CN110699337A - Alpha-amylase mutant BasAmy-4 capable of improving specific activity and coding gene and application thereof - Google Patents
Alpha-amylase mutant BasAmy-4 capable of improving specific activity and coding gene and application thereof Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, in particular to an alpha-amylase BasAmy mutant capable of improving specific activity and a coding gene and application thereof. The mutation sites are: the alpha-amylase BasAmy with the amino acid sequence shown as SEQ ID NO.6 has the following mutations: S267Q, L269F, S270P, K274S, a275Y, Q278S, S279Q and L412C. The specific activity of the mutant obtained by the invention is improved compared with that of proenzyme.
Description
The application is a divisional application of the invented patent application (application number: 2017100308497, application date: 2017-01-16, invention name: alpha-amylase BasAmy mutant for improving specific activity and coding gene and application thereof)
Technical Field
The invention relates to the field of genetic engineering, in particular to an alpha-amylase mutant BasAmy-3 capable of improving specific activity and a coding gene and application thereof.
Background
Alpha-amylase is an important enzyme preparation and can randomly cut alpha-1, 4 glycosidic bonds from the interior of a starch molecule to generate dextrin and reducing sugar. Alpha-amylases are widely used in the food industry, brewing, fermentation and textile industry.
Alpha-amylases are distributed quite widely, ranging from microorganisms to higher plants. Compared with alpha-amylase from other sources, the alpha-amylase from the microorganism has the advantages of wide action temperature, wide pH value range and low production cost, so the alpha-amylase from the microorganism is widely applied to various industrial fields. As the most important class of microbial alpha-amylases, Bacillus alpha-amylases are currently the most widely studied and used.
Bacillus desert alpha-amylase is called BasAmy for short, is a medium temperature alpha-amylase, has wide action pH value, and is suitable for industrial fields of food, paper making, feed and the like. Compared with other bacillus alpha-amylase, the BasAmy specific activity is low, the production cost is high, and the application of the BasAmy specific activity in the industrial fields of food, paper making, feed and the like is limited. Therefore, the improvement of the specific activity of the BasAmy and the reduction of the production cost are problems which are urgently needed to be solved by industrial application of the BasAmy.
Disclosure of Invention
The invention carries out molecular modification on the alpha-amylase BasAmy from the Bacillus somnophilus so as to improve the specific activity of the BasAmy, reduce the production cost and lay a foundation for the industrial application of the alpha-amylase BasAmy.
The invention aims to provide an alpha-amylase BasAmy mutant with improved specific activity.
It is still another object of the present invention to provide a gene encoding an alpha-amylase BasAmy mutant with improved specific activity.
The nucleotide sequence and the amino acid sequence of the alpha-amylase BasAmy of the Bacillus sonolatus are respectively shown as SEQ ID NO.1 and SEQ ID NO. 6.
The invention adopts a site-directed saturation mutagenesis method to carry out molecular modification on the 29 th, 267 th, 270 th, 275 th and 350 th positions of alpha-amylase BasAmy shown in SEQ ID NO.6, and determines the optimal mutant amino acid of the 5 th positions of the 29 th, 267 th, 270 th, 275 th and 350 th positions through high-throughput screening. The mutation from L to A is preferably at position 29, from S to Q at position 267, from S to P at position 270, from A to Y at position 275 and from D to G at position 350.
Meanwhile, the error-prone PCR technology is adopted to modify the nucleotide sequence of alpha-amylase BasAmy shown in SEQ ID NO.1, so that a series of mutation sites are obtained. Through high-throughput screening, 6 effective mutants are obtained, namely A112R, L269F, K274S, Q278S, S279Q and L412C.
On the basis of the effective mutation sites, the four Bsamy mutants with improved specific activity are respectively combined one by one to finally obtain four Bsamy mutants which are named as BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4. The relative activities of these four mutants were 130%, 160%, 180% and 125% of BasAmy, respectively. The nucleotide sequences of the BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 mutants are shown in SEQ ID NO.2 to SEQ ID NO.5, and the amino acid sequences are shown in SEQ ID NO.7 to SEQ ID NO. 10.
Wherein BasAmy-1 comprises mutation sites of L29A, A112R, S267Q, K274S, Q278S, S279Q, D356G and L412C.
Wherein BasAmy-2 comprises mutation sites of L29A, L269F, S270P, A275Y, Q278S, S279Q, D356G and L412C.
Wherein BasAmy-3 comprises mutation sites of L29A, A112R, L269F, K274S, A275Y, Q278S, S279Q and L412C.
Wherein BasAmy-4 comprises mutation sites of S267Q, L269F, S270P, K274S, A275Y, Q278S, S279Q and L412C.
The invention carries out molecular modification on alpha-amylase BasAmy of the Bacillus somnophilus through the protein modification and high-throughput screening technology to obtain four mutants with improved specific activity. Lays a foundation for the industrial application of the alpha-amylase of the Bacillus somnophilus.
Drawings
FIG. 1 optimal reaction pH for original alpha-amylases and mutants BasAmy-1 to BasAmy-4
FIG. 2 pH stability of the original alpha-amylase and the mutants BasAmy-1 to BasAmy-4
FIG. 3 optimal reaction temperatures for the original alpha-amylase and the mutants BasAmy-1 to BasAmy-4
FIG. 4 thermostability of original alpha-Amylase and mutants BasAmy-1 to BasAmy-4
Detailed Description
The molecular biology experiments, which are not specifically described in the following examples, were performed according to the specific methods listed in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to the kit and product instructions; the reagents and biomaterials, if not specifically indicated, are commercially available.
Experimental materials and reagents:
1. bacterial strains and vectors
The alpha-amylase of the Bacillus sonoralis desert (Bacillus sonorensis) is purchased from China center for industrial microorganism culture collection management, the strain number is 10848, the Escherichia coli strain Topl0, Pichia pastoris X33, a vector pPICz alpha A, a vector pGAPz alpha A, and Zeocin is purchased from Invitrogen company.
2. Enzyme and kit
Q5 high fidelity Taq enzyme MIX was purchased from NEB company, plasmid extraction, gel purification, restriction enzyme, kit was purchased from Shanghai Biotech company.
3. Culture medium
The E.coli medium was LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0). LBZ is LB medium plus 25 ug/mLzeocin.
The yeast medium was YPD (1% yeast extract, 2% peptone, 2% glucose). The yeast selection medium was YPDZ (YPD +100mg/L zeocin).
Yeast induction medium BMGY (I% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V)) and BMMY (the remainder was identical to BMGY except that 0.5% methanol was used instead of glycerol).
Example 1 cloning of alpha-Amylase from Bacillus sonoralis (Bacillus sororensis)
Inoculating the Bacillus sonolania desert into LB culture medium, culturing for 24 hr, and extracting its genome DNA. Two primers (R: 5'-CTGAATTCATGGTTTACAAATGCAAACGG-3' and F: 5'-CTTCTAGACTATCGTTGGACATAAATCGA-3') were designed based on the reported sequence of Bacillus sonolatopsis alpha-amylase (Genebank: AOFM01000005.1) for amplification of the Bacillus sonolatopsis alpha-amylase gene. And purifying and recovering the amplified PCR product, and respectively connecting the PCR product to expression vectors pPICz alpha A and pPGAPz alpha A to obtain expression vectors pPICz alpha A-Basamy and pGAPz alpha A-Basamy.
Example 2 rational site-directed mutagenesis
The pPICz alpha A-Basamy is taken as a template, and the primers in the table are used for PCR amplification, and specifically the amplification reaction system is as follows:
| q5 high fidelity Taq enzyme MIX | 23uL |
| Corresponding mutant primers | 1uL |
| Corresponding mutant primers | 1uL |
| pPICzαA-Basamy(20ng) | 2uL |
| Adding water to | 50uL |
The reaction procedure was as follows:
and detecting the PCR amplification result by agarose electrophoresis, and purifying and recovering the PCR product. Decomposing the original plasmid by using restriction endonuclease DpnI, transferring the decomposed product into escherichia coli Top10 by using a heat shock method, verifying a recombinant transformant by using a bacterial liquid PCR, extracting a plasmid of the transformant which is verified to be correct, and sequencing to determine a corresponding mutant. Correctly sequenced mutants were linearized with SacI and transformed into Pichia pastoris X33.
Example 3 high throughput screening of high specific Activity mutant strains
The yeast recombinant transformants obtained in example 2 were picked up one by one with a toothpick into 24-well plates, 1mL of BMGY-containing medium was added to each well, cultured at 30 ℃ and 220rpm for about 24 hours, and the supernatant was centrifuged. Then respectively adding 1.6mLBMMY culture medium to carry out induction culture. After 24h of culture, the supernatant is taken out by centrifugation, 200 mu L of the supernatant is respectively taken out to a 96-pore plate, and the alpha-amylase activity is measured. The detection of the enzyme activity of the alpha-amylase is carried out according to the national standard GB/T24401-2009 of the people's republic of China. After high-throughput screening, 5 effective mutation sites are respectively L29A, S267Q, S270P, A275Y and D350G. The relative specific activities of these 5 mutants are shown in table 1.
TABLE 1 relative specific Activity of original and mutant alpha-amylases
| Numbering | Relative specific activity (%) |
| Primary alpha- |
100 |
| L29A | 115 |
| S267Q | 120 |
| S270P | 125 |
| A275Y | 119 |
| D350G | 128 |
Example 4 error-prone PCR irrational engineering
The pGAPz alpha A-Basamy is taken as a template to carry out error-prone PCR random mutation amplification, and the specific amplification method comprises the following steps:
first round amplification: carrying out PCR amplification by taking vector promoter primers AOX5-F and AOX3-R as primers, wherein the reaction system is as follows:
the reaction procedure was as follows:
recovering the first round PCR product, and removing 1 mu L of diluted 50-100 times to be used as a template for the second round PCR; secondly, the third error-prone PCR takes alpha-amylase specific primers R and F as reaction primers instead of the primers AOX5-F and AOX3-R, and the PCR reaction is repeated. The second and third rounds of the product were double digested with XbaI and EcoRI and ligated into the pGAPz. alpha.A vector between the EcoRI and XbaI sites. The ligation product was transformed into X33 and screened for mutants in YPDZ plate cultures. Through high-throughput screening, 6 effective mutants are obtained, namely A112R, L269F, K274S, Q278S, S279Q and L412C. The relative specific activities of these 6 mutants are shown in table 2.
TABLE 2 relative specific Activity of original alpha-Amylase and mutant alpha-Amylase
| Numbering | Relative specific activity (%) |
| Primary alpha- |
100 |
| A112R | 121 |
| L269F | 130 |
| K274S | 126 |
| Q278S | 131 |
| S279Q | 123 |
| L412C | 125 |
Example 5 combinatorial mutagenesis
And performing combined mutation, and finally obtaining 4 combined mutations which are named as BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 through experiments.
Wherein BasAmy-1 comprises mutation sites of L29A, A112R, S267Q, K274S, Q278S, S279Q, D356G and L412C.
Wherein BasAmy-2 comprises mutation sites of L29A, L269F, S270P, A275Y, Q278S, S279Q, D356G and L412C.
Wherein BasAmy-3 comprises mutation sites of L29A, A112R, L269F, K274S, A275Y, Q278S, S279Q and L412C.
Wherein BasAmy-4 comprises mutation sites of S267Q, L269F, S270P, K274S, A275Y, Q278S, S279Q and L412C.
Example 6 analysis of specific Activity of original alpha-Amylase and alpha-Amylase mutants
Respectively purifying the original alpha-amylase and the mutant alpha-amylase by a nickel column purification method. And respectively measuring the corresponding enzyme activity of the purified alpha-amylase and the mutant alpha-amylase and calculating the specific activity. The relative specific activity of the mutants was calculated as the specific activity of the mutants divided by the specific activity of the original alpha-amylase. The relative specific activities of BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 were finally 130%, 160%, 180% and 125%, respectively.
Example 7 optimal reaction pH and pH stability of original alpha-Amylase and mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4
The optimum reaction pH of the original alpha-amylase BasAmy and the mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 is determined by referring to a national standard method. The optimum reaction pH of BasAmy and mutants of BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 is shown in FIG. 1. As can be seen from FIG. 1, the optimum pH values of the mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 were almost the same as those of BaAmy and were all 6.0.
BasAmy and mutants of BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 were treated at pH4-8 for 2 hours at room temperature, and then enzyme activity was determined by the method of national standard, the results are shown in FIG. 2. As can be seen from FIG. 2, the pH stability of the mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 was consistent with that of BasAmy.
Example 8 optimal reaction temperature and thermal stability of original alpha-Amylase and mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4
The optimum reaction temperature of BasAmy and mutants of BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 was determined by reference to the national standard method, and the results are shown in FIG. 3. As can be seen from FIG. 3, the optimum reaction temperatures of BasAmy and mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 were all 60 ℃.
BasAmy and mutants of BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 were treated in water bath at 50 deg.C-90 deg.C for 30 min, and then enzyme activity was determined by national standard method, and the results are shown in FIG. 4. As can be seen from FIG. 4, the thermostability of the mutants BasAmy-1, BasAmy-2, BasAmy-3 and BasAmy-4 was consistent with that of BasAmy.
Sequence listing
<110> Guangdong overflow Multi-interest Biotech Ltd
<120> alpha-amylase mutant BasAmy-4 with improved specific activity, and coding gene and application thereof
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gcttctaaaa catatgcggc aagcctgaac ggcacgctga tgcagtattt tgaatggaat 120
ctgcctaatg acggccagca ttggaagcgc ttacaaaatg atgcgggata tttatccgac 180
attgggataa cggctgtttg gattccgccc gcctacaagg gaacgagcca ggctgacgtt 240
ggatacggcc catacgattt gtacgattta ggggagttcc tgcaaaaagg gacggtgcgg 300
acgaaatacg ggatgaaaac agagcttcag tcagctgtcg gttcgcttca ttcccagaac 360
atccaagtgt atggcgatgt tgtccttaat cataaggctg gggcggatct gacggaggat 420
gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480
atcaaagcgt ggacaggatt caatttccct ggacgcggca gcacatacag tgattttaaa 540
tggcattggt atcattttga tgggacggat tgggacgaat cccgaaagct gaatcgcatc 600
tacaagttcc gcggagatgg gaaggcatgg gattgggagg tttccagcga aaacggcaac 660
tacgattatt taatgtatgc ggatgtcgat tatgaccacc ccgatgttgt ggcagaaatg 720
aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780
gtcaagcata ttaagttctc ttttctttct gattggttga aggctgtgcg tcagagcact 840
ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900
tacttgcaaa aaaccgattt tcaacattct gtattcgatg tgccgcttca ttttaacctt 960
caggccgcat cttcacacgg aggcagctat gatatgagga atttgctgaa cggaacggtt 1020
gtttccaaac atcctttgaa agcggttaca tttgtcgaca accatgacac acagccgggg 1080
caatcattgg agtcgaccgt ccaaacatgg ttcaagccgc ttgcctacgc ttttattttg 1140
acaagagagg ccgggtaccc gcaggttttt tatggagata tgtatgggac aaaaggtcct 1200
acatcgcggg aaattccttc tcttaaaagt aaactggagc cgattttgaa agcgcgcaag 1260
tattatgctt atggaacaca gcatgattat ttcgatcatc cagatgccat cggctggacg 1320
agggaaggcg atcaatccgt cgctgcatca ggcttggccg ctttaatcac agacggaccg 1380
ggcggatcaa agcggatgta tgtgggcagg cagcatgccg gtgagacatg gcatgacatc 1440
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gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480
atcaaagcgt ggacaggatt caatttccct ggacgcggca gcacatacag tgattttaaa 540
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aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780
gtcaagcata ttaagttcca gtttctttct gattggttgt cggctgtgcg ttctcagact 840
ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900
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caggccgcat cttcacacgg aggcagctat gatatgagga atttgctgaa cggaacggtt 1020
gtttccaaac atcctttgaa agcggttaca tttgtcgaca accatggtac acagccgggg 1080
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gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480
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tacgattatt taatgtatgc ggatgtcgat tatgaccacc ccgatgttgt ggcagaaatg 720
aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780
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ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900
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<211>1536
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
atggtttaca aatgcaaacg gatattatgt tgtgtgctgc tgtttttcat agtgctgccg 60
gcttctaaaa catatgcggc aagcgctaac ggcacgctga tgcagtattt tgaatggaat 120
ctgcctaatg acggccagca ttggaagcgc ttacaaaatg atgcgggata tttatccgac 180
attgggataa cggctgtttg gattccgccc gcctacaagg gaacgagcca ggctgacgtt 240
ggatacggcc catacgattt gtacgattta ggggagttcc tgcaaaaagg gacggtgcgg 300
acgaaatacg ggatgaaaac agagcttcag tcaagagtcg gttcgcttca ttcccagaac 360
atccaagtgt atggcgatgt tgtccttaat cataaggctg gggcggatct gacggaggat 420
gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480
atcaaagcgt ggacaggatt caatttccct ggacgcggca gcacatacag tgattttaaa 540
tggcattggt atcattttga tgggacggat tgggacgaat cccgaaagct gaatcgcatc 600
tacaagttcc gcggagatgg gaaggcatgg gattgggagg tttccagcga aaacggcaac 660
tacgattatt taatgtatgc ggatgtcgat tatgaccacc ccgatgttgt ggcagaaatg 720
aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780
gtcaagcata ttaagttctc ttttttttct gattggttgt cgtatgtgcg ttctcagact 840
ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900
tacttgcaaa aaaccgattt tcaacattct gtattcgatg tgccgcttca ttttaacctt 960
caggccgcat cttcacacgg aggcagctat gatatgagga atttgctgaa cggaacggtt 1020
gtttccaaac atcctttgaa agcggttaca tttgtcgaca accatgacac acagccgggg 1080
caatcattgg agtcgaccgt ccaaacatgg ttcaagccgc ttgcctacgc ttttattttg 1140
acaagagagg ccgggtaccc gcaggttttt tatggagata tgtatgggac aaaaggtcct 1200
acatcgcggg aaattccttc tcttaaaagt aaatgtgagc cgattttgaa agcgcgcaag 1260
tattatgctt atggaacaca gcatgattat ttcgatcatc cagatgccat cggctggacg 1320
agggaaggcg atcaatccgt cgctgcatca ggcttggccg ctttaatcac agacggaccg 1380
ggcggatcaa agcggatgta tgtgggcagg cagcatgccg gtgagacatg gcatgacatc 1440
actgggaacc gttcagattc cgtcgtgatc aattcggacg gctggggaga gttttatgta 1500
aacggcggtt cggtttcgat ttatgtccaa cgatag 1536
<210>5
<211>1536
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
atggtttaca aatgcaaacg gatattatgt tgtgtgctgc tgtttttcat agtgctgccg 60
gcttctaaaa catatgcggc aagcctgaac ggcacgctga tgcagtattt tgaatggaat 120
ctgcctaatg acggccagca ttggaagcgc ttacaaaatg atgcgggata tttatccgac 180
attgggataa cggctgtttg gattccgccc gcctacaagg gaacgagcca ggctgacgtt 240
ggatacggcc catacgattt gtacgattta ggggagttcc tgcaaaaagg gacggtgcgg 300
acgaaatacg ggatgaaaac agagcttcag tcagctgtcg gttcgcttca ttcccagaac 360
atccaagtgt atggcgatgt tgtccttaat cataaggctg gggcggatct gacggaggat 420
gtcaccgcgg ttgaagtgaa tcccggcaat cgaaatcagg aaatatctgg agaatatcga 480
atcaaagcgt ggacaggatt caatttccct ggacgcggca gcacatacag tgattttaaa 540
tggcattggt atcattttga tgggacggat tgggacgaat cccgaaagct gaatcgcatc 600
tacaagttcc gcggagatgg gaaggcatgg gattgggagg tttccagcga aaacggcaac 660
tacgattatt taatgtatgc ggatgtcgat tatgaccacc ccgatgttgt ggcagaaatg 720
aaacggtggg gaacctggta tgcaaaagag cttcaattgg atgggttccg gcttgatgcc 780
gtcaagcata ttaagttcca gttttttcct gattggttgt cgtatgtgcg ttctcagact 840
ggcaaggaaa tgtttacggt tgcggaatac tggcaaaata accttggaga aatcgaaaac 900
tacttgcaaa aaaccgattt tcaacattct gtattcgatg tgccgcttca ttttaacctt 960
caggccgcat cttcacacgg aggcagctat gatatgagga atttgctgaa cggaacggtt 1020
gtttccaaac atcctttgaa agcggttaca tttgtcgaca accatgacac acagccgggg 1080
caatcattgg agtcgaccgt ccaaacatgg ttcaagccgc ttgcctacgc ttttattttg 1140
acaagagagg ccgggtaccc gcaggttttt tatggagata tgtatgggac aaaaggtcct 1200
acatcgcggg aaattccttc tcttaaaagt aaatgtgagc cgattttgaa agcgcgcaag 1260
tattatgctt atggaacaca gcatgattat ttcgatcatc cagatgccat cggctggacg 1320
agggaaggcg atcaatccgt cgctgcatca ggcttggccg ctttaatcac agacggaccg 1380
ggcggatcaa agcggatgta tgtgggcagg cagcatgccg gtgagacatg gcatgacatc 1440
actgggaacc gttcagattc cgtcgtgatc aattcggacg gctggggaga gttttatgta 1500
aacggcggtt cggtttcgat ttatgtccaa cgatag 1536
<210>6
<211>511
<212>PRT
<213> Bacillus sonorensis
<400>6
Met Val Thr Leu Cys Leu Ala Ile Leu Cys Cys Val Leu Leu Pro Pro
1 5 10 15
Ile Val Leu Pro Ala Ser Leu Thr Thr Ala Ala Ser Leu Ala Gly Thr
20 25 30
Leu Met Gly Thr Pro Gly Thr Ala Leu Pro Ala Ala Gly Gly His Thr
35 40 45
Leu Ala Leu Gly Ala Ala Ala Gly Thr Leu Ser Ala Ile Gly Ile Thr
50 55 60
Ala Val Thr Ile Pro Pro Ala Thr Leu Gly Thr Ser Gly Ala Ala Val
65 70 7580
Gly Thr Gly Pro Thr Ala Leu Thr Ala Leu Gly Gly Pro Leu Gly Leu
85 90 95
Gly Thr Val Ala Thr Leu Thr Gly Met Leu Thr Gly Leu Gly Ser Ala
100 105 110
Val Gly Ser Leu His Ser Gly Ala Ile Gly Val Thr Gly Ala Val Val
115 120 125
Leu Ala His Leu Ala Gly Ala Ala Leu Thr Gly Ala Val Thr Ala Val
130 135 140
Gly Val Ala Pro Gly Ala Ala Ala Gly Gly Ile Ser Gly Gly Thr Ala
145 150 155 160
Ile Leu Ala Thr Thr Gly Pro Ala Pro Pro Gly Ala Gly Ser Thr Thr
165 170 175
Ser Ala Pro Leu Thr His Thr Thr His Pro Ala Gly Thr Ala Thr Ala
180 185 190
Gly Ser Ala Leu Leu Ala Ala Ile Thr Leu Pro Ala Gly Ala Gly Leu
195 200 205
Ala Thr Ala Thr Gly Val Ser Ser Gly Ala Gly Ala Thr Ala Thr Leu
210 215 220
Met Thr Ala Ala Val Ala Thr Ala His Pro Ala Val Val Ala Gly Met
225 230 235240
Leu Ala Thr Gly Thr Thr Thr Ala Leu Gly Leu Gly Leu Ala Gly Pro
245 250 255
Ala Leu Ala Ala Val Leu His Ile Leu Pro Ser Pro Leu Ser Ala Thr
260 265 270
Leu Leu Ala Val Ala Gly Ser Thr Gly Leu Gly Met Pro Thr Val Ala
275 280 285
Gly Thr Thr Gly Ala Ala Leu Gly Gly Ile Gly Ala Thr Leu Gly Leu
290 295 300
Thr Ala Pro Gly His Ser Val Pro Ala Val Pro Leu His Pro Ala Leu
305 310 315 320
Gly Ala Ala Ser Ser His Gly Gly Ser Thr Ala Met Ala Ala Leu Leu
325 330 335
Ala Gly Thr Val Val Ser Leu His Pro Leu Leu Ala Val Thr Pro Val
340 345 350
Ala Ala His Ala Thr Gly Pro Gly Gly Ser Leu Gly Ser Thr Val Gly
355 360 365
Thr Thr Pro Leu Pro Leu Ala Thr Ala Pro Ile Leu Thr Ala Gly Ala
370 375 380
Gly Thr Pro Gly Val Pro Thr Gly Ala Met Thr Gly Thr Leu Gly Pro
385 390 395 400
Thr Ser Ala Gly Ile Pro Ser Leu Leu Ser Leu Leu Gly Pro Ile Leu
405 410 415
Leu Ala Ala Leu Thr Thr Ala Thr Gly Thr Gly His Ala Thr Pro Ala
420 425 430
His Pro Ala Ala Ile Gly Thr Thr Ala Gly Gly Ala Gly Ser Val Ala
435 440 445
Ala Ser Gly Leu Ala Ala Leu Ile Thr Ala Gly Pro Gly Gly Ser Leu
450 455 460
Ala Met Thr Val Gly Ala Gly His Ala Gly Gly Thr Thr His Ala Ile
465 470 475 480
Thr Gly Ala Ala Ser Ala Ser Val Val Ile Ala Ser Ala Gly Thr Gly
485 490 495
Gly Pro Thr Val Ala Gly Gly Ser Val Ser Ile Thr Val Gly Ala
500 505 510
<210>7
<211>511
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>7
Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe
1 5 10 15
Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Ala AsnGly Thr
20 25 30
Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp
35 40 45
Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr
50 55 60
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val
65 70 75 80
Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys
85 90 95
Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Arg
100 105 110
Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val
115 120 125
Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val
130 135 140
Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg
145 150 155 160
Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr
165 170 175
Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp
180 185 190
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys
195 200 205
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
210 215 220
Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met
225 230 235 240
Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe
245 250 255
Arg Leu Asp Ala Val Lys His Ile Lys Phe Gln Phe Leu Ser Asp Trp
260 265 270
Leu Ser Ala Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala
275 280 285
Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys
290 295 300
Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu
305 310 315 320
Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu
325 330 335
Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val
340 345 350
Asp Asn His Gly Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln
355 360 365
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala
370 375 380
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro
385 390 395 400
Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu
405 410 415
Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp
420 425 430
His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala
435 440 445
Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys
450 455 460
Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile
465 470 475 480
Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly
485 490 495
Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg
500 505 510
<210>8
<211>511
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>8
Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe
1 5 10 15
Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Ala Asn Gly Thr
20 25 30
Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp
35 40 45
Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr
50 55 60
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val
65 70 75 80
Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys
85 90 95
Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Ala
100 105 110
Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val
115 120 125
Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val
130 135 140
Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg
145 150 155 160
Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr
165 170 175
Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp
180 185 190
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys
195 200 205
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
210 215 220
Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met
225 230 235 240
Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe
245 250 255
Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Phe Pro Asp Trp
260 265 270
Leu Lys Tyr Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala
275 280 285
Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys
290 295 300
Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu
305 310 315 320
Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu
325 330 335
Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val
340 345 350
Asp Asn His Gly Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln
355 360 365
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala
370 375 380
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro
385 390 395 400
Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu
405 410 415
Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp
420 425 430
His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala
435 440 445
Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys
450 455 460
Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile
465 470 475 480
Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly
485 490 495
Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg
500 505 510
<210>9
<211>511
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>9
Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe
1 5 10 15
Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Ala Asn Gly Thr
20 25 30
Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp
35 40 45
Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr
50 55 60
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val
6570 75 80
Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys
85 90 95
Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Arg
100 105 110
Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val
115 120 125
Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val
130 135 140
Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg
145 150 155 160
Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr
165 170 175
Ser Asp Phe Lys Trp His Trp Tyr His Phe Asp Gly Thr Asp Trp Asp
180 185 190
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys
195 200 205
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
210 215 220
Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met
225 230 235 240
Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe
245 250 255
Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe Phe Ser Asp Trp
260 265 270
Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala
275 280 285
Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys
290 295 300
Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu
305 310 315 320
Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu
325 330 335
Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ala Val Thr Phe Val
340 345 350
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln
355 360 365
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala
370 375 380
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro
385 390395 400
Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu
405 410 415
Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp
420 425 430
His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala
435 440 445
Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys
450 455 460
Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile
465 470 475 480
Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly
485 490 495
Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile Tyr Val Gln Arg
500 505 510
<210>10
<211>511
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>10
Met Val Tyr Lys Cys Lys Arg Ile Leu Cys Cys Val Leu Leu Phe Phe
1 5 10 15
Ile Val Leu Pro Ala Ser Lys Thr Tyr Ala Ala Ser Leu Asn Gly Thr
20 25 30
Leu Met Gln Tyr Phe Glu Trp Asn Leu Pro Asn Asp Gly Gln His Trp
35 40 45
Lys Arg Leu Gln Asn Asp Ala Gly Tyr Leu Ser Asp Ile Gly Ile Thr
50 55 60
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Thr Ser Gln Ala Asp Val
65 70 75 80
Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Leu Gln Lys
85 90 95
Gly Thr Val Arg Thr Lys Tyr Gly Met Lys Thr Glu Leu Gln Ser Ala
100 105 110
Val Gly Ser Leu His Ser Gln Asn Ile Gln Val Tyr Gly Asp Val Val
115 120 125
Leu Asn His Lys Ala Gly Ala Asp Leu Thr Glu Asp Val Thr Ala Val
130 135 140
Glu Val Asn Pro Gly Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Arg
145 150 155 160
Ile Lys Ala Trp Thr Gly Phe Asn Phe Pro Gly Arg Gly Ser Thr Tyr
165 170 175
Ser Asp Phe Lys Trp His Trp TyrHis Phe Asp Gly Thr Asp Trp Asp
180 185 190
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys Phe Arg Gly Asp Gly Lys
195 200 205
Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
210 215 220
Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Met
225 230 235 240
Lys Arg Trp Gly Thr Trp Tyr Ala Lys Glu Leu Gln Leu Asp Gly Phe
245 250 255
Arg Leu Asp Ala Val Lys His Ile Lys Phe Gln Phe Phe Pro Asp Trp
260 265 270
Leu Ser Tyr Val Arg Ser Gln Thr Gly Lys Glu Met Phe Thr Val Ala
275 280 285
Glu Tyr Trp Gln Asn Asn Leu Gly Glu Ile Glu Asn Tyr Leu Gln Lys
290 295 300
Thr Asp Phe Gln His Ser Val Phe Asp Val Pro Leu His Phe Asn Leu
305 310 315 320
Gln Ala Ala Ser Ser His Gly Gly Ser Tyr Asp Met Arg Asn Leu Leu
325 330 335
Asn Gly Thr Val Val Ser Lys His Pro LeuLys Ala Val Thr Phe Val
340 345 350
Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr Val Gln
355 360 365
Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ala
370 375 380
Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Pro
385 390 395 400
Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Cys Glu Pro Ile Leu
405 410 415
Lys Ala Arg Lys Tyr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp
420 425 430
His Pro Asp Ala Ile Gly Trp Thr Arg Glu Gly Asp Gln Ser Val Ala
435 440 445
Ala Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys
450 455 460
Arg Met Tyr Val Gly Arg Gln His Ala Gly Glu Thr Trp His Asp Ile
465 470 475 480
Thr Gly Asn Arg Ser Asp Ser Val Val Ile Asn Ser Asp Gly Trp Gly
485 490 495
Glu Phe Tyr Val Asn Gly Gly Ser Val Ser Ile TyrVal Gln Arg
500 505 510
Claims (7)
1. The alpha-amylase BasAmy mutant with improved specific activity is characterized in that the mutant is a mutant of alpha-amylase BasAmy with an amino acid sequence shown as SEQID NO.6, and the mutant has mutation at the following mutation sites,
S267Q, L269F, S270P, K274S, a275Y, Q278S, S279Q and L412C.
2. A gene encoding the specific activity increasing alpha-amylase BasAmy mutant of claim 1.
3. A recombinant vector comprising the gene of claim 2.
4. A host cell comprising the gene of claim 2.
5. The use of the BasAmy mutant of alpha-amylase with increased specific activity of claim 1.
6. The method for improving the specific activity of alpha-amylase BasAmy is characterized in that 267-position of alpha-amylase BasAmy with an amino acid sequence shown as SEQ ID NO.6 is mutated from S to Q; mutation of 269 from L to F; mutation of 274 bit from K to S; mutation of 270 th site from S to P; mutation of 274 bit from K to S; the 275 th site is mutated from A to Y; mutation of 278 bit from Q to S; mutation of position 279 from S to Q; 412 is mutated from L to C.
7. A method for preparing an alpha-amylase BasAmy mutant with increased specific activity, the method comprising the steps of expressing the gene of claim 3 in a host cell and purifying the mutant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911068186.3A CN110699337B (en) | 2017-01-16 | 2017-01-16 | Alpha-amylase mutant BasAmy-4 with improved specific activity as well as coding gene and application thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911068186.3A CN110699337B (en) | 2017-01-16 | 2017-01-16 | Alpha-amylase mutant BasAmy-4 with improved specific activity as well as coding gene and application thereof |
| CN201710030849.7A CN106929495B (en) | 2017-01-16 | 2017-01-16 | α -amylase BasAmy mutant capable of improving specific activity and coding gene and application thereof |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710030849.7A Division CN106929495B (en) | 2017-01-16 | 2017-01-16 | α -amylase BasAmy mutant capable of improving specific activity and coding gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110699337A true CN110699337A (en) | 2020-01-17 |
| CN110699337B CN110699337B (en) | 2023-02-17 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911068182.5A Active CN110713999B (en) | 2017-01-16 | 2017-01-16 | Alpha-amylase mutant BasAmy-3 capable of improving specific activity and coding gene and application thereof |
| CN201911068186.3A Active CN110699337B (en) | 2017-01-16 | 2017-01-16 | Alpha-amylase mutant BasAmy-4 with improved specific activity as well as coding gene and application thereof |
| CN201710030849.7A Active CN106929495B (en) | 2017-01-16 | 2017-01-16 | α -amylase BasAmy mutant capable of improving specific activity and coding gene and application thereof |
| CN201911068115.3A Active CN110628748B (en) | 2017-01-16 | 2017-01-16 | Alpha-amylase mutant BasAmy-2 capable of improving specific activity and coding gene and application thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911068182.5A Active CN110713999B (en) | 2017-01-16 | 2017-01-16 | Alpha-amylase mutant BasAmy-3 capable of improving specific activity and coding gene and application thereof |
Family Applications After (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710030849.7A Active CN106929495B (en) | 2017-01-16 | 2017-01-16 | α -amylase BasAmy mutant capable of improving specific activity and coding gene and application thereof |
| CN201911068115.3A Active CN110628748B (en) | 2017-01-16 | 2017-01-16 | Alpha-amylase mutant BasAmy-2 capable of improving specific activity and coding gene and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (4) | CN110713999B (en) |
| WO (1) | WO2018129985A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110713999B (en) * | 2017-01-16 | 2023-03-21 | 广东溢多利生物科技股份有限公司 | Alpha-amylase mutant BasAmy-3 capable of improving specific activity and coding gene and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5763385A (en) * | 1996-05-14 | 1998-06-09 | Genencor International, Inc. | Modified α-amylases having altered calcium binding properties |
| CN1277258A (en) * | 1999-06-10 | 2000-12-20 | 花王株式会社 | Mutation alph-amylase |
| CN105316300A (en) * | 2015-10-20 | 2016-02-10 | 江西省科学院微生物研究所 | Alpha-amylase mutant ApkA-m with high-temperature activity and thermostability improved and preparation method and application thereof |
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| CN106929495A (en) | 2017-07-07 |
| CN110699337B (en) | 2023-02-17 |
| CN110713999A (en) | 2020-01-21 |
| WO2018129985A1 (en) | 2018-07-19 |
| CN110713999B (en) | 2023-03-21 |
| CN110628748B (en) | 2023-03-31 |
| CN106929495B (en) | 2020-05-22 |
| CN110628748A (en) | 2019-12-31 |
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