CN105950577A - Glucose oxidase mutant with improved thermal stability as well as encoding genes and application thereof - Google Patents
Glucose oxidase mutant with improved thermal stability as well as encoding genes and application thereof Download PDFInfo
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Abstract
The invention discloses a glucose oxidase mutant with improved thermal stability as well as encoding genes and application thereof. According to the invention, an error-prone PCR (Polymerase Chain Reaction) method is used to mutate wild type glucose oxidase genes, and positive mutations are detected by means of a high-throughput screening method. By means of the mutant library constructing and screening method, three glucose oxidase mutants with obviously improved thermal stability are obtained, the thermal stability is improved by 1-3 times compared with the wild type glucose oxidase genes, and therefore the glucose oxidase mutant with improved thermal stability has a good market application prospect and industrial value.
Description
Technical field
The invention belongs to genetic engineering and enzyme engineering field, particular content relates to the glucoseoxidase mutant that heat stability improves
And encoding gene and application.
Background technology
β-D-Portugal can be catalyzed in specific manner under glucoseoxidase (glucose oxidase, E.C.1.1.3.4, GOD) aerobic conditions
Sugar generates gluconic acid and hydrogen peroxide.Glucoseoxidase is one of toolenzyme currently mainly, is widely used at food
The fields such as industry, livestock-raising and medical treatment detection.Glucoseoxidase is applied to food processing work as generally acknowledged safe antioxidant
In skill, in medical industries, glucoseoxidase may be used for the directions such as blood sugar detection, as a kind of feed additive, Fructus Vitis viniferae
Carbohydrate oxidase can improve animal intestinal environment, promotes growth of animal.More and more it is applied to each along with glucoseoxidase
Individual field, industrial especially feed industry has had the highest requirement to its existing performance, as the long period protects at normal temperatures
Hold enzyme activity not decline, hot and extreme pH condition is had toleration, digestive enzyme is had toleration.Wherein enzyme is thermally-stabilised
Property the most crucial for the application of glucoseoxidase, in the preparation process of enzyme and extreme reaction condition under (high temperature), thermostability
Strong enzyme has bigger advantage.
Fallibility PCR (error-prone PCR) technology is a kind of method manufacturing random mutation in DNA sequence, its ultimate principle
It is when PCR amplifying target genes, (as improved enzyme ion concentration, changes by changing the reaction condition of normal PCR process
The concentration etc. of 4 kinds of dNTP in system), use the Taq enzyme of relatively low fidelity, thus be randomly incorporated into false bases with certain frequency,
Thus the mutant library that formation sequence is different.
Summary of the invention
The invention provides glucoseoxidase mutant and encoding gene and application that heat stability improves, the present invention provides
Glucoseoxidase mutant heat stability has had and has been obviously improved.
For achieving the above object, the present invention is achieved by the following technical solutions:
The invention provides the glucoseoxidase mutant GOD-H-2 that a kind of heat stability improves, described glucoseoxidase
The aminoacid sequence of mutant GOD-H-2 is as shown in SEQ ID NO:4, and described mutant GOD-H-2 sequence is by amino
Acid sequence be the glucoseoxidase of SEQ ID NO:1 the 143rd amino acids from alanine become proline, the 461st
Aminoacid is become isoleucine from phenylalanine and obtains.
The invention provides the glucoseoxidase encoding gene of described glucoseoxidase mutant GOD-H-2.
The invention provides the recombinant bacterial strain containing described glucoseoxidase encoding gene.
The invention provides the glucoseoxidase mutant GOD-H-5 that a kind of heat stability improves, described glucoseoxidase
The aminoacid sequence of mutant GOD-H-5 is as shown in SEQ ID NO:7, and described mutant GOD-H-5 sequence is by amino
Acid sequence be the glucoseoxidase of SEQ ID NO:1 the 29th amino acids from isoleucine become leucine, the 143rd
Aminoacid is become proline from alanine, the 390th amino acids is become arginine, the 461st amino acids by phenylpropyl alcohol from glycine
Propylhomoserin becomes isoleucine and obtains.
The invention provides the glucoseoxidase encoding gene of described glucoseoxidase mutant GOD-H-5.
The invention provides the recombinant bacterial strain containing described glucoseoxidase encoding gene.
The invention provides the glucoseoxidase mutant GOD-H-6 that a kind of heat stability improves, described glucoseoxidase
The aminoacid sequence of mutant GOD-H-6 is as shown in SEQ ID NO:8, and described mutant GOD-H-6 sequence is by amino
Acid sequence be the glucoseoxidase of SEQ ID NO:1 the 143rd amino acids from alanine become proline, the 241st
Aminoacid from proline become aspartic acid, the 359th amino acids from threonine become serine, the 461st amino acids by
Phenylalanine becomes isoleucine and obtains.
The invention provides the glucoseoxidase encoding gene of described glucoseoxidase mutant GOD-H-6.
The invention provides the recombinant bacterial strain containing described glucoseoxidase encoding gene.
The invention provides glucoseoxidase mutant GOD-H-2, GOD-H-5 and GOD-H-6 raising for preparing animal
Application in feed additives.
Advantages of the present invention it is an object of the invention to use directed evolution technologies to deriving from aspergillus niger with having the technical effect that
The glucoseoxidase of (Aspergillus niger) carries out protein engineering transformation, and to achieve the above object of the invention, the present invention makes
By fallibility PCR method, glucose oxidase gene is suddenlyd change, then by high-throughput screening method, direct mutation is detected,
The mutant improved to heat stability, compares wild type glucoseoxidase GOD-1,6 mutant GOD-H-1 of the present invention,
GOD-H-2, GOD-H-3, GOD-H-4, GOD-H-5, GOD-H-6 heat stability significantly improves, 80 DEG C of process
After 3min, remnant enzyme activity has been respectively increased 1.3,1.6,1.9,2.1,2.5,2.3 times.The mutant that the present invention provides has
Good market application foreground and industrial value
Accompanying drawing explanation
Fig. 1 is glucoseoxidase mutant GOD-H-1 and wild-type amino acid sequence comparison chart；
Fig. 2 is glucoseoxidase mutant GOD-H-2 and wild-type amino acid sequence comparison chart；
Fig. 3 is glucoseoxidase mutant GOD-H-3 and wild-type amino acid sequence comparison chart；
Fig. 4 is glucoseoxidase mutant GOD-H-4 and wild-type amino acid sequence comparison chart；
Fig. 5 is glucoseoxidase mutant GOD-H-5 and wild-type amino acid sequence comparison chart；
Fig. 6 is glucoseoxidase mutant GOD-H-6 and wild-type amino acid sequence comparison chart；
Fig. 7 is glucoseoxidase mutant GOD-H-1, GOD-H-2, GOD-H-3, GOD-H-4, GOD-H-5,
GOD-H-6 remnant enzyme activity at different temperatures.
Detailed description of the invention
With specific embodiment, technical scheme is further described in detail below in conjunction with the accompanying drawings.
The present invention has used routine techniques and the method that biology field uses.Embodiment is only for explaining the present invention, no
Limit the scope of the invention.
Embodiment 1: fallibility PCR (error prone PCR) method builds glucoseoxidase GOD-1 mutated library
Derive from the glucose oxidase gene GOD-1 of aspergillus niger (Aspergillus niger) by 589 Amino acid profiles (as
Shown in SEQ ID NO:1), use the method for full genome synthesis to synthesize this glucose oxidase gene GOD-1 (such as SEQ ID
Shown in NO:2), the gene two ends of synthesis are also with EcoR I and Not I restriction enzyme site.Expand with this gene of synthesis for template
Increase glucoseoxidase GOD-1 gene, use GeneMorph II random mutation PCR kit (Stratagene) random
Introduce sudden change.
The primer is: 5 '-GCGCGAATTCCGCTGCGGCCCTGCCACACTAC-3 ' (SEQ ID No:9),
5′-TAAAGCGGCCGCTCACTGCATGGAAGCATAATCTTCCAAGATAGCATCC-3 ' (SEQ ID No:
10)。
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, 58 DEG C of annealing 60s and 72 DEG C of extension 2min,
Totally 30 circulations, reclaim genes of interest fragment.
After double digested for purpose fragment EcoR I and Not I, with through identical enzyme action pET 21a (+) carrier (ammonia benzyl
Resistant gene) it is attached reaction with Ligase.Converting the fragment connected to e. coli bl21-DE3, coating contains
The LB flat board of ampicillin, is inverted for 37 DEG C and cultivates, and after there is transformant on flat board, picking monoclonal is to 96 orifice plates, often
Containing 150uL LB culture medium (containing 1mM IPTG, 50ng/mL ampicillin) in hole, 30 DEG C of 220rpm concussion trainings
Support 12h, orifice plate is placed in-20 DEG C, multigelation breaking cellular wall, it is thus achieved that containing the crude enzyme liquid of glucoseoxidase.Take out 5ul respectively
Lysate is to two pieces of 96 new orifice plates, and one of in 80 DEG C of process 3min, two piece of 96 orifice plate all adds containing adjacent connection Fructus Foeniculi
Amine methanol buffer, dextrose buffer liquid, the nitrite ion of horseradish peroxidase solution, add 100uL2M after 37 DEG C of reaction 3min
Sulphuric acid terminates reaction, measures remnant enzyme activity according to chromogenic reaction.
Take the residual 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-H-1 and GOD-H-2, residual activity is 1-3 times of wild type control, suddenlys change the two
Body carries out DNA sequencing.
Sequencing result shows, as depicted in figs. 1 and 2, epicycle fallibility PCR obtains two containing K122N and G492E
The mutant GOD-H-1 of point mutation, its aminoacid sequence is SEQ ID NO:3,2 points containing A143P and F461I
The mutant GOD-H-2 of sudden change, its aminoacid sequence is SEQ ID NO:4.
The lysine K of the 122nd of GOD-H-1: this enzyme becomes agedoite N (AAG becomes AAC), the 492nd
Glycine G become glutamic acid E (GGG becomes GAG).
The alanine A of the 143rd of GOD-H-2: this enzyme becomes proline P (GCC becomes CCC), the 461st
Phenylalanine F becomes isoleucine I (TTC becomes ATC).
Embodiment 2: the second takes turns structure and the structure of screening mutated library of fallibility PCR mutated library
Two mutant GOD-H01 and GOD-H02 that thermostability first round fallibility PCR method screened improves are respectively
Extract plasmid and make the second template taking turns fallibility PCR, the building process of mutated library, the primer of use, PCR reaction condition,
With embodiment 1.
Take turns fallibility PCR by second, obtain substantial amounts of mutant gene fragment equally.The mutant obtained by structure proceeds to greatly
Enterobacteria expression strain BL21-DE3, with GOD-H-1 and GOD-H-2 for comparison during screening thermostability direct mutation, remaining behaviour
Make same as in Example 2, take the residual activity bacterial strain higher than saltant type GOD-H-1 and GOD-H-2 and cultivate to 96 new holes
In plate, carry out repeating screening.
Epicycle screening obtains 4 mutants altogether, is respectively designated as GOD-H-3, GOD-H-4, GOD-H-5 and GOD-H-6.
Wherein GOD-H-3 and GOD-H-4 is the mutant obtained for template with GOD-H01, and its heat stability is higher than GOD-H01,
GOD-H-5 and GOD-H-6 is the mutant obtained for template with GOD-H02, and its heat stability is higher than GOD-H02, chooses
Taking mutants which had send order-checking company to check order.
Sequencing result shows, as shown in Fig. 3, Fig. 4, Fig. 5 and Fig. 6, epicycle fallibility PCR obtains one containing D70Q, K122N
The mutant GOD-H-3 of three point mutation with G492E, its aminoacid sequence is SEQ ID NO:5.One containing K122N,
The mutant GOD-H-4 of four point mutation of Q263P, R341S and G492E, its aminoacid sequence is SEQ ID NO:6.
The mutant GOD-H-5 of one four point mutation containing I29L, A143P, G390R and F461I, its aminoacid sequence is SEQ
ID NO:7.The mutant GOD-H-6 of one four point mutation containing A143P, P241D, T359S and F461I, its amino
Acid sequence is SEQ ID NO:8.
70th aspartic acid D of GOD-H-3: this enzyme becomes glutamic acid Q (DNA sequence is become GAA from GAC),
The lysine K of the 122nd becomes agedoite N (AAG becomes AAC), the glycine G of the 492nd and becomes glutamic acid
E (GGG becomes GAG).
The lysine K of the 122nd of GOD-H-4: this enzyme becomes agedoite N (AAG becomes AAC), and the 263rd is
Glutamine Q become proline P (CAG becomes CCG), the arginine R of the 341st become serine S (CGC become
For AGC), the glycine G of the 492nd becomes glutamic acid E (GGG becomes GAG).
The isoleucine I of the 29th of GOD-H-5: this enzyme becomes leucine L (AUC becomes CUC), the 143rd
Alanine A becomes proline P (GCC becomes CCC), and the glycine G of the 390th becomes arginine R, and (GGC becomes
CGC), the phenylalanine F of the 461st becomes isoleucine I (TTC becomes ATC).
The alanine A of the 143rd of GOD-H-6: this enzyme becomes proline P (GCC becomes CCC), the 241st
Proline P becomes aspartic acid D (CCC becomes GAC), the threonine T of the 359th and becomes serine S (ACC change
For UCC), the phenylalanine F of the 461st becomes isoleucine I (TTC becomes ATC).
Embodiment 3: the structure of pichia pastoris engineered strain
Use primer described in embodiment 1, carry out PCR using the mutant that embodiment 1 and embodiment 2 are obtained as template
Amplification, PCR reaction condition is same as in Example 1.
Glucoseoxidase mutant gene fragment described in the embodiment 1 that amplification is obtained and embodiment 2, and wild type gene
Fragment, is connected with Expression vector pPIC9K by EcoR I and Not I site, construction of expression vector pPIC9K-GOD-1,
pPIC9K-GOD-H-1、pPIC9K-GOD-H-2、pPIC9K-GOD-H-3、pPIC9K-GOD-H-4、pPIC9K-
GOD-H-5 and pPIC9K-GOD-H-6.Expression vector is proceeded to bacillus coli DH 5 alpha competence, a large amount of after picking transformant
Extract plasmid.
Above expression plasmid SalI is carried out linearisation, linearized fragment fragment purification test kit (TaKaRa MiniBEST
DNA Fragment Purifibation Kit) after purified pool, convert Pichia pastoris GS115 by electricity method for transformation, be coated with MD
Flat board.The bacterium colony grown on MD flat board is applied to concentration gradually rise successively (1mg/mL, 2mg/mL, 4mg/mL,
Screen the positive transformant of multicopy on the YPD flat board of Geneticin 8mg/mL), obtain Pichia sp. recombinant bacterial strain.
The transformant of 7 genes is respectively designated as Pichia sp. GOD-1 (Pichia pastoris GOD-1), GOD-H-1
(Pichia pastoris GOD-H-1), Pichia sp. GOD-H-2 (Pichia pastoris GOD-H-2), Pichia sp.
GOD-H-3 (Pichia pastoris GOD-H-3), Pichia sp. GOD-H-4 (Pichia pastoris GOD-H-4), Bi Chi
Yeast GOD-H-5 (Pichia pastoris GOD-H-5) and Pichia sp. GOD-H-6 (Pichia pastoris GOD-H-6),
The transformant of each gene of picking is transferred in BMGY culture medium respectively, 30 DEG C, after 220rpm shaken cultivation 18h, centrifugal
Obtaining thalline, proceed to appropriate thalline, in BMMY culture medium, make cell concentration reach OD600=1,30 DEG C, 220rpm continues
Persistent oscillation is cultivated, and every 24h adds the methanol of volume of culture 1%.After abduction delivering 4d, medium centrifugal is obtained supernatant, will
Supernatant carries out glucoseoxidase vitality test and thermal stability determination.
Embodiment 4: mutant and wild type expression product enzyme activity and the mensuration of heat stability
Enzyme activity determination method:
(0.1mL1% dianisidine methanol storing solution joins 12mL 0.1M pH6.0 to take dianisidine buffer 2.5mL
Phosphate buffer is made into), 18% glucose solution 0.3mL, 0.03% Peroxidase Solution 0.1mL, join in color comparison tube
37 DEG C of insulation 5min, 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, survey at 540nm wavelength
Fixed 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 β-the D-Glucose of 1.0 μm ol can be oxidized to
Gluconic acid and H2O2Enzyme amount be a unit.
The phosphate buffer of fermented supernatant fluid pH 6.0 described in embodiment 3 is diluted to about 20U/mL, under the conditions of 80 DEG C
After processing 3min, measure remnant enzyme activity, live as 100% with the enzyme of untreated samples, calculate relative enzyme and live.Result such as Fig. 7 institute
Showing, wild type glucoseoxidase processes 3min under the conditions of 80 DEG C, and enzyme is lived and only remained 23%, and mutant GOD-H-1,
GOD-H-2, GOD-H-3, GOD-H-4, GOD-H-5 and GOD-H-6 processes 3min equally under the conditions of 80 DEG C and remains to
The enzyme keeping 30-60% is lived.Its thermostability respectively compared with wild type gene, be respectively increased 1.3,1.6,2.2,2.1,2.7,
2.3 again.
As can be seen here, the thermostability of the glucoseoxidase after sudden change is greatly improved compared with wild type, advantageously in it
Application in the industry.
Embodiment 5: the cultivation application experiment of glucoseoxidase
5.1 experimental design
White meat-type chickens Seedling is purchased from certain fowl factory, is divided into 8 hen houses, and 20000 chickens of each hen house, wherein 1,2,3,4 are
Matched group, 5,6,7,8 is experimental group, and experimental group adds the glucoseoxidase that the 0.2U/g present invention provides in basal diet
GOD-H-5, tests 40 days by a definite date.
5.2 performance test
Experiment starts the 2nd, 40 days to weigh each group of chicken on an empty stomach respectively, record just starting weight and end weight.In feeding process, observed and recorded is each
The health status of group chicken, and record weekly every cage feed intake, survival rate, feed-weight ratio and the calculating Europe index in the statistical experiment phase.
Experimental result:
Table 1 matched group production performance data
Table 2 experimental group production performance data
Compared with matched group, the counterpoise of experimental group increased, and survival rate also increased, and feed-weight ratio has declined, and passes through
Calculate Europe index and rise 2.9%, show that with the addition of glucoseoxidase GOD-H-5 in daily ration makes culture benefit increase
Add.
The present embodiment 5 is for the ease of embodying the application of glucoseoxidase of the present invention, however it is not limited to the application of broiler, because of
Can add in basal diet for described glucoseoxidase, may be used for feeding of other fowl poultry kinds.Generally can pig,
The breeding process such as rabbit and milch cow adds in its mixed feed.
Above example is only in order to illustrate technical scheme, rather than is limited；Although with reference to previous embodiment pair
The present invention has been described in detail, for the person of ordinary skill of the art, and still can be to described in previous embodiment
Technical scheme modify, or wherein portion of techniques feature is carried out equivalent；And these amendments or replacement, do not make
The essence of appropriate technical solution departs from the spirit and scope of claimed technical solution of the invention.
SEQUENCE LISTING
<110>Qingdao red cherry Bioisystech Co., Ltd
<120>heat stability improves glucoseoxidase mutant and encoding gene and application
<160> 10
<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
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ttcctcaacg agctggacct gctcggtcag gctgccgcta ctcaactggc
ccgcaacatc 1440
tccaactccg gtgccatgca gacctacttc gctggggaga ctatccccgg
tgataacctc 1500
gcgtatgatg ccgatttgag cgcctggact gagtacatcc cgtaccactt
ccgtcctaac 1560
taccatggcg tgggtacttg ctccatgatg ccgaaggaga tgggcggtgt
tgttgataat 1620
gctgcccgtg tgtatggtgt gcagggactg cgtgtcattg atggttctat
tcctcctacg 1680
caaatgtcgt cccatgtcat gacggtgttc tatgccatgg cgctaaaaat
ttcggatgct 1740
atcttggaag attatgcttc
catgcagtga
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 Asn 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 Glu 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 Pro 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 Ile 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 Gln 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 Asn 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 Glu 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 Asn 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 Pro 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 Ser 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 Glu 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> 589
<212> PRT
<213>artificial sequence
<400> 7
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 Leu 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 Pro 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 Arg 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 Ile 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> 8
<211> 589
<212> PRT
<213>artificial sequence
<400> 8
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 Pro 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
Asp 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 Ser 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 Ile 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> 9
<211> 32
<212> DNA
<213>artificial sequence
<400> 9
gcgcgaattc cgctgcggcc ctgccacact ac
32
<210> 10
<211> 49
<212> DNA
<213>artificial sequence
<400> 10
taaagcggcc gctcactgca tggaagcata atcttccaag
atagcatcc
49
Claims (10)
1. the glucoseoxidase mutant GOD-H-2 that a heat stability improves, it is characterized in that: the aminoacid sequence of described glucoseoxidase mutant GOD-H-2 is as shown in SEQ ID NO:4, and described mutant GOD-H-2 sequence is become proline from the 143rd amino acids of the glucoseoxidase that aminoacid sequence is SEQ ID NO:1 from alanine, the 461st amino acids is become isoleucine from phenylalanine and obtains.
2. the glucoseoxidase encoding gene of the glucoseoxidase mutant GOD-H-2 described in claim 1.
3. contain the recombinant bacterial strain of glucoseoxidase encoding gene described in claim 2.
4. the glucoseoxidase mutant GOD-H-5 that a heat stability improves, it is characterized in that: the aminoacid sequence of described glucoseoxidase mutant GOD-H-5 is as shown in SEQ ID NO:7, and described mutant GOD-H-5 sequence is become leucine from the 29th amino acids of the glucoseoxidase that aminoacid sequence is SEQ ID NO:1 from isoleucine, the 143rd amino acids is become proline from alanine, the 390th amino acids is become arginine from glycine, the 461st amino acids is become isoleucine from phenylalanine and obtains.
5. the glucoseoxidase encoding gene of the glucoseoxidase mutant GOD-H-5 described in claim 4.
6. contain the recombinant bacterial strain of glucoseoxidase encoding gene described in claim 5.
7. the glucoseoxidase mutant GOD-H-6 that a heat stability improves, it is characterized in that: the aminoacid sequence of described glucoseoxidase mutant GOD-H-6 is as shown in SEQ ID NO:8, and described mutant GOD-H-6 sequence is become proline from the 143rd amino acids of the glucoseoxidase that aminoacid sequence is SEQ ID NO:1 from alanine, the 241st amino acids is become aspartic acid from proline, the 359th amino acids is become serine from threonine, the 461st amino acids is become isoleucine from phenylalanine and obtains.
8. the glucoseoxidase encoding gene of the glucoseoxidase mutant GOD-H-6 described in claim 7.
9. contain the recombinant bacterial strain of glucoseoxidase encoding gene described in claim 8.
10. glucoseoxidase mutant GOD-H-2, GOD-H-5 and GOD-H-6 are for preparing the application in animal feed additive.
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| CN108118036A (en) * | 2016-11-28 | 2018-06-05 | 青岛蔚蓝生物集团有限公司 | Novel grape carbohydrate oxidase mutant |
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| CN108251392A (en) * | 2018-03-30 | 2018-07-06 | 广东溢多利生物科技股份有限公司 | It improves than living and the glucose oxidase mutant of thermal stability and its encoding gene and application |
| CN108374001A (en) * | 2018-03-30 | 2018-08-07 | 广东溢多利生物科技股份有限公司 | It improves than glucose oxidase mutant living and its encoding gene and application |
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| CN113403290A (en) * | 2021-05-26 | 2021-09-17 | 广东溢多利生物科技股份有限公司 | Glucose oxidase mutant with improved thermal stability as well as coding gene and application thereof |
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