CN106119219A - Glucoseoxidase mutant that enzymatic activity improves and expression vector and application - Google Patents
Glucoseoxidase mutant that enzymatic activity improves and expression vector and application Download PDFInfo
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- CN106119219A CN106119219A CN201610528933.7A CN201610528933A CN106119219A CN 106119219 A CN106119219 A CN 106119219A CN 201610528933 A CN201610528933 A CN 201610528933A CN 106119219 A CN106119219 A CN 106119219A
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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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
Abstract
The invention provides glucoseoxidase mutant and expression vector and application that enzymatic activity improves.The present invention with according to pichia pastoris phaff (Pichia pastoris) codon preference carried out optimize glucoseoxidase based on, use fallibility PCR method that this enzyme gene is suddenlyd change, direct mutation is picked out again by high-throughput screening method, through two-wheeled fallibility PCR and design and rational afterwards, final 4 enzymes of acquisition mutant significantly improved alive, being respectively GOD M01, GOD M02, GOD M03 and GOD M04, its enzyme work, compared with original gene, has been respectively increased 47%, 55%, 83% and 117%.
Description
Technical field
The invention belongs to genetic engineering and enzyme engineering field, particular content relates to the glucoseoxidase of enzymatic activity raising and dashes forward
Variant and expression vector thereof and application.
Background technology
Can be catalyzed in specific manner under glucoseoxidase (glucose oxidase, E.C.1.1.3.4, GOD) aerobic conditions
β-D-Glucose generates gluconic acid and hydrogen peroxide, has important using value at industrial circle, the most at food
It is used widely in the fields such as industry, livestock-raising and medical treatment detection.Glucoseoxidase is distributed widely in animals and plants and micro-
In organism, microorganism is its main source, and the main bacterial strain that produces is aspergillus niger and penicillium sp.Originate from Penicllium notatum and aspergillus niger
Glucoseoxidase has been put into the big class enzyme preparation of Ministry of Agriculture's " feed additive kind catalogue (2013) " the 4th.Along with glucose
Oxidase is more and more applied to every field, and its existing performance has been had more and more higher by industrial especially feed industry
Requirement, wherein the catalytic efficiency of glucoseoxidase determines its application cost, only has the glucose that higher enzyme is lived
Oxidase could effectively control cost, thus adapts to the requirement of large-scale industrial production, it is achieved product marketization and commercialization.
The natural grape carbohydrate oxidase screening high enzyme alive is an important channel, and uses protein engineering means to natural grape glycosyloxy
Change the artificial reconstructed of enzyme to be employed the most more and more widely.
Fallibility PCR (error-prone PCR) technology is when utilizing Taq enzyme to carry out PCR amplifying target genes, by adjusting
Whole reaction condition (as improved enzyme ion concentration, the concentration etc. of 4 kinds of dNTP in change system), changes the mutation frequency of Taq enzyme, from
And introduce sudden change with certain frequency in genes of interest, build mutation library, then select or screen the mutant of needs.In order to obtain
Obtain more preferable mutation effect, it is possible to use continuous error-prone PCR (sequential error prone PCR) strategy, will be once
The template that the direct mutation gene that PCR amplification obtains expands as PCR next time, carries out random mutagenesis the most repeatedly, makes each
The forward mutation that secondary amplification obtains is accumulated and is produced important beneficial mutation.
Summary of the invention
Glucoseoxidase mutant and expression vector and application that enzymatic activity improves are it is an object of the invention to provide.
The present invention passes through the glucoseoxidase deriving from aspergillus niger (Aspergillus niger) is carried out protein engineering transformation,
Use fallibility PCR method that glucoseoxidase carbohydrase GOD-1 gene is suddenlyd change, then will be the most prominent by high-throughput screening method
Becoming detection, it is thus achieved that mutant protein, compared with wild type, the enzymatic activity of mutant is significantly improved, thus is conducive to real
Existing its reduces production cost in industrialized production, improves using effect.
For achieving the above object, the present invention is achieved by the following technical solutions:
The invention provides the glucoseoxidase mutant GOD-M02 that a kind of enzymatic activity improves, described Fructus Vitis viniferae glycosyloxy
The aminoacid sequence of change enzyme mutant is as shown in SEQ ID NO:4, and described glucoseoxidase mutant GOD-M02 is by amino
Acid sequence is that the 288th amino acids of the glucoseoxidase of SEQ ID NO:1 is become lysine acquisition from glutamic acid.
The invention provides the glucoseoxidase encoding gene of described glucoseoxidase mutant GOD-M02.
The invention provides the recombinant bacterial strain containing described glucoseoxidase encoding gene.
The invention provides the glucoseoxidase mutant GOD-M03 that a kind of enzymatic activity improves, described Fructus Vitis viniferae glycosyloxy
The aminoacid sequence of change enzyme mutant is as shown in SEQ ID NO:5, and described glucoseoxidase mutant GOD-M03 is by amino
Acid sequence be the glucoseoxidase of SEQ ID NO:1 the 207th amino acids from lysine become glutamine, the 288th
Aminoacid is become lysine from glutamic acid and obtains.
The invention provides the glucoseoxidase encoding gene of described glucoseoxidase mutant GOD-M03.
The invention provides the recombinant bacterial strain containing described glucoseoxidase encoding gene.
The invention provides the glucoseoxidase mutant GOD-M04 that a kind of enzymatic activity improves, described Fructus Vitis viniferae glycosyloxy
The aminoacid sequence of change enzyme mutant is as shown in SEQ ID NO:6, and described glucoseoxidase mutant GOD-M04 is by amino
Acid sequence is that the 173rd amino acids of the glucoseoxidase of SEQ ID NO:1 is become valine, the 207th ammonia from threonine
Base acid is become glutamine from lysine, the 288th amino acids is become lysine from glutamic acid and obtains.
The invention provides the glucoseoxidase encoding gene of described glucoseoxidase mutant GOD-M04.
The invention provides the recombinant bacterial strain containing described glucoseoxidase encoding gene.
Present invention also offers glucoseoxidase mutant GOD-M02, GOD-M03, GOD-M04 for preparing animal
Application in feed additive.
Advantages of the present invention and have the technical effect that first the present invention has synthesized glucoseoxidase GOD-1 complete genome sequence,
According to pichia pastoris phaff codon preference, its DNA sequence is optimized, it is thus achieved that the glucose oxidation of optimization simultaneously
Enzyme gene order, and use fallibility PCR method that it is carried out random mutation, with pET 28a (+) the efficient mutation library of vector construction,
Again the plasmid containing mutant gene is proceeded in expressive host E.coli BL21-DE3, select monoclonal expressing protein, the most every
Individual orifice plate inoculation wild type gene expression strain is as comparison.Centrifugal resuspended after, take part bacterium solution and measure enzymatic activity.Activity is higher than
The bacterial strain of comparison is transferred in new culture plate, carries out repeating screening.Enzymatic activity screening obtained, higher than the bacterial strain of comparison, is sent
Gene sequencing company checks order, it is thus achieved that mutant DNA sequence.
Then with the first round obtain mutant DNA sequence as template, it is carried out second and takes turns fallibility PCR, from build
Mutant library continues the mutant of screening direct mutation.The mutational site that two-wheeled screening obtains is overlapped, it is thus achieved that 4 enzymes are lived
The mutant that power improves, respectively GOD-M01, GOD-M02, GOD-M03, GOD-M04 sequence information is shown in SEQ IDNO:3, SEQ
IDNO:4, SEQ IDNO:5 and SEQ ID NO:6, compares wild type glucoseoxidase carbohydrase GOD-1, and 4 of the present invention dash forward
The enzyme of variant is lived and has been respectively increased 47%, 55%, 83% and 117%.
Accompanying drawing explanation
Fig. 1 is glucoseoxidase mutant GOD-M01 and wild-type amino acid sequence comparative result；
Fig. 2 is glucoseoxidase mutant GOD-M02 and wild-type amino acid sequence comparative result；
Fig. 3 is glucoseoxidase mutant GOD-M03 and wild-type amino acid sequence comparative result；
Fig. 4 is glucoseoxidase mutant GOD-M04 and wild-type amino acid sequence comparative result；
Fig. 5 is that glucoseoxidase mutant GOD-M01, GOD-M02, GOD-M03, GOD-M04 are alive with the enzyme of wild type
Comparative measurements result.
Detailed description of the invention
With specific embodiment, technical scheme is further described in detail below in conjunction with the accompanying drawings.Below in conjunction with
The present invention will be further described for embodiment, it should be pointed out that the present embodiment is only used for explaining the present invention, rather than to this
The restriction of bright scope.
Embodiment 1: fallibility PCR (error prone PCR) method builds glucoseoxidase GOD-1 mutated library
The glucoseoxidase GOD-1 deriving from aspergillus niger is formed (see SEQ ID NO:1) by 589 aminoacid.For fast
Speed obtains GOD-1 complete genome sequence and is beneficial to the transformation to GOD-1 enzyme molecule, according to the GOD-1 gene sequence information reported,
Full genome synthetic method is used to synthesize glucoseoxidase GOD-1 complete genome sequence (such as Genbank ID KJ774107.1 institute
Show), the gene two ends of synthesis are also with EcoR I and Not I restriction enzyme site.The most inclined according to pichia pastoris phaff codon
Its DNA sequence is optimized by good property, and using with the GOD-1 (see SEQ ID NO:2) after optimizing is template amplification Fructus Vitis viniferae glycosyloxy
Change enzyme carbohydrase GOD-1 gene, use GeneMorph II random mutation PCR kit (Stratagene) to be randomly incorporated into sudden change.
The primer is:
5′-GCGCGAATTCTTGCCACATTACATTAGATCCAACGGTAT-3 ' (SEQ ID No:7), 5 '-
TAAAGCGGCCGCTTATTGCATAGAAGCGTAATCTTCCAAAATAGC-3′(SEQ ID No:8)
EcoR I and Not I restriction enzyme site it is respectively at underscore.
Reaction condition is: 94 DEG C of denaturations 10min, 94 DEG C of degeneration 60s, and 58 DEG C of annealing 60s and 72 DEG C extend 2min, totally 30
Individual circulation, 1% sepharose electrophoresis, test kit reclaims genes of interest fragment.
After double digested for purpose fragment EcoR I and Not I, with through identical enzyme action pET 21a (+) carrier
(ampicillin resistance) is attached reaction with Ligase.The fragment connected is converted to e. coli bl21-DE3, coating
LB flat board containing ampicillin, 37 DEG C be inverted cultivate, after transformant occurring on flat board, picking monoclonal to 96 orifice plates,
Containing 150uL LB culture medium (containing 1mM IPTG, 50ng/mL ampicillin) in every hole, the inoculation of the most each orifice plate is wild
Type gene expression bacterial strain is as comparison, and 12h is cultivated in 30 DEG C of 220rpm concussions, and orifice plate is placed in-20 DEG C, and multigelation breaking cellular wall obtains
Must be containing the crude enzyme liquid of glucoseoxidase.Take out 5uL crude enzyme liquid to delay to 96 new orifice plates, addition containing dianisidine methanol
Rush the nitrite ion 145uL altogether of liquid, dextrose buffer liquid, horseradish peroxidase solution, after 37 DEG C of reaction 3min, add 100uL2M
Sulphuric acid terminates reaction, measures enzyme according to chromogenic reaction and lives.
Take the enzyme activity bacterial strain higher than wild type GOD-1 in 96 new well culture plates, carry out repeating screening.Screen 2
Individual mutant, respectively GOD-M01 and GOD-M02, enzyme activity is 1-2 times of wild type control, and picking monoclonal send base respectively
Because of the order-checking of order-checking company.
Sequencing result shows, as depicted in figs. 1 and 2, epicycle fallibility PCR obtains dashing forward containing T173V simple point mutation
Variant GOD-M01 (its aminoacid sequence is a SEQ ID NO:3) and mutant GOD-M02 containing E288K simple point mutation
(its aminoacid sequence is SEQ ID NO:4).
(the 173rd amino acids is become valine from threonine to GOD-M01:Mutation:173T → V, the DNA of its correspondence
Sequence is become GTT from ACT),
(the 288th amino acids is become lysine from glutamic acid to GOD-M02:Mutation:288E → K, the DNA of its correspondence
Sequence is become AAG from GAA).
Embodiment 2: the second takes turns structure and the structure of screening mutated library of fallibility PCR mutated library
Two mutant GOD-M01 and GOD-M02 first round fallibility PCR method screened extract plasmid respectively and make the
Two templates taking turns fallibility PCR, the building process of mutated library, the primer of use, PCR reaction condition, with embodiment 1.By with
Upper process, obtains substantial amounts of mutant gene fragment equally.The mutant obtained by structure proceeds to E. coli expression strains
BL21-DE3, with GOD-M01 and GOD-M02 for comparison during screening high activity direct mutation, remaining operation is same as in Example 2, takes
Specific activity saltant type GOD-M01 and the high bacterial strain of GOD-M02, in 96 new well culture plates, carry out repeating screening.Screen 1
The mutant that enzymatic activity improves, named GOD-M03.Picking monoclonal send gene sequencing company to check order.
Sequencing result shows, as it is shown on figure 3, epicycle fallibility PCR obtains the prominent of K207Q and E288K two point mutation
Variant GOD-M03, its aminoacid sequence is SEQ ID NO:5.
(the 207th amino acids is become glutamine from lysine to GOD-M03:Mutation:207K → Q, its correspondence
DNA sequence is become CAA from AAG)；
(the 288th amino acids is become lysine from glutamic acid to Mutation:288E → K, and the DNA sequence of its correspondence is by GAA
Become AAG).
Embodiment 3: mutational site superposition builds new mutant
The mutant sequence obtained according to embodiment 1 and embodiment 2, folds the direct mutation site that two-wheeled sudden change obtains
Add, specifically the threonine (Thr) of 173 of mutant GOD-M03 is sported valine (Val), build one and contain
T173V, K207Q and E288K tri-mutant of point mutation, as shown in Figure 4.This mutant sequence directly transfers to gene chemical synthesis company
Synthesis.The gene two ends of synthesis are with EcoR I and Not I site.The named GOD-M04 of this mutant, its aminoacid sequence is
SEQ ID NO:6.
(the 173rd amino acids is become valine from threonine to GOD-M04:173T → V, and the DNA sequence of its correspondence is by ACT
Become GTT)；
Mutation:207K → Q (the 207th amino acids is become glutamine from lysine, the DNA sequence of its correspondence by
AAG becomes CAA)；
(the 288th amino acids is become lysine from glutamic acid to Mutation:288E → K, and the DNA sequence of its correspondence is by GAA
Become AAG).
The mutant obtained by structure proceeds to E. coli expression strains BL21-DE3, assesses it and expresses when enzyme is lived with GOD-
M03 is comparison, and remaining operation is same as in Example 2, and compared with final certification this mutant GOD-M04 with GOD-M03, enzyme is lived to be had
Improve further.
Embodiment 4: the structure of pichia pastoris engineered strain
Use primer described in embodiment 1, carry out PCR expansion using the mutant that embodiment 1 and embodiment 2 obtain as template
Increase, obtained 3 two ends with EcoR I and the glucoseoxidase mutant gene of Not I restriction enzyme site.PCR reaction condition
For: 94 DEG C of degeneration 5min；94 DEG C of degeneration 30s, 56 DEG C of renaturation 30s, 72 DEG C extend 2min, 30 circulations, and 72 DEG C extend 10min.
The mutant being directly synthesized in glucoseoxidase mutant gene fragment that above-mentioned clone is obtained, embodiment 3
Genetic fragment and raw glucose oxidation carbohydrase gene GOD-1 genetic fragment are through the double enzyme of restricted enzyme EcoR I and Not I
Cut, be connected with the pPIC9k carrier also passing through double digestion, construction of expression vector pPIC9K-GOD-M01, pPIC9K-GOD-
M02, pPIC9K-GOD-M03, pPIC9K-GOD-M04 and pPIC9K-GOD-1.
Above expression vector SalI is carried out linearisation, linearized fragment fragment purification test kit (TaKaRa
MiniBEST DNA Fragment Purifibation Kit) after purified pool, convert Pichia sp. by electricity method for transformation
GS115, is coated with MD flat board.The bacterium colony grown on MD flat board is applied to concentration and gradually rises (1mg/mL, 2mg/ successively
ML, 4mg/mL, 8mg/mL) Geneticin YPD flat board on screen the positive transformant of multicopy, obtain Pichia sp. restructuring
Bacterial strain.
The transformant of 5 genes is respectively designated as Pichia sp. GOD-M01 (Pichia pastoris GOD-M01),
Pichia sp. GOD-M02 (Pichia pastoris GOD-M02), Pichia sp. GOD-M03 (Pichia pastoris GOD-
M03), Pichia sp. GOD-M04 (Pichia pastoris GOD-M04) and Pichia sp. GOD-1 (Pichia pastoris
GOD-1), the transformant of each gene of picking is transferred in BMGY culture medium respectively, 30 DEG C, after 220rpm shaken cultivation 18h, from
The heart obtains thalline, proceeds to appropriate thalline, in BMMY culture medium, make cell concentration reach OD600=1,30 DEG C, and 220rpm continues
Shaken cultivation, every 24h adds the methanol of volume of culture 1%.After abduction delivering 4d, medium centrifugal is obtained supernatant, by supernatant
Liquid carries out glucoseoxidase vitality test.
Glucoseoxidase enzyme activity detection method:
(0.1mL1% dianisidine methanol storing solution joins 12mL 0.1M to take dianisidine buffer 2.5mL
PH6.0 phosphate buffer is made into), 18% glucose solution 0.3mL, 0.03% Peroxidase Solution 0.1mL, join colorimetric
37 DEG C of insulation 5min in pipe, add 0.1mL glucoseoxidase enzyme liquid (blank tube adds 0.1mL distilled water), react 3min
After, add 2M sulphuric acid 2mL, mix to terminate reaction.With standard blank sample as blank, at 540nm wavelength, measure blank
(A0) and sample solution (A1) light absorption value.Draw Δ A=A1-A0
Sample enzyme activity calculates:
X=(Δ A × n × 3)/(11.3 × t × 0.1)
T minute, min
0.1 sample volume, mL
11.3 extinction coefficient
N extension rate
3 reactant liquor volumes, mL
Enzyme is lived unit and definition: at pH5.5, under conditions of 37 DEG C, per minute can be the β of 1.0 μm ol-D-Glucose oxidation
Become gluconic acid and H2O2Enzyme amount be a unit.
Enzyme activity determination result: as shown in Figure 5.Measure according to the method described above and obtain Pichia sp. GOD-1 fermented supernatant fluid
It is 53U/mL that enzyme is lived, and Pichia sp. GOD-M01, Pichia sp. GOD-M02, Pichia sp. GOD-M03 and Pichia sp. GOD-
The enzyme of M04 fermented supernatant fluid is lived and is respectively 78U/mL, 82U/mL, 97U/mL and 115U/mL, and enzyme running water is flat to be respectively increased
47%, 55%, 83% and 117%.
Embodiment 5: the cultivation application experiment of glucoseoxidase
5.1 experimental design
Yellow-feather broiler Seedling is purchased from certain fowl factory of Shandong, uses full random test design, by young for 1000 0 age in days Huang plumage meat
Chicken is divided into 5 process, each process 4 repetition, each repetition 50, wherein experiment component four groups, numbered experiment 1 respectively
Group, test 2 groups, test 3 groups and experiment 4 groups, respectively corresponding add in basal diet GOD-M01, GOD-M02, GOD-M03 and
GOD-M04 glucose oxidase preparation, addition is 0.2U/g daily ration, remains one and is processed as matched group.Experiment by a definite date 70
My god, raise the phase the most in advance 2 days, 68 days formal phases.
5.2 performance test
Start the 1st, 35,70 days each group of chicken is weighed on an empty stomach in experiment respectively, record just starting weight and end weight.Feeding process
In, observed and recorded respectively organizes the health status of chicken, and records weekly every cage feed intake, average daily gain in the statistical experiment phase, flat
All daily ingestion amount and feed-weight ratio.
5.3 experiment statisticses and analysis
During 35 age in days, its daily ingestion amount, daily gain and feed-weight ratio are not all made significant difference (P ＞ by each group glucoseoxidase
0.05).During 70 age in days, compared with matched group, the glucose oxidase preparation of each group all has becoming of rising to the daily ingestion amount of chicken
Gesture, increases 3.2%, 4.1%, 6.4% and 2.0% respectively, but each group difference is not notable (P ＞ 0.05).Each experimental group
Daily gain is significantly higher than matched group (P ＜ 0.05), has been respectively increased 12.4%, 13.0%, 25.3% and 7.3%.Each experimental group
Feed-weight ratio is substantially less than matched group (P ＜ 0.05), reduces 9.7%, 13.3%, 16.5% and 6.2% respectively.
From above experimental result it can be seen that with the addition of each experimental group of glucoseoxidase compared with matched group, adopting
In the case of appetite is consistent, average daily gain all increased, and feed-weight ratio has declined, and adds glucose oxidation in daily ration
In the case of enzyme, feed conversion rate can be effectively improved, increase culture benefit.
The present embodiment 5 is for the ease of embodying the application of glucoseoxidase of the present invention, however it is not limited to answering of broiler
With, because described glucoseoxidase can add in basal diet, may be used for feeding of other fowl poultry kinds.Generally may be used
To add in its mixed feed in the breeding process such as pig, rabbit and milch cow.
Above example is only in order to illustrate technical scheme, rather than is limited；Although with reference to aforementioned reality
Execute example the present invention has been described in detail, for the person of ordinary skill of the art, still can be to aforementioned enforcement
Technical scheme described in example is modified, or wherein portion of techniques feature is carried out equivalent；And these are revised or replace
Change, do not make the essence of appropriate technical solution depart from the spirit and scope of claimed technical solution of the invention.
SEQUENCE LISTING
<110>Qingdao red cherry Bioisystech Co., Ltd
<120>enzymatic activity improves glucoseoxidase carbohydrase mutant and expression vector and application
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 589
<212> PRT
<213>aspergillus niger
<400> 1
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Val
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Thr Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys
195 200 205
Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Glu
275 280 285
His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 2
<211> 1770
<212> DNA
<213>artificial sequence
<400> 2
ttgccacatt acattagatc caacggtatt gaagcatcct tgttgactga tcctaaggat 60
gtttcaggta gaactgttga ttacattatt gctggaggtg gattgactgg tttgactact 120
gctgctagat tgactgaaaa cccaaacatt tcagttttgg ttattgaatc aggttcttac 180
gaatccgata gaggtcctat tattgaagat ttgaacgctt acggggacat ttttggttct 240
tccgttgatc atgcttacga aactgttgaa ttggctacta acaaccaaac tgctttgatt 300
agatccggta acggattggg tggttccact ttggttaacg gaggtacttg gactagacct 360
cataaggctc aagttgattc ctgggaaact gtttttggta acgaaggatg gaactgggat 420
aacgttgctg cttactcctt gcaagctgaa cgagcgcggg cccctaacgc taagcaaatt 480
gctgctggtc attactttaa cgcttcctgt catggaacta acggaactgt tcatgctggt 540
ccaagagata ctggagacga ttactcccca attgttaagg ctttgatgtc cgctgttgaa 600
gataggggtg ttccaactaa gaaggatttt ggatgtggcg acccacatgg agtttctatg 660
tttccaaaca ctttgcatga agatcaagtt agatccgatg ctgctagaga atggttgttg 720
cctaactacc aaagacctaa cttgcaagtt ttgactggtc aatacgttgg taaagttttg 780
ttgtcacaaa acggaactac tcctagagct gttggagttg aatttggtac tcataagggt 840
aacactcata acgtttacgc tgaacatgaa gttttgttgg ctgctggttc cgctgtttca 900
ccaactattt tggaatactc cggtattggt atgaagtcta ttttggaacc attgggtatt 960
gatactgttg ttgatttgcc agttggattg aacttgcaag atcaaactac tgctactgtt 1020
agatccagaa ttacttccgc tggagctgga caaggacaag ctgcttggtt tgctactttt 1080
aacgaaactt ttggagatta ctccgaaaag gctcatgaat tgttgaacac taagttggaa 1140
caatgggctg aagaagctgt tgctagaggt ggttttcata acactactgc tttgttgatt 1200
caatacgaaa actacagaga ttggattgtt aaccataacg ttgcttactc agaattgttt 1260
ttggatactg ctggagttgc ttcctttgat gtttgggatt tgttgccctt cactagagga 1320
tacgttcata ttttggataa agacccatac ttgcatcatt ttgcttacga tccacaatac 1380
tttttgaacg aattggattt gttgggacaa gctgctgcta ctcaattggc tagaaacatt 1440
tctaactcgg gtgctatgca aacttacttt gctggagaaa ctattccagg tgacaacttg 1500
gcttacgatg ctgatttgtc cgcttggact gaatacattc cataccattt tagacctaac 1560
taccacggag ttggtacttg ttctatgatg ccaaaggaaa tgggaggtgt tgttgataac 1620
gctgctagag tttacggagt tcaaggattg agagttattg atggttcaat tccaccaact 1680
caaatgtctt cacatgttat gactgtgttt tacgctatgg ctttgaagat ttcagatgct 1740
attttggaag attacgcttc tatgcaataa 1770
<210> 3
<211> 589
<212> PRT
<213>artificial sequence
<400> 3
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Val
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Val Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys
195 200 205
Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Glu
275 280 285
His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 4
<211> 589
<212> PRT
<213>artificial sequence
<400> 4
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Val
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Thr Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Lys Lys
195 200 205
Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Lys
275 280 285
His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 5
<211> 589
<212> PRT
<213>artificial sequence
<400> 5
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Val
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Thr Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Gln Lys
195 200 205
Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Lys
275 280 285
His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 6
<211> 589
<212> PRT
<213>artificial sequence
<400> 6
Leu Pro His Tyr Ile Arg Ser Asn Gly Ile Glu Ala Ser Leu Leu Thr
1 5 10 15
Asp Pro Lys Asp Val Ser Gly Arg Thr Val Asp Tyr Ile Ile Ala Gly
20 25 30
Gly Gly Leu Thr Gly Leu Thr Thr Ala Ala Arg Leu Thr Glu Asn Pro
35 40 45
Asn Ile Ser Val Leu Val Ile Glu Ser Gly Ser Tyr Glu Ser Asp Arg
50 55 60
Gly Pro Ile Ile Glu Asp Leu Asn Ala Tyr Gly Asp Ile Phe Gly Ser
65 70 75 80
Ser Val Asp His Ala Tyr Glu Thr Val Glu Leu Ala Thr Asn Asn Gln
85 90 95
Thr Ala Leu Ile Arg Ser Gly Asn Gly Leu Gly Gly Ser Thr Leu Val
100 105 110
Asn Gly Gly Thr Trp Thr Arg Pro His Lys Ala Gln Val Asp Ser Trp
115 120 125
Glu Thr Val Phe Gly Asn Glu Gly Trp Asn Trp Asp Asn Val Ala Ala
130 135 140
Tyr Ser Leu Gln Ala Glu Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile
145 150 155 160
Ala Ala Gly His Tyr Phe Asn Ala Ser Cys His Gly Val Asn Gly Thr
165 170 175
Val His Ala Gly Pro Arg Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val
180 185 190
Lys Ala Leu Met Ser Ala Val Glu Asp Arg Gly Val Pro Thr Gln Lys
195 200 205
Asp Phe Gly Cys Gly Asp Pro His Gly Val Ser Met Phe Pro Asn Thr
210 215 220
Leu His Glu Asp Gln Val Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu
225 230 235 240
Pro Asn Tyr Gln Arg Pro Asn Leu Gln Val Leu Thr Gly Gln Tyr Val
245 250 255
Gly Lys Val Leu Leu Ser Gln Asn Gly Thr Thr Pro Arg Ala Val Gly
260 265 270
Val Glu Phe Gly Thr His Lys Gly Asn Thr His Asn Val Tyr Ala Lys
275 280 285
His Glu Val Leu Leu Ala Ala Gly Ser Ala Val Ser Pro Thr Ile Leu
290 295 300
Glu Tyr Ser Gly Ile Gly Met Lys Ser Ile Leu Glu Pro Leu Gly Ile
305 310 315 320
Asp Thr Val Val Asp Leu Pro Val Gly Leu Asn Leu Gln Asp Gln Thr
325 330 335
Thr Ala Thr Val Arg Ser Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly
340 345 350
Gln Ala Ala Trp Phe Ala Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ser
355 360 365
Glu Lys Ala His Glu Leu Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu
370 375 380
Glu Ala Val Ala Arg Gly Gly Phe His Asn Thr Thr Ala Leu Leu Ile
385 390 395 400
Gln Tyr Glu Asn Tyr Arg Asp Trp Ile Val Asn His Asn Val Ala Tyr
405 410 415
Ser Glu Leu Phe Leu Asp Thr Ala Gly Val Ala Ser Phe Asp Val Trp
420 425 430
Asp Leu Leu Pro Phe Thr Arg Gly Tyr Val His Ile Leu Asp Lys Asp
435 440 445
Pro Tyr Leu His His Phe Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu
450 455 460
Leu Asp Leu Leu Gly Gln Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile
465 470 475 480
Ser Asn Ser Gly Ala Met Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro
485 490 495
Gly Asp Asn Leu Ala Tyr Asp Ala Asp Leu Ser Ala Trp Thr Glu Tyr
500 505 510
Ile Pro Tyr His Phe Arg Pro Asn Tyr His Gly Val Gly Thr Cys Ser
515 520 525
Met Met Pro Lys Glu Met Gly Gly Val Val Asp Asn Ala Ala Arg Val
530 535 540
Tyr Gly Val Gln Gly Leu Arg Val Ile Asp Gly Ser Ile Pro Pro Thr
545 550 555 560
Gln Met Ser Ser His Val Met Thr Val Phe Tyr Ala Met Ala Leu Lys
565 570 575
Ile Ser Asp Ala Ile Leu Glu Asp Tyr Ala Ser Met Gln
580 585
<210> 7
<211> 39
<212> DNA
<213>artificial sequence
<400> 7
gcgcgaattc ttgccacatt acattagatc caacggtat 39
<210> 8
<211> 45
<212> DNA
<213>artificial sequence
<400> 8
taaagcggcc gcttattgca tagaagcgta atcttccaaa atagc 45
Claims (10)
1. the glucoseoxidase mutant GOD-M02 that an enzymatic activity improves, it is characterised in that described glucoseoxidase
The aminoacid sequence of mutant is as shown in SEQ ID NO:4, and described glucoseoxidase mutant GOD-M02 is by aminoacid sequence
288th amino acids of the glucoseoxidase being classified as SEQ ID NO:1 is become lysine from glutamic acid and obtains.
2. the glucoseoxidase encoding gene of the glucoseoxidase mutant GOD-M02 described in claim 1.
3. contain the recombinant bacterial strain of glucoseoxidase encoding gene described in claim 2.
4. the glucoseoxidase mutant GOD-M03 that an enzymatic activity improves, it is characterised in that described glucoseoxidase
The aminoacid sequence of mutant is as shown in SEQ ID NO:5, and described glucoseoxidase mutant GOD-M03 is by aminoacid sequence
207th amino acids of the glucoseoxidase being classified as SEQ ID NO:1 is become glutamine, the 288th bit amino from lysine
Acid is become lysine from glutamic acid and obtains.
5. the glucoseoxidase encoding gene of the glucoseoxidase mutant GOD-M03 described in claim 4.
6. contain the recombinant bacterial strain of glucoseoxidase encoding gene described in claim 5.
7. the glucoseoxidase mutant GOD-M04 that an enzymatic activity improves, it is characterised in that described glucoseoxidase
The aminoacid sequence of mutant is as shown in SEQ ID NO:6, and described glucoseoxidase mutant GOD-M04 is by aminoacid sequence
173rd amino acids of the glucoseoxidase being classified as SEQ ID NO:1 is become valine, the 207th amino acids from threonine
Become glutamine from lysine, the 288th amino acids is become lysine from glutamic acid and obtains.
8. the glucoseoxidase encoding gene of the glucoseoxidase mutant GOD-M04 described in claim 7.
9. contain the recombinant bacterial strain of glucoseoxidase encoding gene described in claim 8.
10. glucoseoxidase mutant GOD-M02, GOD-M03, GOD-M04 are for preparing in animal feed additive
Application.
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| CN201610528933.7A CN106119219A (en) | 2016-07-06 | 2016-07-06 | Glucoseoxidase mutant that enzymatic activity improves and expression vector and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108251391A (en) * | 2017-08-18 | 2018-07-06 | 青岛蔚蓝生物集团有限公司 | Novel grape carbohydrate oxidase mutant |
| CN111004786A (en) * | 2019-12-25 | 2020-04-14 | 广东溢多利生物科技股份有限公司 | Glucose oxidase and carrier and application thereof |
| CN114376071A (en) * | 2021-12-24 | 2022-04-22 | 青岛根源生物技术集团有限公司 | Application of glucose oxidase in preparation of feed additive for improving intestinal flora structure and increasing survival rate of crustacean |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525778A (en) * | 2013-10-18 | 2014-01-22 | 江南大学 | Glucose oxidase mutant with high catalytic activity |
| CN103981159A (en) * | 2014-06-05 | 2014-08-13 | 青岛蔚蓝生物集团有限公司 | Glucose oxidase mutant and application thereof |
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- 2016-07-06 CN CN201610528933.7A patent/CN106119219A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525778A (en) * | 2013-10-18 | 2014-01-22 | 江南大学 | Glucose oxidase mutant with high catalytic activity |
| CN103981159A (en) * | 2014-06-05 | 2014-08-13 | 青岛蔚蓝生物集团有限公司 | Glucose oxidase mutant and application thereof |
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| JULIA MARÍN-NAVARRO ET AL.: "Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase", 《PLOS ONE》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108251391A (en) * | 2017-08-18 | 2018-07-06 | 青岛蔚蓝生物集团有限公司 | Novel grape carbohydrate oxidase mutant |
| CN111004786A (en) * | 2019-12-25 | 2020-04-14 | 广东溢多利生物科技股份有限公司 | Glucose oxidase and carrier and application thereof |
| CN111004786B (en) * | 2019-12-25 | 2021-12-07 | 广东溢多利生物科技股份有限公司 | Glucose oxidase and carrier and application thereof |
| CN114376071A (en) * | 2021-12-24 | 2022-04-22 | 青岛根源生物技术集团有限公司 | Application of glucose oxidase in preparation of feed additive for improving intestinal flora structure and increasing survival rate of crustacean |
| 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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