CN107058264B - Alpha-amylase JcAmy mutant with improved specific activity and coding gene and application thereof - Google Patents

Abstract

The invention relates to the field of genetic engineering, in particular to an alpha-amylase JcAmy mutant with improved specific activity and a coding gene and application thereof. The amino acid sequence of the mutant is any one or more of the 6 th position, the 53 th position, the 173 th position, the 245 th position and/or the 281 th position of the amino acid sequence shown as SEQ ID No.2 as a substituent group. Compared with the specific activity of the original alpha-amylase, the improvement range of the specific activity of the mutated alpha-amylase is 21-92%, and the method lays a foundation for the industrial application of the halophilous bacillus amyloliquefaciens alpha-amylase.

Description

Alpha-amylase JcAmy mutant with improved 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 JcAmy mutant with improved specific activity and a coding gene and application thereof.

Background

Alpha-amylase, the systematic name of which is 1, 4-alpha-D-glucan hydrolase, is an endo-hydrolase, mainly plays a role in catalyzing 1, 4-alpha-D-glucan of starch to generate reducing dextrin and saccharides, and plays an important role in the fields of starch, detergents, beverages, textiles and the like.

Currently, in the fields of alcohol brewing, starch sugar and the like, a starchy raw material is generally subjected to two stages of liquefaction and saccharification in the processing process. The enzymes used in the liquefaction and saccharification processes are mainly alpha-amylases and saccharifying enzymes. Commercial α -amylase and saccharifying enzymes currently in wide use in industry have optimal pH values of about 6.5 and 4.5, and thus require the addition of acid or alkali to adjust pH during the liquefaction and saccharification processes. The addition of large amounts of acid and base during liquefaction and saccharification not only complicates the processing process, but also increases the production cost. If the alpha-amylase which is stable under the acidic condition can be developed, acid and alkali are not required to be additionally added for pH adjustment in the liquefying and saccharifying processes, so that the reagent consumption can be reduced, the processing technology is simplified, the production cost can be reduced, and the grain is saved. Has great significance for the starch processing field.

The alpha-amylase of the halophilous Bacillus halodurans (Jeotgalibacillus campisalis) is called JcAmy for short. JcAmy is an acid resistant amylase with an optimum pH of 5.0 and good stability in the pH range of 4 to 8, enabling it to perform well under acidic liquefaction conditions. Although JcAmy has good pH characteristics, it has low specific activity and high production cost, which limits its industrial application. Therefore, the improvement of the specific enzyme activity of JcAmy and the reduction of the production cost are problems to be solved urgently in the industrial application of JcAmy.

In recent years, a series of microbial alpha-amylases have been expressed heterologously in Escherichia coli or yeast. However, the activity of the alpha-amylase produced by wild bacteria screened from nature is generally lower, so that the alpha-amylase cannot be directly applied to industrial fermentation production. In the prior art, the enzyme production capability of the strain is improved by technical means such as mutagenesis, crossbreeding and the like on the wild strain, but the technologies such as mutagenesis, crossbreeding and the like have large workload and have higher probability of uncontrollably generating negative mutation.

The invention improves the specific activity of the halophilic marine bacillus alpha-amylase JcAmy by a site-directed mutagenesis technology, greatly reduces the production cost and lays a foundation for further industrial application.

Disclosure of Invention

The invention aims to modify the molecular of the alpha-amylase JcAmy derived from the salt-land fresh-sea-bud bacillus, so that the modified alpha-amylase has higher specific activity, the production cost is reduced, and a foundation is laid for the industrial application of the salt-land fresh-sea-bud bacillus alpha-amylase.

The invention aims to provide a mutant of an alpha-amylase JcAmy with improved specific activity.

It is still another object of the present invention to provide a gene encoding the above-described JcAmy mutant of α -amylase.

The nucleotide sequence and the amino acid sequence of the salty seafood bacillus alpha-amylase JcAmy are shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

The invention adopts a site-directed saturation mutagenesis method to carry out molecular modification on the 6 th, 53 th, 173 th, 245 th and/or 281 th sites of the alpha-amylase JcAmy shown in SEQ ID NO.2, and obtains the alpha-amylase mutant with improved specific activity through high-throughput screening. The amino acid sequences of the mutants are shown as SEQ ID NO.3 to SEQ ID NO.10, and the nucleotide sequences of the coding mutants are shown as SEQ ID NO.11 to SEQ ID NO. 18.

An optimized and improved alpha-amylase JcAmy mutant according to a specific embodiment of the invention has an amino acid sequence in which at least one amino acid atposition 6, 53, 173, 245 and/or 281 of the alpha-amylase JcAmy of SEQ ID No.2 is replaced by a corresponding amino acid selected from the group consisting of:

the 6 th position of the alpha-amylase JcAmy is replaced with G6F, G6M, G6P, G6N or G6S;

the 53 rd position of the alpha-amylase JcAmy is replaced by N35S or N35A;

the 173-position of the alpha-amylase JcAmy was replaced with N173K or N173S;

the 245 th position of the alpha-amylase JcAmy is replaced by Q245G, Q245P or Q245R;

the 281 th position of the alpha-amylase JcAmy is replaced by G281N, G281D, G281S or G281K.

The optimized and improved alpha-amylase JcAmy mutant JcAmy-1 according to the embodiment of the invention has the mutation sites of: G6F, N35S, N173K, Q245G and G281N, and the amino acid sequence is shown as SEQ ID NO. 3.

The optimized and improved alpha-amylase JcAmy mutant JcAmy-2 according to the embodiment of the invention has the mutation sites of: G6M, N35S, N173S, Q245P and G281D, and the amino acid sequence is shown as SEQ ID NO. 4.

The optimized and improved alpha-amylase JcAmy mutant JcAmy-3 according to the embodiment of the invention has the mutation sites of: G6P, N35A, N173K, Q245R and G281K, and the amino acid sequence is shown as SEQ ID NO. 5.

The optimized and improved alpha-amylase JcAmy mutant JcAmy-4 according to the embodiment of the invention has the mutation sites of: G6N, N35S, N173K, Q245P and G281S, and the amino acid sequence is shown as SEQ ID NO. 6.

The optimized and improved alpha-amylase JcAmy mutant JcAmy-5 according to the embodiment of the invention has the mutation sites of: G6S, N35A, N173S, Q245P and G281K, and the amino acid sequence is shown as SEQ ID NO. 7.

The optimized and improved alpha-amylase JcAmy mutant JcAmy-6 according to the embodiment of the invention has the mutation sites of: G6P, N35A, N173K, Q245G and G281S, and the amino acid sequence is shown as SEQ ID NO. 8.

The optimized and improved alpha-amylase JcAmy mutant JcAmy-7 according to the embodiment of the invention has the mutation sites of: G6S, N35A, N173K, Q245P and G281N, and the amino acid sequence is shown as SEQ ID NO. 9.

The optimized and improved alpha-amylase JcAmy mutant JcAmy-8 according to the embodiment of the invention has the mutation sites of: G6M, N35A, N173K, Q245G and G281S, and the amino acid sequence is shown as SEQ ID NO. 10.

The invention carries out molecular modification on a-amylase JcAmy of a halophilum halioticum (Jeotgalibacillus campisalis) by protein rational modification and high-throughput screening technology. Compared with the specific activity of the original alpha-amylase, the improvement range of the specific activity of the mutated alpha-amylase is 21-92%, and the method lays a foundation for the industrial application of the halophilous bacillus amyloliquefaciens alpha-amylase.

Drawings

FIG. 1 shows the optimum pH for the original alpha-amylase and the alpha-amylase mutant, JcAmy1-8, according to embodiments of the invention.

FIG. 2 shows the pH stability of the original alpha-amylase and the alpha-amylase mutant, JcAmy1-8, according to embodiments of the invention.

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. strain and carrier: coli strain Topl0, Pichia pastoris X33, vector pPICz. alpha.A, Zeocin were purchased from Invitrogen.

2. Gene: alpha-amylase JcAmy (Sequence ID: WP-052476631.1) of the published salty seafood bacillus (Jeotgalibacillus campisalis) is subjected to gene synthesis after codon optimization according to pichia pastoris.

3. 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.

4. Culture medium: the Escherichia coli culture medium is LB, and the formula is as follows: 1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0. LBZ is LB medium plus 25ug/mL Zeocin.

The yeast culture medium is YPD, and the formula is as follows: 1% yeast extract, 2% peptone, 2% glucose. The yeast screening medium is YPDZ, and the formula is YPD +100mg/L zeocin.

Yeast induction medium BMGY, formula 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 1% glycerol (V/V)) and BMMY, except that 0.5% methanol was used instead of glycerol, the other components were identical to BMGY.

Example 1 cloning of the alpha-amylase JcAmy from Haemophilus halodurans (Jeotgalibacillus campisalis) salt

Two primers (F: 5'-GATCGAATTCGCTACTCCTCAAAACGGTACTATGA-3' and R: 5'-TAGCGCGGCCGCCTACTCACCATAAATGGAAACAGAA-3') were designed based on the sequence of the synthesized a-amylase JcAmy gene for amplification of the C.halophilus alpha-amylase gene. And purifying and recovering the amplified PCR product, and connecting the product to an expression vector pPICz alpha A to obtain the expression vector pPICz alpha A-JcAmy.

Example 2 site-directed mutagenesis

The 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

Taking the pPICz alpha A-JcAmy as a template, and carrying out PCR amplification by using corresponding primers, wherein the specific amplification reaction system is as follows:

q5 high fidelity Taq enzyme MIX	23uL
		Corresponding mutant primers	1uL
Corresponding mutant primers	1uL
		pPICzαA-JcAmy(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. A series of single-site mutants with improved specific activities are obtained by screening, and the relative specific activities of the mutants are shown in Table 1.

TABLE 1 relative specific Activity of original alpha-Amylase and Single Point mutant alpha-Amylase

Numbering	Relative specific activity (%)
		Primary alpha-amylases	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

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. And respectively adding 1.6mL of BMMY culture medium for 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.

Example 4 combinatorial mutagenesis

Position 6: G6F, G6M, G6P, G6N, G6S;

position 53: N35S, N35A;

173 th site: N173K, N173S;

at position 245: Q245G, Q245P, Q245R;

281 th position: G281N, G281D, G281S, G281K.

The single mutation sites of improved specific enzyme activity in example 2, G6F, G6M, G6P, G6N, G6S, N35S, N35A, N173K, N173S, Q245G, Q245P, Q245R, G281N, G281D, G281S and G281K were combined, and the experimental procedure was the same as in example 2. The 8 combined mutations finally obtained by experiments are respectively named as JcAmy-1, JcAmy-2, JcAmy-3, JcAmy-4, JcAmy-5, JcAmy-6, JcAmy-7 and JcAmy-8

Wherein JcAmy-1 comprises the following mutation sites: G6F, N35S, N173K, Q245G, G281N.

Wherein the JcAmy-2 comprises mutation sites of: G6M, N35S, N173S, Q245P, G281D.

Wherein the JcAmy-3 comprises mutation sites of: G6P, N35A, N173K, Q245R, G281K.

Wherein JcAmy-4 comprises mutation sites of: G6N, N35S, N173K, Q245P, G281S.

Wherein JcAmy-5 comprises the following mutation sites: G6S, N35A, N173S, Q245P, G281K.

Wherein the JcAmy-6 comprises mutation sites of: G6P, N35A, N173K, Q245G, G281S.

Wherein JcAmy-7 comprises the following mutation sites: G6S, N35A, N173K, Q245P, G281N.

Wherein JcAmy-8 comprises the following mutation sites: G6M, N35A, N173K, Q245G, G281S.

Example 5 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 improvement of the specific activity of the mutant was calculated by dividing the specific activity of the mutant by the specific activity of the original alpha-amylase. Compared with the original JcAmy, the specific activity of the mutated JcAmy is improved by 21-92% (the specific result is shown in Table 2).

TABLE 2 relative specific Activity of original alpha-Amylase and mutant alpha-Amylase

Numbering	Relative specific activity (%)
		Primary alpha-amylases	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

Example 6 optimum pH and pH stability of the original alpha-Amylase and the alpha-Amylase mutant JcAmy1-8

The optimum pH of the original alpha-amylase and the alpha-amylase mutant JcAmy1-8 was determined by reference to the national standard method. The optimum pH for the original alpha-amylase and the alpha-amylase mutant, JcAmy1-8, are shown in FIG. 1. As can be seen from FIG. 1, the optimum pH of the mutant JcAmy1-8 did not change much, almost as much as the original alpha-amylase.

The original alpha-amylase and the alpha-amylase mutant JcAmy1-8 are respectively treated for 3 hours at room temperature under the condition of pH4-8, and then the enzyme activity is determined by referring to the national standard method. The pH stability of the original α -amylase and the α -amylase mutant, JcAmy1-8, is shown in FIG. 2. As can be seen from FIG. 2, the mutants JcAmy1, JcAmy3 and JcAmy7 were more stable than the original α -amylase at pH4, while the mutants JcAmy2, JcAmy4, JcAmy5, JcAmy6 and JcAmy8 were pH stable in agreement with the original α -amylase.

<110> Guangdong overflow Multi-interest Biotech Ltd

Alpha-amylase JcAmy mutant with improved specific activity and coding gene and application thereof

<160>18

<210>1

<211>1458

<212>DNA

<213> Bacillus salina seafood

<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 taactcttcc1380

aacgccgtta ccatcaactc tgacggttgg ggacagtttt ttgtcaatgg tggttctgtt 1440

tccatttatg gtgagtag

1458

<210>2

<211>460

<212>PRT

<213> Bacillus salina seafood

<400>2

ATPQNGTMMQ YFEWYLPNDG LHWNRLTNDASNLKNLGVTT 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 LHWNRLTNDASNLKSLGVTT 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 LHWNRLTNDASNLKSLGVTT 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

SIYGE485

<210>5

<211>485

<212>PRT

<213> Artificial sequence

<400>5

ATPQNPTMMQ YFEWYLPNDG LHWNRLTNDASNLKALGVTT 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 LHWNRLTNDASNLKSLGVTT 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 LHWNRLTNDASNLKALGVTT 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 LHWNRLTNDASNLKALGVTT 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 LHWNRLTNDASNLKALGVTT 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 LHWNRLTNDASNLKALGVTT 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 gtaagagaaacgccggagag acctggagag acaagaccgg taactcttcc 1380

aacgccgtta ccatcaactc tgacggttgg ggacagtttt ttgtcaatgg tggttctgtt 1440

tccatttatg gtgagtag 1458

Claims (4)

1. An alpha-amylase JcAmy mutant characterized in that the amino acid sequence of the mutant is a substituent at any one or more of position 6, position 35, position 173, position 245 and/or position 281 of the amino acid sequence shown as SEQ ID No.2,

position 6 is replaced with G6F, G6M, G6P, G6N or G6S;

position 35 is replaced with N35S or N35A;

substitution at position 173 with N173K or N173S;

position 245 is replaced with Q245G, Q245P or Q245R;

position 281 is replaced by G281N, G281D, G281S or G281K.

2. The α -amylase JcAmy mutant according to claim 1, wherein the mutant has the following mutation sites:

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。

3. A gene encoding the JcAmy mutant alpha-amylase of claim 1.

4. Use of the α -amylase JcAmy mutant according to claim 1 or 2 for catalyzing the hydrolysis of 1,4- α -D-glucan of starch.

Images