CN109370973A - A kind of maltogenic amylase production bacterial strain - Google Patents
A kind of maltogenic amylase production bacterial strain Download PDFInfo
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- CN109370973A CN109370973A CN201811395721.1A CN201811395721A CN109370973A CN 109370973 A CN109370973 A CN 109370973A CN 201811395721 A CN201811395721 A CN 201811395721A CN 109370973 A CN109370973 A CN 109370973A
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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)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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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)
- C12Y302/01133—Glucan 1,4-alpha-maltohydrolase (3.2.1.133), i.e. maltogenic alpha-amylase
Abstract
The invention discloses a kind of maltogenic amylases to produce bacterial strain, belongs to microorganisms technical field.The present invention obtains the maltogenic amylase enzyme mutant of transformation efficiency raising by rite-directed mutagenesis and combinatorial mutagenesis, and the mutant is expressed in Escherichia coli.Enzyme solution is used for maltose production, under the additive amount of 20U/g dried starch, mutant K198E/D290T can make maltose content to 97% or more, trisaccharide, four sugared contents level off to 0 when reacting 5h for raising when conversion reaction carries out 10h during dextrine conversion.Compared with wild type maltogenic amylase, there are higher transformation efficiency and less conversion byproducts, there is important commercial application potentiality.
Description
Technical field
The present invention relates to a kind of maltogenic amylases to produce bacterial strain, belongs to microorganisms technical field.
Background technique
Maltogenic amylase (maltogenic amylase or maltogenase, EC 3.2.1.133) is glycoside hydrolysis
Enzyme GH-H set member.Currently, the main bacterial origin of maltogenic amylase is bacillus stearothermophilus (Bacillus
Stearothermophilus), Bacillus cercus (Bacillus cereus), bacillus subtilis (Bacillus
Subtilis), bacillus licheniformis (Bacillus licheniformis), thermophilic actinomycete (Thermus vulgaris) with
And Thermus (Thermus sp.) etc..The maltogenic amylase of separate sources, also makes a big difference in nature.At present
Applied to the maltogenic amylase of malt syrup preparation and anti-bread staling, it is mainly derived from bacillus stearothermophilus.
The reducing disaccharides that maltose is made of two glucose units through α-Isosorbide-5-Nitrae glucosides key connection, chemical name are
Six ring glucosyl group-D- of 4-O-D-, six ring glucose.Its sugariness is soft, and because of low viscosity, agent of low hygroscopicity and good thermostabilization
Property feature, can be used as food enhancing agent replace dextrose and saccharose, field of food industry have huge application potential.Industry
The preparation of upper maltose is using starchiness as raw material, by alpha-amylase, malt (or beta amylase, fungal amylase) hydrolysis
The syrup of a kind of based on maltose (40%-60%) is made in technique, if maltose content be more than 45% (preferably 50% with
On), then referred to as high maltose syrup.In the food industry the purposes of high maltose syrup first is that production cake, the products such as candy.
Syrup infusion temperature be much higher than maltose, generally more than 140 DEG C.Maltose content be greater than 70%, even as high as 90% or more, then by
Referred to as superhigh maltose syrup.Maltose can avoid blood glucose rise compared to glucose, and the preparation for being applied to antibody, vaccine etc. has
There is the application advantage better than glucose.Therefore the malt syrup of ultra-high purity also results in more and more in the application of field of medicaments
Concern.
Current maltose production technology is more mature, when using alpha-amylase and beta amylase production maltose, product
Middle maltose content may be up to 90%, and glucose, trisaccharide, tetrose and part oligosaccharide and dextrin are main conversion by-products
Object.Wherein dextrin and part oligosaccharide, can be removed by ethanol precipitation.The preparation of ultra-high purity maltose will then pass through chromatography
The methods of separation and crystallization obtain.Due to maltose large viscosity, difficult crystallization, usually require that maltose purity exists in crystallization raw material
90% or more, therefore the purity of chromatographic isolation, maltose crystallization is played a crucial role.Chromatographic isolation can remove substantially
The small molecule carbohydrate of glucose and pentasaccharides or more is smaller to maltose impurities affect.But the trisaccharide and tetrose in product due to
It is more close with maltose property, often become the major impurity in isolating and purifying, not only directly reduces product purity, return
The moisture content of maltose crystallinity, Viscosity of Syrup and final products brings great disadvantage to influence, and makes maltose ultimate yield
It substantially reduces.
Maltogenic amylase has small molecular sugar hydrolysing activity, and the small molecular sugars such as hydrolyzable trisaccharide, tetrose form glucose
And maltose, therefore be usually used in compounding with alpha-amylase, beta amylase and Pullulanase etc. in the production of superelevation maltose with
Proportion of by-product is reduced, maltose is made to be more conducive to crystallize.It is reported that deriving from bacillus stearothermophilus (Bacillus
Stearothermophilus the optimal reactive temperature with higher of maltogenic amylase) and lower optimal pH react item
Part can meet more harsh industrial process conditions, and maltose ratio in product is improved to 92%, is industrially had greatly
Application advantage.
Summary of the invention
The first purpose of the invention is to provide a kind of genetic engineering bacteriums for producing maltogenic amylase, express SEQ ID
Maltogenic amylase shown in NO.1~8 are any.
In one embodiment of the invention, the genetic engineering bacterium is recombination bacillus coli.
In one embodiment of the invention, the recombination bacillus coli with E.coli BL21, E.coli JM109,
E.coli DH5 α or E.coli TOP10 is host.
In one embodiment of the invention, the recombination bacillus coli is with E.coli BL21 (DE3) for host.
In one embodiment of the invention, the maltogenic amylase is expressed with pET serial carrier.
In one embodiment of the invention, using pET24a as expression vector.
A second object of the present invention is to provide a kind of method for producing maltogenic amylase, the method is by the base
Because engineering bacteria is seeded in culture medium, enzymatic production.
In one embodiment of the invention, the culture medium is TB culture medium.
In one embodiment of the invention, final concentration is added after 35~37 DEG C of 1~4h of culture in the Escherichia coli
The IPTG of 0.01mM is induced, and continues 36~48h of cultivation and fermentation in 25~28 DEG C of shaking tables.
The application in terms of the product of the genetic engineering bacterium preparation containing maltose is also claimed in the present invention.
The utility model has the advantages that the present invention obtains the maltogenic amylase of transformation efficiency raising by rite-directed mutagenesis and combinatorial mutagenesis
Enzyme mutant, and the mutant is expressed in Escherichia coli.Enzyme solution is used for maltose production, in 20U/g dried starch
Additive amount under, during dextrine conversion mutant K198E/D290T can make maltose content conversion reaction carry out 10h when mention
Up to 97% or more, trisaccharide, four sugared contents level off to 0 when reacting 5h.Compared with wild type maltogenic amylase, have higher
Transformation efficiency and less conversion byproducts, have important commercial application potentiality.
Detailed description of the invention
Maltose during Fig. 1 wild type maltogenic amylase (WT) and mutant (K198E/D290T) production maltose
Content (A), three sugared contents (B), four sugared contents (C) variation.
Specific embodiment
Glucose, maltose, trisaccharide and tetrose content analyzed using HPLC.The chromatographic condition of measurement is: Elite
2000HPLC chromatograph, Elite autosampler, chromatographic column Thermo APS-2HYPERSIL 13286 (4.6mm ×
250mm), HITACHI L-2490 Composition distribution；Mobile phase is 70% (v/v) acetonitrile solution, flow velocity 0.8mL/min；Column
40 DEG C of temperature.
Embodiment 1: the preparation of wild maltogenic amylase.
(1) building of maltogenic amylase recombinant bacterium
According to the amyM amino acid sequence (NCBI number: AAA22233.1) on NCBI, codon optimization is carried out to sequence,
Using the gene order amyM of chemical total synthesis method synthesis maltogenic amylase.For constructing the matter of coli expression carrier
Grain is pET24a (+).PET24a (+) plasmid and the plasmid with amyM gene are subjected to III double digestion of Nco I and Hind respectively,
Digestion products are connected overnight after glue recycles with T4 ligase, and connection product is converted to escherichia coli jm109 competent cell,
Converted product is coated on the LB plate of the kanamycins containing 100mg/L, and through 37 DEG C of overnight incubations, 2 single colonies of picking on plate are connect
Enter LB liquid medium, extracting plasmid verifying after 8h, as a result correctly, the amyM/pET24a plasmid being enriched with.By plasmid
AmyM/pET24a converts e. coli bl21 (DE3) competent cell, and picking transformant (contains 100mg/L in LB liquid medium
Kanamycins) in 37 DEG C of overnight incubations, save glycerol tube, be named as amyM/pET24a/BL21 (DE3).
(2) expression and purification of maltogenic amylase
It is raw in LB liquid medium (kanamycins containing 100mg/L) from glycerol tube inoculation amyM/pET24a/BL21 (DE3)
Seed is accessed TB liquid fermentation medium (kanamycins containing 100mg/L) by 5% inoculum concentration by long 8h.Escherichia coli are at 37 DEG C
After cultivating 2h, the IPTG that 0.01mM final concentration is added is induced, and after 25 DEG C of shaking tables continue cultivation and fermentation 48h, will be fermented
Liquid removes thallus in 4 DEG C, 8000rpm centrifugation 10min, collects fermentation supernatant, after measured, enzyme activity is up to 4892U/mL.
50% (NH is slowly added in supernatant4)2SO4, 4 DEG C stand overnight, and 4 DEG C, 8000rpm centrifugation 20min are collected
Precipitating.After redissolving precipitating with the 20mM citrate buffer solution of pH7.5, the dialysed overnight in 20mM citrate buffer solution.Period is more
Change 2-3 buffer.Loading sample is made after filtering by 0.22 μm of film, recombinant protein is carried out using avant protein purification instrument
Purifying.Anion-exchange chromatography purification step: (1) it balances: balancing DEAE anion exchange with the 20mM buffer of 5 times of volumes
Chromatographic column；(2) loading: the sample pre-processed is with the flow velocity loading of 1mL/min；(3) elute: flow velocity 1mL/min carries out ladder
Degree elution, Detection wavelength 290nm, the eluent of Fraction collection enzyme activity containing maltogenic amylase.The wild malt purified
Saccharogenic amylase.
Embodiment 2: maltogenic amylase enzyme mutant preparation
(1) single mutation
The 198th in maltogenic amylase lysine (Lys) is mutated into glutamic acid (Glu) and tyrosine respectively
(Tyr), K198E, K198Y are labeled as；The 290th in maltogenic amylase aspartic acid (Asp) is mutated into sweet ammonia respectively
Sour (G, ly) and threonine (Thr), are respectively labeled as D290G, D290T.
Introduce the rite-directed mutagenesis primer of K198E mutation are as follows:
Forward primer: 5 '-CGACGACGCTACCGAAGGTTACTTCCACCA-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TGGTGGAAGTAACCTTCGGTAGCGTCGTCG-3 ' (underscore is mutating alkali yl)
Introduce the rite-directed mutagenesis primer of K198Y mutation are as follows:
Forward primer: 5 '-CGACGACGCTACCTATGGTTACTTCCACCA-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TGGTGGAAGTAACCATAGGTAGCGTCGTCG-3 ' (underscore is mutating alkali yl)
Introduce the rite-directed mutagenesis primer of D290G mutation are as follows:
Forward primer: 5 '-GAATGGTACGGTGACGGTCCGGGTACCGCT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-AGCGGTACCCGGACCGTCACCGTACCATTC-3 ' (underscore is mutating alkali yl)
Introduce the rite-directed mutagenesis primer of D290T mutation are as follows:
Forward primer: 5 '-GAATGGTACGGTGACACGCCGGGTACCGCT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-AGCGGTACCCGGCGTGTCACCGTACCATTC-3 ' (underscore is mutating alkali yl)
Using above-mentioned primer, using amyM/pET24a plasmid as template, PCR reaction is carried out.Reaction in 50 μ L systems into
Row, reaction condition are as follows: 94 DEG C of initial denaturation 4min；Then carry out 30 circulations (94 DEG C of 10s, 55 DEG C of 10s, 72 DEG C of 7min20s)；72
DEG C extend 10min；Last 4 DEG C of heat preservations.PCR product is digested through Dpn I (Fermentas company), converts Escherichia coli respectively
JM109 competent cell, converted product are coated on the LB plate of the kanamycins containing 100mg/L, through 37 DEG C of overnight incubations, on plate
2 single colonies of picking access LB liquid medium, extract plasmid after 8h, and correct, preservation glycerol tube is sequenced.
(2) expression and purification of mutant enzyme
Mutant expression and purification process are as described in Example 1.
(3) double mutation
On the basis of single mutation, it is combined mutation, constructs mutant K198E/D290G, K198Y/D290G respectively,
K198E/D290T, K198Y/D290T.
Embodiment 3: maltogenic amylase enzyme activity analysis
(1) enzyme-activity unit defines
When measuring maltogenic amylase enzyme activity using 3,5- dinitro bigcatkin willow acid system (DNS method), catalysis generates 1 μm of ol per minute
Enzyme amount needed for reduced sugar is as a unit of activity.
(2) enzyme activity determination step
Preheating: it takes the 0.5% soluble starch solution (50mM pH5.5 citrate buffer solution) of 2mL in test tube, is placed in
10min is preheated in 60 DEG C of water-baths.
Reaction: being added 0.1mL sample enzyme solution, shaken well, and accurate timing 10min is added 3mL DNS shaken well, puts
Enter and terminate reaction in ice water, boiling water bath boils 7min.It is cooling.
Measurement: distilled water is added into above-mentioned reaction system and is settled to 15mL, mixes.It measures and inhales under 540nm wavelength
Light value simultaneously calculates enzyme activity.
The Rate activity of wild type maltogenic amylase and mutant enzyme is listed in the table below (by the ratio of wild type maltogenic amylase
Vigor is set as 100%, and each mutant Rate activity is opposite Rate activity relative to the ratio of wild specific activity of enzyme):
The Rate activity and opposite Rate activity of 1 wild type maltogenic amylase of table and mutant enzyme
Embodiment 4: Production by Enzymes maltose
2L 20% (w/v) potato starch solution is prepared, pH to 5.5 is adjusted, acid α-shallow lake of 30U/g dried starch is added
Powder enzyme (is purchased from Jie Neng section), and the Pullulanase (being purchased from Jie Neng section) and 10U/g of 24U/g dried starch are added after injection liquefaction processing
The beta amylase (extracting from sweet potato) of dried starch, 60 DEG C of stir process for 24 hours, are once saccharified.
By being once saccharified, glucose, maltose, trisaccharide and four sugared contents are respectively 0.26% in reaction system,
89.73%, 9.21% and 0.8%.
Then, it is separately added into the wild type maltogenic amylase of 20U/g dried starch into primary saccharification reaction system and dashes forward
Variant enzyme K198E, K198E/D290G, K198E/D290T.60 DEG C of stirrings, carry out dextrine conversion.Reaction is measured by sampling to 25h and produces
Object composition.
Each component content is as shown in table 2 in reaction system:
The saccharification result (reaction 20h) of the different maltogenic amylases of table 2
Content of material each in reaction process is monitored, as a result as shown in Figure 1, mutant K198E/D290T can make wheat
When conversion reaction carries out 10h, to 97% or more, trisaccharide, four sugared contents level off to 0 when reacting 5h to bud sugared content for raising.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Hunan Huisheng Bio-Technology Co., Ltd.
<120>a kind of maltogenic amylase produces bacterial strain
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<170> PatentIn version 3.3
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Asn Asn Ser Gly Val Asn Val Leu Asp Phe Asp Leu Asn Thr Val Ile
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Gly Leu Leu Phe Ser Gly Ser Leu Pro Tyr Asn Pro Asn Ala Ala Glu
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Asp Phe Lys Gln Ile Glu Glu His Phe Gly Asn Trp Thr Thr Phe Asp
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Phe Val Pro Asn His Ser Thr Pro Phe Lys Ala Asn Asp Ser Thr Phe
165 170 175
Ala Glu Gly Gly Ala Leu Tyr Asn Asn Gly Thr Tyr Met Gly Asn Tyr
180 185 190
Phe Asp Asp Ala Thr Lys Gly Tyr Phe His His Asn Gly Asp Ile Ser
195 200 205
Asn Trp Asp Asp Arg Tyr Glu Ala Gln Trp Lys Asn Phe Thr Asp Pro
210 215 220
Ala Gly Phe Ser Leu Ala Asp Leu Ser Gln Glu Asn Gly Thr Ile Ala
225 230 235 240
Gln Tyr Leu Thr Asp Ala Ala Val Gln Leu Val Ala His Gly Leu Arg
245 250 255
Ile Asp Ala Val Lys His Phe Asn Ser Gly Phe Ser Lys Ser Leu Ala
260 265 270
Asp Lys Leu Tyr Gln Lys Lys Asp Ile Phe Leu Val Gly Glu Trp Tyr
275 280 285
Gly Gly Asp Pro Gly Thr Ala Asn His Leu Glu Lys Val Arg Tyr Ala
290 295 300
Asn Asn Ser Gly Val Asn Val Leu Asp Phe Asp Leu Asn Thr Val Ile
305 310 315 320
Arg Asn Val Phe Gly Thr Phe Thr Gln Thr Met Tyr Asp Leu Asn Asn
325 330 335
Met Val Asn Gln Thr Gly Asn Glu Tyr Lys Tyr Lys Glu Asn Leu Ile
340 345 350
Thr Phe Ile Asp Asn His Asp Met Ser Arg Phe Leu Ser Val Asn Ser
355 360 365
Lys Asn Lys Ala Asn Leu His Gln Arg Leu Leu Ser Phe Ser Leu Arg
370 375 380
Gly Val Arg Pro Pro Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Ala Gly
385 390 395 400
Gly Asn Asp Pro Tyr Asn Arg Gly Met Met Pro Ala Phe Asp Thr Thr
405 410 415
Thr Thr Ala Phe Lys Glu Val Ser Thr Leu Ala Gly Leu Arg Arg Asn
420 425 430
Asn Ala Ala Ile Gln Tyr Gly Thr Thr Thr Gln Arg Trp Ile Asn Asn
435 440 445
Asp Val Tyr Ile Tyr Glu Arg Lys Phe Phe Asn Asp Val Val Leu Val
450 455 460
Ala Ile Asn Arg Asn Thr Gln Ser Ser Tyr Ser Ile Ser Gly Leu Gln
465 470 475 480
Thr Ala Leu Pro Asn Gly Ser Tyr Ala Asp Tyr Leu Ser Gly Leu Leu
485 490 495
Gly Gly Asn Gly Ile Ser Val Ser Asn Gly Ser Val Ala Ser Phe Thr
500 505 510
Leu Ala Pro Gly Ala Val Ser Val Trp Gln Tyr Ser Thr Ser Ala Ser
515 520 525
Ala Pro Gln Ile Gly Ser Val Ala Pro Asn Met Gly Ile Pro Gly Asn
530 535 540
Val Val Thr Ile Asp Gly Lys Gly Phe Gly Thr Thr Gln Gly Thr Val
545 550 555 560
Thr Phe Gly Gly Val Thr Ala Thr Val Lys Ser Trp Thr Ser Asn Arg
565 570 575
Ile Glu Val Tyr Val Pro Asn Met Ala Ala Gly Leu Thr Asp Val Lys
580 585 590
Val Thr Ala Gly Gly Val Ser Ser Asn Leu Tyr Ser Tyr Asn Ile Leu
595 600 605
Ser Gly Thr Gln Thr Ser Val Val Phe Thr Val Lys Ser Ala Pro Pro
610 615 620
Thr Asn Leu Gly Asp Lys Ile Tyr Leu Thr Gly Asn Ile Pro Glu Leu
625 630 635 640
Gly Asn Trp Ser Thr Asp Thr Ser Gly Ala Val Asn Asn Ala Gln Gly
645 650 655
Pro Leu Leu Ala Pro Asn Tyr Pro Asp Trp Phe Tyr Val Phe Ser Val
660 665 670
Pro Ala Gly Lys Thr Ile Gln Phe Lys Phe Phe Ile Lys Arg Ala Asp
675 680 685
Gly Thr Ile Gln Trp Glu Asn Gly Ser Asn His Val Ala Thr Thr Pro
690 695 700
Thr Gly Ala Thr Gly Asn Ile Thr Val Thr Trp Gln Asn
705 710 715
<210> 4
<211> 717
<212> PRT
<213>artificial sequence
<400> 4
Met Lys Lys Lys Thr Leu Ser Leu Phe Val Gly Leu Met Leu Leu Ile
1 5 10 15
Gly Leu Leu Phe Ser Gly Ser Leu Pro Tyr Asn Pro Asn Ala Ala Glu
20 25 30
Ala Ser Ser Ser Ala Ser Val Lys Gly Asp Val Ile Tyr Gln Ile Ile
35 40 45
Ile Asp Arg Phe Tyr Asp Gly Asp Thr Thr Asn Asn Asn Pro Ala Lys
50 55 60
Ser Tyr Gly Leu Tyr Asp Pro Thr Lys Ser Lys Trp Lys Met Tyr Trp
65 70 75 80
Gly Gly Asp Leu Glu Gly Val Arg Gln Lys Leu Pro Tyr Leu Lys Gln
85 90 95
Leu Gly Val Thr Thr Ile Trp Leu Ser Pro Val Leu Asn Asn Leu Asp
100 105 110
Thr Leu Ala Gly Thr Asp Asn Thr Gly Tyr His Gly Tyr Trp Thr Arg
115 120 125
Asp Phe Lys Gln Ile Glu Glu His Phe Gly Asn Trp Thr Thr Phe Asp
130 135 140
Thr Leu Val Asn Asp Ala His Gln Asn Gly Ile Lys Val Ile Val Asp
145 150 155 160
Phe Val Pro Asn His Ser Thr Pro Phe Lys Ala Asn Asp Ser Thr Phe
165 170 175
Ala Glu Gly Gly Ala Leu Tyr Asn Asn Gly Thr Tyr Met Gly Asn Tyr
180 185 190
Phe Asp Asp Ala Thr Lys Gly Tyr Phe His His Asn Gly Asp Ile Ser
195 200 205
Asn Trp Asp Asp Arg Tyr Glu Ala Gln Trp Lys Asn Phe Thr Asp Pro
210 215 220
Ala Gly Phe Ser Leu Ala Asp Leu Ser Gln Glu Asn Gly Thr Ile Ala
225 230 235 240
Gln Tyr Leu Thr Asp Ala Ala Val Gln Leu Val Ala His Gly Leu Arg
245 250 255
Ile Asp Ala Val Lys His Phe Asn Ser Gly Phe Ser Lys Ser Leu Ala
260 265 270
Asp Lys Leu Tyr Gln Lys Lys Asp Ile Phe Leu Val Gly Glu Trp Tyr
275 280 285
Gly Thr Asp Pro Gly Thr Ala Asn His Leu Glu Lys Val Arg Tyr Ala
290 295 300
Asn Asn Ser Gly Val Asn Val Leu Asp Phe Asp Leu Asn Thr Val Ile
305 310 315 320
Arg Asn Val Phe Gly Thr Phe Thr Gln Thr Met Tyr Asp Leu Asn Asn
325 330 335
Met Val Asn Gln Thr Gly Asn Glu Tyr Lys Tyr Lys Glu Asn Leu Ile
340 345 350
Thr Phe Ile Asp Asn His Asp Met Ser Arg Phe Leu Ser Val Asn Ser
355 360 365
Lys Asn Lys Ala Asn Leu His Gln Arg Leu Leu Ser Phe Ser Leu Arg
370 375 380
Gly Val Arg Pro Pro Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Ala Gly
385 390 395 400
Gly Asn Asp Pro Tyr Asn Arg Gly Met Met Pro Ala Phe Asp Thr Thr
405 410 415
Thr Thr Ala Phe Lys Glu Val Ser Thr Leu Ala Gly Leu Arg Arg Asn
420 425 430
Asn Ala Ala Ile Gln Tyr Gly Thr Thr Thr Gln Arg Trp Ile Asn Asn
435 440 445
Asp Val Tyr Ile Tyr Glu Arg Lys Phe Phe Asn Asp Val Val Leu Val
450 455 460
Ala Ile Asn Arg Asn Thr Gln Ser Ser Tyr Ser Ile Ser Gly Leu Gln
465 470 475 480
Thr Ala Leu Pro Asn Gly Ser Tyr Ala Asp Tyr Leu Ser Gly Leu Leu
485 490 495
Gly Gly Asn Gly Ile Ser Val Ser Asn Gly Ser Val Ala Ser Phe Thr
500 505 510
Leu Ala Pro Gly Ala Val Ser Val Trp Gln Tyr Ser Thr Ser Ala Ser
515 520 525
Ala Pro Gln Ile Gly Ser Val Ala Pro Asn Met Gly Ile Pro Gly Asn
530 535 540
Val Val Thr Ile Asp Gly Lys Gly Phe Gly Thr Thr Gln Gly Thr Val
545 550 555 560
Thr Phe Gly Gly Val Thr Ala Thr Val Lys Ser Trp Thr Ser Asn Arg
565 570 575
Ile Glu Val Tyr Val Pro Asn Met Ala Ala Gly Leu Thr Asp Val Lys
580 585 590
Val Thr Ala Gly Gly Val Ser Ser Asn Leu Tyr Ser Tyr Asn Ile Leu
595 600 605
Ser Gly Thr Gln Thr Ser Val Val Phe Thr Val Lys Ser Ala Pro Pro
610 615 620
Thr Asn Leu Gly Asp Lys Ile Tyr Leu Thr Gly Asn Ile Pro Glu Leu
625 630 635 640
Gly Asn Trp Ser Thr Asp Thr Ser Gly Ala Val Asn Asn Ala Gln Gly
645 650 655
Pro Leu Leu Ala Pro Asn Tyr Pro Asp Trp Phe Tyr Val Phe Ser Val
660 665 670
Pro Ala Gly Lys Thr Ile Gln Phe Lys Phe Phe Ile Lys Arg Ala Asp
675 680 685
Gly Thr Ile Gln Trp Glu Asn Gly Ser Asn His Val Ala Thr Thr Pro
690 695 700
Thr Gly Ala Thr Gly Asn Ile Thr Val Thr Trp Gln Asn
705 710 715
<210> 5
<211> 717
<212> PRT
<213>artificial sequence
<400> 5
Met Lys Lys Lys Thr Leu Ser Leu Phe Val Gly Leu Met Leu Leu Ile
1 5 10 15
Gly Leu Leu Phe Ser Gly Ser Leu Pro Tyr Asn Pro Asn Ala Ala Glu
20 25 30
Ala Ser Ser Ser Ala Ser Val Lys Gly Asp Val Ile Tyr Gln Ile Ile
35 40 45
Ile Asp Arg Phe Tyr Asp Gly Asp Thr Thr Asn Asn Asn Pro Ala Lys
50 55 60
Ser Tyr Gly Leu Tyr Asp Pro Thr Lys Ser Lys Trp Lys Met Tyr Trp
65 70 75 80
Gly Gly Asp Leu Glu Gly Val Arg Gln Lys Leu Pro Tyr Leu Lys Gln
85 90 95
Leu Gly Val Thr Thr Ile Trp Leu Ser Pro Val Leu Asn Asn Leu Asp
100 105 110
Thr Leu Ala Gly Thr Asp Asn Thr Gly Tyr His Gly Tyr Trp Thr Arg
115 120 125
Asp Phe Lys Gln Ile Glu Glu His Phe Gly Asn Trp Thr Thr Phe Asp
130 135 140
Thr Leu Val Asn Asp Ala His Gln Asn Gly Ile Lys Val Ile Val Asp
145 150 155 160
Phe Val Pro Asn His Ser Thr Pro Phe Lys Ala Asn Asp Ser Thr Phe
165 170 175
Ala Glu Gly Gly Ala Leu Tyr Asn Asn Gly Thr Tyr Met Gly Asn Tyr
180 185 190
Phe Asp Asp Ala Thr Tyr Gly Tyr Phe His His Asn Gly Asp Ile Ser
195 200 205
Asn Trp Asp Asp Arg Tyr Glu Ala Gln Trp Lys Asn Phe Thr Asp Pro
210 215 220
Ala Gly Phe Ser Leu Ala Asp Leu Ser Gln Glu Asn Gly Thr Ile Ala
225 230 235 240
Gln Tyr Leu Thr Asp Ala Ala Val Gln Leu Val Ala His Gly Leu Arg
245 250 255
Ile Asp Ala Val Lys His Phe Asn Ser Gly Phe Ser Lys Ser Leu Ala
260 265 270
Asp Lys Leu Tyr Gln Lys Lys Asp Ile Phe Leu Val Gly Glu Trp Tyr
275 280 285
Gly Gly Asp Pro Gly Thr Ala Asn His Leu Glu Lys Val Arg Tyr Ala
290 295 300
Asn Asn Ser Gly Val Asn Val Leu Asp Phe Asp Leu Asn Thr Val Ile
305 310 315 320
Arg Asn Val Phe Gly Thr Phe Thr Gln Thr Met Tyr Asp Leu Asn Asn
325 330 335
Met Val Asn Gln Thr Gly Asn Glu Tyr Lys Tyr Lys Glu Asn Leu Ile
340 345 350
Thr Phe Ile Asp Asn His Asp Met Ser Arg Phe Leu Ser Val Asn Ser
355 360 365
Lys Asn Lys Ala Asn Leu His Gln Arg Leu Leu Ser Phe Ser Leu Arg
370 375 380
Gly Val Arg Pro Pro Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Ala Gly
385 390 395 400
Gly Asn Asp Pro Tyr Asn Arg Gly Met Met Pro Ala Phe Asp Thr Thr
405 410 415
Thr Thr Ala Phe Lys Glu Val Ser Thr Leu Ala Gly Leu Arg Arg Asn
420 425 430
Asn Ala Ala Ile Gln Tyr Gly Thr Thr Thr Gln Arg Trp Ile Asn Asn
435 440 445
Asp Val Tyr Ile Tyr Glu Arg Lys Phe Phe Asn Asp Val Val Leu Val
450 455 460
Ala Ile Asn Arg Asn Thr Gln Ser Ser Tyr Ser Ile Ser Gly Leu Gln
465 470 475 480
Thr Ala Leu Pro Asn Gly Ser Tyr Ala Asp Tyr Leu Ser Gly Leu Leu
485 490 495
Gly Gly Asn Gly Ile Ser Val Ser Asn Gly Ser Val Ala Ser Phe Thr
500 505 510
Leu Ala Pro Gly Ala Val Ser Val Trp Gln Tyr Ser Thr Ser Ala Ser
515 520 525
Ala Pro Gln Ile Gly Ser Val Ala Pro Asn Met Gly Ile Pro Gly Asn
530 535 540
Val Val Thr Ile Asp Gly Lys Gly Phe Gly Thr Thr Gln Gly Thr Val
545 550 555 560
Thr Phe Gly Gly Val Thr Ala Thr Val Lys Ser Trp Thr Ser Asn Arg
565 570 575
Ile Glu Val Tyr Val Pro Asn Met Ala Ala Gly Leu Thr Asp Val Lys
580 585 590
Val Thr Ala Gly Gly Val Ser Ser Asn Leu Tyr Ser Tyr Asn Ile Leu
595 600 605
Ser Gly Thr Gln Thr Ser Val Val Phe Thr Val Lys Ser Ala Pro Pro
610 615 620
Thr Asn Leu Gly Asp Lys Ile Tyr Leu Thr Gly Asn Ile Pro Glu Leu
625 630 635 640
Gly Asn Trp Ser Thr Asp Thr Ser Gly Ala Val Asn Asn Ala Gln Gly
645 650 655
Pro Leu Leu Ala Pro Asn Tyr Pro Asp Trp Phe Tyr Val Phe Ser Val
660 665 670
Pro Ala Gly Lys Thr Ile Gln Phe Lys Phe Phe Ile Lys Arg Ala Asp
675 680 685
Gly Thr Ile Gln Trp Glu Asn Gly Ser Asn His Val Ala Thr Thr Pro
690 695 700
Thr Gly Ala Thr Gly Asn Ile Thr Val Thr Trp Gln Asn
705 710 715
<210> 6
<211> 717
<212> PRT
<213>artificial sequence
<400> 6
Met Lys Lys Lys Thr Leu Ser Leu Phe Val Gly Leu Met Leu Leu Ile
1 5 10 15
Gly Leu Leu Phe Ser Gly Ser Leu Pro Tyr Asn Pro Asn Ala Ala Glu
20 25 30
Ala Ser Ser Ser Ala Ser Val Lys Gly Asp Val Ile Tyr Gln Ile Ile
35 40 45
Ile Asp Arg Phe Tyr Asp Gly Asp Thr Thr Asn Asn Asn Pro Ala Lys
50 55 60
Ser Tyr Gly Leu Tyr Asp Pro Thr Lys Ser Lys Trp Lys Met Tyr Trp
65 70 75 80
Gly Gly Asp Leu Glu Gly Val Arg Gln Lys Leu Pro Tyr Leu Lys Gln
85 90 95
Leu Gly Val Thr Thr Ile Trp Leu Ser Pro Val Leu Asn Asn Leu Asp
100 105 110
Thr Leu Ala Gly Thr Asp Asn Thr Gly Tyr His Gly Tyr Trp Thr Arg
115 120 125
Asp Phe Lys Gln Ile Glu Glu His Phe Gly Asn Trp Thr Thr Phe Asp
130 135 140
Thr Leu Val Asn Asp Ala His Gln Asn Gly Ile Lys Val Ile Val Asp
145 150 155 160
Phe Val Pro Asn His Ser Thr Pro Phe Lys Ala Asn Asp Ser Thr Phe
165 170 175
Ala Glu Gly Gly Ala Leu Tyr Asn Asn Gly Thr Tyr Met Gly Asn Tyr
180 185 190
Phe Asp Asp Ala Thr Glu Gly Tyr Phe His His Asn Gly Asp Ile Ser
195 200 205
Asn Trp Asp Asp Arg Tyr Glu Ala Gln Trp Lys Asn Phe Thr Asp Pro
210 215 220
Ala Gly Phe Ser Leu Ala Asp Leu Ser Gln Glu Asn Gly Thr Ile Ala
225 230 235 240
Gln Tyr Leu Thr Asp Ala Ala Val Gln Leu Val Ala His Gly Leu Arg
245 250 255
Ile Asp Ala Val Lys His Phe Asn Ser Gly Phe Ser Lys Ser Leu Ala
260 265 270
Asp Lys Leu Tyr Gln Lys Lys Asp Ile Phe Leu Val Gly Glu Trp Tyr
275 280 285
Gly Gly Asp Pro Gly Thr Ala Asn His Leu Glu Lys Val Arg Tyr Ala
290 295 300
Asn Asn Ser Gly Val Asn Val Leu Asp Phe Asp Leu Asn Thr Val Ile
305 310 315 320
Arg Asn Val Phe Gly Thr Phe Thr Gln Thr Met Tyr Asp Leu Asn Asn
325 330 335
Met Val Asn Gln Thr Gly Asn Glu Tyr Lys Tyr Lys Glu Asn Leu Ile
340 345 350
Thr Phe Ile Asp Asn His Asp Met Ser Arg Phe Leu Ser Val Asn Ser
355 360 365
Lys Asn Lys Ala Asn Leu His Gln Arg Leu Leu Ser Phe Ser Leu Arg
370 375 380
Gly Val Arg Pro Pro Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Ala Gly
385 390 395 400
Gly Asn Asp Pro Tyr Asn Arg Gly Met Met Pro Ala Phe Asp Thr Thr
405 410 415
Thr Thr Ala Phe Lys Glu Val Ser Thr Leu Ala Gly Leu Arg Arg Asn
420 425 430
Asn Ala Ala Ile Gln Tyr Gly Thr Thr Thr Gln Arg Trp Ile Asn Asn
435 440 445
Asp Val Tyr Ile Tyr Glu Arg Lys Phe Phe Asn Asp Val Val Leu Val
450 455 460
Ala Ile Asn Arg Asn Thr Gln Ser Ser Tyr Ser Ile Ser Gly Leu Gln
465 470 475 480
Thr Ala Leu Pro Asn Gly Ser Tyr Ala Asp Tyr Leu Ser Gly Leu Leu
485 490 495
Gly Gly Asn Gly Ile Ser Val Ser Asn Gly Ser Val Ala Ser Phe Thr
500 505 510
Leu Ala Pro Gly Ala Val Ser Val Trp Gln Tyr Ser Thr Ser Ala Ser
515 520 525
Ala Pro Gln Ile Gly Ser Val Ala Pro Asn Met Gly Ile Pro Gly Asn
530 535 540
Val Val Thr Ile Asp Gly Lys Gly Phe Gly Thr Thr Gln Gly Thr Val
545 550 555 560
Thr Phe Gly Gly Val Thr Ala Thr Val Lys Ser Trp Thr Ser Asn Arg
565 570 575
Ile Glu Val Tyr Val Pro Asn Met Ala Ala Gly Leu Thr Asp Val Lys
580 585 590
Val Thr Ala Gly Gly Val Ser Ser Asn Leu Tyr Ser Tyr Asn Ile Leu
595 600 605
Ser Gly Thr Gln Thr Ser Val Val Phe Thr Val Lys Ser Ala Pro Pro
610 615 620
Thr Asn Leu Gly Asp Lys Ile Tyr Leu Thr Gly Asn Ile Pro Glu Leu
625 630 635 640
Gly Asn Trp Ser Thr Asp Thr Ser Gly Ala Val Asn Asn Ala Gln Gly
645 650 655
Pro Leu Leu Ala Pro Asn Tyr Pro Asp Trp Phe Tyr Val Phe Ser Val
660 665 670
Pro Ala Gly Lys Thr Ile Gln Phe Lys Phe Phe Ile Lys Arg Ala Asp
675 680 685
Gly Thr Ile Gln Trp Glu Asn Gly Ser Asn His Val Ala Thr Thr Pro
690 695 700
Thr Gly Ala Thr Gly Asn Ile Thr Val Thr Trp Gln Asn
705 710 715
<210> 7
<211> 717
<212> PRT
<213>artificial sequence
<400> 7
Met Lys Lys Lys Thr Leu Ser Leu Phe Val Gly Leu Met Leu Leu Ile
1 5 10 15
Gly Leu Leu Phe Ser Gly Ser Leu Pro Tyr Asn Pro Asn Ala Ala Glu
20 25 30
Ala Ser Ser Ser Ala Ser Val Lys Gly Asp Val Ile Tyr Gln Ile Ile
35 40 45
Ile Asp Arg Phe Tyr Asp Gly Asp Thr Thr Asn Asn Asn Pro Ala Lys
50 55 60
Ser Tyr Gly Leu Tyr Asp Pro Thr Lys Ser Lys Trp Lys Met Tyr Trp
65 70 75 80
Gly Gly Asp Leu Glu Gly Val Arg Gln Lys Leu Pro Tyr Leu Lys Gln
85 90 95
Leu Gly Val Thr Thr Ile Trp Leu Ser Pro Val Leu Asn Asn Leu Asp
100 105 110
Thr Leu Ala Gly Thr Asp Asn Thr Gly Tyr His Gly Tyr Trp Thr Arg
115 120 125
Asp Phe Lys Gln Ile Glu Glu His Phe Gly Asn Trp Thr Thr Phe Asp
130 135 140
Thr Leu Val Asn Asp Ala His Gln Asn Gly Ile Lys Val Ile Val Asp
145 150 155 160
Phe Val Pro Asn His Ser Thr Pro Phe Lys Ala Asn Asp Ser Thr Phe
165 170 175
Ala Glu Gly Gly Ala Leu Tyr Asn Asn Gly Thr Tyr Met Gly Asn Tyr
180 185 190
Phe Asp Asp Ala Thr Tyr Gly Tyr Phe His His Asn Gly Asp Ile Ser
195 200 205
Asn Trp Asp Asp Arg Tyr Glu Ala Gln Trp Lys Asn Phe Thr Asp Pro
210 215 220
Ala Gly Phe Ser Leu Ala Asp Leu Ser Gln Glu Asn Gly Thr Ile Ala
225 230 235 240
Gln Tyr Leu Thr Asp Ala Ala Val Gln Leu Val Ala His Gly Leu Arg
245 250 255
Ile Asp Ala Val Lys His Phe Asn Ser Gly Phe Ser Lys Ser Leu Ala
260 265 270
Asp Lys Leu Tyr Gln Lys Lys Asp Ile Phe Leu Val Gly Glu Trp Tyr
275 280 285
Gly Thr Asp Pro Gly Thr Ala Asn His Leu Glu Lys Val Arg Tyr Ala
290 295 300
Asn Asn Ser Gly Val Asn Val Leu Asp Phe Asp Leu Asn Thr Val Ile
305 310 315 320
Arg Asn Val Phe Gly Thr Phe Thr Gln Thr Met Tyr Asp Leu Asn Asn
325 330 335
Met Val Asn Gln Thr Gly Asn Glu Tyr Lys Tyr Lys Glu Asn Leu Ile
340 345 350
Thr Phe Ile Asp Asn His Asp Met Ser Arg Phe Leu Ser Val Asn Ser
355 360 365
Lys Asn Lys Ala Asn Leu His Gln Arg Leu Leu Ser Phe Ser Leu Arg
370 375 380
Gly Val Arg Pro Pro Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Ala Gly
385 390 395 400
Gly Asn Asp Pro Tyr Asn Arg Gly Met Met Pro Ala Phe Asp Thr Thr
405 410 415
Thr Thr Ala Phe Lys Glu Val Ser Thr Leu Ala Gly Leu Arg Arg Asn
420 425 430
Asn Ala Ala Ile Gln Tyr Gly Thr Thr Thr Gln Arg Trp Ile Asn Asn
435 440 445
Asp Val Tyr Ile Tyr Glu Arg Lys Phe Phe Asn Asp Val Val Leu Val
450 455 460
Ala Ile Asn Arg Asn Thr Gln Ser Ser Tyr Ser Ile Ser Gly Leu Gln
465 470 475 480
Thr Ala Leu Pro Asn Gly Ser Tyr Ala Asp Tyr Leu Ser Gly Leu Leu
485 490 495
Gly Gly Asn Gly Ile Ser Val Ser Asn Gly Ser Val Ala Ser Phe Thr
500 505 510
Leu Ala Pro Gly Ala Val Ser Val Trp Gln Tyr Ser Thr Ser Ala Ser
515 520 525
Ala Pro Gln Ile Gly Ser Val Ala Pro Asn Met Gly Ile Pro Gly Asn
530 535 540
Val Val Thr Ile Asp Gly Lys Gly Phe Gly Thr Thr Gln Gly Thr Val
545 550 555 560
Thr Phe Gly Gly Val Thr Ala Thr Val Lys Ser Trp Thr Ser Asn Arg
565 570 575
Ile Glu Val Tyr Val Pro Asn Met Ala Ala Gly Leu Thr Asp Val Lys
580 585 590
Val Thr Ala Gly Gly Val Ser Ser Asn Leu Tyr Ser Tyr Asn Ile Leu
595 600 605
Ser Gly Thr Gln Thr Ser Val Val Phe Thr Val Lys Ser Ala Pro Pro
610 615 620
Thr Asn Leu Gly Asp Lys Ile Tyr Leu Thr Gly Asn Ile Pro Glu Leu
625 630 635 640
Gly Asn Trp Ser Thr Asp Thr Ser Gly Ala Val Asn Asn Ala Gln Gly
645 650 655
Pro Leu Leu Ala Pro Asn Tyr Pro Asp Trp Phe Tyr Val Phe Ser Val
660 665 670
Pro Ala Gly Lys Thr Ile Gln Phe Lys Phe Phe Ile Lys Arg Ala Asp
675 680 685
Gly Thr Ile Gln Trp Glu Asn Gly Ser Asn His Val Ala Thr Thr Pro
690 695 700
Thr Gly Ala Thr Gly Asn Ile Thr Val Thr Trp Gln Asn
705 710 715
<210> 8
<211> 717
<212> PRT
<213>artificial sequence
<400> 8
Met Lys Lys Lys Thr Leu Ser Leu Phe Val Gly Leu Met Leu Leu Ile
1 5 10 15
Gly Leu Leu Phe Ser Gly Ser Leu Pro Tyr Asn Pro Asn Ala Ala Glu
20 25 30
Ala Ser Ser Ser Ala Ser Val Lys Gly Asp Val Ile Tyr Gln Ile Ile
35 40 45
Ile Asp Arg Phe Tyr Asp Gly Asp Thr Thr Asn Asn Asn Pro Ala Lys
50 55 60
Ser Tyr Gly Leu Tyr Asp Pro Thr Lys Ser Lys Trp Lys Met Tyr Trp
65 70 75 80
Gly Gly Asp Leu Glu Gly Val Arg Gln Lys Leu Pro Tyr Leu Lys Gln
85 90 95
Leu Gly Val Thr Thr Ile Trp Leu Ser Pro Val Leu Asn Asn Leu Asp
100 105 110
Thr Leu Ala Gly Thr Asp Asn Thr Gly Tyr His Gly Tyr Trp Thr Arg
115 120 125
Asp Phe Lys Gln Ile Glu Glu His Phe Gly Asn Trp Thr Thr Phe Asp
130 135 140
Thr Leu Val Asn Asp Ala His Gln Asn Gly Ile Lys Val Ile Val Asp
145 150 155 160
Phe Val Pro Asn His Ser Thr Pro Phe Lys Ala Asn Asp Ser Thr Phe
165 170 175
Ala Glu Gly Gly Ala Leu Tyr Asn Asn Gly Thr Tyr Met Gly Asn Tyr
180 185 190
Phe Asp Asp Ala Thr Glu Gly Tyr Phe His His Asn Gly Asp Ile Ser
195 200 205
Asn Trp Asp Asp Arg Tyr Glu Ala Gln Trp Lys Asn Phe Thr Asp Pro
210 215 220
Ala Gly Phe Ser Leu Ala Asp Leu Ser Gln Glu Asn Gly Thr Ile Ala
225 230 235 240
Gln Tyr Leu Thr Asp Ala Ala Val Gln Leu Val Ala His Gly Leu Arg
245 250 255
Ile Asp Ala Val Lys His Phe Asn Ser Gly Phe Ser Lys Ser Leu Ala
260 265 270
Asp Lys Leu Tyr Gln Lys Lys Asp Ile Phe Leu Val Gly Glu Trp Tyr
275 280 285
Gly Thr Asp Pro Gly Thr Ala Asn His Leu Glu Lys Val Arg Tyr Ala
290 295 300
Asn Asn Ser Gly Val Asn Val Leu Asp Phe Asp Leu Asn Thr Val Ile
305 310 315 320
Arg Asn Val Phe Gly Thr Phe Thr Gln Thr Met Tyr Asp Leu Asn Asn
325 330 335
Met Val Asn Gln Thr Gly Asn Glu Tyr Lys Tyr Lys Glu Asn Leu Ile
340 345 350
Thr Phe Ile Asp Asn His Asp Met Ser Arg Phe Leu Ser Val Asn Ser
355 360 365
Lys Asn Lys Ala Asn Leu His Gln Arg Leu Leu Ser Phe Ser Leu Arg
370 375 380
Gly Val Arg Pro Pro Ile Tyr Tyr Gly Thr Glu Gln Tyr Met Ala Gly
385 390 395 400
Gly Asn Asp Pro Tyr Asn Arg Gly Met Met Pro Ala Phe Asp Thr Thr
405 410 415
Thr Thr Ala Phe Lys Glu Val Ser Thr Leu Ala Gly Leu Arg Arg Asn
420 425 430
Asn Ala Ala Ile Gln Tyr Gly Thr Thr Thr Gln Arg Trp Ile Asn Asn
435 440 445
Asp Val Tyr Ile Tyr Glu Arg Lys Phe Phe Asn Asp Val Val Leu Val
450 455 460
Ala Ile Asn Arg Asn Thr Gln Ser Ser Tyr Ser Ile Ser Gly Leu Gln
465 470 475 480
Thr Ala Leu Pro Asn Gly Ser Tyr Ala Asp Tyr Leu Ser Gly Leu Leu
485 490 495
Gly Gly Asn Gly Ile Ser Val Ser Asn Gly Ser Val Ala Ser Phe Thr
500 505 510
Leu Ala Pro Gly Ala Val Ser Val Trp Gln Tyr Ser Thr Ser Ala Ser
515 520 525
Ala Pro Gln Ile Gly Ser Val Ala Pro Asn Met Gly Ile Pro Gly Asn
530 535 540
Val Val Thr Ile Asp Gly Lys Gly Phe Gly Thr Thr Gln Gly Thr Val
545 550 555 560
Thr Phe Gly Gly Val Thr Ala Thr Val Lys Ser Trp Thr Ser Asn Arg
565 570 575
Ile Glu Val Tyr Val Pro Asn Met Ala Ala Gly Leu Thr Asp Val Lys
580 585 590
Val Thr Ala Gly Gly Val Ser Ser Asn Leu Tyr Ser Tyr Asn Ile Leu
595 600 605
Ser Gly Thr Gln Thr Ser Val Val Phe Thr Val Lys Ser Ala Pro Pro
610 615 620
Thr Asn Leu Gly Asp Lys Ile Tyr Leu Thr Gly Asn Ile Pro Glu Leu
625 630 635 640
Gly Asn Trp Ser Thr Asp Thr Ser Gly Ala Val Asn Asn Ala Gln Gly
645 650 655
Pro Leu Leu Ala Pro Asn Tyr Pro Asp Trp Phe Tyr Val Phe Ser Val
660 665 670
Pro Ala Gly Lys Thr Ile Gln Phe Lys Phe Phe Ile Lys Arg Ala Asp
675 680 685
Gly Thr Ile Gln Trp Glu Asn Gly Ser Asn His Val Ala Thr Thr Pro
690 695 700
Thr Gly Ala Thr Gly Asn Ile Thr Val Thr Trp Gln Asn
705 710 715
Claims (10)
1. a kind of genetic engineering bacterium for producing maltogenic amylase, which is characterized in that shown in expression NO.1~8 SEQ ID are any
Maltogenic amylase.
2. genetic engineering bacterium according to claim 1, which is characterized in that the genetic engineering bacterium is recombination bacillus coli.
3. genetic engineering bacterium according to claim 2, which is characterized in that the recombination bacillus coli with E.coli BL21,
E.coli JM109, E.coli DH5 α or E.coli TOP10 are host.
4. genetic engineering bacterium according to claim 2 or 3, which is characterized in that the recombination bacillus coli is with E.coli
BL21 (DE3) is host.
5. genetic engineering bacterium according to any one of claims 1 to 4, which is characterized in that express the wheat with pET serial carrier
Bud saccharogenic amylase.
6. genetic engineering bacterium according to claim 5, which is characterized in that using pET24a as expression vector.
7. a kind of method for producing maltogenic amylase, which is characterized in that the method is to be seeded to the genetic engineering bacterium
In culture medium, enzymatic production.
8. the method according to the description of claim 7 is characterized in that the culture medium is TB culture medium.
9. method according to claim 7 or 8, which is characterized in that the Escherichia coli are in 35~37 DEG C of 1~4h of culture
Afterwards, the IPTG that final concentration 0.01mM is added is induced, and continues 36~48h of cultivation and fermentation in 25~28 DEG C of shaking tables.
10. application of any genetic engineering bacterium of claim 1~6 in terms of preparing the product containing maltose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811395721.1A CN109370973B (en) | 2018-11-22 | 2018-11-22 | Maltogenic amylase producing strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811395721.1A CN109370973B (en) | 2018-11-22 | 2018-11-22 | Maltogenic amylase producing strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109370973A true CN109370973A (en) | 2019-02-22 |
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| 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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| 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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