CN107058264A - Than the alpha amylase JcAmy mutant living improved and its encoding gene and application - Google Patents

Than the alpha amylase JcAmy mutant living improved and its encoding gene and application Download PDF

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CN107058264A
CN107058264A CN201710032298.8A CN201710032298A CN107058264A CN 107058264 A CN107058264 A CN 107058264A CN 201710032298 A CN201710032298 A CN 201710032298A CN 107058264 A CN107058264 A CN 107058264A
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jcamy
amylase
alpha
mutant
amino acid
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CN107058264B (en
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李阳源
黄江
王建荣
聂金梅
陈丽芝
何小梅
杨玲
黄佳乐
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Hunan Kangjie Biotechnology Co., Ltd
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Yiduoli Biological Science & Tech Co Ltd Guangdong
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Priority to PCT/CN2017/107576 priority patent/WO2018129981A1/en
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    • C—CHEMISTRY; METALLURGY
    • 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
    • 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 present invention relates to genetic engineering field, and in particular to than the alpha amylase JcAmy mutant living improved and its encoding gene and application.The amino acid sequence of the mutant is substituted radical for any one in the 6th, the 53rd, the 173rd, the 245th and/or the 281st of the amino acid sequence as shown in SEQ ID No.2 an or more position.Ratio relative to original alpha amylase is lived, and alpha amylase is 21% 92% than increase rate living after mutation, is that the industrial applications of the saline land fresh bacillus alpha amylase in the Saltwater Sea lay the foundation.

Description

Than the alpha-amylase JcAmy mutant living improved and its encoding gene and application
Technical field
The present invention relates to genetic engineering field, and in particular to than the alpha-amylase JcAmy mutant and its coding living improved Gene and application.
Background technology
Alpha-amylase, systematic name is Isosorbide-5-Nitrae-α-D- glucan hydrolases, is a kind of endo hydrolysis enzyme, its main function is Isosorbide-5-Nitrae-α-D- glucans generation reproducibility the dextrin and carbohydrate of catalytic starch, in the fields such as starch, cleaning agent, beverage and weaving tool Play an important role.
, typically will be by liquefaction and sugar in starchy material process at present in the fields such as brewing alcohol, starch sugar Change two stages.Enzyme used in liquefaction and saccharifying is mainly alpha-amylase and carbohydrase.It is industrial at present extensive The commercialization alpha-amylase and the optimum pH of carbohydrase used is about 6.5 and 4.5, therefore between liquefaction and saccharifying mid-term Need to add soda acid to adjust pH.A large amount of soda acids that add not only complicate processing technology in liquefaction and saccharifying, and Add production cost.If alpha-amylase stable in acid condition can be developed, in liquefaction and saccharifying Soda acid need not additionally be added and carry out pH regulations, reagent consumption, simplified processing process can be not only reduced, and life can be reduced Cost is produced, is saved food.Starch manufacture field is significant.
The saline land fresh bacillus in the Saltwater Sea (Jeotgalibacillus campisalis) alpha-amylase abbreviation JcAmy. JcAmy is a kind of acid resistance amylase, and its optimal pH is 5.0, has good stability in the range of pH 4 to 8, can It is enough that effect well is played under the conditions of acidity liquefaction.Although JcAmy has good pH characteristics, it is lower than work, production Cost is high, limits its industrial applications.Therefore, JcAmy specific enzyme activity is improved, its production cost is reduced, is JcAmy industrialization Using the topic for being badly in need of solving.
In recent years, a series of microbial source alpha-amylase realizes the heterogenous expression in Escherichia coli or yeast.But Be due to the alpha-amylase that obtained wild mushroom production is screened from nature vigor it is general all than relatively low, it is impossible to directly apply to work The fermenting and producing of industry.Prior art is improved generally by the technological means such as mutagenesis, crossbreeding are carried out to wild strain The enzymatic productivity of bacterial strain, but the workload of the technology such as mutagenesis hybridization is big and uncontrollably the negative probability being mutated of appearance compares Greatly.
The present invention improves the fresh Bacillus alpha-amylase JcAmy in saline land Saltwater Sea Rate activity by site-directed mutagenesis technique, Its production cost is greatly reduced, is that its further industrial applications lays the foundation.
The content of the invention
The purpose of the present invention is by changing to carrying out molecule from the fresh Bacillus alpha-amylase JcAmy in the saline land Saltwater Sea Make, make improved alpha-amylase that there is higher ratio to live, reduce production cost, be the saline land fresh Bacillus alpha-amylase in the Saltwater Sea Industrial applications lay the foundation.
It is an object of the invention to provide than the alpha-amylase JcAmy mutant living improved.
A further object of the present invention is to provide the encoding gene of above-mentioned alpha-amylase JcAmy mutant.
The saline land fresh Bacillus alpha-amylase JcAmy in Saltwater Sea nucleotide sequence and amino acid sequence such as SEQ ID NO.1, Its amino acid sequence is as shown in SEQ ID NO.2.
The present invention using the method for fixed point saturation mutation to the 6th of the alpha-amylase JcAmy shown in SEQ ID NO.2, 53rd, the 173rd, the 245th and/or the 281st progress molecular modification, by high flux screening be improved than α living- Amylase mutant.The amino acid sequence of these mutant is as shown in SEQ ID NO.3 to SEQ ID NO.10, encoding mutant body Nucleotide sequence as shown in SEQ ID NO.11 to SEQ ID NO.18.
According to the alpha-amylase JcAmy mutant of the optimization improvement of the embodiment of the present invention, its amino acid sequence For in the 6th of SEQ ID NO.2 alpha-amylase JcAmy, the 53rd, the 173rd, the 245th and/or the 281st at least One amino acid is replaced into one of following amino acid accordingly：
The 6th of alpha-amylase JcAmy is replaced by G6F, G6M, G6P, G6N or G6S；
The 53rd of alpha-amylase JcAmy is replaced by N35S or N35A；
The 173rd of alpha-amylase JcAmy is replaced by N173K or N173S；
The 245th of alpha-amylase JcAmy is replaced by Q245G, Q245P or Q245R；
The 281st of alpha-amylase JcAmy is replaced by G281N, G281D, G281S or G281K.
According to the alpha-amylase JcAmy mutant JcAmy-1 of the optimization improvement of the embodiment of the present invention, it is mutated Site is：G6F, N35S, N173K, Q245G, G281N, amino acid sequence is as shown in SEQ ID NO.3.
According to the alpha-amylase JcAmy mutant JcAmy-2 of the optimization improvement of the embodiment of the present invention, it is mutated Site is：G6M, N35S, N173S, Q245P, G281D, amino acid sequence is as shown in SEQ ID NO.4.
According to the alpha-amylase JcAmy mutant JcAmy-3 of the optimization improvement of the embodiment of the present invention, it is mutated Site is：G6P, N35A, N173K, Q245R, G281K, amino acid sequence is as shown in SEQ ID NO.5.
According to the alpha-amylase JcAmy mutant JcAmy-4 of the optimization improvement of the embodiment of the present invention, it is mutated Site is：G6N, N35S, N173K, Q245P, G281S, amino acid sequence is as shown in SEQ ID NO.6.
According to the alpha-amylase JcAmy mutant JcAmy-5 of the optimization improvement of the embodiment of the present invention, it is mutated Site is：G6S, N35A, N173S, Q245P, G281K, amino acid sequence is as shown in SEQ ID NO.7.
According to the alpha-amylase JcAmy mutant JcAmy-6 of the optimization improvement of the embodiment of the present invention, it is mutated Site is：G6P, N35A, N173K, Q245G, G281S, amino acid sequence is as shown in SEQ ID NO.8.
According to the alpha-amylase JcAmy mutant JcAmy-7 of the optimization improvement of the embodiment of the present invention, it is mutated Site is：G6S, N35A, N173K, Q245P, G281N, amino acid sequence is as shown in SEQ ID NO.9.
According to the alpha-amylase JcAmy mutant JcAmy-8 of the optimization improvement of the embodiment of the present invention, it is mutated Site is：G6M, N35A, N173K, Q245G, G281S, amino acid sequence is as shown in SEQ ID NO.10.
The present invention is by the transformation of albumen rationality and High Throughput Screening Assay to the fresh bacillus in the saline land Saltwater Sea (Jeotgalibacillus campisalis) a- amylase JcAmy carries out molecular modification.Relative to the ratio of original alpha-amylase Living, alpha-amylase is 21%-92% than increase rate living after mutation, is the industry of the saline land fresh Bacillus alpha-amylase in the Saltwater Sea Change application to lay the foundation.
Brief description of the drawings
Fig. 1 is shown according to the original alpha-amylase of the specific embodiment of the invention and alpha-amylase mutant JcAmy1-8 Optimal pH.
Fig. 2 is shown according to the original alpha-amylase of the specific embodiment of the invention and alpha-amylase mutant JcAmy1-8 PH stability.
Embodiment
Do not make the experimental methods of molecular biology illustrated, equal reference in following examples《Molecular Cloning:A Laboratory guide》 Listed specific method is carried out in the book of (third edition) J. Pehanorm Brookers one, or is carried out according to kit and product description； The reagent and biomaterial, unless otherwise specified, are commercially obtained.
Experiment material and reagent：
1st, bacterial strain and carrier：Coli strain Topl0, Pichia pastoris X33, carrier pPICz α A, Zeocin are purchased from Invitrogen companies.
2nd, gene：By the fresh bacillus in the saline land Saltwater Sea (Jeotgalibacillus campisalis) α-shallow lake announced Powder enzyme JcAmy (Sequence ID:WP_052476631.1), according to carrying out synthetic gene after Pichia pastoris codon optimization.
3rd, enzyme and kit：Q5 high-fidelity Taq enzymes MIX is purchased from NEB companies, plasmid extraction, glue purification, restriction enzyme Enzyme, kit are purchased from Shanghai Sheng Gong companies.
4th, culture medium：Escherichia coli culture medium is LB, formula：1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0.LBZ is that LB culture mediums add 25ug/mL Zeocin.
Yeast culture medium is YPD, is formulated and is：1% yeast extract, 2% peptone, 2% glucose.Yeast screening assay culture Base is YPDZ, is formulated as YPD+100mg/L zeocin.
Yeast inducing culture BMGY, is formulated as 1% yeast extract, 2% peptone, 1.34%YNB, 0.00004% Biotin, 1% glycerine (V/V)) and BMMY, divided by 0.5% methanol replaces glycerine, remaining composition phase is identical with BMGY.
The fresh bacillus in embodiment 1, the saline land Saltwater Sea (Jeotgalibacillus campisalis) alpha-amylase JcAmy Clone
According to two primer (F of sequences Design of the a- amylase JcAmy genes of synthesis：5'- GATCGAATTCGCTACTCCTCAAAACGGTACTATGA-3' and R：5'- TAGCGCGGCCGCCTACTCACCATAAATGGAAACAGAA-3') it is used to expand the saline land fresh Bacillus alpha-amylase base in the Saltwater Sea Cause.The PCR primer of amplification is purified and reclaimed, expression vector pPICz α A is connected to, obtains expression vector pPICz α A-JcAmy.
Embodiment 2, rite-directed mutagenesis
Single mutation site is
G6F, G6M, G6P, G6N or G6S；Or
N35S, N35A, N173K or N173S；Or
Q245G, Q245P or Q245R；Or
G281N, G281D, G281S or G281K
Using above-mentioned pPICz α A-JcAmy as template, performing PCR amplification is entered with corresponding primer, specifically amplification reaction system It is as follows：
Q5 high-fidelity Taq enzymes MIX 23uL
Correspondence mutant primer 1uL
Correspondence mutant primer 1uL
pPICzαA-JcAmy(20ng) 2uL
Add water to 50uL
Response procedures are as follows：
Agarose electrophoresis detects PCR amplifications, and PCR primer is reclaimed in purifying.With restriction enzyme DpnI by original matter Grain is decomposed, and the product decomposed is just transferred into Escherichia coli Top10 with heat shock method, is verified recombinant conversion by bacterium solution PCR, is carried The plasmid for verifying correct transformant is taken to be sequenced, so that it is determined that corresponding mutant.Correct mutant will be sequenced, will use SacI is linearized, and is transferred to Pichia pastoris X33.A series of raisings are obtained than single-site mutant body living, these mutation by screening Comparing for body is living as shown in table 1.
The original alpha-amylase of table 1 and single-point mutants alpha-amylase compare work
Numbering Compare (%) living
Original alpha-amylase 100
G6F 115
G6M 121
G6P 130
G6N 119
G6S 116
N35S 135
N35A 141
N173K 126
N173S 136
Q245G 123
Q245P 128
Q245R 121
G281N 142
G281D 136
G281S 115
G281K 127
Embodiment 3, high flux screening height are than mutant strain living
Yeast recombinant conversion in embodiment 2 is chosen to 24 orifice plates one by one with toothpick, 1mL is added in each hole and is contained BMGY culture mediums, 30 DEG C, 220rpm cultures 24h or so, supernatant is removed in centrifugation.1.6mL BMMY culture mediums are separately added into again to be lured Lead culture.Cultivate after 24h, centrifuging and taking supernatant, above-mentioned supernatant is taken out into 200 μ L to 96 orifice plates respectively, carry out alpha-amylase enzyme It is living to determine.Alpha-amylase Enzyme activity assay is with reference to National Standard of the People's Republic of China《GB/T 24401-2009》It is measured.
Embodiment 4, combinatorial mutagenesis
6th：G6F, G6M, G6P, G6N, G6S；
53rd：N35S, N35A；
173rd：N173K, N173S；
245th：Q245G, Q245P, Q245R；
281st：G281N, G281D, G281S, G281K.
By enzyme in embodiment 2 than single mutation site G6F, G6M, G6P, G6N, G6S, N35S, N35A, the N173K living improved, N173S, Q245G, Q245P, Q245R, G281N, G281D, G281S, G281K are respectively combined, experimentation be the same as Example 2 It is identical.By experiment finally give 8 combinatorial mutagenesises be respectively designated as JcAmy-1, JcAmy-2, JcAmy-3, JcAmy-4, JcAmy-5、JcAmy-6、JcAmy-7、JcAmy-8
The mutational site that wherein JcAmy-1 is included is：G6F, N35S, N173K, Q245G, G281N.
The mutational site that wherein JcAmy-2 is included is：G6M, N35S, N173S, Q245P, G281D.
The mutational site that wherein JcAmy-3 is included is：G6P, N35A, N173K, Q245R, G281K.
The mutational site that wherein JcAmy-4 is included is：G6N, N35S, N173K, Q245P, G281S.
The mutational site that wherein JcAmy-5 is included is：G6S, N35A, N173S, Q245P, G281K.
The mutational site that wherein JcAmy-6 is included is：G6P, N35A, N173K, Q245G, G281S.
The mutational site that wherein JcAmy-7 is included is：G6S, N35A, N173K, Q245P, G281N.
The mutational site that wherein JcAmy-8 is included is：G6M, N35A, N173K, Q245G, G281S.
The ratio of embodiment 5, original alpha-amylase and alpha-amylase mutant, which is lived, to be analyzed
Original alpha-amylase and mutant alpha-amylases are purified respectively, purification process is ni-sepharose purification.Will purifying Good alpha-amylase and mutant alpha-amylases determines corresponding enzyme activity and calculated than work respectively.With mutant than living divided by former Beginning alpha-amylase is than living, to calculate mutant than increase rate living.Improved compared to original JcAmy, the JcAmy after mutation than living Amplitude is 21%-92% (concrete outcome is shown in Table 2).
The original alpha-amylase of table 2 and mutant alpha-amylases compare work
Numbering Compare (%) living
Original alpha-amylase 100
JcAmy-1 135
JcAmy-2 159
JcAmy-3 142
JcAmy-4 121
JcAmy-5 130
JcAmy-6 150
JcAmy-7 192
JcAmy-8 160
Embodiment 6, the optimal pH of original alpha-amylase and alpha-amylase mutant JcAmy1-8 and pH stability
Original alpha-amylase and alpha-amylase mutant JcAmy1-8 optimal pH are determined with reference to national standard method.Original α-shallow lake Powder enzyme and alpha-amylase mutant JcAmy1-8 optimal pH are as shown in Figure 1.As shown in Figure 1, mutant JcAmy1-8's is most suitable Too big change does not occur for pH, almost as original alpha-amylase.
By original alpha-amylase and alpha-amylase mutant JcAmy1-8 room temperature treatment 3 hours under the conditions of pH4-8 respectively, Enzyme activity is determined referring next to state's calibration method.Original alpha-amylase and alpha-amylase mutant JcAmy1-8 pH stability are as schemed Shown in 2.As shown in Figure 2, stability of mutant JcAmy1, JcAmy3 and the JcAmy7 under the conditions of pH4 is better than original α-shallow lake Powder enzyme, and mutant JcAmy2, JcAmy4, JcAmy5, JcAmy6, JcAmy8 pH stability it is consistent with original alpha-amylase.
<110>GuangDong YiDuoLi Biology Science Co., Ltd
<120>Than the alpha-amylase JcAmy mutant living improved and its encoding gene and application
<160> 18
<210> 1
<211> 1458
<212> DNA
<213>The saline land fresh bacillus in the Saltwater Sea
<400> 1
gctactcctc aaaacggtac tatgatgcaa tactttgaat ggtacttgcc aaacgatggt 60
ttgcattgga accgtttgac caacgacgcc tccaacctta agaacttggg tgtcactacc 120
gtttggatcc ctcctgccta caagggtact tcccaaaacg acgtcggata cggagcctac 180
gacttgtacg accttggtga gttcaaccaa aagggtaccg tccgtaccaa gtacggtact 240
agaggtcagt tgcagaccgc tatcaacacc cttaagaacc agggtatcgg tacttacgga 300
gacgtcgtca tgaaccataa gggaggtgct gactttaccg agtccgtcca agctgtcgaa 360
gtcaacccaa acaacagatc tcaagaaact tccggtgaat acaccatttc cgcttggacc 420
ggattcaact tcgccggtcg taacaacttg cactccgcct tcaagtggag atggtaccac 480
ttcgacggta ctgactggga ccagtccaga tccttgaaca gaatttacaa gttcagagga 540
tctggtaagt cctgggacac cgaagtttcc aacgagttcg gtaactacga ctatcttatg 600
tatgccgacg ttgacttcga tcacccagag gtcaaggccg agttgaagaa ctggggtaag 660
tggtatgttc aatctttgaa ccttgatggt tttagattgg atgccgttaa acatatcaag 720
cacgattaca tccaagagtg gttggccgac gtcagacgta ctactggtaa agagttgttc 780
accgttgccg aatactggca gaacgacttg ggtgccatta acaattactt ggctaagacc 840
ggatattctc actccgtctt cgacgtccca cttcactaca acttccagcg tgctgccaac 900
tccggaggta atttcgatat gagaactatt ttcaacggat ccgttgtcca gcaacaccca 960
actttggccg tcaccatcgt cgacaaccat gactcccagc caggtcaatc cttggagtcc 1020
accgttgacg cttggttcaa accacttgcc tacgctatga tcatgaccag agagcagggt 1080
taccctaact tgttctacgg tgacttttac ggaaccaagg gttcctctaa tagagagatc 1140
ccaaatttgt cttctaaatt gactcctatt ttgaaggcca gaaaagacat ggcctacggt 1200
acccagcatg actaccttaa tcaccaagac gttatcggtt ggaccagaga gggtgttact 1260
gaccgttcca agtccggttt ggccactatc ttgtctgacg gaccaggagg aaacaagtgg 1320
atgtatgtcg gtaagagaaa cgccggagag acctggagag acaagaccgg taactcttcc 1380
aacgccgtta ccatcaactc tgacggttgg ggacagtttt ttgtcaatgg tggttctgtt 1440
tccatttatg gtgagtag
1458
<210> 2
<211> 460
<212> PRT
<213>The saline land fresh bacillus in the Saltwater Sea
<400> 2
ATPQNGTMMQ YFEWYLPNDG LHWNRLTNDA SNLKNLGVTT VWIPPAYKGT SQNDVGYGAY 60
DLYDLGEFNQ KGTVRTKYGT RGQLQTAINT LKNQGIGTYG DVVMNHKGGA DFTESVQAVE 120
VNPNNRSQET SGEYTISAWT GFNFAGRNNL HSAFKWRWYH FDGTDWDQSR SLNRIYKFRG 180
SGKSWDTEVS NEFGNYDYLM YADVDFDHPE VKAELKNWGK WYVQSLNLDG FRLDAVKHIK 240
HDYIQEWLAD VRRTTGKELF TVAEYWQNDL GAINNYLAKT GYSHSVFDVP LHYNFQRAAN 300
SGGNFDMRTI FNGSVVQQHP TLAVTIVDNH DSQPGQSLES TVDAWFKPLA YAMIMTREQG 360
YPNLFYGDFY GTKGSSNREI PNLSSKLTPI LKARKDMAYG TQHDYLNHQD VIGWTREGVT 420
DRSKSGLATI LSDGPGGNKW MYVGKRNAGE TWRDKTGNSS 460
<210> 3
<211> 485
<212> PRT
<213>Artificial sequence
<400> 3
ATPQNFTMMQ YFEWYLPNDG LHWNRLTNDA SNLKSLGVTT VWIPPAYKGT SQNDVGYGAY 60
DLYDLGEFNQ KGTVRTKYGT RGQLQTAINT LKNQGIGTYG DVVMNHKGGA DFTESVQAVE 120
VNPNNRSQET SGEYTISAWT GFNFAGRNNL HSAFKWRWYH FDGTDWDQSR SLKRIYKFRG 180
SGKSWDTEVS NEFGNYDYLM YADVDFDHPE VKAELKNWGK WYVQSLNLDG FRLDAVKHIK 240
HDYIGEWLAD VRRTTGKELF TVAEYWQNDL GAINNYLAKT NYSHSVFDVP LHYNFQRAAN 300
SGGNFDMRTI FNGSVVQQHP TLAVTIVDNH DSQPGQSLES TVDAWFKPLA YAMIMTREQG 360
YPNLFYGDFY GTKGSSNREI PNLSSKLTPI LKARKDMAYG TQHDYLNHQD VIGWTREGVT 420
DRSKSGLATI LSDGPGGNKW MYVGKRNAGE TWRDKTGNSS NAVTINSDGW GQFFVNGGSV 480
SIYGE 485
<210> 4
<211> 485
<212> PRT
<213>Artificial sequence
<400>4
ATPQNMTMMQ YFEWYLPNDG LHWNRLTNDA SNLKSLGVTT VWIPPAYKGT SQNDVGYGAY 60
DLYDLGEFNQ KGTVRTKYGT RGQLQTAINT LKNQGIGTYG DVVMNHKGGA DFTESVQAVE 120
VNPNNRSQET SGEYTISAWT GFNFAGRNNL HSAFKWRWYH FDGTDWDQSR SLSRIYKFRG 180
SGKSWDTEVS NEFGNYDYLM YADVDFDHPE VKAELKNWGK WYVQSLNLDG FRLDAVKHIK 240
HDYIPEWLAD VRRTTGKELF TVAEYWQNDL GAINNYLAKT DYSHSVFDVP LHYNFQRAAN 300
SGGNFDMRTI FNGSVVQQHP TLAVTIVDNH DSQPGQSLES TVDAWFKPLA YAMIMTREQG 360
YPNLFYGDFY GTKGSSNREI PNLSSKLTPI LKARKDMAYG TQHDYLNHQD VIGWTREGVT 420
DRSKSGLATI LSDGPGGNKW MYVGKRNAGE TWRDKTGNSS NAVTINSDGW GQFFVNGGSV 480
SIYGE 485
<210> 5
<211> 485
<212> PRT
<213>Artificial sequence
<400> 5
ATPQNPTMMQ YFEWYLPNDG LHWNRLTNDA SNLKALGVTT VWIPPAYKGT SQNDVGYGAY 60
DLYDLGEFNQ KGTVRTKYGT RGQLQTAINT LKNQGIGTYG DVVMNHKGGA DFTESVQAVE 120
VNPNNRSQET SGEYTISAWT GFNFAGRNNL HSAFKWRWYH FDGTDWDQSR SLKRIYKFRG 180
SGKSWDTEVS NEFGNYDYLM YADVDFDHPE VKAELKNWGK WYVQSLNLDG FRLDAVKHIK 240
HDYIREWLAD VRRTTGKELF TVAEYWQNDL GAINNYLAKT KYSHSVFDVP LHYNFQRAAN 300
SGGNFDMRTI FNGSVVQQHP TLAVTIVDNH DSQPGQSLES TVDAWFKPLA YAMIMTREQG 360
YPNLFYGDFY GTKGSSNREI PNLSSKLTPI LKARKDMAYG TQHDYLNHQD VIGWTREGVT 420
DRSKSGLATI LSDGPGGNKW MYVGKRNAGE TWRDKTGNSS NAVTINSDGW GQFFVNGGSV 480
SIYGE 485
<210> 6
<211> 485
<212> PRT
<213>Artificial sequence
<400> 6
ATPQNNTMMQ YFEWYLPNDG LHWNRLTNDA SNLKSLGVTT VWIPPAYKGT SQNDVGYGAY 60
DLYDLGEFNQ KGTVRTKYGT RGQLQTAINT LKNQGIGTYG DVVMNHKGGA DFTESVQAVE 120
VNPNNRSQET SGEYTISAWT GFNFAGRNNL HSAFKWRWYH FDGTDWDQSR SLKRIYKFRG 180
SGKSWDTEVS NEFGNYDYLM YADVDFDHPE VKAELKNWGK WYVQSLNLDG FRLDAVKHIK 240
HDYIPEWLAD VRRTTGKELF TVAEYWQNDL GAINNYLAKT SYSHSVFDVP LHYNFQRAAN 300
SGGNFDMRTI FNGSVVQQHP TLAVTIVDNH DSQPGQSLES TVDAWFKPLA YAMIMTREQG 360
YPNLFYGDFY GTKGSSNREI PNLSSKLTPI LKARKDMAYG TQHDYLNHQD VIGWTREGVT 420
DRSKSGLATI LSDGPGGNKW MYVGKRNAGE TWRDKTGNSS NAVTINSDGW GQFFVNGGSV 480
SIYGE 485
<210> 7
<211> 485
<212> PRT
<213>Artificial sequence
<400> 7
ATPQNSTMMQ YFEWYLPNDG LHWNRLTNDA SNLKALGVTT VWIPPAYKGT SQNDVGYGAY 60
DLYDLGEFNQ KGTVRTKYGT RGQLQTAINT LKNQGIGTYG DVVMNHKGGA DFTESVQAVE 120
VNPNNRSQET SGEYTISAWT GFNFAGRNNL HSAFKWRWYH FDGTDWDQSR SLSRIYKFRG 180
SGKSWDTEVS NEFGNYDYLM YADVDFDHPE VKAELKNWGK WYVQSLNLDG FRLDAVKHIK 240
HDYIPEWLAD VRRTTGKELF TVAEYWQNDL GAINNYLAKT KYSHSVFDVP LHYNFQRAAN 300
SGGNFDMRTI FNGSVVQQHP TLAVTIVDNH DSQPGQSLES TVDAWFKPLA YAMIMTREQG 360
YPNLFYGDFY GTKGSSNREI PNLSSKLTPI LKARKDMAYG TQHDYLNHQD VIGWTREGVT 420
DRSKSGLATI LSDGPGGNKW MYVGKRNAGE TWRDKTGNSS NAVTINSDGW GQFFVNGGSV 480
SIYGE 485
<210> 8
<211> 485
<212> PRT
<213>Artificial sequence
<400> 8
ATPQNPTMMQ YFEWYLPNDG LHWNRLTNDA SNLKALGVTT VWIPPAYKGT SQNDVGYGAY 60
DLYDLGEFNQ KGTVRTKYGT RGQLQTAINT LKNQGIGTYG DVVMNHKGGA DFTESVQAVE 120
VNPNNRSQET SGEYTISAWT GFNFAGRNNL HSAFKWRWYH FDGTDWDQSR SLKRIYKFRG 180
SGKSWDTEVS NEFGNYDYLM YADVDFDHPE VKAELKNWGK WYVQSLNLDG FRLDAVKHIK 240
HDYIGEWLAD VRRTTGKELF TVAEYWQNDL GAINNYLAKT SYSHSVFDVP LHYNFQRAAN 300
SGGNFDMRTI FNGSVVQQHP TLAVTIVDNH DSQPGQSLES TVDAWFKPLA YAMIMTREQG 360
YPNLFYGDFY GTKGSSNREI PNLSSKLTPI LKARKDMAYG TQHDYLNHQD VIGWTREGVT 420
DRSKSGLATI LSDGPGGNKW MYVGKRNAGE TWRDKTGNSS NAVTINSDGW GQFFVNGGSV 480
SIYGE 485
<210> 9
<211> 485
<212> PRT
<213>Artificial sequence
<400> 9
ATPQNSTMMQ YFEWYLPNDG LHWNRLTNDA SNLKALGVTT VWIPPAYKGT SQNDVGYGAY 60
DLYDLGEFNQ KGTVRTKYGT RGQLQTAINT LKNQGIGTYG DVVMNHKGGA DFTESVQAVE 120
VNPNNRSQET SGEYTISAWT GFNFAGRNNL HSAFKWRWYH FDGTDWDQSR SLKRIYKFRG 180
SGKSWDTEVS NEFGNYDYLM YADVDFDHPE VKAELKNWGK WYVQSLNLDG FRLDAVKHIK 240
HDYIPEWLAD VRRTTGKELF TVAEYWQNDL GAINNYLAKT NYSHSVFDVP LHYNFQRAAN 300
SGGNFDMRTI FNGSVVQQHP TLAVTIVDNH DSQPGQSLES TVDAWFKPLA YAMIMTREQG 360
YPNLFYGDFY GTKGSSNREI PNLSSKLTPI LKARKDMAYG TQHDYLNHQD VIGWTREGVT 420
DRSKSGLATI LSDGPGGNKW MYVGKRNAGE TWRDKTGNSS NAVTINSDGW GQFFVNGGSV 480
SIYGE 485
<210> 10
<211> 485
<212> PRT
<213>Artificial sequence
<400> 10
ATPQNMTMMQ YFEWYLPNDG LHWNRLTNDA SNLKALGVTT VWIPPAYKGT SQNDVGYGAY 60
DLYDLGEFNQ KGTVRTKYGT RGQLQTAINT LKNQGIGTYG DVVMNHKGGA DFTESVQAVE 120
VNPNNRSQET SGEYTISAWT GFNFAGRNNL HSAFKWRWYH FDGTDWDQSR SLKRIYKFRG 180
SGKSWDTEVS NEFGNYDYLM YADVDFDHPE VKAELKNWGK WYVQSLNLDG FRLDAVKHIK 240
HDYIGEWLAD VRRTTGKELF TVAEYWQNDL GAINNYLAKT SYSHSVFDVP LHYNFQRAAN 300
SGGNFDMRTI FNGSVVQQHP TLAVTIVDNH DSQPGQSLES TVDAWFKPLA YAMIMTREQG 360
YPNLFYGDFY GTKGSSNREI PNLSSKLTPI LKARKDMAYG TQHDYLNHQD VIGWTREGVT 420
DRSKSGLATI LSDGPGGNKW MYVGKRNAGE TWRDKTGNSS NAVTINSDGW GQFFVNGGSV 480
SIYGE 485
<210> 11
<211> 1458
<212> DNA
<213>Artificial sequence
<400> 11
gctactcctc aaaactttac tatgatgcaa tactttgaat ggtacttgcc aaacgatggt 60
ttgcattgga accgtttgac caacgacgcc tccaacctta agtctttggg tgtcactacc 120
gtttggatcc ctcctgccta caagggtact tcccaaaacg acgtcggata cggagcctac 180
gacttgtacg accttggtga gttcaaccaa aagggtaccg tccgtaccaa gtacggtact 240
agaggtcagt tgcagaccgc tatcaacacc cttaagaacc agggtatcgg tacttacgga 300
gacgtcgtca tgaaccataa gggaggtgct gactttaccg agtccgtcca agctgtcgaa 360
gtcaacccaa acaacagatc tcaagaaact tccggtgaat acaccatttc cgcttggacc 420
ggattcaact tcgccggtcg taacaacttg cactccgcct tcaagtggag atggtaccac 480
ttcgacggta ctgactggga ccagtccaga tccttgaaaa gaatttacaa gttcagagga 540
tctggtaagt cctgggacac cgaagtttcc aacgagttcg gtaactacga ctatcttatg 600
tatgccgacg ttgacttcga tcacccagag gtcaaggccg agttgaagaa ctggggtaag 660
tggtatgttc aatctttgaa ccttgatggt tttagattgg atgccgttaa acatatcaag 720
cacgattaca tcggtgagtg gttggccgac gtcagacgta ctactggtaa agagttgttc 780
accgttgccg aatactggca gaacgacttg ggtgccatta acaattactt ggctaagacc 840
aactattctc actccgtctt cgacgtccca cttcactaca acttccagcg tgctgccaac 900
tccggaggta atttcgatat gagaactatt ttcaacggat ccgttgtcca gcaacaccca 960
actttggccg tcaccatcgt cgacaaccat gactcccagc caggtcaatc cttggagtcc 1020
accgttgacg cttggttcaa accacttgcc tacgctatga tcatgaccag agagcagggt 1080
taccctaact tgttctacgg tgacttttac ggaaccaagg gttcctctaa tagagagatc 1140
ccaaatttgt cttctaaatt gactcctatt ttgaaggcca gaaaagacat ggcctacggt 1200
acccagcatg actaccttaa tcaccaagac gttatcggtt ggaccagaga gggtgttact 1260
gaccgttcca agtccggttt ggccactatc ttgtctgacg gaccaggagg aaacaagtgg 1320
atgtatgtcg gtaagagaaa cgccggagag acctggagag acaagaccgg taactcttcc 1380
aacgccgtta ccatcaactc tgacggttgg ggacagtttt ttgtcaatgg tggttctgtt 1440
tccatttatg gtgagtag 1458
<210> 12
<211> 1458
<212> DNA
<213>Artificial sequence
<400> 12
gctactcctc aaaacatgac tatgatgcaa tactttgaat ggtacttgcc aaacgatggt 60
ttgcattgga accgtttgac caacgacgcc tccaacctta agtctttggg tgtcactacc 120
gtttggatcc ctcctgccta caagggtact tcccaaaacg acgtcggata cggagcctac 180
gacttgtacg accttggtga gttcaaccaa aagggtaccg tccgtaccaa gtacggtact 240
agaggtcagt tgcagaccgc tatcaacacc cttaagaacc agggtatcgg tacttacgga 300
gacgtcgtca tgaaccataa gggaggtgct gactttaccg agtccgtcca agctgtcgaa 360
gtcaacccaa acaacagatc tcaagaaact tccggtgaat acaccatttc cgcttggacc 420
ggattcaact tcgccggtcg taacaacttg cactccgcct tcaagtggag atggtaccac 480
ttcgacggta ctgactggga ccagtccaga tccttgtcta gaatttacaa gttcagagga 540
tctggtaagt cctgggacac cgaagtttcc aacgagttcg gtaactacga ctatcttatg 600
tatgccgacg ttgacttcga tcacccagag gtcaaggccg agttgaagaa ctggggtaag 660
tggtatgttc aatctttgaa ccttgatggt tttagattgg atgccgttaa acatatcaag 720
cacgattaca tcccagagtg gttggccgac gtcagacgta ctactggtaa agagttgttc 780
accgttgccg aatactggca gaacgacttg ggtgccatta acaattactt ggctaagacc 840
gattattctc actccgtctt cgacgtccca cttcactaca acttccagcg tgctgccaac 900
tccggaggta atttcgatat gagaactatt ttcaacggat ccgttgtcca gcaacaccca 960
actttggccg tcaccatcgt cgacaaccat gactcccagc caggtcaatc cttggagtcc 1020
accgttgacg cttggttcaa accacttgcc tacgctatga tcatgaccag agagcagggt 1080
taccctaact tgttctacgg tgacttttac ggaaccaagg gttcctctaa tagagagatc 1140
ccaaatttgt cttctaaatt gactcctatt ttgaaggcca gaaaagacat ggcctacggt 1200
acccagcatg actaccttaa tcaccaagac gttatcggtt ggaccagaga gggtgttact 1260
gaccgttcca agtccggttt ggccactatc ttgtctgacg gaccaggagg aaacaagtgg 1320
atgtatgtcg gtaagagaaa cgccggagag acctggagag acaagaccgg taactcttcc 1380
aacgccgtta ccatcaactc tgacggttgg ggacagtttt ttgtcaatgg tggttctgtt 1440
tccatttatg gtgagtag 1458
<210> 13
<211> 1458
<212> DNA
<213>Artificial sequence
<400> 13
gctactcctc aaaacccaac tatgatgcaa tactttgaat ggtacttgcc aaacgatggt 60
ttgcattgga accgtttgac caacgacgcc tccaacctta aggctttggg tgtcactacc 120
gtttggatcc ctcctgccta caagggtact tcccaaaacg acgtcggata cggagcctac 180
gacttgtacg accttggtga gttcaaccaa aagggtaccg tccgtaccaa gtacggtact 240
agaggtcagt tgcagaccgc tatcaacacc cttaagaacc agggtatcgg tacttacgga 300
gacgtcgtca tgaaccataa gggaggtgct gactttaccg agtccgtcca agctgtcgaa 360
gtcaacccaa acaacagatc tcaagaaact tccggtgaat acaccatttc cgcttggacc 420
ggattcaact tcgccggtcg taacaacttg cactccgcct tcaagtggag atggtaccac 480
ttcgacggta ctgactggga ccagtccaga tccttgaaga gaatttacaa gttcagagga 540
tctggtaagt cctgggacac cgaagtttcc aacgagttcg gtaactacga ctatcttatg 600
tatgccgacg ttgacttcga tcacccagag gtcaaggccg agttgaagaa ctggggtaag 660
tggtatgttc aatctttgaa ccttgatggt tttagattgg atgccgttaa acatatcaag 720
cacgattaca tcagagagtg gttggccgac gtcagacgta ctactggtaa agagttgttc 780
accgttgccg aatactggca gaacgacttg ggtgccatta acaattactt ggctaagacc 840
aagtattctc actccgtctt cgacgtccca cttcactaca acttccagcg tgctgccaac 900
tccggaggta atttcgatat gagaactatt ttcaacggat ccgttgtcca gcaacaccca 960
actttggccg tcaccatcgt cgacaaccat gactcccagc caggtcaatc cttggagtcc 1020
accgttgacg cttggttcaa accacttgcc tacgctatga tcatgaccag agagcagggt 1080
taccctaact tgttctacgg tgacttttac ggaaccaagg gttcctctaa tagagagatc 1140
ccaaatttgt cttctaaatt gactcctatt ttgaaggcca gaaaagacat ggcctacggt 1200
acccagcatg actaccttaa tcaccaagac gttatcggtt ggaccagaga gggtgttact 1260
gaccgttcca agtccggttt ggccactatc ttgtctgacg gaccaggagg aaacaagtgg 1320
atgtatgtcg gtaagagaaa cgccggagag acctggagag acaagaccgg taactcttcc 1380
aacgccgtta ccatcaactc tgacggttgg ggacagtttt ttgtcaatgg tggttctgtt 1440
tccatttatg gtgagtag 1458
<210> 14
<211> 1458
<212> DNA
<213>Artificial sequence
<400> 14
gctactcctc aaaacaacac tatgatgcaa tactttgaat ggtacttgcc aaacgatggt 60
ttgcattgga accgtttgac caacgacgcc tccaacctta agtctttggg tgtcactacc 120
gtttggatcc ctcctgccta caagggtact tcccaaaacg acgtcggata cggagcctac 180
gacttgtacg accttggtga gttcaaccaa aagggtaccg tccgtaccaa gtacggtact 240
agaggtcagt tgcagaccgc tatcaacacc cttaagaacc agggtatcgg tacttacgga 300
gacgtcgtca tgaaccataa gggaggtgct gactttaccg agtccgtcca agctgtcgaa 360
gtcaacccaa acaacagatc tcaagaaact tccggtgaat acaccatttc cgcttggacc 420
ggattcaact tcgccggtcg taacaacttg cactccgcct tcaagtggag atggtaccac 480
ttcgacggta ctgactggga ccagtccaga tccttgaaga gaatttacaa gttcagagga 540
tctggtaagt cctgggacac cgaagtttcc aacgagttcg gtaactacga ctatcttatg 600
tatgccgacg ttgacttcga tcacccagag gtcaaggccg agttgaagaa ctggggtaag 660
tggtatgttc aatctttgaa ccttgatggt tttagattgg atgccgttaa acatatcaag 720
cacgattaca tcccagagtg gttggccgac gtcagacgta ctactggtaa agagttgttc 780
accgttgccg aatactggca gaacgacttg ggtgccatta acaattactt ggctaagacc 840
tcttattctc actccgtctt cgacgtccca cttcactaca acttccagcg tgctgccaac 900
tccggaggta atttcgatat gagaactatt ttcaacggat ccgttgtcca gcaacaccca 960
actttggccg tcaccatcgt cgacaaccat gactcccagc caggtcaatc cttggagtcc 1020
accgttgacg cttggttcaa accacttgcc tacgctatga tcatgaccag agagcagggt 1080
taccctaact tgttctacgg tgacttttac ggaaccaagg gttcctctaa tagagagatc 1140
ccaaatttgt cttctaaatt gactcctatt ttgaaggcca gaaaagacat ggcctacggt 1200
acccagcatg actaccttaa tcaccaagac gttatcggtt ggaccagaga gggtgttact 1260
gaccgttcca agtccggttt ggccactatc ttgtctgacg gaccaggagg aaacaagtgg 1320
atgtatgtcg gtaagagaaa cgccggagag acctggagag acaagaccgg taactcttcc 1380
aacgccgtta ccatcaactc tgacggttgg ggacagtttt ttgtcaatgg tggttctgtt 1440
tccatttatg gtgagtag 1458
<210> 15
<211> 1458
<212> DNA
<213>Artificial sequence
<400> 15
gctactcctc aaaactctac tatgatgcaa tactttgaat ggtacttgcc aaacgatggt 60
ttgcattgga accgtttgac caacgacgcc tccaacctta aggctttggg tgtcactacc 120
gtttggatcc ctcctgccta caagggtact tcccaaaacg acgtcggata cggagcctac 180
gacttgtacg accttggtga gttcaaccaa aagggtaccg tccgtaccaa gtacggtact 240
agaggtcagt tgcagaccgc tatcaacacc cttaagaacc agggtatcgg tacttacgga 300
gacgtcgtca tgaaccataa gggaggtgct gactttaccg agtccgtcca agctgtcgaa 360
gtcaacccaa acaacagatc tcaagaaact tccggtgaat acaccatttc cgcttggacc 420
ggattcaact tcgccggtcg taacaacttg cactccgcct tcaagtggag atggtaccac 480
ttcgacggta ctgactggga ccagtccaga tccttgtcta gaatttacaa gttcagagga 540
tctggtaagt cctgggacac cgaagtttcc aacgagttcg gtaactacga ctatcttatg 600
tatgccgacg ttgacttcga tcacccagag gtcaaggccg agttgaagaa ctggggtaag 660
tggtatgttc aatctttgaa ccttgatggt tttagattgg atgccgttaa acatatcaag 720
cacgattaca tcccagagtg gttggccgac gtcagacgta ctactggtaa agagttgttc 780
accgttgccg aatactggca gaacgacttg ggtgccatta acaattactt ggctaagacc 840
aagtattctc actccgtctt cgacgtccca cttcactaca acttccagcg tgctgccaac 900
tccggaggta atttcgatat gagaactatt ttcaacggat ccgttgtcca gcaacaccca 960
actttggccg tcaccatcgt cgacaaccat gactcccagc caggtcaatc cttggagtcc 1020
accgttgacg cttggttcaa accacttgcc tacgctatga tcatgaccag agagcagggt 1080
taccctaact tgttctacgg tgacttttac ggaaccaagg gttcctctaa tagagagatc 1140
ccaaatttgt cttctaaatt gactcctatt ttgaaggcca gaaaagacat ggcctacggt 1200
acccagcatg actaccttaa tcaccaagac gttatcggtt ggaccagaga gggtgttact 1260
gaccgttcca agtccggttt ggccactatc ttgtctgacg gaccaggagg aaacaagtgg 1320
atgtatgtcg gtaagagaaa cgccggagag acctggagag acaagaccgg taactcttcc 1380
aacgccgtta ccatcaactc tgacggttgg ggacagtttt ttgtcaatgg tggttctgtt 1440
tccatttatg gtgagtag 1458
<210> 16
<211> 1458
<212> DNA
<213>Artificial sequence
<400> 16
gctactcctc aaaacccaac tatgatgcaa tactttgaat ggtacttgcc aaacgatggt 60
ttgcattgga accgtttgac caacgacgcc tccaacctta aggctttggg tgtcactacc 120
gtttggatcc ctcctgccta caagggtact tcccaaaacg acgtcggata cggagcctac 180
gacttgtacg accttggtga gttcaaccaa aagggtaccg tccgtaccaa gtacggtact 240
agaggtcagt tgcagaccgc tatcaacacc cttaagaacc agggtatcgg tacttacgga 300
gacgtcgtca tgaaccataa gggaggtgct gactttaccg agtccgtcca agctgtcgaa 360
gtcaacccaa acaacagatc tcaagaaact tccggtgaat acaccatttc cgcttggacc 420
ggattcaact tcgccggtcg taacaacttg cactccgcct tcaagtggag atggtaccac 480
ttcgacggta ctgactggga ccagtccaga tccttgaaga gaatttacaa gttcagagga 540
tctggtaagt cctgggacac cgaagtttcc aacgagttcg gtaactacga ctatcttatg 600
tatgccgacg ttgacttcga tcacccagag gtcaaggccg agttgaagaa ctggggtaag 660
tggtatgttc aatctttgaa ccttgatggt tttagattgg atgccgttaa acatatcaag 720
cacgattaca tcggtgagtg gttggccgac gtcagacgta ctactggtaa agagttgttc 780
accgttgccg aatactggca gaacgacttg ggtgccatta acaattactt ggctaagacc 840
tcttattctc actccgtctt cgacgtccca cttcactaca acttccagcg tgctgccaac 900
tccggaggta atttcgatat gagaactatt ttcaacggat ccgttgtcca gcaacaccca 960
actttggccg tcaccatcgt cgacaaccat gactcccagc caggtcaatc cttggagtcc 1020
accgttgacg cttggttcaa accacttgcc tacgctatga tcatgaccag agagcagggt 1080
taccctaact tgttctacgg tgacttttac ggaaccaagg gttcctctaa tagagagatc 1140
ccaaatttgt cttctaaatt gactcctatt ttgaaggcca gaaaagacat ggcctacggt 1200
acccagcatg actaccttaa tcaccaagac gttatcggtt ggaccagaga gggtgttact 1260
gaccgttcca agtccggttt ggccactatc ttgtctgacg gaccaggagg aaacaagtgg 1320
atgtatgtcg gtaagagaaa cgccggagag acctggagag acaagaccgg taactcttcc 1380
aacgccgtta ccatcaactc tgacggttgg ggacagtttt ttgtcaatgg tggttctgtt 1440
tccatttatg gtgagtag 1458
<210> 17
<211> 1458
<212> DNA
<213>Artificial sequence
<400> 17
gctactcctc aaaactctac tatgatgcaa tactttgaat ggtacttgcc aaacgatggt 60
ttgcattgga accgtttgac caacgacgcc tccaacctta aggctttggg tgtcactacc 120
gtttggatcc ctcctgccta caagggtact tcccaaaacg acgtcggata cggagcctac 180
gacttgtacg accttggtga gttcaaccaa aagggtaccg tccgtaccaa gtacggtact 240
agaggtcagt tgcagaccgc tatcaacacc cttaagaacc agggtatcgg tacttacgga 300
gacgtcgtca tgaaccataa gggaggtgct gactttaccg agtccgtcca agctgtcgaa 360
gtcaacccaa acaacagatc tcaagaaact tccggtgaat acaccatttc cgcttggacc 420
ggattcaact tcgccggtcg taacaacttg cactccgcct tcaagtggag atggtaccac 480
ttcgacggta ctgactggga ccagtccaga tccttgaaga gaatttacaa gttcagagga 540
tctggtaagt cctgggacac cgaagtttcc aacgagttcg gtaactacga ctatcttatg 600
tatgccgacg ttgacttcga tcacccagag gtcaaggccg agttgaagaa ctggggtaag 660
tggtatgttc aatctttgaa ccttgatggt tttagattgg atgccgttaa acatatcaag 720
cacgattaca tcccagagtg gttggccgac gtcagacgta ctactggtaa agagttgttc 780
accgttgccg aatactggca gaacgacttg ggtgccatta acaattactt ggctaagacc 840
aactattctc actccgtctt cgacgtccca cttcactaca acttccagcg tgctgccaac 900
tccggaggta atttcgatat gagaactatt ttcaacggat ccgttgtcca gcaacaccca 960
actttggccg tcaccatcgt cgacaaccat gactcccagc caggtcaatc cttggagtcc 1020
accgttgacg cttggttcaa accacttgcc tacgctatga tcatgaccag agagcagggt 1080
taccctaact tgttctacgg tgacttttac ggaaccaagg gttcctctaa tagagagatc 1140
ccaaatttgt cttctaaatt gactcctatt ttgaaggcca gaaaagacat ggcctacggt 1200
acccagcatg actaccttaa tcaccaagac gttatcggtt ggaccagaga gggtgttact 1260
gaccgttcca agtccggttt ggccactatc ttgtctgacg gaccaggagg aaacaagtgg 1320
atgtatgtcg gtaagagaaa cgccggagag acctggagag acaagaccgg taactcttcc 1380
aacgccgtta ccatcaactc tgacggttgg ggacagtttt ttgtcaatgg tggttctgtt 1440
tccatttatg gtgagtag 1458
<210> 18
<211> 1458
<212> DNA
<213>Artificial sequence
<400> 18
gctactcctc aaaacatgac tatgatgcaa tactttgaat ggtacttgcc aaacgatggt 60
ttgcattgga accgtttgac caacgacgcc tccaacctta aggctttggg tgtcactacc 120
gtttggatcc ctcctgccta caagggtact tcccaaaacg acgtcggata cggagcctac 180
gacttgtacg accttggtga gttcaaccaa aagggtaccg tccgtaccaa gtacggtact 240
agaggtcagt tgcagaccgc tatcaacacc cttaagaacc agggtatcgg tacttacgga 300
gacgtcgtca tgaaccataa gggaggtgct gactttaccg agtccgtcca agctgtcgaa 360
gtcaacccaa acaacagatc tcaagaaact tccggtgaat acaccatttc cgcttggacc 420
ggattcaact tcgccggtcg taacaacttg cactccgcct tcaagtggag atggtaccac 480
ttcgacggta ctgactggga ccagtccaga tccttgaaga gaatttacaa gttcagagga 540
tctggtaagt cctgggacac cgaagtttcc aacgagttcg gtaactacga ctatcttatg 600
tatgccgacg ttgacttcga tcacccagag gtcaaggccg agttgaagaa ctggggtaag 660
tggtatgttc aatctttgaa ccttgatggt tttagattgg atgccgttaa acatatcaag 720
cacgattaca tcggtgagtg gttggccgac gtcagacgta ctactggtaa agagttgttc 780
accgttgccg aatactggca gaacgacttg ggtgccatta acaattactt ggctaagacc 840
tcttattctc actccgtctt cgacgtccca cttcactaca acttccagcg tgctgccaac 900
tccggaggta atttcgatat gagaactatt ttcaacggat ccgttgtcca gcaacaccca 960
actttggccg tcaccatcgt cgacaaccat gactcccagc caggtcaatc cttggagtcc 1020
accgttgacg cttggttcaa accacttgcc tacgctatga tcatgaccag agagcagggt 1080
taccctaact tgttctacgg tgacttttac ggaaccaagg gttcctctaa tagagagatc 1140
ccaaatttgt cttctaaatt gactcctatt ttgaaggcca gaaaagacat ggcctacggt 1200
acccagcatg actaccttaa tcaccaagac gttatcggtt ggaccagaga gggtgttact 1260
gaccgttcca agtccggttt ggccactatc ttgtctgacg gaccaggagg aaacaagtgg 1320
atgtatgtcg gtaagagaaa cgccggagag acctggagag acaagaccgg taactcttcc 1380
aacgccgtta ccatcaactc tgacggttgg ggacagtttt ttgtcaatgg tggttctgtt 1440
tccatttatg gtgagtag 1458

Claims (9)

1. alpha-amylase JcAmy mutant, it is characterised in that the amino acid sequence of the mutant is such as SEQ ID No.2 institutes The 6th of the amino acid sequence shown, in the 53rd, the 173rd, the 245th and/or the 281st any one or more Position is substituted radical.
2. alpha-amylase JcAmy mutant according to claim 1, it is characterised in that the amino acid sequence of the mutant The 6th of the amino acid sequence being classified as shown in SEQ ID No.2 is replaced by G6F, G6M, G6P, G6N or G6S.
3. alpha-amylase JcAmy mutant according to claim 1, it is characterised in that the amino acid sequence of the mutant The 53rd of the amino acid sequence being classified as shown in SEQ ID No.2 is replaced by N35S or N35A.
4. alpha-amylase JcAmy mutant according to claim 1, it is characterised in that the amino acid sequence of the mutant The 173rd of the amino acid sequence being classified as shown in SEQ ID No.2 is replaced by N173K or N173S.
5. alpha-amylase JcAmy mutant according to claim 1, it is characterised in that the amino acid sequence of the mutant The 245th of the amino acid sequence being classified as shown in SEQ ID No.2 is replaced by Q245G, Q245P or Q245R.
6. alpha-amylase JcAmy mutant according to claim 1, it is characterised in that the amino acid sequence of the mutant The 281st of the amino acid sequence being classified as shown in SEQ ID No.2 is replaced by G281N, G281D, G281S or G281K.
7. alpha-amylase JcAmy mutant according to claim 1, it is characterised in that the mutational site of the mutant For：G6F, N35S, N173K, Q245G, G281N；
G6M, N35S, N173S, Q245P, G281D；
G6P, N35A, N173K, Q245R, G281K；
G6N, N35S, N173K, Q245P, G281S；
G6S, N35A, N173S, Q245P, G281K；
G6P, N35A, N173K, Q245G, G281S；
G6S, N35A, N173K, Q245P, G281N；Or
G6M, N35A, N173K, Q245G, G281S.
8. encode the gene of alpha-amylase JcAmy mutant described in any one in claim 1-8.
9. the application of alpha-amylase JcAmy mutant described in any one in claim 1-8.
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WO2018129981A1 (en) * 2017-01-16 2018-07-19 广东溢多利生物科技股份有限公司 α-AMYLASE JCAMY MUTANT WITH INCREASED 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

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