1% using 0.1M buffer solutions of different pH values ​​(pH 3.5-4: citrate-disodium hydrogen phosphate; pH 4.5-6: acetic acid-sodium acetate; pH 6.5-7: disodium hydrogen phosphate-sodium dihydrogen phosphate). The measurement system consisted of 900 μL of substrate and 100 μL of appropriately diluted enzyme solution. The optimal pH was determined by reacting the solution in a 95°C water bath for 30 min. Subsequent procedures were the same as in Example 4. Figure 2 As shown, the optimal pH for parental M1 is 5, and the optimal pH for mutant A262D is 5-5.5.

[0096] Example 6: Determination of the specific activities of α-amylase M1 and A262D

[0097] The prepared 2% soluble starch solution was diluted to a final concentration of 1% with a pH 5.5 buffer solution (0.1M HAc-NaAc) as the substrate. The activities of M1 and A262D were determined at 95°C using the same method as in Example 4. As shown in Table 2, the specific activities of M1 and A262D were 12648.07 and 18594.79 U / mg, respectively, with A262D showing a 47% increase compared to the parent.

[0098] Example 7: Determination of the kinetics of α-amylase M1 and A262D

[0099] Soluble starch at concentrations of 0.5, 0.8, 1.0, 1.3, 1.5, 2.0, 2.5, 5, 8, 10, 12, and 15 mg / mL was prepared using a pH 5.5 buffer solution (0.1 M acetate-sodium acetate) as substrate. Kinetic parameters were determined at their respective optimal pH and temperature, with a reaction time of 15 min. Results are as follows: Figure 3 As shown in Table 2, the V of A262D max Above M1, substrate affinity (K m It is smaller than M1, but k cat / K m Slightly higher than M1.

[0100] Table 2

[0101]

[0102] The above embodiments are only used to explain the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. Mutants of α-amylase having high specific activity, characterized in that, The amino acid sequence of the alpha-amylase mutant is shown as SEQ ID NO:

2.

2. An α-amylase gene, characterized in that, The alpha-amylase mutant having high specific activity according to claim 1.

3. A recombinant vector comprising the alpha-amylase gene according to claim 2.

4. A recombinant strain comprising the alpha-amylase gene according to claim 2.

5. A method for preparing an α-amylase having a high specific activity, characterized in that, The method comprises the following steps: 1) preparing a recombinant vector comprising the alpha-amylase gene according to claim 2; 2) transforming a host cell with the recombinant vector obtained in step 1); 3) fermenting the host cell and isolating the alpha-amylase.

6. Use of the alpha-amylase mutant having high specific activity according to claim 1 for hydrolyzing starch.

7. Use of the alpha-amylase mutant according to claim 1 in energy, food and feed.

8. A method of increasing the specific activity of an α-amylase, characterized in that, The method comprises the following steps: performing single-point mutation of A262D on the alpha-amylase M1 having the amino acid sequence shown as SEQ ID NO. 1.

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