CN110699337B - Alpha-amylase mutant BasAmy-4 with improved specific activity as well as coding gene and application thereof - Google Patents
Alpha-amylase mutant BasAmy-4 with improved specific activity as well as coding gene and application thereof Download PDFInfo
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- CN110699337B CN110699337B CN201911068186.3A CN201911068186A CN110699337B CN 110699337 B CN110699337 B CN 110699337B CN 201911068186 A CN201911068186 A CN 201911068186A CN 110699337 B CN110699337 B CN 110699337B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- 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)
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 in 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 with improved specific activity, and a coding gene and application thereof.
Background
Alpha-amylase is an important enzyme preparation which 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 very widely distributed, 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 cereus (Bacillus sonorensis) alpha-amylase is called BasAmy for short, is a moderate temperature alpha-amylase, has wide action pH value, and is suitable for the 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 sonola desert, thereby improving the specific activity of the BasAmy, reducing the production cost and laying 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 mutated amino acid of the 5 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, namely A112R, L269F, K274S, Q278S, S279Q and L412C are obtained.
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 contains mutation sites of L29A, A112R, S267Q, K274S, Q278S, S279Q, D356G and L412C.
Wherein BasAmy-2 contains mutation sites of L29A, L269F, S270P, A275Y, Q278S, S279Q, D356G and L412C.
Wherein BasAmy-3 contains 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 Bacillus sonoralis (Bacillus sonorensis) is purchased from China center for Industrial microbial culture Collection, and 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 is purchased from NEB company, plasmid extraction, gel purification, restriction enzyme and a kit are 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 25ug/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. 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 recombinant transformants by using bacterial liquid PCR, extracting plasmids of transformants verified to be correct, and sequencing to determine corresponding mutants. 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 the original alpha-Amylase and the mutant alpha-Amylase
| 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 to replace the primers AOX5-F and AOX3-R as reaction primers, and the PCR reaction is repeated. The second and third rounds of the product were double digested with XbaI and EcoRI and ligated between the EcoRI and XbaI sites on the pGAPz. Alpha.A vector. The ligation product was transformed into X33, and the mutant strain was screened in YPDZ plate culture. Through high-throughput screening, 6 effective mutants of A112R, L269F, K274S, Q278S, S279Q and L412C are obtained. 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 contains mutation sites of L29A, L269F, S270P, A275Y, Q278S, S279Q, D356G and L412C.
Wherein BasAmy-3 contains 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, wherein the purification method is nickel column purification. 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 130%,160%,180% and 125%, respectively, at the end.
Example 7 optimal reaction pH and pH stability of Primary 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 for BasAmy and mutants 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 BaAmy, and were all 6.0.
BasAmy, mutants of BasAmy-1, basAmy-2, basAmy-3 and BasAmy-4 are respectively treated for 2 hours at room temperature under the condition of pH4-8, and then enzyme activity is determined by referring to a national standard method, and the result is shown in figure 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, mutants BasAmy-1, basAmy-2, basAmy-3 and BasAmy-4 were all 60 ℃.
BasAmy, mutants of BasAmy-1, basAmy-2, basAmy-3 and BasAmy-4 are respectively treated in water bath at 50-90 ℃ for 30 minutes, and then enzyme activity is determined by referring to the national standard method, and the result is shown in figure 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 Yiduoli Biotechnology corporation
<120> alpha-amylase mutant BasAmy-4 with improved specific activity, and coding gene and application thereof
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1536
<212> DNA
<213> Bacillus sonoralis desert (Bacillus sonorensis)
<400> 1
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 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
actgggaacc gttcagattc cgtcgtgatc aattcggacg gctggggaga gttttatgta 1500
aacggcggtt cggtttcgat ttatgtccaa cgatag 1536
<210> 2
<211> 1536
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
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 ttaagttcca gtttctttct gattggttgt cggctgtgcg 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 accatggtac 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> 3
<211> 1536
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atggtttaca aatgcaaacg gatattatgt tgtgtgctgc tgtttttcat agtgctgccg 60
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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 tttttttcct gattggttga agtatgtgcg 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 accatggtac 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> 4
<211> 1536
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atggtttaca aatgcaaacg gatattatgt tgtgtgctgc tgtttttcat agtgctgccg 60
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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 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 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 Leu Ser Asp Trp
260 265 270
Leu Lys Ala Val Arg Gln Ser 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
385 390 395 400
Thr Ser Arg Glu Ile Pro Ser Leu Lys Ser Lys Leu 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> 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 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 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
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 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
385 390 395 400
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> 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 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 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 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
385 390 395 400
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
Claims (7)
1. The alpha-amylase BasAmy mutant capable of improving specific activity is characterized in that the mutant is a mutant of alpha-amylase BasAmy with an amino acid sequence shown as SEQ ID NO.6, and mutation sites are as follows:
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. Use of the specific activity increasing alpha-amylase BasAmy mutant according to claim 1 for hydrolysis of starch.
6. The method for improving the specific activity of the BasAmy of the alpha-amylase is characterized in that 267 of the BasAmy of the alpha-amylase with an amino acid sequence shown as SEQ ID NO.6 is mutated from S to Q; mutation from L to F at position 269; mutation of 274 bit from K to S; the 270 site is mutated from S to P, and the 275 site is mutated from A to Y; mutation of 278 from Q to S; mutation of position 279 from S to Q; mutation at position 412 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 2 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 |
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| 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 |
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| 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 |
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| CN110699337A CN110699337A (en) | 2020-01-17 |
| CN110699337B true 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 |
| 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 |
| 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 |
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| Application Number | Title | Priority Date | Filing Date |
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| 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 |
| 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 |
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| Application Number | Title | Priority Date | Filing Date |
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| 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 |
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| Country | Link |
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| CN (4) | CN110713999B (en) |
| WO (1) | WO2018129985A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| 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 |
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 |
| CN105483099A (en) * | 2008-06-06 | 2016-04-13 | 丹尼斯科美国公司 | Geobacillus stearothermophilus [alpha]-amylase (AMYS) variants with improved properties |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2432519T3 (en) * | 1996-04-30 | 2013-12-04 | Novozymes A/S | Alpha-amylase mutants |
| JP4417532B2 (en) * | 1999-06-10 | 2010-02-17 | 花王株式会社 | Mutant α-amylase |
| US20080280328A1 (en) * | 2005-11-18 | 2008-11-13 | Novozymes A/S | Glucoamylase Variants |
| CN105229147B (en) * | 2013-03-11 | 2020-08-11 | 丹尼斯科美国公司 | Alpha-amylase combinatorial variants |
| PL3034588T3 (en) * | 2014-12-17 | 2019-09-30 | The Procter And Gamble Company | Detergent composition |
| 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 |
-
2017
- 2017-01-16 CN CN201911068182.5A patent/CN110713999B/en active Active
- 2017-01-16 CN CN201911068115.3A patent/CN110628748B/en active Active
- 2017-01-16 CN CN201911068186.3A patent/CN110699337B/en active Active
- 2017-01-16 CN CN201710030849.7A patent/CN106929495B/en active Active
- 2017-10-26 WO PCT/CN2017/107821 patent/WO2018129985A1/en active Application Filing
Patent 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 |
| CN105483099A (en) * | 2008-06-06 | 2016-04-13 | 丹尼斯科美国公司 | Geobacillus stearothermophilus [alpha]-amylase (AMYS) variants with improved properties |
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| ACCESSION No.：WP_006637292.1，alpha-amylase [Bacillus sonorensis];无;《GenBank》;20150614;Features和Origin部分 * |
| Improving the thermostability of alpha-amylase by combinatorial coevolving-site saturation mutagenesis;Chenghua Wang等;《BMC Bioinformatics》;20121011(第13期);第1-7页 * |
| 地芽孢杆菌Geobacillus sp. GXS1α-淀粉酶的饱和突变及酶学性质研究;薛蓓等;《生物技术通讯》;20131031(第10期);摘要，第156页右栏第2段，第157页左栏第1段和表2 * |
Also Published As
| Publication number | Publication date |
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| WO2018129985A1 (en) | 2018-07-19 |
| CN106929495B (en) | 2020-05-22 |
| CN110713999A (en) | 2020-01-21 |
| CN110628748A (en) | 2019-12-31 |
| CN110713999B (en) | 2023-03-21 |
| CN110699337A (en) | 2020-01-17 |
| CN110628748B (en) | 2023-03-31 |
| CN106929495A (en) | 2017-07-07 |
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