CN113201518A

CN113201518A - High-activity alpha-amylase

Info

Publication number: CN113201518A
Application number: CN202110479064.4A
Authority: CN
Inventors: 刘庚敬
Original assignee: Guangzhou Boshi Biotechnology Co ltd
Current assignee: Guangzhou Boshi Biotechnology Co ltd
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-08-03

Abstract

The invention relates to a high-activity alpha-amylase, which is characterized in that an alpha-amylase gene sequence (alpha-amylase, GenBank: KX216807.1) is obtained from NCBI, error-prone PCR is carried out to obtain a mutation product, the mutation product is recombined into pichia pastoris to obtain expression, mutant seeds with higher enzyme activity are screened, 1 mutant seed with the enzyme activity being 258.99% higher than that of a wild type is obtained, the gene sequence is 542 th position, original C is respectively mutated into G, and the high-activity alpha-amylase has higher specific enzyme activity and can save cost.

Description

High-activity alpha-amylase

Technical Field

The invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to high-activity alpha-amylase.

Background

Alpha-amylases, alpha-1, 4-glucan-4-glucanohydrolases (ec.3.2.1.1), act randomly on the alpha-1, 4-glucosidic linkages within starch, glycogen, oligosaccharides or polysaccharides and hydrolyze starch into small molecules such as glucose, maltose, oligosaccharides. The amylase is a kind of enzyme preparation with the widest application and the largest yield. Industrial alpha-amylases are classified into two major classes, bacterial alpha-amylases and fungal alpha-amylases, depending on the microbial origin. The main products of starch hydrolysis by bacterial alpha-amylase are dextrin and a small amount of oligosaccharide and glucose, the starch and maltotriose can be hydrolyzed by fungal alpha-amylase, and the final products are mainly maltose, partial oligosaccharide and a small amount of glucose. Maltose is widely applied to the pharmaceutical industry and the food industry due to the excellent characteristics of maltose, and the fungal alpha-amylase has special high maltose generation capacity, so that the application of the fungal alpha-amylase in various industries is increased year by year, such as high maltose syrup, baking and brewing industries and the like, but the fungal alpha-amylase still has the problem of insufficient enzyme activity at present. The invention obtains the mutant with high activity by large-flux screening.

Disclosure of Invention

The invention aims to provide high-activity alpha-amylase, which can save cost.

The preparation process of the invention is as follows:

1. the alpha-amylase sequence (alpha-amylase, GenBank: KX216807.1, Seq No.1) was obtained from NCBI and synthesized by Chinesian corporation to design PCR primers: 5'-TATCGGAATTAATTCGGATCCATGAAGTCTTTCTTAAGTCTCCTTTGC-3' (Seq No.2), 5'-TGGTGGTGCTCGAGTGCGGCCGCTTAGTTCTTTTGGAATATGGCAGG-3' (Seq No.3), after the PCR reaction, adding 20 μ l of cloning Enhancer into a PCR system, incubating at 37 ℃ for 15min, incubating at 80 ℃ for 15min, digesting the pET20b (+) plasmid by using BamH I and Not I, carrying out 0.75% agarose gel electrophoresis on the digested product, recovering, dissolving In sterilized double distilled water, uniformly mixing the incubated PCR product with a purified linear vector, adding In-Fusion enzyme, reacting at 50 ℃ for 15min, transforming E.coli JM109, selecting positive clone, sending to a biological company for sequencing, transforming the correctly identified plasmid into a host E.coli BL21, and expressing to obtain the genetically engineered bacterium containing wild type sequence plasmid;

2. extraction of plasmid DNA

E.coli BL21 genetically engineered bacteria carrying plasmid pET20b (+)/alpha-amylase are inoculated in LB/Amp (Amp final concentration 100 mug/mL) liquid culture medium, after overnight culture at 37 ℃ and 200r/min, plasmids are extracted by using a plasmid miniprep kit, and the specific operation is carried out according to the instruction;

3. error-prone PCR amplification and construction of mutant libraries

Taking the plasmid obtained in the step 2 as a template, carrying out enzyme digestion by Not I to linearize the plasmid, carrying out error-prone PCR amplification on the gene by using primer sequences 5'-TATCGGAATTAATTCGGATCCATGAAGTCTTTCTTAAGTCTCCTTTGC-3' (Seq No.2) and 5'-TGGTGGTGCTCGAGTGCGGCCGCTTAGTTCTTTTGGAATATGGCAGG-3' (Seq No.3), wherein the error-prone PCR amplification system (50 mu L) is 10 XTaKaRa Taq Buffer and dNTPs mix, the primers are respectively 0.2 mu mol/L, and the template DNA is 200ng, Taq DNA polymerase 2.5U, 5mmol/L Mn²⁺0.5U/. mu.l Taq DNA polymerase 2.5. mu.l, 7mmol/L Mg²⁺And PCR reaction conditions are as follows: 5min at 95 ℃ (94 ℃ 1min, 55 ℃ 1min, 72 ℃ 2min, 35 cycles In total), 10min at 72 ℃, adding 20 mul of Cloning Enhancer into a PCR system, incubating at 37 ℃ for 15min, incubating at 80 ℃ for 15min, carrying out enzyme digestion linearization on pET20b (+) plasmid by BamH I and Not I, carrying out 0.75% agarose gel electrophoresis on enzyme digestion products, recovering, dissolving In sterilized double distilled water, uniformly mixing incubated PCR products with purified linear vectors, adding In-Fusion enzyme, reacting at 50 ℃ for 15min, and transforming E.coli BL 21;

4. high throughput screening of mutant libraries

Incubating the transformed E.coli BL21 obtained in the step 3 at 37 ℃ for 1h, collecting thallus, coating an ampicillin resistant culture medium (100 mu g/mL), incubating at 37 ℃ for 12h, scraping all colonies on a plate, inoculating the colonies into a 100mL LB culture medium containing 100 mu g/mL ampicillin, performing shaking culture at 37 ℃ for 14h, extracting a mixed plasmid, performing Xba I enzyme digestion on the mixed plasmid, performing electric shock transformation on the mixed plasmid into Pichia pastoris GS115, coating an MD plate, after colonies appear on the MD plate, selecting the colonies onto an MM culture medium coated with X-gal to ensure that the strains changing the X-gal into blue are positive mutants, selecting the positive strains, inoculating into a 48-hole culture plate, adding 500 mu L and 2% of YPD culture medium into each hole, culturing at 28 ℃ and 200r/min for 48h, centrifugally collecting the thallus, suspending the thallus by using 500 mu L BMMY culture medium, culturing at 28 ℃ and, Culturing at 200r/min for 48h, supplementing methanol every 12h until the final concentration is 0.5%, and centrifuging after induction to obtain supernatant as crude enzyme solution;

5. enzyme protein purification

Preparing a buffer solution used for AKTA, wherein the ratio of solution A: 20mmol/L disodium hydrogen phosphate-citric acid buffer (pH7.5), solution B: after the column was equilibrated with 1mol/L sodium chloride solution (20mmol/L disodium hydrogenphosphate-citric acid buffer solution, pH7.5) and 5 bed volumes of solution A, the crude enzyme solution was passed through solution A to a captorQ (1mL) anion column, and gradient elution was carried out by solution B: eluting the hybrid protein by using 11% of liquid B with the volume 5 times that of the column bed, and eluting the target protein by using 5% of liquid B;

6. determination of protein content in crude enzyme solution

Drawing a standard curve: bovine Serum Albumin (BSA) is used as a standard substance, 10mg/mL BSA mother solution is prepared, and the BSA mother solution is diluted into the following gradients: 10.0mg/mL, 8.0mg/mL, 6.0mg/mL, 4.0mg/mL, 2.0mg/mL, 1.0mg/mL, 0.5mg/mL, taking 40 muL of BSA solution or ultrapure water with different concentrations, adding 260 muL of Bio-Rad Coomassie brilliant blue protein staining solution diluted by 5 times, shaking and uniformly mixing, standing at room temperature for 15min, taking 200 muL, adding the 200 muL into an ELISA plate, measuring absorbance at 595nm, recording numerical values, drawing a standard curve, taking 40 muL of crude enzyme solution or purified enzyme solution, adding 260 muL of diluted staining solution, standing at room temperature for 15min after uniformly mixing, taking 200 muL, adding the 200 muL into the ELISA plate, measuring the absorbance at 595nm, recording the numerical values, and calculating the protein concentration of the enzyme solution according to the standard curve;

7. determination of enzyme activity and specific activity

Measuring enzyme activity by using a DNS (3, 5-dinitrosalicylic acid) method, wherein the specific enzyme activity is equal to enzyme activity/protein concentration;

8. and (3) sending the mutant with the highest specific enzyme activity to a biological company for sequencing.

The invention has the beneficial effects that:

the obtained mutant alpha-amylase has higher specific enzyme activity and can save cost.

Drawings

FIG. 1 error-prone PCR electrophoretogram

FIG. 2 protein Standard Curve

FIG. 3 DNS Standard Curve

FIG. 4 three-dimensional modeling of mutant alpha-amylase and wild-type alpha-amylase

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1

Materials and reagents related to the present example are shown in table 1, and experimental instruments are shown in table 2;

TABLE 1 Experimental materials and reagents

TABLE 2 Experimental instrumentation

Gene electric shock leading-in instrument	Bio-Rad Ltd
		Berle T100 PCR instrument	Beijing century Kexin scientific instruments Co., Ltd
BBS-H1100 clean bench	BIOBASE BIODUSTRY (SHANDONG) Co.,Ltd.
		DYY-6D type nucleic acid electrophoresis apparatus	BEIJING LIUYI INSTRUMENT FACTORY
Mini Protein 3 Protein electrophoresis system	Bio-Rad Inc. of USA
		GelDoc gel imaging system	Bio-Rad Inc. of USA
UV1900 ultraviolet visible light spectrophotometer	Jean Qi (Shanghai) Instrument Technology Co.,Ltd.
		TGL-16MB high-speed freezing centrifuge	CHANGSHA XIANGZHI CENTRIFUGE INSTRUMENT Co.,Ltd.
THZ-300 constant temperature culture shaking table	Shanghai Meteorological Instrument Co Ltd
		SPX-100B-Z biochemical incubator	Suzhou Jiandong precision instruments Co., Ltd

The LB medium formula: 10g/l of tryptone, 5g/l of yeast powder, 10g/l of NaCl and 7.0 of pHs;

solid LB medium: adding 2g of agar powder into each 100mL of liquid LB culture medium, and autoclaving;

YPD medium: 1% yeast extract, 2% peptone, 2% glucose, ph 6.0;

MD solid medium: 0.00004% Biotin, 1.34% YNB, 2% glucose, 1.5% agarose;

MM solid medium: 0.00004% Biotin, 1.34% YNB, 0.5% methanol, 1.5% agarose;

BMMY medium: 2% peptone, 1% yeast extract, 0.3% K₂HPO₄，1.18％KH₂PO₄1.34% YNB, 0.5% methanol (V/V), 0.00004% Biotin;

PTM trace salts: 0.6% CuSO₄，0.008％NaI₂，0.3％MnSO₄，0.02％Na₂MoO₄，0.002％H₃BO₃，0.05％CoCl₂，2％ZnCl₂，6.5％FeSO₄0.5% sulfuric acid (V/V);

yeast fermentation basal salt culture medium: 0.5% KH₂PO₄，5％NH₄H₂PO₄，1.485％MgSO₄，1.82％K₂SO₄，0.093％CaSO₄0.15% KOH, 0.00011% Biotin, 0.44% PTM trace salt, 2% glucose;

yeast fermentation basic salt induction medium: 0.5% KH₂PO₄，5％NH₄H₂PO₄，1.485％MgSO₄，1.82％K₂SO₄，0.093％CaSO₄0.15% KOH, 0.00011% Biotin, 0.44% PTM trace salt, 0.5% methanol;

IPTG stock (200 mg/mL): preparing 200mg/mL isopropyl sulfo-beta-D-galactoside by deionized water, filtering and sterilizing by using a 0.22 mu m filter, subpackaging into 1mL per tube, and storing at-20 ℃;

ampicillin stock (200 mg/mL): preparing 200mg/mL ampicillin (Amp) stock solution with deionized water, filtering with 0.22 μm filter for sterilization, subpackaging into 1mL per tube, and storing at-20 deg.C;

50 × TAE electrophoresis buffer: 242g of Tris, 57.1mL of glacial acetic acid and 100mL of 0.5mol/L EDTA (pH8.0), and the volume is fixed to 1L by using distilled water;

agarose solution (1.2%): weighing 0.60g of agarose, and dissolving in 50mL of barbital sodium-HCl buffer solution;

1. the alpha-amylase sequence (alpha-amylase, GenBank: KX216807.1, Seq No.1) was obtained from NCBI and synthesized by Chinesian corporation to design PCR primers: 5'-TATCGGAATTAATTCGGATCCATGAAGTCTTTCTTAAGTCTCCTTTGC-3' (Seq No.2), 5'-TGGTGGTGCTCGAGTGCGGCCGCTTAGTTCTTTTGGAATATGGCAGG-3' (Seq No.3), after the PCR reaction is finished, adding 20 mul of Cloning Enhancer into a PCR system, incubating at 37 ℃ for 15min, incubating at 80 ℃ for 15min, digesting the pET20b (+) plasmid by using BamH I and Not I, carrying out 0.75% agarose gel electrophoresis on the digested product, recovering, dissolving In sterilized double distilled water, uniformly mixing the incubated PCR product with a purified linear vector, adding In-Fusion enzyme, reacting at 50 ℃ for 15min, transforming E.coli JM109, selecting positive clone, sending the positive clone to a biological company for sequencing, transforming the correctly identified plasmid into a host E.coli BL21, and expressing to obtain the genetically engineered bacterium containing wild type sequence plasmids;

2. extraction of plasmid DNA

3. error-prone PCR amplification and construction of mutant libraries

Taking the plasmid obtained in the step 2 as a template, carrying out enzyme digestion by Not I to linearize the plasmid, carrying out error-prone PCR amplification on the gene (shown in figure 1) by using a primer sequence 5'-TATCGGAATTAATTCGGATCCATGAAGTCTTTCTTAAGTCTCCTTTGC-3' (Seq No.2), 5 '-TGGTGGTGCTCGAGTGCGGCCGCTTAGTTCTTTTGGAATATGGCAGG-3' (Seq No.3), wherein a 50 mu L error-prone PCR amplification system is 10 XTaKaRa Taq Buffer, dNTPs mix, 0.2 mu mol/L of each primer, 200ng of template DNA, 2.5U of Taq DNA polymerase, and 5mmol/L of Mn²⁺0.5U/. mu.l Taq DNA polymerase 2.5. mu.l, 7mmol/L Mg² ⁺And PCR reaction conditions are as follows: 5min at 95 ℃ (94 ℃ 1min, 55 ℃ 1min, 72 ℃ 2min, 35 cycles In total), 10min at 72 ℃, adding 20 mul of Cloning Enhancer into a PCR system, incubating at 37 ℃ for 15min, incubating at 80 ℃ for 15min, carrying out enzyme digestion linearization on pET20b (+) plasmid by BamH I and Not I, carrying out 0.75% agarose gel electrophoresis on enzyme digestion products, recovering, dissolving In sterilized double distilled water, uniformly mixing incubated PCR products with purified linear vectors, adding In-Fusion enzyme, reacting at 50 ℃ for 15min, and transforming E.coli BL 21;

4. high throughput screening of mutant libraries

Incubating the transformed E.coli BL21 obtained in the step 3 at 37 ℃ for 1h, collecting thallus, coating an ampicillin resistant culture medium (100 mu g/mL), incubating at 37 ℃ for 12h, scraping all colonies on a plate, inoculating the colonies into a 100mL LB culture medium containing 100 mu g/mL ampicillin, performing oscillation culture at 37 ℃ for 14h, extracting a mixed plasmid, performing Xba I enzyme digestion on the mixed plasmid, performing electric shock transformation on the mixed plasmid into Pichia pastoris GS115, coating an MD plate, after colonies appear on the MD plate, selecting the colonies onto an MM culture medium coated with X-gal to ensure that the strains with blue X-gal are positive mutants, selecting the positive strains, inoculating into a 48-hole culture plate, adding 500 mu L of YPD culture medium and 2% of inoculum size into each hole, culturing at 28 ℃ and 200r/min for 48h, centrifugally collecting the thallus, culturing and suspending the thallus in 500 mu L BMMY at 28 ℃ and 200r/min for 48h, methanol is supplemented every 12h until the final concentration is 0.5%, and after induction is finished, the crude enzyme solution is obtained by centrifuging and taking supernatant;

5. enzyme protein purification

6. determination of protein content in crude enzyme solution

Drawing a standard curve: bovine Serum Albumin (BSA) is used as a standard substance, 10mg/mL BSA mother solution is prepared, and the BSA mother solution is diluted into the following gradients: 10.0mg/mL, 8.0mg/mL, 6.0mg/mL, 4.0mg/mL, 2.0mg/mL, 1.0mg/mL, 0.5mg/mL, taking 40 μ L of BSA solution or ultrapure water with different concentrations, adding 260 μ L of the Bio-Rad Coomassie brilliant blue protein staining solution diluted by 5 times, shaking and uniformly mixing, standing at room temperature for 15min, taking 200 μ L, adding the solution into an ELISA plate, measuring the absorbance at 595nm, recording the numerical value, drawing a standard curve (see figure 2), taking 40 μ L of crude enzyme solution or purified enzyme solution, adding 260 μ L of the diluted staining solution, standing at room temperature for 15min after uniformly mixing, taking 200 μ L, adding the solution into the ELISA plate, measuring the absorbance at 595nm, recording the numerical value, and calculating the protein concentration of the enzyme solution according to the standard curve (see figure 3);

TABLE 3 protein concentration of enzyme solution (mg/mL)

Group of	OD_595nm	Protein concentration	Group of	OD_595nm	Protein concentration
						Wild type	0.1009	0.79	Mutant 17	0.1016	0.82
Mutant 1	0.1020	0.84	Mutant 18	0.1025	0.86
						Mutant 2	0.0993	0.72	Mutant 19	0.1020	0.84
Mutant 3	0.1009	0.79	Mutant 20	0.1031	0.89
						Mutant 4	0.1002	0.76	Mutant 21	0.1029	0.88
Mutant 5	0.1000	0.75	Mutant 22	0.0996	0.73
						Mutant 6	0.1018	0.83	Mutant 23	0.1016	0.82
Mutant 7	0.1009	0.79	Mutant 24	0.1018	0.83
						Mutant 8	0.1018	0.83	Mutant 25	0.1025	0.86
Mutant 9	0.1002	0.76	Mutant 26	0.1011	0.8
						Mutant 10	0.1016	0.82	Mutant 27	0.0998	0.74
Mutant 11	0.0998	0.74	Mutant 28	0.1029	0.88
						Mutant 12	0.1007	0.78	Mutant 29	0.1002	0.76
Mutant 13	0.1020	0.84	Mutant 30	0.1013	0.81
						Mutant 14	0.0998	0.74	Mutant 31	0.1025	0.86
Mutant 15	0.1009	0.79	Mutant 32	0.1036	0.91
						Mutant 16	0.1002	0.76	Mutant 33	0.1047	0.96

7. Determination of enzyme activity and specific activity

7.1 preparation of maltose Standard solution (10 mmol/L): weighing 34.2mg of maltose, and adding distilled water to a constant volume of 10 mL;

7.2 formulation of DNS reagent: weighing 0.63g of DNSS, dissolving in 50mL of water, placing in a water bath at 45 ℃, continuously stirring, adding 26.2mL of 2mol/L NaOH, slowly dropwise adding, continuously stirring, weighing 18.2g of sodium potassium tartrate, 0.5g of phenol and 0.5g of sodium sulfite, and dissolving in water; continuously stirring and uniformly mixing the substances in a water bath kettle at 45 ℃ to completely dissolve the substances; cooling, adding water to a constant volume of 100mL, storing in a brown bottle, and storing in a refrigerator at 4 ℃ for at least one week;

7.3 plotting DNS standard curve: taking 6 test tubes with numbers of 1-6, adding maltose standard solution (10mmol/L), distilled water and DNS reagent according to the following table 4, mixing well, and placing into boiling water bath to boil for 15 min; immediately after the reaction, the mixture was cooled with cold running water, 10.5mL of distilled water was added, the mixture was mixed by reversing the top and bottom, and the absorbance at 550nm of the mixture was measured using tube No. 6 as a control (see FIG. 3);

TABLE 4 DNS Standard Curve reagent ratio

Numbering	Maltose standard liquid (mL)	Maltose content (μmol)	Distilled water (mL)	DNS(mL)
					1	0.1	1	0.4	1.5
2	0.2	2	0.3	1.5
					3	0.3	3	0.2	1.5
4	0.4	4	0.1	1.5
					5	0.5	5	0	1.5
6	0	0	0.5	1.5

7.4 enzyme Activity assay

Adding 400 mu L of 1% (W/V) soluble starch solution prepared by distilled water into an EP tube; adding 100 μ L enzyme solution, and reacting at 45 deg.C for 30 min; immediately sucking 250 mu L of reaction solution into a test tube, adding 750 mu L of LDNS reagent into the test tube, uniformly mixing, and boiling for 15min in boiling water; taking out the test tube, immediately cooling the test tube with flowing water, adding 5.25mL of distilled water, and uniformly mixing; measuring the light absorption value of the mixed solution at 550nm by using a spectrophotometer; one enzyme activity unit (U): the amount of enzyme required to hydrolyze starch to 1. mu. mol maltose per minute under certain conditions (45 ℃); the calculation formula of the enzyme activity is as follows: (OD)_550nm-0.3247)/0.5454; specific enzyme activity/protein concentration;

TABLE 5 alpha-Amylase Activity

The mutants with the highest specific enzyme activity were sent to the organism company for sequencing (see Table 6).

TABLE 6 comparison of mutant sequences with highest specific enzyme activity with wild type sequences

The invention obtains the mutant with 258.99% higher specific enzyme activity.

9. At https: wild-type alpha-amylase and mutant-enzyme alpha-amylase were modeled three-dimensionally (see FIG. 4).

Sequence listing

<110> Guangzhou Bojii Biotechnology Ltd

<120> a highly active alpha-amylase

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1389

<212> DNA

<213> Rhizopus oryzae

<400> 1

atgaagtctt tcttaagtct cctttgcagc gtcttccttt tacctttggt tgtacagtct 60

gtgcctgtca tcaagcgagc ctcagccagc gactgggaga accgagtcat ctaccaattg 120

ttaactgatc gatttgcaaa atcgaccgat gataccaatg gctgcaataa cctgagtgac 180

tactgtggcg gaacatttca aggaatcatt aatcacttgg attacattgc cggaatggga 240

tttgatgcta tctggatatc acctatcccc aaaaatgcga atggaggtta ccatggctat 300

tgggctactg acttttctca aataaatgag cattttggaa ctgctgatga cttgaaaaag 360

ttggttgcag ctgctcatgc aaagaacatg tacgttatgc tggacgttgt tgccaatcat 420

gctggcattc cttcatcagg tggcgactac tctggctaca cgttcggtca aagctctgaa 480

taccacacag cctgtgatat caattacaac agccagacct ctattgagca gtgctggatt 540

tctggtttgc ctgatatcaa cactgaagac tcggccattg ttagcaaatt gaattcgatt 600

gtttctggtt gggtatctga ttatggcttt gacggtcttc gaatcgacac tgtgaagcac 660

attcgtaaag atttctggga cggctatgtc tctgctgctg gtgtatttgc taccggagaa 720

gtgcttagcg gcgatgtttc ttatgtctca ccctatcagc agcatgttcc ttctttactc 780

aactacccat tgtattatcc agtctatgat gtattcacca aatcccgtac catgagccgt 840

ttaagctctg gcttttctga tattaaaaat ggaaacttta aagacattga tgtcttggtc 900

aactttattg acaatcacga tcagcctcgt ttgttatcca aagctgatca aagtctcgtc 960

aagaatgctc ttgcttattc tttcatggtc caaggtatcc ctgtcttgta ctatggtaca 1020

gaacaatcct tcaagggtgg taacgatcct aacaacagag aggtcttatg gaccactggt 1080

tactcgacca catctgatat gtacaagttt gtcactactc ttgtcaaggc acgcaagggc 1140

tcaaactcca cagtaaatat gggaattgct caaaccgata acgtctatgt gttccaaaga 1200

ggtggctctc tggttgttgt caataactat ggtcaaggat caacaaacac aattactgta 1260

aaggctggct cgttctctaa tggagatact ttgactgatg tgttctccaa caaatctgtt 1320

actgttcaaa ataaccagat cacattccaa ttgcagaatg gaaaccctgc catattccaa 1380

aagaactaa 1389

<210> 3

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tatcggaatt aattcggatc catgaagtct ttcttaagtc tcctttgc 48

<210> 4

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tggtggtgct cgagtgcggc cgcttagttc ttttggaata tggcagg 47

Claims

1. A high-activity alpha-amylase is characterized in that the gene sequence (alpha-amylase, GenBank: KX216807.1) of the enzyme is mutated into G from original C at the 542 th site, and the construction method comprises the following steps:

1) the alpha-amylase sequence was obtained from NCBI (alpha-amylase, GenBank: KX216807.1), synthesized by yokogaku corporation, and designed PCR primers: 5'-TATCGGAATTAATTCGGATCCATGAAGTCTTTCTTAAGTCTCCTTTGC-3', 5'-TGGTGGTGCTCGAGTGCGGCCGCTTAGTTCTTTTGGAATATGGCAGG-3', PCR after the reaction, adding 20 μ l of Cloning Enhancer into the PCR system, incubating at 37 ℃ for 15min, incubating at 80 ℃ for 15min, carrying out enzyme digestion on pET20b (+) plasmid by using BamH I and Not I, recovering the enzyme digestion product after 0.75% agarose gel electrophoresis, dissolving the enzyme digestion product In sterilized double distilled water, uniformly mixing the incubated PCR product with a purified linear vector, adding In-Fusion enzyme, reacting at 50 ℃ for 15min, transforming E.coli JM109, selecting positive clone, carrying out sequencing by a biological company, transforming the correctly identified plasmid into a host E.coli BL21 for expression, and obtaining the genetically engineered bacterium containing wild type sequence plasmid;

2) extraction of plasmid DNA

3) error-prone PCR amplification and construction of mutant libraries

Taking the plasmid obtained in the step 2 as a template, carrying out enzyme digestion by Not I to linearize the plasmid, carrying out error-prone PCR amplification gene by using a primer sequence 5'-TATCGGAATTAATTCGGATCCATGAAGTCTTTCTTAAGTCTCCTTTGC-3', 5'-TGGTGGTGCTCGAGTGCGGCCGCTTAGTTCTTTTGGAATATGGCAGG-3', wherein an error-prone PCR amplification system (50 mu L) is 10 XTaKaRa Taq Buffer and dNTPs mix, each primer is 0.2 mu mol/L, template DNA is 200ng, Taq DNA polymerase is 2.5U, and 5mmol/L Mn is²⁺0.5U/. mu.l Taq DNA polymerase 2.5. mu.l, 7mmol/L Mg²⁺And PCR reaction conditions are as follows: 5min at 95 ℃ (94 ℃ 1min, 55 ℃ 1min, 72 ℃ 2min, 35 cycles total), 10min at 72 ℃, 20 μ l of Cloning Enhancer is added into a PCR system, incubation is carried out for 15min at 37 ℃, incubation is carried out for 15min at 80 ℃, and pET20b (+) plasmid is applied with BamH I and Not I enzymeCutting and linearizing, recovering the enzyme digestion product after 0.75% agarose gel electrophoresis, dissolving In sterilized double distilled water, uniformly mixing the incubated PCR product and a purified linear carrier, adding In-Fusion enzyme, reacting for 15min at 50 ℃, and converting E.coli BL 21;

4) high throughput screening of mutant libraries

Incubating the transformed E.coli BL21 obtained in the step 3 at 37 ℃ for 1h, collecting thallus, coating an ampicillin resistant culture medium (100 mu g/mL), incubating at 37 ℃ for 12h, scraping all colonies on a plate, inoculating the colonies into a 100mL LB culture medium containing 100 mu g/mL ampicillin, performing oscillation culture at 37 ℃ for 14h, extracting a mixed plasmid, performing XbaI enzyme digestion on the mixed plasmid, performing electric shock transformation on the mixed plasmid into Pichia pastoris GS115, coating an MD plate, after colonies appear on the MD plate, selecting the colonies onto an MM culture medium coated with X-gal to ensure that the strains with blue X-gal are positive mutants, selecting the positive strains, inoculating into a 48-hole culture plate, adding 500 mu L of YPD culture medium and 2% of inoculum size into each hole, culturing at 28 ℃ and 200r/min for 48h, centrifugally collecting the thallus, culturing and suspending the thallus in 500 mu L BMMY for 48h at 28 ℃ and 200r/min, methanol is supplemented every 12h until the final concentration is 0.5%, and after induction is finished, the crude enzyme solution is obtained by centrifuging and taking supernatant;

5) enzyme protein purification

6) determination of protein content in crude enzyme solution

7) determination of enzyme activity and specific activity

8) and (3) sending the mutant with the highest specific enzyme activity to a biological company for sequencing.

2. The use of the highly active α -amylase according to claim 1, wherein said highly active α -amylase is used in the pharmaceutical industry and the food industry.