CN109370973B - Maltogenic amylase producing strain - Google Patents

Maltogenic amylase producing strain Download PDF

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CN109370973B
CN109370973B CN201811395721.1A CN201811395721A CN109370973B CN 109370973 B CN109370973 B CN 109370973B CN 201811395721 A CN201811395721 A CN 201811395721A CN 109370973 B CN109370973 B CN 109370973B
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谢艳萍
何球山
钟红霞
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Hunan Jindai Technology Development Co.,Ltd.
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    • 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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Abstract

The invention discloses a maltogenic amylase producing strain, belonging to the technical field of microorganisms. The invention obtains the maltogenic amylase mutant with improved transformation efficiency through site-directed mutagenesis and combined mutagenesis, and expresses the mutant in escherichia coli. When the enzyme solution is used for producing maltose, under the condition of the addition amount of 20U/g dry starch, the mutant K198E/D290T can improve the maltose content to more than 97 percent when the conversion reaction is carried out for 10 hours in the secondary saccharification process, and the content of trisaccharide and tetrasaccharide approaches to 0 when the reaction is carried out for 5 hours. Compared with wild maltogenic amylase, the method has higher transformation efficiency and fewer transformation byproducts, and has important industrial application potential.

Description

Maltogenic amylase producing strain
Technical Field
The invention relates to a maltogenic amylase producing strain, belonging to the technical field of microorganisms.
Background
Maltogenic amylases (maltogenic or maltogenic, EC 3.2.1.133) are members of the glycoside hydrolase GH-H family. Currently, main bacterial sources of maltogenic amylase are Bacillus stearothermophilus (Bacillus stearothermophilus), Bacillus cereus (Bacillus cereus), Bacillus subtilis (Bacillus subtilis), Bacillus licheniformis (Bacillus licheniformis), Thermus vulgaris (Thermus vulgaris), Thermus sp. Maltogenic amylases of different origins differ greatly in their properties. The maltogenic amylase currently applied to the preparation of maltose syrup and the resistance to bread aging is mainly derived from bacillus stearothermophilus.
Maltose is a reducing disaccharide composed of two glucose units connected by alpha-1, 4 glycosidic bonds, and has the chemical name of 4-O-D-hexacyclic glucose. The sweet taste is soft, and the sweet taste can be used as a food sweetener to replace glucose and sucrose due to the characteristics of low viscosity, low hygroscopicity and good thermal stability, and has great application potential in the field of food industry. In the industrial production of maltose, a syrup based on maltose (40% -60%) is prepared from starchy material by alpha-amylase and malt (or beta-amylase, fungal amylase) hydrolysis, and if the maltose content exceeds 45% (preferably above 50%), the syrup is called high maltose syrup. One of the uses of high maltose syrup in the food industry is in the manufacture of products such as cakes, candies, etc. The syrup is boiled at a temperature far higher than that of maltose, generally over 140 ℃. Maltose contents of greater than 70%, and even up to 90% or more, are known as ultra-high maltose syrups. Compared with glucose, maltose can avoid the rise of blood sugar, and has application advantages superior to glucose in the preparation of antibodies, vaccines and the like. The use of ultra-high purity maltose syrups in the medical field has therefore also attracted increasing attention.
The existing maltose production process is mature, when alpha-amylase and beta-amylase are used for producing maltose, the content of maltose in the product can reach 90%, and glucose, trisaccharide, tetrasaccharide and part of oligosaccharide and dextrin are main conversion byproducts. The dextrin and part of oligosaccharide can be removed by ethanol precipitation. The ultra-high purity maltose is prepared by the methods of chromatographic separation, crystallization and the like. Since maltose has a high viscosity and is difficult to crystallize, the purity of maltose in a crystallization raw material is generally required to be 90% or more, and thus the purity of chromatographic separation plays an important role in maltose crystallization. The chromatographic separation can basically remove glucose, pentasaccharide and small molecular saccharides above, and has little influence on the purity of maltose. However, the trisaccharide and tetrasaccharide in the product are similar to maltose in property, and are often main impurities in separation and purification, so that the purity of the product is directly reduced, the crystallinity of maltose, the viscosity of syrup and the moisture content of the final product are greatly influenced, and the final yield of maltose is greatly reduced.
The maltogenic amylase has micromolecule sugar hydrolysis activity, can hydrolyze micromolecule sugar such as trisaccharide, tetrasaccharide and the like to form glucose and maltose, so that the maltogenic amylase is usually compounded with alpha-amylase, beta-amylase, pullulanase and the like in the production of ultrahigh maltose to reduce the proportion of byproducts, so that the maltose is more beneficial to crystallization. The maltogenic amylase derived from Bacillus stearothermophilus (Bacillus stearothermophilus) is reported to have higher optimal reaction temperature and lower optimal pH reaction condition, can meet more rigorous industrial production conditions, increases the proportion of maltose in the product to 92 percent, and has great application advantage in industry.
Disclosure of Invention
The first purpose of the invention is to provide a genetic engineering bacterium for producing the maltogenic amylase, which expresses the maltogenic amylase shown in any one of SEQ ID NO. 1-8.
In one embodiment of the invention, the genetically engineered bacterium is recombinant escherichia coli.
In one embodiment of the present invention, the recombinant escherichia coli is hosted by e.coli BL21, e.coli JM109, e.coli DH5 α or e.coli TOP 10.
In one embodiment of the invention, the recombinant escherichia coli is hosted in e.coli BL21(DE 3).
In one embodiment of the invention, the maltogenic amylase is expressed as a pET series vector.
In one embodiment of the invention, pET24a is used as an expression vector.
The second purpose of the invention is to provide a method for producing the maltogenic amylase, which is to inoculate the genetically engineered bacteria into a culture medium and ferment to produce enzyme.
In one embodiment of the invention, the medium is TB medium.
In one embodiment of the invention, after the Escherichia coli is cultured at 35-37 ℃ for 1-4 h, IPTG with a final concentration of 0.01mM is added for induction, and the Escherichia coli is further cultured and fermented for 36-48 h at 25-28 ℃ in a shaking table.
The invention also claims the application of the genetically engineered bacteria in preparing maltose-containing products.
Has the advantages that: the invention obtains the maltogenic amylase mutant with improved transformation efficiency through site-directed mutagenesis and combined mutagenesis, and expresses the mutant in escherichia coli. When the enzyme solution is used for producing maltose, under the condition of the addition amount of 20U/g dry starch, the mutant K198E/D290T can improve the maltose content to more than 97 percent when the conversion reaction is carried out for 10 hours in the secondary saccharification process, and the content of trisaccharide and tetrasaccharide approaches to 0 when the reaction is carried out for 5 hours. Compared with wild maltogenic amylase, the method has higher transformation efficiency and fewer transformation byproducts, and has important industrial application potential.
Drawings
FIG. 1 shows the changes in maltose content (A), trisaccharide content (B) and tetrasaccharide content (C) in the production of maltose by wild-type maltogenic amylase (WT) and mutant (K198E/D290T).
Detailed Description
The contents of glucose, maltose, trisaccharide and tetrasaccharide were analyzed by HPLC. The chromatographic conditions determined were: elite 2000HPLC chromatograph, Elite autosampler, column Thermo APS-2HYPERSIL 13286(4.6 mm. times.250 mm), HITACHI L-2490 differential detector; the mobile phase is 70% (v/v) acetonitrile water solution, and the flow rate is 0.8 mL/min; the column temperature was 40 ℃.
Example 1: preparation of wild maltogenic amylase.
(1) Construction of recombinant maltogenic amylase
The sequence was codon optimized based on the amino acid sequence of amyM at NCBI (NCBI accession No.: AAA22233.1), and the gene sequence amyM of maltogenic amylase was synthesized using a chemical total synthesis method. The plasmid used for constructing the E.coli expression vector was pET24a (+). The plasmid pET24a (+) and the plasmid with amyM gene are subjected to double enzyme digestion with Nco I and Hind III respectively, after enzyme digestion products are recovered by glue, T4 ligase is used for connecting overnight, the connecting products are transformed into escherichia coli JM109 competent cells, the transformation products are coated on an LB plate containing 100mg/L kanamycin and cultured at 37 ℃ for overnight, 2 single colonies are picked from the plate and inoculated into an LB liquid culture medium, and after 8h, the plasmid is extracted for verification, so that the result is correct, and the enriched amyM/pET24a plasmid is obtained. Plasmid amyM/pET24a was transformed into E.coli BL21(DE3) competent cells, and transformants were picked and cultured overnight at 37 ℃ in LB liquid medium (containing 100mg/L kanamycin), and the tubes were stored and named amyM/pET24a/BL21(DE 3).
(2) Expression and purification of maltogenic amylase
The seed amyM/pET24a/BL21(DE3) was grown for 8h in LB broth (containing 100mg/L kanamycin) from a glycerol tube, and the seed was inoculated into TB broth (containing 100mg/L kanamycin) at 5% inoculum size. After the escherichia coli is cultured for 2h at 37 ℃, 0.01mM IPTG with final concentration is added for induction, after the escherichia coli is cultured and fermented continuously for 48h in a shaker at 25 ℃, the fermentation liquor is centrifuged for 10min at 8000rpm and 4 ℃ to remove thalli, fermentation supernatant is collected, and the enzyme activity can reach 4892U/mL through determination.
Slowly adding 50% (NH) into the supernatant4)2SO4Standing at 4 deg.C overnight, centrifuging at 8000rpm at 4 deg.C for 20min, and collecting precipitate. After the pellet was reconstituted with 20mM citrate buffer, pH7.5, the pellet was dialyzed overnight against 20mM citrate buffer. During which the buffer was changed 2-3 times. After filtration through a 0.22 μm membrane, a sample was prepared and recombinant protein was purified using an avant protein purifier. Anion exchange chromatography purification step: (1) balancing: equilibrating the DEAE anion exchange chromatography column with 5 volumes of 20mM buffer; (2) loading: sampling the pretreated sample at the flow rate of 1 mL/min; (3) and (3) elution: gradient elution is carried out at the flow rate of 1mL/min, the detection wavelength is 290nm, and the eluate containing the activity of the maltogenic amylase is collected step by step. Obtaining the purified wild maltogenic amylase.
Example 2: preparation of maltogenic amylase mutants
(1) Single mutation
The 198 th lysine (Lys) of maltogenic amylase is mutated into glutamic acid (Glu) and tyrosine (Tyr) marked as K198E and K198Y; the maltogenic amylase has aspartic acid at position 290 (Asp) mutated to glycine (G, ly) and threonine (Thr), labeled D290G and D290T, respectively.
The site-directed mutagenesis primers for introducing the K198E mutation were:
a forward primer: 5' -CGACGACGCTACCGAAGGTTACTTCCACCA-3' (the mutated base is underlined)
Reverse primer: 5' -TGGTGGAAGTAACCTTCGGTAGCGTCGTCG-3' (the mutated base is underlined)
The site-directed mutagenesis primers for introducing the K198Y mutation were:
a forward primer: 5' -CGACGACGCTACCTATGGTTACTTCCACCA-3' (the mutated base is underlined)
Reverse primer: 5' -TGGTGGAAGTAACCATAGGTAGCGTCGTCG-3' (the mutated base is underlined)
Site-directed mutagenesis primers for introducing the D290G mutation were:
a forward primer: 5' -GAATGGTACGGTGACGGTCCGGGTACCGCT-3' (the mutated base is underlined)
Reverse primer: 5' -AGCGGTACCCGGACCGTCACCGTACCATTC-3' (the mutated base is underlined)
Site-directed mutagenesis primers for introducing the D290T mutation were:
a forward primer: 5' -GAATGGTACGGTGACACGCCGGGTACCGCT-3' (the mutated base is underlined)
Reverse primer: 5' -AGCGGTACCCGGCGTGTCACCGTACCATTC-3' (the mutated base is underlined)
PCR was carried out using the above primers and amyM/pET24a plasmid as a template. The reaction is carried out in a 50-mu-L system under the following conditions: pre-denaturation at 94 ℃ for 4 min; followed by 30 cycles (94 ℃ 10s, 55 ℃ 10s, 72 ℃ 7min20 s); extending for 10min at 72 ℃; finally, keeping the temperature at 4 ℃. The PCR products are digested by Dpn I (Fermentas company), escherichia coli JM109 competent cells are respectively transformed, the transformed products are coated on an LB plate containing 100mg/L kanamycin and cultured overnight at 37 ℃, 2 single colonies are selected from the plate and inoculated into an LB liquid culture medium, plasmids are extracted after 8h, sequencing is correct, and the glycerin tube is preserved.
(2) Expression and purification of mutant enzymes
Mutant expression and purification procedures were as described in example 1.
(3) Double mutation
On the basis of single mutation, combined mutation is carried out to respectively construct mutants K198E/D290G, K198Y/D290G, K198E/D290T and K198Y/D290T.
Example 3: enzyme activity assay for maltogenic amylase
(1) Definition of enzyme Activity Unit
The amount of enzyme required to catalyze the production of 1. mu. mol of reducing sugars per minute when maltogenic amylase was activated was measured using the 3, 5-dinitrosalicylic acid method (DNS method) as a unit of activity.
(2) Enzyme activity determination procedure
Preheating: 2mL of 0.5% soluble starch solution (50mM pH5.5 citrate buffer) was placed in a test tube and preheated in a 60 ℃ water bath for 10 min.
Reaction: adding 0.1mL sample enzyme solution, shaking uniformly, timing for 10min accurately, adding 3mL DNS, shaking uniformly, adding into ice water to terminate the reaction, and boiling in boiling water bath for 7 min. And (6) cooling.
Measurement: adding distilled water into the reaction system, fixing the volume to 15mL, and uniformly mixing. The absorbance was measured at a wavelength of 540nm and the enzyme activity was calculated.
The specific activities of the wild-type maltogenic amylase and the mutant enzyme are listed in the following table (the specific activity of the wild-type maltogenic amylase is set as 100%, and the ratio of the specific activity of each mutant to the specific activity of the wild enzyme is relative activity):
TABLE 1 specific Activity and relative Activity of wild-type maltogenic amylase and mutant enzymes
Figure BDA0001875128020000051
Example 4: enzyme method for producing maltose
Preparing 2L of 20% (w/v) potato starch solution, adjusting pH to 5.5, adding 30U/g dry starch acidic alpha-amylase (from Jenenaceae), spray liquefying, adding 24U/g dry starch pullulanase (from Jenenaceae) and 10U/g dry starch beta-amylase (extracted from sweet potato), stirring at 60 deg.C for 24 hr, and saccharifying.
After primary saccharification, the contents of glucose, maltose, trisaccharide and tetrasaccharide in the reaction system are respectively 0.26%, 89.73%, 9.21% and 0.8%.
Then, 20U/g dry starch of wild type maltogenic amylase and mutant enzymes K198E, K198E/D290G, K198E/D290T were added to the primary saccharification reaction system, respectively. Stirring at 60 ℃ for secondary saccharification. The reaction time is 25h, and a sample is taken to determine the product composition.
The contents of the components in the reaction system are shown in table 2:
TABLE 2 saccharification effect of different maltogenic amylases (reaction 20h)
Figure BDA0001875128020000061
The content of each substance in the reaction process is monitored, and the result is shown in figure 1, the mutant K198E/D290T can improve the maltose content to more than 97% when the conversion reaction is carried out for 10 hours, and the content of trisaccharide and tetrasaccharide approaches to 0 when the reaction is carried out for 5 hours.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
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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> 3
<211> 717
<212> PRT
<213> Artificial sequence
<400> 3
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 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 genetic engineering bacterium of maltogenic amylase is characterized in that the maltogenic amylase shown in SEQ ID NO.8 is expressed.
2. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium is a recombinant escherichia coli.
3. The genetically engineered bacterium of claim 2, wherein the recombinant Escherichia coli is selected from the group consisting of Escherichia coli, and Escherichia coliE. coliBL21、E. coli JM109、E. coliDH5 alpha orE. coliTOP10 is a host cell.
4. The genetically engineered bacterium of claim 2 or 3, wherein the Escherichia coli is recombinedE. coliBL21(DE3) is a host cell.
5. The genetically engineered bacterium of claim 2 or 3, wherein the maltogenic amylase is expressed as a pET series vector.
6. The genetically engineered bacterium of claim 4, wherein the maltogenic amylase is expressed as a pET series vector.
7. The genetically engineered bacterium of claim 6, wherein pET24a is used as an expression vector.
8. A method for producing maltogenic amylase, which comprises inoculating the genetically engineered bacterium of any one of claims 1 to 7 into a culture medium, and fermenting to produce the enzyme.
9. The method of claim 8, wherein the medium is TB medium.
10. Use of the genetically engineered bacterium of any one of claims 1 to 7 for the preparation of a maltose-containing product.
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