CN108841801A - A kind of method of amino acid residue relevant to enzyme activity in screening enzyme - Google Patents
A kind of method of amino acid residue relevant to enzyme activity in screening enzyme Download PDFInfo
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Abstract
The invention discloses a kind of methods of amino acid residue relevant to enzyme activity in screening enzyme, belong to protein engineering field.The method is：Firstly, the electrostatic interaction at zymoprotein different aminoacids site is classified；Secondly, from the various dimensions such as gene order, amino acid sequence, three-D space structure and genetic evolution, sequence-structure-evolution relationship of electrostatic interaction in zymoprotein and its locating amino acid residue region is analyzed, using statistical method, the regularity of distribution of electrostatic interaction at different aminoacids site in zymoprotein is summarized；Again, genetic stability, occurrence probability, the position etc. of electrostatic interaction in zymoprotein and its locating amino acid residue region in zymoprotein evolutionary process are analyzed；Finally, summarize zymoprotein different location at electrostatic interaction during evolution possessed by hereditary feature.
Description
Technical field
The present invention relates to a kind of methods of amino acid residue relevant to enzyme activity in screening enzyme, belong to protein engineering skill
Art field.
Background technique
Thermal stability is a key factor of enzyme, the thermal stability of enzyme not only contribute to extend by force the storage time of enzyme,
The loss of enzyme vigor in preservation, transportational process is reduced, and enzyme can be made to keep higher vigor at a higher temperature, from
And reaction efficiency is improved, shorten the production cycle, and then reduce production cost.
Electrostatic interaction in zymoprotein between amino acid, the ionic bond that is otherwise known as or salt bridge, are considered for a long time
It is an important factor for influencing protein stability, is one of the core internal factor for determining zymoprotein structure and function, therefore, enzyme egg
White internal electrostatic interaction is all the focus of research all the time for the contribution of protein molecular overall stability.
But the amino acid present position that electrostatic interaction is generated in zymoprotein is different, the shadow caused by zymoprotein
Ringing may be different, and the research of the existing influence about salt bridge to protein molecular stability has certain dispute.
Wherein, having had research is analyzed for the amino acid distribution for generating electrostatic interaction in zymoprotein
It, although this research makes some progress, is not yet obtained with statistics to obtain influence of the salt bridge to protein molecular stability
The conclusion being of universal significance, can not be accurately to instruct the building of electrostatic interaction in zymoprotein to provide effective guidance.
More accurately to obtain the amino acid distribution impacted to the stability of zymoprotein, we are attempted to place
Electrostatic interaction at zymoprotein different aminoacids site is classified, from gene order, amino acid sequence, three-dimensional space
On the various dimensions such as structure and genetic evolution, to the sequence-of electrostatic interaction in zymoprotein and its locating amino acid residue region
Structure-evolution relationship is analyzed, and using statistical method, summarizes in zymoprotein that electrostatic is mutual at different aminoacids site
The regularity of distribution of effect；In addition, evolving to electrostatic interaction in zymoprotein and its locating amino acid residue region in zymoprotein
Genetic stability, occurrence probability, position in the process etc. are analyzed, and are summarized electrostatic interaction at zymoprotein different location and are existed
Possessed hereditary feature in evolutionary process.
By the above two aspects analysis, the formation rule of zymoprotein intramolecular electrostatic interaction is formed more comprehensive
Understand, to propose that there is the electrostatic interaction screening technique for improving zymoprotein molecular structure stabilized in zymoprotein, for building
Electrostatic interaction mutant provides thinking and direction in zymoprotein.
Summary of the invention
To solve the above problems, the present invention provides a kind of sides of amino acid residue relevant to enzyme activity in screening enzyme
Method.The method is：Firstly, the electrostatic interaction at zymoprotein different aminoacids site is classified；Secondly, from base
Because on the various dimensions such as sequence, amino acid sequence, three-D space structure and genetic evolution, to electrostatic interaction in zymoprotein and its
The sequence in locating amino acid residue region-structure-evolution relationship is analyzed, and using statistical method, summarizes zymoprotein
The regularity of distribution of electrostatic interaction at interior different aminoacids site；Again, to electrostatic interaction in zymoprotein and its locating
It is analyzed genetic stability, occurrence probability, position etc. of the amino acid residue region in zymoprotein evolutionary process；Finally, total
Tie zymoprotein different location at electrostatic interaction during evolution possessed by hereditary feature.Using the method can more compared with
To be accurately obtained in enzyme the relevant amino acid residue with enzyme activity, especially enzyme heat stability.
Technical scheme is as follows：
The present invention provides a kind of methods of amino acid residue relevant to enzyme activity in screening enzyme, and the method is screening
All electrically charged amino acid residues are as reference amino acid residue in enzyme；
Amino acid residue by the difference of the number of sites with reference amino acid residue less than 5 screens, as with the base
The quasi- corresponding alternative amino acid residue of amino acid residue；
It closes position between reference amino acid residue and alternative amino acid residue corresponding with the reference amino acid residue
System need at least meet one in claimed below：
(1) reference amino acid residue and alternative amino acid residue are located in same secondary structure；The secondary structure includes
Alpha-helix or beta sheet；
(5) the reference amino acid residue in the same α-helixstructure and the alternative amino elected is deleted on the basis of it
The number of sites difference of sour residue is between 2-10；Or the reference amino acid residue in same beta sheet structure with using it as base
The number of sites difference that standard deletes the alternative amino acid residue elected is no more than 4；
(6) C of reference amino acid residueαWith the C for deleting the alternative amino acid residue elected on the basis of itαBetween away from
FromBetween；
(7) reference amino acid residue is protected at least one in the alternative amino acid residue elected is deleted on the basis of it
Keep amino acid residue；
The alternative amino acid residue for meeting above-mentioned requirements is amino acid residue relevant to enzyme activity in enzyme；
Or analyze the alternative amino acid residue for meeting above-mentioned requirements, salt bridge possibility is formed after filtering out mutation
Amino acid residue, which is amino acid residue relevant to enzyme activity in enzyme；
The number of sites is obtained according to following methods：From N-terminal meter, by all amino acid residues in amino acid sequence
It being numbered, the position where first amino acid residue is first, and so on, it obtains in whole amino acid sequences, institute
There is the number of sites of amino acid residue；
The CαRefer to the carbon atom being connected in amino acid with carboxyl；
The conservative amino acid residues refer to, in cell succeeding generations, the amino acid residue that remains unchanged.
In one embodiment of the invention, the method is all electrically charged amino acid residue conducts in screening enzyme
Reference amino acid residue；
It will be screened with the reference amino acid Residue positions number difference without containing glutamic acid for 4 or 5 amino acid residue,
Alternately amino acid residue；Or it is the amino acid for being 3 or 4 with the reference amino acid Residue positions number difference containing glutamic acid is residual
Base screens, alternately amino acid residue；
It closes position between reference amino acid residue and alternative amino acid residue corresponding with the reference amino acid residue
System need at least meet one in claimed below：
(5) reference amino acid residue and alternative amino acid residue are located in same secondary structure；The secondary structure includes
Alpha-helix or beta sheet；
(6) the reference amino acid residue in the same α-helixstructure and the alternative amino elected is deleted on the basis of it
The number of sites difference of sour residue is between 2-10；Or the reference amino acid residue in same beta sheet structure with using it as base
The number of sites difference that standard deletes the alternative amino acid residue elected is no more than 4；
(7) C of reference amino acid residueαWith the C for deleting the alternative amino acid residue elected on the basis of itαBetween away from
FromBetween；
(8) reference amino acid residue is protected at least one in the alternative amino acid residue elected is deleted on the basis of it
Keep amino acid residue；
The alternative amino acid residue for meeting above-mentioned requirements is amino acid residue relevant to enzyme activity in enzyme；
Or analyze the alternative amino acid residue for meeting above-mentioned requirements, salt bridge possibility is formed after filtering out mutation
Amino acid residue, which is amino acid residue relevant to enzyme activity in enzyme.
In one embodiment of the invention, the activity that enzyme is referred to enzyme activity, thermal stability, pH stability,
Optimum temperature and optimal pH.
In one embodiment of the invention, the enzyme activity refers to the thermal stability of enzyme.
In one embodiment of the invention, the conservative amino acid residues are that conservative is residual greater than 70% amino acid
Base；
The conservative refers to, in cell succeeding generations, ability that amino acid residue remains unchanged.
The conservative is greater than 70% and refers to that in cell succeeding generations, the probability that amino acid residue remains unchanged is greater than
70%.
The conservative can be obtained by Jevtrace2 (v3.12b) software detection.
In one embodiment of the invention, the analysis is to be analyzed using PyMol molecular graphics software system.
In one embodiment of the invention, the enzyme is amylase.
In one embodiment of the invention, the amylase is Q-enzyrne.
In one embodiment of the invention, the Q-enzyrne is from Geobacillus
The Q-enzyrne of thermoglucosidans STB02,
In one embodiment of the invention, the amino acid sequence of the Q-enzyrne is SEQ ID NO.1.
The present invention provides the methods of relevant to enzyme activity amino acid residue in a kind of above-mentioned screening enzyme to change enzyme
Application in terms of enzyme activity.
In one embodiment of the invention, the activity that enzyme is referred to enzyme activity, thermal stability, pH stability,
Optimum temperature and optimal pH.
In one embodiment of the invention, the thermal stability that enzyme is referred to enzyme activity.
The present invention provides a kind of method for changing enzyme heat stability, the method is that will screen to obtain using the above method
Amino acid residue be mutated.
In one embodiment of the invention, the method is the amino acid residue that will be screened using the above method
Sport the amino acid residue that opposite charges is had with its corresponding reference amino acid residue.
In one embodiment of the invention, in the method, the electrically charged atom of the amino acid residue after mutation with
The distance between electrically charged atom of its corresponding reference amino acid residue exists
In one embodiment of the invention, the enzyme is amylase.
In one embodiment of the invention, the amylase is Q-enzyrne.
In one embodiment of the invention, the Q-enzyrne is from Geobacillus
The Q-enzyrne of thermoglucosidans STB02,
In one embodiment of the invention, the amino acid sequence of the Q-enzyrne is SEQ ID NO.1.
The present invention provides apply a kind of above-mentioned method for changing enzyme heat stability that enzyme mutant is prepared.
In one embodiment of the invention, the amino acid sequence of the mutant is SEQ ID NO.2, SEQ ID
NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.
Beneficial effect：
(1) it more can accurately be obtained using screening technique of the invention hot steady with enzyme activity, especially enzyme in enzyme
Qualitative relevant amino acid residue；
(2) present invention, which passes through, sports charge residue residue for important amino acid Residue positions in the enzyme filtered out,
Electrostatic interaction is formed with neighbouring endogenous, oppositely charged amino acid residue, constructs the enzyme with more high thermal stability,
1.5-2.0 times of Increased Plasma Half-life after keeping the temperature enzyme at 60~65 DEG C；
(3) present invention has very important significance to the industrial application value for improving enzyme, and operation of the present invention is simple, peace
Atoxic, has good hereditability, and application prospect is good.
Detailed description of the invention
Fig. 1 is that the Thermal inactivation rate of each Q-enzyrne mutant and wild type starch branching enzyme is bent in the embodiment of the present invention
Line；
Fig. 2 is that the Thermal inactivation rate of each Q-enzyrne mutant and wild type starch branching enzyme is bent in the embodiment of the present invention
Line；
Fig. 3 is that the Thermal inactivation rate of each Q-enzyrne mutant and wild type starch branching enzyme is bent in the embodiment of the present invention
Line.
Specific embodiment
Zymoprotein as used in the following examples is Q-enzyrne (1,4- α-glucan branching enzyme；
It EC2.4.1.18), is a kind of glycosyl transferase for belonging to glycoside hydrolase Families 13 (GH 13), it being capable of catalytic starch molecule alpha-
Isosorbide-5-Nitrae-glycosidic bond breaks to form the short chain that dissociates, and passes through transglucosidation for the short chain under cutting with α -1,6- glycosidic bond shape
Formula is connected on receptor chain, and new α -1,6- branch point are formed on starch molecule original main chain.By this kind of Transglycosylation, form sediment
Powder branching enzyme can increase the degree of branching of starch, improve the anti-digestibility of starch and slowly digestible, delay the retrogradation process of starch,
Enhance the stability of starch and improve the service performance of starch, can be used for producing the starch derivatives with applications well value.
Detection method of the present invention is as follows：
The analysis method of Q-enzyrne thermal stability：
Q-enzyrne is kept the temperature at a certain temperature, different time points sampling is rapidly cooled to 0 DEG C, measures the residual of enzyme
Remaining vigor, not keep the temperature the vigor of enzyme solution as 100%.Draw opposite enzyme activity-time graph.
The measuring method of Q-enzyrne vigor：
The amylopectin potato solution of 0.25% (w/v) is prepared with 10mM phosphate buffer (pH7.5), and 0.01g/ is added
It is uniformly mixed to be placed under 50 DEG C of water bath conditions after mL Q-enzyrne and reacts 15min.Boiling water bath enzyme deactivation after reaction.It takes
175L reaction mixture addition 2.5mL developing solution (0.05% (w/v) KI, 0.005% (w/v) I2, pH7.5) is placed in quiet at room temperature
15min is set sufficiently to develop the color.Light absorption value is measured at 530nm after colour developing 15min.One enzyme activity unit is defined as：In 530nm
Place, enzyme is added in light absorption value amount needed for reducing by 1% per minute is an enzyme activity unit.
Culture medium used in the present invention is as follows：
LB culture medium：Yeast powder 5g/L, tryptone 10g/L, NaCl 10g/L, pH 7.0.
TB culture medium：Yeast powder 24g/L, tryptone 12g/L, glycerol 5g/L, KH2PO417mM, K2HPO472mM, pH
7.0。
Embodiment 1：The screening of acid residues sites
Screening technique is：
All electrically charged amino acid residues are as reference amino acid residue in screening enzyme；
It will be screened with the reference amino acid Residue positions number difference without containing glutamic acid for 4 or 5 amino acid residue,
Alternately amino acid residue；Or it is the amino acid for being 3 or 4 with the reference amino acid Residue positions number difference containing glutamic acid is residual
Base screens, alternately amino acid residue；
It closes position between reference amino acid residue and alternative amino acid residue corresponding with the reference amino acid residue
System need at least meet one in claimed below：
(1) reference amino acid residue and alternative amino acid residue are located in same secondary structure；The secondary structure includes
Alpha-helix or beta sheet；
(2) the reference amino acid residue in the same α-helixstructure and the alternative amino elected is deleted on the basis of it
The number of sites difference of sour residue is between 2-10；Or the reference amino acid residue in same beta sheet structure with using it as base
The number of sites difference that standard deletes the alternative amino acid residue elected is no more than 4；
(3) C of reference amino acid residueαWith the C for deleting the alternative amino acid residue elected on the basis of itαBetween away from
FromBetween；
(4) reference amino acid residue is protected at least one in the alternative amino acid residue elected is deleted on the basis of it
Keep amino acid residue；
The alternative amino acid residue for meeting above-mentioned requirements is analyzed, has to form salt bridge possibility after filtering out mutation
Amino acid residue, the alternative amino acid residue are amino acid residue relevant to enzyme activity in enzyme.
Above-mentioned screening technique is applied in the Q-enzyrne that amino acid sequence is SEQ ID NO.1, obtains the 231st
Point, the 339th site, the 37th site and the 571st site.
Embodiment 2：The building of enzyme mutant
The amino acid residue screened in embodiment 1 is sported with its corresponding reference amino acid residue with phase
The amino acid residue of counter charges, amino acid residue and neighbouring charge residue residue after mutation form salt bridge, electrically charged
Between atom distance betweenBetween.
These salt bridges are sequentially formed in：Between Q231R, Q231K and D227, between T339E, T339D and K335, V37E,
Between V37D and K32, between I571D and R569.
Specific step is as follows：
With expression vector gbe/pET-20 (+) for template, complementary primer chain (being shown in Table 1) needed for contrived experiment, primer by
The synthesis of Jin Wei intelligence Biotechnology Co., Ltd, the method referring to shown in TaKaRa company STAR Primer GXL kit specification
Carry out rite-directed mutagenesis.PCR reaction system is according to set fixed condition in STAR Primer kit specification：5×PrimeSTAR
Buffer(Mg2+Plus) 10 μ L, 1 μ L of template DNA, forward and reverse primer (10 μM) are 1 μ L, PrimeSTAR HS
0.5 4 μ L of μ L, dNTPs (each 2.5mM) of DNAPolymerase (2.5U/ μ L), is eventually adding 32.5 μ L of ultrapure water.PCR amplification item
Part is：Initial denaturation 5min under the conditions of 98 DEG C；Then with 98 DEG C of 10s, 55 DEG C of 10s, 72 DEG C of 7min are a circulation, above
Under the conditions of carry out 35 circulation；15min is kept the temperature at last 72 DEG C.
The introducing in 1 Q-enzyrne mutational site of table
1Underscore base corresponds to corresponding mutating acid.
Embodiment 3：The building of genetic engineering bacterium containing expression enzyme mutant gene
The building of engineering bacteria containing Q-enzyrne mutant gene carries out as follows：
(1) PCR product is transferred in E.coli DH5 α according to E.coli DH5 α competence method for transformation, and by receptor
Bacterium is applied to containing on 100 μ g/mL ampicillin LB solid mediums；
(2) the LB solid medium after coating is inverted in 37 DEG C of constant incubators and cultivates 12h；
(3) picking positive monoclonal is inoculated in the LB liquid medium containing 100 μ g/mL ampicillins and at 37 DEG C
Cultivate 10~12h；
(4) thallus being collected into is extracted into plasmid and is identified using double digestion electrophoresis and sequencing；
(5) at 37 DEG C, PCR product 2h is handled with DpnI, the PCR product handled well is then transformed into E.coli
In JM109, the E.coli JM 109 of conversion is applied in the LB agar medium containing 100 μ g/mL ammonia benzyl mycins, 37
It is incubated overnight 12h in DEG C insulating box, single colonie is selected and is inoculated into the LB Liquid Culture containing 100 μ g/mL ammonia benzyl mycins
In base, at 37 DEG C, 200r/min overnight incubation and according to method shown in plasmid extraction kit specification extract plasmid identification
Sequencing.The purpose plasmid built is transferred in expressive host E.coli BL21 (DE 3) competence by chemical transformation.Most
Genetically engineered E.coli BL21 (DE 3) (pET-22b (+)/gbe) is obtained eventually.
Embodiment 4：The expression of enzyme mutant
Host strain activation culture：It will exist containing expression vector plasmid pET-20b (+)/gbe E.coli BL21 (DE 3)
Scribing line separation is carried out on LB solid medium, is placed in 37 DEG C of constant incubators and is incubated overnight, and picking positive single colonie is inoculated in
In sterilizing 50mL centrifuge tube containing 15mLLB fluid nutrient medium.The centrifuge tube is placed in the rotary shaking table of 200r/min,
12h is cultivated at 37 DEG C.
Fermented and cultured：Activated 200 μ L of bacteria culture fluid is inoculated in the 250mL containing 50mL TB fluid nutrient medium
In triangular flask, it is placed in the rate in rotary shaking table with 200r/min, is cultivated at 37 DEG C to OD600Reach 1.0-1.5 it
Between.The IPTG of final concentration of 0.01mM/L is added and reduces cultivation temperature to 25 DEG C, continues to induce 16h with this condition.
Each culture medium is in the ampicillin using the preceding final concentration of 100 μm of g/mL of addition.
Embodiment 5：The heat stability test of enzyme mutant
Be Q-enzyrne mutant Q231R, Q231K of 1 μm of ol/L by enzyme concentration, Q231A, T339E, T339D,
T339A, V37E, V37D, V37A, I571D and I571A keep the temperature a period of time, separated in time at 60 DEG C and 65 DEG C respectively
Sampling, measures the vigor of this kind of enzyme solutions at 50 DEG C according to Q-enzyrne vigour-testing method.
Compared with wild type starch branching enzyme, Q231R, Q231K, T339E, T339D, V37E, V37D and I571D mutant
Half-life period t at 60 DEG C1/2(min, 60 DEG C) extends 40%, 38%, 21%, 26%, 16% and 21% respectively, at 65 DEG C
Half-life period t1/21.5-2.0 times of extension of (min, 65 DEG C)；Q231A, T339A, V37A and I571A mutant are at 60 DEG C and 65
Half-life period at DEG C is similar to wild type starch branching enzyme.(heat stability test result such as Fig. 1 and table 2)
The half-life period of table 2 wild type and mutant GBE
1Each value is the average value of 3 parallel laboratory tests.
Embodiment 6：The Enzyme activity assay of enzyme mutant
The amylopectin potato solution for being 0-5mg/mL with 10mM phosphate buffer (pH 7.5) compound concentration is added one
The quantitative mutant of Q-enzyrne after purification Q231R, Q231K, Q231A, T339E, T339D, T339A, V37E, V37D,
V37A, I571D and I571A carry out reaction at 50 DEG C and respectively according to above-mentioned shown method measurement Q-enzyrne mutant
Vigor.The data obtained is imported into SigmaPlot program (Systat Software Inc., version 10.0for
Windows Michaelis (Michaelis-Menten) equation is obtained in).
Wherein：V is that the ratio of Q-enzyrne mutant under different concentration of substrate is living；S is concentration of substrate；VmaxIt is maximum anti-
Answer rate；KMFor Michaelis constant.(Enzyme activity assay result such as table 3)
The enzyme activity and K of 3 wild type of table and mutant GBEMValue
1Each value is the average value of 3 parallel laboratory tests.
Embodiment 7：The deactivation kinetics of enzyme mutant are analyzed
The amylopectin potato solution for being 0-5mg/mL with 10mM phosphate buffer (pH 7.5) compound concentration is added one
Quantitative Q-enzyrne after purification carries out reaction at 50 DEG C and measures vigor according to above method.It calculates wild type and dashes forward
Gibbs energy Δ G (kJmol of the variant Q-enzyrne in heat inactivation process-1), enthalpy change Δ H (kJmol-1), Entropy Changes Δ
S(J·K-1·mol-1), these parameters can be obtained by following formula：
Δ H=Ea-RT
Wherein, KBIndicate Boltzmann constant (1.381 × 10-23J·K-1), h indicates Planck's constant (1.104 × 10- 35Jmin), R indicates gas constant (8.314JK-1·mol-1), the unit of T is absolute temperature (K).(deactivation kinetics point
Analyse result such as table 4)
4 wild type of table and the analysis of mutant GBE deactivation kinetics
1Each value is the average value of 3 parallel laboratory tests.
Embodiment 8：The enzyme concentration of enzyme mutant is analyzed
The potato starch solution for preparing 10% is substrate, adjusts pH 7.5, and starch branch is added by 25U/g enzyme concentration
Enzyme, at 60 DEG C reaction modifying for 24 hours, boiling water bath enzyme deactivation after reaction.It is identical having with wild type starch branching enzyme to starch
When modified effect, less amount is can be used in the Q-enzyrne mutant with high thermal stability.(enzyme concentration analyzes result
Such as table 5)
The application of table 5 wild type and mutant GBE
1Each value is the average value of 3 parallel laboratory tests.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Sequence table
<110>Southern Yangtze University
<120>A kind of method of amino acid residue relevant to enzyme activity in screening enzyme
<160> 30
<170> PatentIn version 3.3
<210> 1
<211> 643
<212> PRT
<213>Artificial sequence
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Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
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Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
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Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
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Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 2
<211> 643
<212> PRT
<213>Artificial sequence
<400> 2
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Arg Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 3
<211> 643
<212> PRT
<213>Artificial sequence
<400> 3
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Lys Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 4
<211> 643
<212> PRT
<213>Artificial sequence
<400> 4
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Glu Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 5
<211> 643
<212> PRT
<213>Artificial sequence
<400> 5
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Asp Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 6
<211> 643
<212> PRT
<213>Artificial sequence
<400> 6
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Glu Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 7
<211> 643
<212> PRT
<213>Artificial sequence
<400> 7
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Asp Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Ile Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 8
<211> 643
<212> PRT
<213>Artificial sequence
<400> 8
Met Ser Val Val Pro Pro Thr Asp Leu Glu Ile Tyr Leu Phe His Glu
1 5 10 15
Gly Ser Leu Tyr Lys Ser Tyr Glu Leu Phe Gly Ala His Val Ile Lys
20 25 30
Gln Asn Asp Val Val Gly Thr Arg Phe Cys Val Trp Ala Pro His Ala
35 40 45
Arg Gln Val Arg Leu Val Gly Ser Phe Asn Asp Trp Asn Gly Thr Asn
50 55 60
Phe Asn Leu Val Lys Val Ser Asn Gln Gly Val Trp Thr Ile Phe Ile
65 70 75 80
Pro Glu Asn Leu Glu Gly His Leu Tyr Lys Tyr Glu Ile Thr Thr Ser
85 90 95
Asp Gly Asn Val Val Leu Lys Ala Asp Pro Tyr Ala Phe His Ser Glu
100 105 110
Leu Arg Pro Arg Thr Ala Ser Ile Val Tyr Asp Ile Lys Gly Tyr Gln
115 120 125
Trp Asn Asp Gln Thr Trp Arg Arg Lys Lys Gln Arg Lys Arg Ile Tyr
130 135 140
Asp Gln Pro Leu Phe Ile Tyr Glu Leu His Phe Gly Ser Trp Lys Lys
145 150 155 160
Lys Glu Asn Gly Asn Phe Tyr Thr Tyr Arg Glu Met Ala Asp Glu Leu
165 170 175
Leu Pro Tyr Val Met Glu His Gly Phe Thr His Ile Glu Leu Leu Pro
180 185 190
Leu Val Glu His Pro Leu Asp Arg Ser Trp Gly Tyr Gln Gly Thr Gly
195 200 205
Tyr Tyr Ser Ala Thr Ser Arg Tyr Gly Thr Pro His Asp Leu Met His
210 215 220
Phe Ile Asp Arg Phe His Gln Ala Gly Ile Gly Val Ile Phe Asp Trp
225 230 235 240
Val Pro Gly His Phe Cys Lys Asp Glu His Gly Leu Tyr Met Phe Asp
245 250 255
Gly Ala Pro Thr Tyr Glu Tyr Asp Asn Ile Gln Asp Arg Glu Asn Gly
260 265 270
Glu Trp Gly Thr Ala Asn Phe Asp Leu Gly Lys Pro Glu Val Arg Ser
275 280 285
Phe Leu Ile Ser Asn Ala Leu Phe Trp Met Glu Tyr Phe His Val Asp
290 295 300
Gly Phe Arg Val Asp Ala Val Ala Asn Met Leu Tyr Trp Pro Asn Arg
305 310 315 320
Glu Ala Ala Gln Gln Asn Pro His Ala Val Gln Phe Leu Gln Lys Leu
325 330 335
Asn Glu Thr Val Phe Ala His Asp Pro Gly Ile Leu Met Ile Ala Glu
340 345 350
Asp Ser Thr Glu Trp Pro Leu Val Thr Ala Pro Thr Tyr Ala Gly Gly
355 360 365
Leu Gly Phe Asn Tyr Lys Trp Asn Met Gly Trp Met Asn Asp Ile Leu
370 375 380
Thr Tyr Met Glu Thr Ala Pro Glu Lys Arg Lys His Val His Asn Lys
385 390 395 400
Val Thr Phe Ser Leu Leu Tyr Ala Tyr Ser Glu Asn Phe Ile Leu Pro
405 410 415
Phe Ser His Asp Glu Val Val His Gly Lys Lys Ser Leu Leu Asn Lys
420 425 430
Met Pro Gly Thr Tyr Glu Glu Lys Phe Ala Gln Leu Arg Leu Leu Tyr
435 440 445
Gly Tyr Leu Leu Thr His Pro Gly Lys Lys Leu Leu Phe Met Gly Gly
450 455 460
Glu Phe Ala Gln Phe Asp Glu Trp Lys Asp Ala Glu Gln Leu Asp Trp
465 470 475 480
Met Leu Phe Asp Phe Glu Met His Gln Lys Met Asn Met Tyr Val Lys
485 490 495
Ala Leu Leu Lys Cys Tyr Lys Arg Cys Lys Ser Leu Tyr Glu Leu Asp
500 505 510
His Ser Pro Asp Gly Phe Glu Trp Ile Asp Val His Asn Ala Glu Gln
515 520 525
Ser Ile Phe Ser Phe Val Arg Arg Gly Lys Lys Glu Asn Asp Leu Leu
530 535 540
Val Val Val Cys Asn Phe Thr Ser Lys Val Tyr His Asp Tyr Lys Val
545 550 555 560
Gly Val Pro Leu Phe Ala Lys Tyr Arg Glu Asp Ile Ser Ser Asp Ala
565 570 575
Ala Lys Phe Gly Gly Trp Gly Asn Val Asn Ala Lys Pro Val Ala Ala
580 585 590
Ser Lys Glu Pro Phe His Gly Lys Pro Tyr His Ile Arg Met Thr Val
595 600 605
Pro Pro Phe Gly Ile Ser Ile Leu Arg Pro Val Lys Lys Arg Gly Glu
610 615 620
Arg Ser Val Asp Gly Lys Glu Lys Val His Arg His Val Ile Gly Gly
625 630 635 640
Arg Ala Arg
<210> 9
<211> 25
<212> DNA
<213>Artificial sequence
<400> 9
atcgcttcca tcgagcgggc attgg 25
<210> 10
<211> 25
<212> DNA
<213>Artificial sequence
<400> 10
ccaatgcccg ctcgatggaa gcgat 25
<210> 11
<211> 25
<212> DNA
<213>Artificial sequence
<400> 11
atcgcttcca taaagcgggc attgg 25
<210> 12
<211> 25
<212> DNA
<213>Artificial sequence
<400> 12
ccaatgcccg ctttatggaa gcgat 25
<210> 13
<211> 25
<212> DNA
<213>Artificial sequence
<400> 13
atcgcttcca tgcagcgggc attgg 25
<210> 14
<211> 25
<212> DNA
<213>Artificial sequence
<400> 14
ccaatgcccg ctgcatggaa gcgat 25
<210> 15
<211> 23
<212> DNA
<213>Artificial sequence
<400> 15
attaaatgag gaggtatttg cgc 23
<210> 16
<211> 23
<212> DNA
<213>Artificial sequence
<400> 16
gcgcaaatac ctcctcattt aat 23
<210> 17
<211> 23
<212> DNA
<213>Artificial sequence
<400> 17
attaaatgag gacgtatttg cgc 23
<210> 18
<211> 23
<212> DNA
<213>Artificial sequence
<400> 18
gcgcaaatac gtcctcattt aat 23
<210> 19
<211> 23
<212> DNA
<213>Artificial sequence
<400> 19
attaaatgag gcagtatttg cgc 23
<210> 20
<211> 23
<212> DNA
<213>Artificial sequence
<400> 20
gcgcaaatac tgcctcattt aat 23
<210> 21
<211> 25
<212> DNA
<213>Artificial sequence
<400> 21
caaaacgacg ttgaaggaac ccggt 25
<210> 22
<211> 25
<212> DNA
<213>Artificial sequence
<400> 22
accgggttcc ttcaacgtcg ttttg 25
<210> 23
<211> 25
<212> DNA
<213>Artificial sequence
<400> 23
caaaacgacg ttgacggaac ccggt 25
<210> 24
<211> 25
<212> DNA
<213>Artificial sequence
<400> 24
accgggttcc gtcaacgtcg ttttg 25
<210> 25
<211> 25
<212> DNA
<213>Artificial sequence
<400> 25
caaaacgacg ttgcaggaac ccggt 25
<210> 26
<211> 25
<212> DNA
<213>Artificial sequence
<400> 26
accgggttcc tgcaacgtcg ttttg 25
<210> 27
<211> 24
<212> DNA
<213>Artificial sequence
<400> 27
aataccggga agacatcagc agcg 24
<210> 28
<211> 24
<212> DNA
<213>Artificial sequence
<400> 28
cgctgctgat gtcttcccgg tatt 24
<210> 29
<211> 24
<212> DNA
<213>Artificial sequence
<400> 29
aataccggga agcaatcagc agcg 24
<210> 30
<211> 24
<212> DNA
<213>Artificial sequence
<400> 30
cgctgctgat tgcttcccgg tatt 24
Claims (13)
1. a kind of method of amino acid residue relevant to enzyme activity in screening enzyme, which is characterized in that the method is screening enzyme
In all electrically charged amino acid residues as reference amino acid residue；
Amino acid residue by the difference of the number of sites with reference amino acid residue less than 5 screens, as with the reference ammonia
The corresponding alternative amino acid residue of base acid residue；
Positional relationship between reference amino acid residue and alternative amino acid residue corresponding with the reference amino acid residue needs
At least meet one in claimed below：
(1) reference amino acid residue and alternative amino acid residue are located in same secondary structure；The secondary structure includes α-spiral shell
Rotation or beta sheet；
(2) the reference amino acid residue in the same α-helixstructure with that the alternative amino acid elected is deleted on the basis of it is residual
The number of sites difference of base is between 2-10；Or the reference amino acid residue in same beta sheet structure is deleted on the basis of it
The number of sites difference for the alternative amino acid residue elected is no more than 4；
(3) C of reference amino acid residueαWith the C for deleting the alternative amino acid residue elected on the basis of itαThe distance betweenBetween；
(4) reference amino acid residue is conservative ammonia at least one in the alternative amino acid residue elected is deleted on the basis of it
Base acid residue；
The alternative amino acid residue for meeting above-mentioned requirements is amino acid residue relevant to enzyme activity in enzyme；
Or analyze the alternative amino acid residue for meeting above-mentioned requirements, have to form the ammonia of salt bridge possibility after filtering out mutation
Base acid residue, the alternative amino acid residue are amino acid residue relevant to enzyme activity in enzyme.
2. the method for amino acid residue relevant to enzyme activity in a kind of screening enzyme as described in claim 1, which is characterized in that
The method be screening enzyme in all electrically charged amino acid residues as reference amino acid residue；
It will be screened with the reference amino acid Residue positions number difference without containing glutamic acid for 4 or 5 amino acid residue, as
Alternative amino acid residue；Or the amino acid residue for being 3 or 4 with the reference amino acid Residue positions number difference containing glutamic acid is sieved
It elects, alternately amino acid residue；
Positional relationship between reference amino acid residue and alternative amino acid residue corresponding with the reference amino acid residue needs
At least meet one in claimed below：
(1) reference amino acid residue and alternative amino acid residue are located in same secondary structure；The secondary structure includes α-spiral shell
Rotation or beta sheet；
(2) the reference amino acid residue in the same α-helixstructure with that the alternative amino acid elected is deleted on the basis of it is residual
The number of sites difference of base is between 2-10；Or the reference amino acid residue in same beta sheet structure is deleted on the basis of it
The number of sites difference for the alternative amino acid residue elected is no more than 4；
(3) C of reference amino acid residueαWith the C for deleting the alternative amino acid residue elected on the basis of itαThe distance betweenBetween；
(4) reference amino acid residue is conservative ammonia at least one in the alternative amino acid residue elected is deleted on the basis of it
Base acid residue；
The alternative amino acid residue for meeting above-mentioned requirements is amino acid residue relevant to enzyme activity in enzyme；
Or analyze the alternative amino acid residue for meeting above-mentioned requirements, have to form the ammonia of salt bridge possibility after filtering out mutation
Base acid residue, the alternative amino acid residue are amino acid residue relevant to enzyme activity in enzyme.
3. the method for amino acid residue relevant to enzyme activity, feature exist in a kind of screening enzyme as claimed in claim 1 or 2
In activity, thermal stability, pH stability, optimum temperature or the optimal pH for referring to enzyme with enzyme activity.
4. the method for amino acid residue relevant to enzyme activity, special in a kind of screening enzyme a method according to any one of claims 1-3
Sign is that the enzyme activity refers to the thermal stability of enzyme.
5. the method for amino acid residue relevant to enzyme activity in a kind of screening enzyme as described in claim 1-4 is any, special
Sign is that the conservative amino acid residues are the amino acid residue that conservative is greater than 70%.
6. the method for amino acid residue relevant to enzyme activity, special in a kind of screening enzyme a method as claimed in any one of claims 1 to 5
Sign is that the analysis is to be analyzed using PyMol molecular graphics software system.
7. the method for amino acid residue relevant to enzyme activity in a kind of screening enzyme as described in claim 1-6 is any, special
Sign is that the enzyme is amylase.
8. the method for amino acid residue relevant to enzyme activity is changing enzyme in a kind of screening enzyme as claimed in claim 1 to 7
Enzyme activity in terms of application.
9. a kind of method for changing enzyme heat stability, which is characterized in that the method is will be any described using claim 1-7
The amino acid residue that screens of method be mutated.
10. a kind of method for changing enzyme heat stability as claimed in claim 9, which is characterized in that the method is that will apply
The amino acid residue that method as claimed in claim 1 to 7 is screened sports and its corresponding reference amino acid residue
Amino acid residue with opposite charges.
11. a kind of method of change enzyme heat stability as described in claim 9 or 10, which is characterized in that in the method, dash forward
The distance between the electrically charged atom of the electrically charged atom of amino acid residue after change and its corresponding reference amino acid residue
?
12. enzyme mutant is prepared in a kind of method of any change enzyme heat stability of application claim 9-11.
13. enzyme mutant as claimed in claim 12, which is characterized in that the amino acid sequence of the mutant is SEQ ID
NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID
NO.8。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684751A (en) * | 2019-10-23 | 2020-01-14 | 江南大学 | Starch branching enzyme mutant with improved catalytic capability |
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