CN116286748A - Heat-resistant alpha-amylase mutant with high specific activity, and gene and application thereof - Google Patents
Heat-resistant alpha-amylase mutant with high specific activity, and gene and application thereof Download PDFInfo
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- 102000004139 alpha-Amylases Human genes 0.000 title claims abstract description 51
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- 230000000694 effects Effects 0.000 title claims abstract description 23
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- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- FYGDTMLNYKFZSV-BYLHFPJWSA-N β-1,4-galactotrioside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-BYLHFPJWSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01001—Alpha-amylase (3.2.1.1)
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Abstract
The invention relates to the technical field of agricultural biology, in particular to a heat-resistant alpha-amylase mutant with high specific activity, and a gene and application thereof. The invention obtains the mutant of the alpha-amylase by carrying out A262D single-point mutation on mutant alpha-amylase K152H/A166C/E168H (M1) with the amino acid sequence shown as SEQ ID NO. 1. Compared with the alpha-amylase M1, the specific activity of the alpha-amylase mutant is greatly improved, and the optimal temperature is not changed and is 95 ℃.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a heat-resistant alpha-amylase mutant with high specific activity, and a gene and application thereof.
Background
Enzymes are the most important biological products required to sustain life of the earth. Alpha-amylase is one of the most industrially used enzyme preparations. Alpha-amylase is a starch hydrolase that randomly cleaves alpha-1, 4-D-glucosidic bonds in starch molecules, thereby releasing glucose, short-chain oligosaccharides, limit dextrins, maltotriose and maltose. Starch consists of two polymers, amylose (consisting of alpha-1, 4-glycosidic linkages) and amylopectin (containing alpha-1, 4-glycosidic linkages and alpha-1, 6-glycosidic linkages). The use of inexpensive starch as a raw material to produce high value added products is an economically viable way.
Most industrial applications of alpha-amylase are mainly from microbial sources. The main advantages of microbial derived alpha-amylase as an industrial process are more stable, more economical, easier handling at both genetic and protein level. Alpha-amylase is very widely used, for example in the sugar industry for liquefaction and gelatinization of starch, in the textile industry for paper desizing, biofuel production, pharmaceutical and analytical chemistry. While wild-type alpha-amylase requires some modification to withstand the harsh industrial production environment. Further improvement of the catalytic efficiency is also a major challenge in cases where stability is satisfied. Therefore, the thermostable alpha-amylase is subjected to molecular improvement by means of protein engineering to improve the catalytic efficiency, so that mutants capable of improving the production efficiency are obtained, and the production cost is reduced.
Disclosure of Invention
It is an object of the present invention to provide a mutant obtained by point mutation using an alpha-amylase mutant K152H/A166C/E168H (M1) derived from archaea thermophilum Thermococcus eurythermalis as a female parent.
It is a further object of the present invention to provide a gene encoding the above mutant.
It is a further object of the present invention to provide the amino acid sequences of the above mutants.
It is still another object of the present invention to provide a recombinant vector comprising the above mutant gene.
It is still another object of the present invention to provide a recombinant strain comprising the above mutant gene.
According to a specific embodiment of the invention, site-directed mutagenesis is performed on the alpha-amylase mutant K152H/A166C/E168H (M1) with the amino acid sequence shown in SEQ ID NO. 1.
SEQ ID NO:1
AKYLELEEGGVIMQAFYWDVPSGGIWDTIRQKIPEWYDAGISAIWIPPASKGMGG
AYSMGYDPYDFFDLGEYDQKGTVETRFGSKQELVNMINTAHAYGIKVIADIVINH
RHRAGGDLEWNPFVNDYTWTDFSKVASGKYTANYLDFHPNEVHCCDEGTFGGFP
DICHHKSWDQYWLWASNESYAAYLRSIGVDAWRFDYVKGYGAWVVKDWLDW
WGGWAVGEYWDTNVDALLNWAYSSDAKVFDFPLYYKMDAAFDNKNIPALVEAL
KNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNK
DRLKNLIWIHDHLAGGSTDIVYYDNDELIFVRNGYGDKPGLITYINLGSSKAGRW
VYVPKFAGACIHEYTGNLGGWVDKWVDSSGWVYLEAPAHDPANGYYGYSVWSYCGVG。
According to a specific embodiment of the present invention, a wild-type alpha-amylase having an amino acid sequence shown in SEQ ID NO.1 is subjected to mutation at position 262 from alanine to aspartic acid, thereby obtaining an alpha-amylase mutant.
The alpha-amylase mutant with high thermostability according to the present invention has the amino acid sequence shown in SEQ ID NO. 2 and consists of 435 amino acids.
SEQ ID NO:2：
AKYLELEEGGVIMQAFYWDVPSGGIWDTIRQKIPEWYDAGISAIWIPPASKGMGG
AYSMGYDPYDFFDLGEYDQKGTVETRFGSKQELVNMINTAHAYGIKVIADIVINH
RHRAGGDLEWNPFVNDYTWTDFSKVASGKYTANYLDFHPNEVHCCDEGTFGGFP
DICHHKSWDQYWLWASNESYAAYLRSIGVDAWRFDYVKGYGAWVVKDWLDW
WGGWAVGEYWDTNVDALLNWAYSSDAKVFDFPLYYKMDAAFDNKNIPDLVEAL
KNGGTVVSRDPFKAVTFVANHDTDIIWNKYPAYAFILTYEGQPTIFYRDYEEWLNK
DRLKNLIWIHDHLAGGSTDIVYYDNDELIFVRNGYGDKPGLITYINLGSSKAGRW
VYVPKFAGACIHEYTGNLGGWVDKWVDSSGWVYLEAPAHDPANGYYGYSVWSYCGVG。
According to a specific embodiment of the present invention, there is also provided a gene encoding the above-mentioned alpha-amylase mutant having high thermostability, the nucleotide sequence of which is as shown in SEQ ID NO:3, which is 1305bp in total.
SEQ ID NO：3：
GCTAAATACCTGGAACTGGAAGAAGGTGGTGTTATCATGCAGGCTTTCTACTGG
GACGTTCCGTCTGGTGGTATCTGGTGGGACACCATCCGTCAGAAAATCCCGGAA
TGGTACGACGCTGGTATCTCTGCTATCTGGATCCCGCCGGCTTCTAAAGGTATGG
GTGGTGCTTACTCTATGGGTTACGACCCGTACGACTTCTTCGACCTGGGTGAAT
ACGACCAGAAAGGTACCGTTGAAACCCGTTTCGGTTCTAAACAGGAACTGGTT
AACATGATCAACACCGCTCACGCTTACGGTATCAAAGTTATCGCTGACATCGTTA
TCAACCACCGTGCTGGTGGTGACCTGGAATGGAACCCGTTCGTTAACGACTAC
ACCTGGACCGACTTCTCTAAAGTTGCTTCTGGTAAATACACCGCTAACTACCTG
GACTTCCACCCGAACGAAGTTCATTGCTGCGACGAAGGTACCTTCGGTGGTTT
CCCGGACATCTGTCACCATAAATCTTGGGACCAGTACTGGCTGTGGGCTTCTAA
CGAATCTTACGCTGCTTACCTGCGTTCTATCGGTGTTGACGCTTGGCGTTTCGAC
TACGTTAAAGGTTACGGTGCTTGGGTTGTTAAAGACTGGCTGGACTGGTGGGG
TGGTTGGGCTGTTGGTGAATACTGGGACACCAACGTTGACGCTCTGCTGAACT
GGGCTTACTCTTCTGACGCTAAAGTTTTCGACTTCCCGCTGTACTACAAAATGG
ACGCTGCTTTCGACAACAAAAACATCCCGGATCTGGTTGAAGCTCTGAAAAAC
GGTGGTACCGTTGTTTCTCGTGACCCGTTCAAAGCTGTTACCTTCGTTGCTAAC
CACGACACCGACATCATCTGGAACAAATACCCGGCTTACGCTTTCATCCTGACC
TACGAAGGTCAGCCGACCATCTTCTACCGTGACTACGAAGAATGGCTGAACAA
AGACCGTCTGAAAAACCTGATCTGGATCCACGACCACCTGGCTGGTGGTTCTA
CCGACATCGTTTACTACGACAACGACGAACTGATCTTCGTTCGTAACGGTTACG
GTGACAAACCGGGTCTGATCACCTACATCAACCTGGGTTCTTCTAAAGCTGGTC
GTTGGGTTTACGTTCCGAAATTCGCTGGTGCTTGCATCCACGAATACACCGGTA
ACCTGGGTGGTTGGGTTGACAAATGGGTTGACTCTTCTGGTTGGGTTTACCTGG
AAGCTCCGGCTCACGACCCGGCTAACGGTTACTACGGTTACTCTGTTTGGTCTTACTGCGGTGTTGGT。
According to a specific embodiment of the present invention, there is also provided a recombinant vector comprising the above-mentioned alpha-amylase mutant gene, the starting vector of which is specifically pET-22b (+).
According to a specific embodiment of the invention, there is also provided a recombinant strain comprising the above-mentioned alpha-amylase mutant gene, the starting strain of which is specifically E.coli BL21 (DE 3).
The recombinant expression vector is specifically pET-22b (+); the recombinant strain is specifically E.coli BL21 (DE 3).
The method for preparing the alpha-amylase with high heat resistance according to the 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 the alpha-amylase.
Compared with alpha-amylase M1, the alpha-amylase mutant has the advantages that the optimal action temperature of alpha-amylase hydrolase is unchanged, the specific activity is improved, the specific activity at 95 ℃ is 18594.79U/mg, the M1 is 12648.07U/mg, and the mutant is improved by 47% compared with a parent.
The invention provides application of the alpha-amylase mutant with high specific activity, and can be particularly applied to the fields of energy, food and feed.
The invention overcomes the defects of the prior art and provides the alpha-amylase mutant which has high heat resistance and high specific activity and is suitable for being applied to the fields of energy sources, foods, feeds and the like. The optimum acting temperature of the mutant enzyme provided by the invention is 95 ℃, the optimum pH is 5.5 ℃, and the specific activity at 95 ℃ is improved by 47% compared with that of the parent M1. Therefore, the alpha-amylase mutant provided by the invention can well meet the requirements of the application in the fields of energy, food, feed and the like on the hydrolysis activity of the alpha-amylase at high temperature, and has very broad application prospect.
Drawings
FIG. 1 shows the optimum temperature results for the purified alpha-amylase parent and mutant;
FIG. 2 shows the optimum pH results for the purified alpha-amylase parent and mutant;
FIG. 3 shows the kinetic results of the purified alpha-amylase parent and mutant.
Detailed Description
Test materials and reagents
1. Strains and vectors: expression host E.coli BL21 (DE 3), expression plasmid vector pET-22b (+).
2. Enzymes and other biochemical reagents: endoenzymes, ligases, substrate soluble starches;
3. coli culture medium LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH natural).
Description: the molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.
Example 1 preparation of recombinant Strain BL21 (pET-22 b (+) -teamy)
1. Construction of recombinant Strain BL21 (pET-22 b (+) -teamy)
The pre-obtained mutant K152H/A166C/E168H (M1) recombinant E.coli strain BL21 (pET-22 b (+) -teamy-K152H/A166C/E168H) was coated on LB (containing 100. Mu.g/mLAmp) for activation. And then carrying out verification by nucleic acid gel electrophoresis.
EXAMPLE 2 preparation of recombinant Strain BL21 (pET-22 b (+) -m 1-A262D)
1. Construction of recombinant plasmid pET-22b (+) -m1-A262D
The optimized mutation site is designed to mutate 262 alanine into aspartic acid, the mutation site is introduced by a point mutation kit method, and sequencing verification is carried out on the mutation site, so that the alpha-amylase mutant plasmid pET-22b (+) -m1-A262D is finally obtained. The primers used are shown in Table 1:
TABLE 1 mutant alpha-amylase specific primers
2. Construction of recombinant Strain BL21 (pET-22 b (+) -m 1-A262D)
The correctly sequenced monoclonal was inoculated into 50mL LB medium, incubated overnight (37 ℃) in a shaker, and plasmids were extracted using a plasmid miniprep kit. The plasmid was transferred into E.coli BL21 (DE 3) as an expression host, and the plasmid was spread on LB (100. Mu.g/mL Amp) for selection. Recombinant E.coli strain BL21 (pET-22 b (+) -m 1-A262D) was obtained.
Example 3 acquisition of the alpha-Amylase protein parent M1 and mutant A262D
1. Inducible expression of protein M1 and A262D
The obtained recombinant expression strains BL21 (pET-22 b (+) -teamy-m 1) and BL21 (pET-22 b (+) -m 1-A262D) are inoculated into 50mL LB culture medium for seed culture, and after culturing for 16 hours at the temperature of 37 ℃, the strain is transferred into 400mL LB culture medium with the inoculum size of 1 percent, and cultured for 2-4 hours at the temperature of 37 ℃, the concentration of the strain is measured, an enzyme-labeled instrument is used for reading the absorbance at the wavelength of 600nm, and when reaching 0.6-0.8, IPTG is added to the final concentration of 1Mm,200rpm and 16 ℃ for induction expression.
2. Purification of protein M1 and A262D
The bacterial liquid after the induction of expression was centrifuged at 12000rpm for 10min, the bacterial cells were collected, resuspended in 10mM Tris-HCl solution (pH 7.6), and then sonicated, and the supernatant was collected by centrifugation. The protein was purified by nickel affinity chromatography, eluting with 1M imidazole, 20mM Tris-HCl,0.5M NaCl, and collecting the eluate to give purified protein, which was desalted.
Example 4 detection of optimal action temperature of alpha-Amylase M1 and A262D
After induction of expression, M1 and A262D were purified and the enzyme activity was measured.
Method for measuring enzyme activity (DNS (3, 5-dinitrosalicylic acid): the prepared 2% soluble starch was diluted with Ph5.5 buffer solution (0.1M HAc-NaAc) to 1% final concentration to give a starch solution as substrate, and the measurement system comprised 900. Mu.L of substrate and 100. Mu.L of appropriately diluted enzyme solution at 60, 70, 80, 85, 90, 95, respectively,
And (3) reacting for 30min in a water bath kettle at the temperature of 100 ℃, adding 1.5mL of DNS reagent to terminate the reaction, placing the reaction product in a boiling water bath to treat the reaction product for 5min, rapidly cooling the reaction product to the room temperature, taking 250 mu L of mixed solution, reading the light absorption value at the wavelength of 540nm by using an enzyme-labeling instrument, and setting 1 blank control and 3 parallel reactions in each group. As a result, as shown in FIG. 1, the optimum temperature of the parent M1 was 95℃and the optimum temperature of the mutant A262D was 95 ℃.
Definition of enzyme activity unit (U): under optimal conditions, the amount of enzyme required to hydrolyze soluble starch to 1. Mu. MoL glucose per minute is one unit of enzyme activity.
Example 5 detection of optimal pH for alpha-Amylase M1 and A262D
The prepared 2% soluble starch was diluted to 1% final concentration of starch solution as substrate with 0.1M buffer solutions of different pH (pH 3.5-4 citric acid-disodium hydrogen phosphate: pH 4.5-6 acetic acid-sodium acetate, pH 6.5-7: disodium hydrogen phosphate-sodium dihydrogen phosphate), the measurement system comprising 900. Mu.L of substrate and 100. Mu.L of appropriately diluted enzyme solution, and the optimum pH was measured by reacting in a 95 water bath for 30min, and the subsequent procedure was the same as in example 4. As shown in FIG. 2, the parent M1 has an optimum pH of 5 and mutant A262D has an optimum pH of 5-5.5.
EXAMPLE 6 determination of specific Activity of alpha-amylase M1 and A262D
The prepared 2% soluble starch was diluted to 1% final concentration with a buffer solution (0.1M HAc-NaAc) at pH5.5 to serve as a substrate, and the activity of M1 and A262D was measured at 95℃in the same manner as in example 4. As shown in Table 2, the specific activities of M1 and A262D are 12648.07 and 18594.79U/mg, respectively, and A262D is improved by 47% compared with the parent.
Example 7 determination of kinetics of alpha-Amylase M1 and A262D
The dynamic parameters are measured with buffer solution (0.1M acetic acid-sodium acetate) with pH of 5.5 and concentration of 0.5, 0.8, 1.0, 1.3, 1.5, 2.0, 2.5, 5, 8, 10, 12 and 15mg/mL of soluble starch as substrate at the optimal pH and optimal temperature, and the reaction time is 15min. The results are shown in FIG. 3 and Table 2, V of A262D max Above M1, substrate affinity (K m ) Smaller than M1, but k cat /K m Slightly higher than M1.
TABLE 2
The above embodiments are only used for explaining the technical solution of the present application, and do not limit the protection scope of the present application.
Claims (8)
1. The alpha-amylase mutant with high specific activity is characterized in that the amino acid sequence of the alpha-amylase mutant is shown as SEQ ID NO. 2.
2. An alpha-amylase gene encoding the alpha-amylase mutant of claim 1 having a high specific activity.
3. A recombinant vector comprising the α -amylase gene of claim 2.
4. A recombinant strain comprising the α -amylase gene of claim 2.
5. A method for preparing an alpha-amylase having a high specific activity, comprising the steps of:
1) Preparing a recombinant vector comprising the α -amylase gene of claim 2;
2) Transforming a host cell with the recombinant vector obtained in step 1);
3) Culturing the host cell by fermentation, and isolating the alpha-amylase.
6. Use of an alpha-amylase mutant with high specific activity according to claim 1.
7. Use of the alpha-amylase mutant according to claim 1 in energy, food and feed.
8. A method for increasing the specific activity of an alpha-amylase, which comprises the step of carrying out a262D single point mutation on an alpha-amylase M1 having an amino acid sequence as shown in SEQ ID No. 1.
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| CN117821426A (en) * | 2024-01-16 | 2024-04-05 | 山西晋龙集团饲料有限公司 | Mutant polypeptide of alpha-amylase and application thereof |
| CN117904079A (en) * | 2024-02-23 | 2024-04-19 | 南京大学 | Alpha-amylase mutant, gene, vector and preparation method thereof |
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| CN117821426A (en) * | 2024-01-16 | 2024-04-05 | 山西晋龙集团饲料有限公司 | Mutant polypeptide of alpha-amylase and application thereof |
| CN117821426B (en) * | 2024-01-16 | 2024-06-04 | 山西晋龙集团饲料有限公司 | Mutant polypeptide of alpha-amylase and application thereof |
| CN117904079A (en) * | 2024-02-23 | 2024-04-19 | 南京大学 | Alpha-amylase mutant, gene, vector and preparation method thereof |
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