An alpha-amylase mutant and its gene, vector and preparation method

Abstract

The present invention discloses an alpha-amylase mutant and its gene, vector and preparation method. The point mutation of the alpha-amylase mutant includes substitution mutations at positions 63, 115, 261, 262, 266, 316 and 317 of the original enzyme amino acid sequence. The preparation method of the mutant is as follows: designing a point mutation primer, taking the original enzyme gene as a template, and using the point mutation primer to perform a PCR reaction to obtain a mutant gene and a linearized plasmid; the mutant gene is cloned and connected with a linearized expression vector in one step, and then transferred into a host bacterium for fermentation expression; after collecting the recombinant bacteria, the cells are crushed, and the supernatant is taken by centrifugation to obtain a mutant enzyme. Based on this method, a gene encoding the above-mentioned alpha-amylase mutant protein is provided, and a cloning vector or expression vector containing the above-mentioned gene is constructed based on the gene sequence. The thermal stability and pH tolerance of the alpha-amylase mutant in the present invention are good, and it is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to an alpha-amylase and a preparation method thereof, in particular to an alpha-amylase mutant and a gene, a vector and a preparation method thereof. Background Art

[0002] Amylase is a hydrolase that can hydrolyze the glycosidic bonds present in starch molecules and produce dextrins and oligosaccharides. Amylase is mainly divided into three subtypes, namely α, β and γ types, among which α-amylase is the most intensively studied and widely used starch hydrolase. α-Amylase (α-1,4-glucan-4-glucoside hydrolase, EC 3.2.1.1) can cut the α-1,4-D-glucosidic bonds between glucose residues in starch molecules and other similar α-glucans to produce glucose, short-chain oligosaccharides, limited dextrins, maltotriose and maltose. Starch is composed of two polymers, namely amylose (linked by α-1,4-D-glucosidic bonds) and amylopectin (linked by α-1,4-D-glucosidic bonds and α-1,6-D-glucosidic bonds), and its aqueous solution often has a high viscosity. α-Amylase can reduce the viscosity of starch aqueous solution, so it is also called liquefying amylase.

[0003] α-Amylase mainly comes from the expression of microorganisms, including bacteria and fungi. α-Amylase from microorganisms has lower production cost, is easy to be artificially edited at the gene level and protein level, has high stability and specificity, and is widely used in food, pharmaceutical and chemical industries. For example, it is used for starch gelatinization and syrup production in the sugar industry, paper desizing in the textile industry, agricultural waste production as animal feed or biofuel, and analysis and detection in the pharmaceutical and chemical industries, urban sewage purification, etc. At present, the research direction of α-amylase is not only its production, but also the artificial modification of wild-type α-amylase to improve its stability and make it suitable for application under extreme conditions.

[0004] Existing microbial derived α-amylases mainly come from Bacillus amyloliquefaciens and Bacillus licheniformis, and the α-amylases produced by them are optimally at pH 5.0-6.0 and at a temperature between 35-80°C. When the enzyme solution catalyzes starch hydrolysis at a pH of 8.0-10.0 or at a temperature of 85 to 95°C, the enzyme activity gradually decreases or even becomes inactivated. Due to the harsh conditions in actual industrial production environments, the use of genetic engineering methods to perform site-directed mutagenesis on α-amylases improves their catalytic efficiency, protein stability, and pH tolerance, which helps to improve the availability of enzymes and reduce production costs in the industry. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide an α-amylase mutant with high thermal stability and pH stability. The second purpose of the present invention is to provide a recombinant gene capable of expressing an α-amylase mutant. The third purpose of the present invention is to provide a vector capable of amplifying or expressing a mutant gene. The fourth purpose of the present invention is to propose a method for preparing an α-amylase mutant to solve the problem of how to transform a wild-type α-amylase by point mutation.

[0006] Technical solution: The α-amylase mutant described in the present invention comprises the following point mutations: the proline at position 261 of the amino acid sequence shown in SEQ ID No. 1 is mutated to glycine or histidine and the leucine at position 262 is mutated to isoleucine or arginine.

[0007] The present invention artificially optimizes the alpha-amylase sequence from Cordyceps farinosa, performs site-directed saturation mutation on amino acids with close spatial positions between adjacent beta-folded structures in the original alpha-amylase, increases their interaction force, and improves the stability of the enzyme mutant.

[0008] The present invention is based on the original α-amylase sequence, and one or more amino acid substitution mutations are performed. The original α-amylase is derived from the genome of the fungus Cordyceps farinosa, and the mutations include: the 63rd leucine is mutated to isoleucine, the 63rd leucine is mutated to tryptophan, the 63rd leucine is mutated to valine, the 63rd leucine is mutated to histidine, the 115th leucine is mutated to glutamine, the 115th leucine is mutated to methionine, the 115th leucine is mutated to arginine, the 115th leucine is mutated to asparagine, the 261st proline is mutated to glycine, the 261st proline is mutated to histidine, the 262nd leucine is mutated to isoleucine, the 262nd leucine is mutated to arginine, the 262nd leucine is mutated to arginine, the 262nd leucine is mutated to glutamine, the 115th leucine is mutated to methionine, the 115th leucine is mutated to arginine, the 261st proline is mutated to glycine, the 261st proline is mutated to histidine, the 262nd leucine is mutated to isoleucine, the 262nd leucine is mutated to argin ... Tyrosine at position 66 mutated to aspartic acid, tyrosine at position 266 mutated to methionine, tyrosine at position 266 mutated to leucine, asparagine at position 316 mutated to arginine, asparagine at position 316 mutated to methionine, asparagine at position 316 mutated to phenylalanine, asparagine at position 316 mutated to valine, asparagine at position 316 mutated to isoleucine, alanine at position 317 mutated to cysteine, alanine at position 317 mutated to serine, alanine at position 317 mutated to leucine, alanine at position 317 mutated to tryptophan, and alanine at position 317 mutated to isoleucine.

[0009] Furthermore, based on the above-mentioned mutations at positions 261 and 262, the point mutations also include mutating the aspartic acid at position 316 of the amino acid sequence shown in SEQ ID No. 1 to arginine, methionine or phenylalanine; and mutating the alanine at position 317 to serine, tryptophan or leucine.

[0010] Furthermore, based on the above-mentioned mutations at positions 261, 262, 316 and 317, the point mutation also includes mutating the tyrosine at position 266 of the amino acid sequence shown in SEQ ID No. 1 to leucine or aspartic acid.

[0011] Furthermore, based on the above-mentioned mutations at positions 261 and 262, the point mutation also includes mutating the leucine at position 63 of the amino acid sequence shown in SEQ ID No. 1 to isoleucine or tryptophan.

[0012] Furthermore, based on the above-mentioned mutations at positions 261, 262 and 63, the point mutation also includes mutating the leucine at position 115 of the amino acid sequence shown in SEQ ID No. 1 to arginine.

[0013] Preferably, the amino acid sequence of the α-amylase mutant is specifically one of the sequences shown in SEQ ID No. 2-7, which are:

[0014] The proline at position 261 and the leucine at position 262 in the original enzyme sequence were mutated to glycine and isoleucine;

[0015] The leucine at position 63, proline at position 261, and leucine at position 262 in the original enzyme sequence were mutated to isoleucine, glycine, and isoleucine;

[0016] The leucine at position 63, leucine at position 115, proline at position 261, and leucine at position 262 in the original enzyme sequence were mutated to tryptophan, arginine, glycine, and isoleucine;

[0017] The proline at position 261, the leucine at position 262, the tyrosine at position 266, the aspartic acid at position 316, and the alanine at position 317 in the original enzyme sequence were mutated to glycine, isoleucine, leucine, methionine, and tryptophan.

[0018] The proline at position 261, the leucine at position 262, the aspartic acid at position 316, and the alanine at position 317 in the original enzyme sequence were mutated to histidine, arginine, arginine, and serine.

[0019] The proline at position 261, the leucine at position 262, the tyrosine at position 266, the aspartic acid at position 316, and the alanine at position 317 in the original enzyme sequence were mutated to histidine, arginine, aspartic acid, phenylalanine, and leucine.

[0020] In order to recombinantly express the above α-amylase mutant, the present invention provides a gene encoding the above α-amylase mutant protein.

[0021] Based on the gene sequence of the above α-amylase mutant protein, the present invention constructs a cloning vector or expression vector containing the above gene. The vector can be a plasmid or a virus.

[0022] In order to prepare the above-mentioned α-amylase mutant, the present invention provides a method for preparing the α-amylase mutant, comprising the following steps:

[0023] (1) Designing point mutation primers, using the original C. farinose α-amylase gene as a template, and using the point mutation primers to perform PCR reaction to obtain the mutant gene;

[0024] (2) inserting the mutant gene into an expression vector and then transferring it into a host bacterium for fermentation expression;

[0025] (3) Collecting host bacteria expressing the α-amylase mutant, resuspending the bacteria and breaking the cells, and centrifuging and taking the supernatant to obtain a crude enzyme solution containing the α-amylase mutant.

[0026] Preferably, the sequence of the point mutation primer includes:

[0027]

[0028]

[0029] Note: The underlined sites in the primers are mutation sites

[0030] The system of the PCR reaction is:

[0031]

[0032] The conditions for the PCR reaction were:

[0033]

[0034] The annealing temperature can be selected between 56-58°C.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention recombinantly constructs a series of α-amylase mutants with good thermal stability and pH tolerance by performing site-directed mutagenesis on multiple amino acid sites in the original α-amylase sequence. Compared with the original enzyme, the enzyme activity of the mutant enzyme is increased by more than 60% at temperatures above 85°C. At the same time, under overacid or overalkaline conditions, the enzyme activity of the mutant enzyme is also significantly higher than that of the original enzyme. This feature effectively improves the availability of α-amylase under extremely harsh conditions in actual industrial production environments, has a wide range of applications, strong durability, and can be used on a large scale in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The protein structure diagram of C. farinose α-amylase and the amino acid positions 63 and 115;

[0037] Figure 2 The protein structure diagram of C. farinose α-amylase and the amino acid positions 261 and 262;

[0038] Figure 3 The protein structure diagram of C. farinose α-amylase and the amino acid positions 266, 316 and 317;

[0039] Figure 4 Glucose-OD 540 Standard curve;

[0040] Figure 5 To determine the specific enzyme activity of the original enzyme and the mutant enzyme after heat treatment;

[0041] Figure 6 The specific enzyme activity of the original enzyme and the mutant enzyme was determined under different pH conditions. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0043] Example 1: In this example, the point mutation of the α-amylase mutant is P261G / L262I, and the preparation method thereof is as follows:

[0044] 1. Strains and plasmids

[0045] The strain E. coli DH5α was used for plasmid construction, and the strain E. coli BL21 (DE3) was used for exogenous protein expression, both of which were obtained from commercial sources. The pET-22b (+) plasmid carrying the original C. farinose α-amylase gene was synthesized by Jin Weizhi (Suzhou). The pET-22b (+) plasmid expressing the α-amylase with the site-directed mutation P261G / L262I was constructed by the applicant. The gene sequence in the plasmid was sequenced by Jin Weizhi (Suzhou).

[0046] 2. Experimental reagents and culture medium

[0047] The main experimental reagents include: KOD-Plus-enzyme (TOYOBO), DNA gel recovery kit (Shanghai Bioengineering), plasmid extraction kit (Shanghai Bioengineering), ClonExpress II one-step cloning kit (Novozyme), DNA Marker (Shanghai Bioengineering), DNS reagent (Phygene), endonuclease DpnI (Adamas), and other conventional reagents are domestic analytical grade. The LB medium used is composed of: yeast powder 5g / L, tryptone 10g / L, NaCl 5g / L, and if solid culture medium is used, 15g / L agar powder is added. TB liquid culture medium: yeast powder 12g / L, tryptone 12g / L, glycerol 4ml / L, dipotassium hydrogen phosphate 12.5g / L, potassium dihydrogen phosphate 2.3g / L.

[0048] 3. Construction of recombinant plasmid of α-amylase mutant

[0049] Through computer modeling, the 3D model of the original C. farinose α-amylase was predicted, and primers for the relevant mutation sites were designed, as shown in Table 1. The model structure of the mutation site is shown in Figure 1-3 As shown. The mutation primers and plasmid construction primers were synthesized by Jinweizhi (Suzhou). PCR was performed using the pET-22b(+) plasmid carrying the original C. farinose α-amylase gene as a template. The PCR system is shown in Table 2, and the PCR reaction conditions are shown in Table 3. The amino acid sequence of the original α-amylase is:

[0050] MKLTASLTLLTQALAVLGADTNSWKSRTIYFALTDRVARSASDNGGDGCGQLQDYCGGTFKG L EGKLDYIKGMGFDAIWITPVVQNSARGYHGYWASNLYATNSHYGTSDELKG L VNAAHGKGIYIMVDVVANHVGNGPLNEMQPAPLNQGSSYHPACGINYNDQHSIETCRVASDLPDLDTTDPKIRTLYKDWIKWLMSTYKFDGVRIDTVKHVEKDFWPDFAWASGSYTIGEVFSGDPNYVAGYSKLMGGLLNYPVYF PL NRF Y QQQNSSQALVDMHNQIGSLVPDPTTLGTFLDNHDNPRFLSQKNDVSLFK NALTYVLLARGIPIVYYGSEQAYAGGGDPQNREDLWRSRFNTNSDMYKFFQALGGVRKSHGGLPGNDHVHLFVESDAYAWSRQDGAVMALTSNIGKGQQRQFCFFTQKNNKTWRGIFDGKTYTSGGDGKLCATVNNGEPIVFVAQ. ​​Wherein, the underlined part is the possible point mutation site, including the 63rd, 115th, 261st, 262nd, 266th, 316th, and 317th amino acid residues of the original sequence, and the mutation sites in this embodiment are the 261st and 262nd amino acid residues.

[0051] The nucleic acid sequence of the original α-amylase is:

[0052] ATGAAACTGACCGCGAGCCTGACCCTGCTGACCCAAGCGCTGGCGGTGCTGGGCG

[0053] CGGATACCAACAGCTGGAAAAGCCGCACCATTTATTTTGCGCTGACCGATCGCGTG

[0054] GCGCGCAGCGCGAGCGATAACGGCGGCGATGGCTGCGGTCAGCTGCAAGATTATT

[0055] GCGGCGGCACCTTTAAAGGCCTGGAAGGCAAACTGGATTATATTAAAGGCATGGG

[0056] CTTTGATGCGATTTGGATTACCCCGGTGGTGCAGAACAGCGCGCGCGGCTATCATG

[0057] GCTATTGGGCGAGCAACCTGTATGCGACCAACAGCCATTATGGCACGAGCGATGAA

[0058] CTGAAAGGCCTGGTGAACGCGGCGCATGGCAAAGGCATTTATATTATGGTGGATGT

[0059] GGTGGCGAACCATGTGGGCAACGGCCCGCTGAACGAAATGCAGCCGGCGCCGCT

[0060] GAACCAAGGCAGCAGCTATCATCCGGCGTGCGGCATTAACTATAACGATCAGCATA

[0061] GCATTGAAACCTGCCGCGTGGCGAGCGATCTGCCGGATCTGGATACCACCGATCCG

[0062] AAAATTCGCACCCTGTATAAAGATTGGATTAAATGGCTGATGAGCACCTATAAATTT

[0063] GATGGCGTGCGCATTGATACCGTGAAACATGTGGAAAAAGATTTTTGGCCGGATTT

[0064] TGCGTGGGCGAGCGGCAGCTATACCATTGGCGAAGTGTTTAGCGGCGATCCGAAC

[0065] TATGTGGCGGGCTATAGCAAACTGATGGGCGGCCTGCTGAACTATCCGGTGTATTTT

[0066] CCGCTGAACCGCTTTTATCAGCAACAGAACAGCAGCCAAGCGCTGGTGGATATGC

[0067] ATAATCAGATTGGCAGCCTGGTGCCGGACCCGACCACCCTGGGCACCTTTCTGGAT

[0068] AACCATGATAACCCGCGCTTTCTGAGTCAGAAAAACGATGTGAGCCTGTTTAAAA

[0069] ACGCGCTGACCTATGTGCTGCTGGCGCGCGGCATTCCGATTGTGTATTATGGCAGC

[0070] GAACAAGCGTATGCGGGCGGTGGCGATCCGCAGAACCGCGAAGATCTGTGGCGCA

[0071] GCCGCTTTAACACCAACAGCGATATGTATAAATTTTTTCAAGCGCTGGGCGGCGTG

[0072] CGCAAAAGTCATGGCGGCCTGCCGGGCAACGATCATGTGCATCTGTTTGTGGAAA

[0073] GCGATGCGTATGCGTGGAGCCGCCAAGATGGCGCGGTGATGGCGCTGACGAGCAA

[0074] CATTGGCAAAGGTCAGCAGCGTCAGTTTTGCTTTTTTACGCAGAAAAACAACAAA

[0075] ACCTGGCGCGGCATTTTTGATGGCAAAACCTATACGAGCGGCGGCGACGGCAAAC

[0076] TGTGCGCGACCGTGAACAACGGCGAACCGATTGTGTTTGTGGCGCAG

[0077] Table 1 Primers for point mutation and plasmid construction

[0078]

[0079] Note: The underlined part in the primers indicates the mutation site, “F” represents the upstream primer and “R” represents the downstream primer.

[0080] Table 2 PCR reaction system

[0081]

[0082] Table 3 PCR reaction conditions

[0083]

[0084] In this embodiment, the annealing temperature is 58°C.

[0085] After the PCR product was verified by nucleic acid electrophoresis, it was purified using a DNA gel recovery kit. The reaction system was prepared as shown in Table 4, and the reaction was carried out at 37°C for 30 minutes for one-step cloning to construct the expression plasmid of the mutant enzyme.

[0086] Table 4 One-step cloning system

[0087]

[0088] The obtained plasmid was transformed into the engineered bacteria E. coli DH5α for amplification and extraction, and then sequenced and verified by GeneWeizhi (Suzhou). The gene sequence of the mutant is shown in SEQ ID NO.8.

[0089] 4. Recombinant bacteria expressing α-amylase mutants

[0090] Transfer the recombinant plasmid into the expression host E.coli BL21 (DE3), and spread it on an LB plate containing 100 ng / mL ampicillin for screening. Inoculate the transformant into 5 mL of LB liquid medium containing 100 ng / mL ampicillin, and culture it overnight at 37°C and 140 rpm in a shaker. Take 2.5 mL of the bacterial solution and inoculate it into 50 mL of TB liquid medium containing 100 ng / mL ampicillin, and culture it at 37°C and 140 rpm in a shaker for 6 hours. Add the filtered and sterilized IPTG to the fermentation broth to a final concentration of 0.4 mM, and continue to culture it at 22°C and 140 rpm for 22 hours.

[0091] 5. Preparation of crude enzyme solution of α-amylase mutant

[0092] Transfer the fermentation liquid of the recombinant bacteria to a centrifuge tube and collect the cells by centrifugation at 11,000 rpm. Resuspend the cells with 20 mL of the corresponding buffer, break the cells by ultrasonication for 10 min, and centrifuge again at 11,000 rpm to remove the cell debris. The resulting supernatant is the crude enzyme solution.

[0093] Embodiment 2: The rest is the same as Embodiment 1, except that:

[0094] The point mutation of the α-amylase mutant is L63I / P261G / L262I. The mutant enzyme plasmid constructed in Example 1 is used as a template, and the point mutation primers and plasmid construction primers are as follows:

[0095] Table 5 Point mutation primers and plasmid construction primers

[0096]

[0097] Note: The underlined sites in the primers are mutation sites

[0098] In this embodiment, the annealing temperature is 58°C.

[0099] The gene sequence of the α-amylase mutant L63I / P261G / L262I is shown in SEQ ID NO.9.

[0100] Embodiment 3: The rest is the same as Embodiment 1, except that:

[0101] The point mutation of the α-amylase mutant is L63W / L115R / P261G / L262I. The mutant enzyme plasmid constructed in Example 1 is used as a template, and the point mutation primers and plasmid construction primers are as follows:

[0102] Table 6 Point mutation primers and plasmid construction primers

[0103]

[0104] Note: The underlined sites in the primers are mutation sites

[0105] In this embodiment, the annealing temperature is 57°C.

[0106] The gene sequence of the α-amylase mutant L63W / L115R / P261G / L262I is shown in SEQ ID NO.10.

[0107] Embodiment 4: The rest is the same as Embodiment 1, except that:

[0108] The point mutation of the α-amylase mutant is P261G / L262I / Y266L / N316M / A317W. The mutant enzyme plasmid constructed in Example 1 is used as a template. The point mutation primers and plasmid construction primers are as follows:

[0109] Table 7 Point mutation primers and plasmid construction primers

[0110]

[0111] Note: The underlined sites in the primers are mutation sites

[0112] In this embodiment, the annealing temperature is 56°C.

[0113] The gene sequence of the α-amylase mutant P261G / L262I / Y266L / N316M / A317W is shown in SEQ ID NO.11.

[0114] Embodiment 5: The rest is the same as Embodiment 1, except that:

[0115] The point mutation of the α-amylase mutant is P261H / L262R / N316R / A317S, and its point mutation primers and plasmid construction primers are as follows:

[0116] Table 8 Point mutation primers and plasmid construction primers

[0117]

[0118] Note: The underlined sites in the primers are mutation sites

[0119] In this embodiment, the annealing temperature is 56°C.

[0120] The gene sequence of the α-amylase mutant P261H / L262R / N316R / A317S is shown in SEQ ID NO.12.

[0121] Embodiment 6: The rest is the same as Embodiment 1, except that:

[0122] The point mutation of the α-amylase mutant is P261H / L262R / Y266D / N316F / A317L, and its point mutation primers and plasmid construction primers are as follows:

[0123] Table 9 Point mutation primers and plasmid construction primers

[0124]

[0125] Note: The underlined sites in the primers are mutation sites

[0126] In this embodiment, the annealing temperature is 56°C.

[0127] The gene sequence of the α-amylase mutant P261H / L262R / Y266D / N316F / A317L is shown in SEQ ID NO.13.

[0128] The DNS method was used to determine the enzyme activity of the crude enzyme solution of the α-amylase mutant prepared in Examples 1-6. The DNS method is one of the commonly used methods for determining enzyme activity. It is based on the barbituric acid coloring reaction between the reducing sugar produced by the enzyme-catalyzed reaction and dithiobarbituric acid (DNS) under alkaline conditions. The specific method is as follows:

[0129] Take 30 μL of crude enzyme solution and add it to 120 μL of 1% starch solution and blow it evenly. After reacting at 65°C for 5 minutes, add 150 μL of DNS solution. Treat the mixed solution in a boiling water bath for 10 minutes, immediately cool it to room temperature in an ice bath, and measure its absorbance at 540 nm. In the negative control experiment, the crude enzyme solution was replaced with buffer solution, and the rest of the operations were the same. Prepare 0.5000, 0.3750, 0.2500, 0.1875, and 0.1250 mg / mL glucose standard solutions, take 150 μL and use the above treatment method to obtain the glucose-540nm standard curve (y=7.1794x-0.4505), as shown in the figure. Figure 4 As shown. Definition of crude enzyme activity: Under enzymatic reaction conditions, the amount of enzyme required to catalyze starch hydrolysis to produce 1 nmol of glucose per 1 min is one enzyme activity unit (U). Specific enzyme activity is the catalytic ability of the enzyme per unit volume of crude enzyme solution (U / mL); the specific enzyme activity of crude enzyme solution is calculated based on the absorbance value at 540nm and the standard curve.

[0130] The enzyme activity determination method and results of the original α-amylase and the mutant enzymes prepared in Examples 1-6 after high temperature treatment are as follows:

[0131] The crude enzyme solution prepared in Examples 1-6 was incubated at 75°C, 85°C, and 95°C for 3 hours, and the residual enzyme activity was measured. The results are as follows: Figure 5After high temperature treatment at 85 and 95 °C, the mutants L63W / L115R / P261G / L262I, L63I / P261G / L262I, P261G / L262I, P261H / L262R / N316R / A317S, P261G / L262I / Y266L / N316M / A317W, and P261H / L262R / Y266D / N316F / A317L still had higher enzyme activity than the original enzyme. After incubation at 85°C for 3 hours, the enzyme activity of the mutant enzyme L63I / P261G / L262I was 62.5% higher than that of the original enzyme; after incubation at 95°C for 3 hours, the enzyme activity of the mutant enzyme P261H / L262R / N316R / A317S was 71.2% higher than that of the original enzyme.

[0132] The enzyme activity determination methods and results of the original α-amylase and the mutant enzymes prepared in Examples 1-6 under different pH conditions are as follows:

[0133] The fermentation broth of the recombinant bacteria in Examples 1-6 was centrifuged and the cells were collected, and the cells were resuspended in phosphate buffers with pH values ​​of 4.0, 5.5, 7.0, 8.5, and 10.0, respectively. The crude extraction and enzyme activity determination steps of the remaining enzymes were the same as above. The results of the beneficial mutations are shown in FIG. Figure 6 As shown, some mutants of the original enzyme: L63W / L115R / P261G / L262I, P261G / L262I / Y266L / N316M / A317W, P261H / L262R / N316R / A317S, P261H / L262R / Y266D / N316F / A317L, have improved enzymatic activity compared to the original enzyme under peracidic or peralkaline conditions. Among them, in the buffer solution with a pH of 4.0, the residual enzyme activity of the mutant enzyme P261H / L262R / Y266D / N316F / A317L was 44.3% higher than that of the original enzyme; in the buffer solutions with a pH of 8.5 and 10.0, the enzyme activity of P261G / L262I / Y266L / N316M / A317W was increased by 78.2% and 114.2% compared with that of the original enzyme.

Claims

1. An α-amylase mutant, characterized in that The amino acid sequence of the α-amylase mutant is one of the sequences shown in SEQ ID No. 4-7.

2. A gene encoding the α-amylase mutant according to claim 1.

3. A cloning vector or expression vector containing the gene according to claim 2.

4. The method for preparing an α-amylase mutant according to claim 1, characterized in that: The steps include: (1) Designing point mutation primers, using the original C. farinose α-amylase gene as a template, and using the point mutation primers to perform PCR reaction to obtain the mutant gene and linearized plasmid; (2) using a one-step cloning method to insert the mutant gene into an expression vector and then transfer it into a host bacterium for fermentation expression; (3) Collecting host bacteria expressing the α-amylase mutant, resuspending the bacteria and breaking the cells, and centrifuging and taking the supernatant to obtain a crude enzyme solution containing the α-amylase mutant.

5. The method for preparing an α-amylase mutant according to claim 4, characterized in that: The sequence of the point mutation primer includes: The PCR reaction system is: The PCR reaction conditions were:

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