CN115295076A - Design and application of ketoreductase mutant - Google Patents
Design and application of ketoreductase mutant Download PDFInfo
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Abstract
The present disclosure provides ketoreductase mutants that are active on both (R) - (-) -1, 3-butanediol and (S) - (-) -1, 3-butanediol, and computational design methods for obtaining such mutants. The ketoreductase mutant disclosed by the application can catalyze the reaction of synthesizing 4-hydroxy-2-butanone from racemic 1, 3-butanediol, and has good industrial application value.
Description
Technical Field
The invention relates to computer-aided design and virtual screening of protease mutants, in particular to a calculation method combining virtual screening of protease stable mutants and virtual screening of catalytic activity mutants so as to realize design of protease mutants with non-natural substrate activity.
Background
The protease catalyst plays an important role in modern synthesis industry, and with the continuous expansion of the application range of enzyme catalysis, the catalytic performance of natural enzymes existing in nature cannot meet the requirements of enzyme research and industrial application. Directed evolution is one of important technical means for modifying protease, is a strategy of protein engineering closer to a natural evolution mode, is also called laboratory evolution, is a method for rapidly modifying protein with a certain purpose by simulating a natural evolution process in vitro, and can complete the evolution process which originally needs millions of years in nature in a short time under the condition of unknown three-dimensional structure information and action mechanism of a target protein, thereby obtaining the enzyme with expected functions. In recent years, the directed evolution technology is widely applied to the development of enzyme catalysts needed in the fields of pharmacy, food, chemistry and chemical engineering and the like, so that another revolution in the technical field of biological catalysis is initiated, and the research and application range of protein engineering is greatly expanded. The applicants have been working on the research and application of directed evolution techniques for enzymes and have succeeded in developing a large number of protease catalysts for the production of pharmaceuticals and fine chemicals. However, directed evolution in laboratories often requires screening of large numbers of enzyme mutation libraries, the construction of these mutation libraries themselves and the subsequent screening work being burdensome tasks for the researchers. On the basis of the research on a large number of directed evolution samples in the early stage, the applicant combines computational biology and bioinformatics technologies to summarize and design a set of feasible calculation method, can effectively perform computer virtual screening on the assumed enzyme mutants, reliably predict the enzyme mutants with expected performance, and further greatly reduce the range of the mutant library required to be constructed and screened in the directed evolution process of the laboratory. The calculation method disclosed by the application is not only powerful supplement to the directed evolution process of the laboratory, but also combined with the directed evolution technology of the laboratory, and can break through the limitation of the library range which can be constructed and screened by the laboratory, reduce the research and development cost, improve the research and development efficiency, and more effectively obtain the enzyme mutants with expected performance.
Many computational methods have now been disclosed for studying proteins. For example, molecular docking algorithms are used to observe the binding mode of small molecule substrates and proteins, homology modeling and de novo modeling algorithms are used to construct three-dimensional models of proteins, and tools for calculating structural stability of proteins, such as Foldx, I-mutant, rosetta, etc., have been widely used in protein mutation design. However, since the algorithms themselves all belong to empirical mathematical calculation forms, the calculation formulas contain energy calculation terms of some physical principles and statistical energy terms obtained by statistics of the existing database, so far, computer-aided mutation design methods all have respective limitations, and the performance of mutants obtained through calculation and prediction is difficult to be reliably matched with experimental verification results. Currently, no computational design procedure for protease mutants is presented to reliably predict enzyme mutants with desired properties.
Disclosure of Invention
The invention particularly develops a computational method for efficient computer virtual screening of enzyme mutants for reliable prediction of enzyme mutants with desired properties. The invention constructs a special processing method of the stability calculation result, creatively increases the calculation process of the reaction energy barrier, can improve the accuracy of the virtual screening of the protease mutation, not only can greatly reduce the number of the mutations required to be screened in the experiment and save manpower and material resources, but also can unexpectedly realize the effect of engineering modification on the enzyme which cannot be achieved by the traditional enzyme directed evolution means.
The calculation method of the invention is shown in figure 1 and comprises the following 4 specific steps:
(1) Protein structure model acquisition: and obtaining a three-dimensional structure model of the protein according to the amino acid sequence of the target protease. The structural model can be obtained from experiments recorded in a PDB (protein data bank) database, or can be virtually constructed by using a homology modeling method or a de novo modeling method according to a protein sequence, and the homology modeling with higher homology is more accurate than the model obtained by de novo modeling. It is necessary for protein structures of different origin to be in a catalytic conformation, i.e. substrate molecule (product or transition state) -protein complex.
(2) Substrate docking analysis: the amino acid sites on the three-dimensional structure of the target protease that bind to the substrate molecule are determined, and then the natural substrate of the enzyme or the target substrate (including the unnatural substrate) is docked with the protein. Selecting the site suitable for mutation in the active site of the protein structure according to different conformation of the natural substrate or the target substrate. More computing software is available to implement molecular docking, such as Discovery studio, schrodinger, yasara (including Autodock and Autodock vina plug-ins), and the like. The amino acid sites that require mutation are screened out by alignment to the alignment conformation.
(3) Mutant stability calculation: and (3) performing stability calculation on the single-point mutant and/or the multipoint combined mutant according to the site obtained by screening the docking result. Firstly, a mutant set required to be virtually screened is generated in batches by using a python script, then a structural model of each mutant can be generated by using software such as Yasara or Rosetta, the structural stability of each mutant is calculated by using algorithms such as ddg _ monomer, cartesian _ ddg, foldX, provean, ELASPIC or Amber TI, and finally the free energy difference (delta G) between each mutant structure and a wild-type enzyme structure is calculated by using a python analysis script.
There may be two processing strategies for the results of the above stability calculations: simple ordering method [ 3a ] and statistical method [ 3b ].
【3a】 A simple sorting method: simply sorting the delta Delta G results of the mutants from low to high, and selecting the mutants with the top sorting as stable mutants obtained by computer virtual screening prediction.
【3b】 The statistical method comprises the following steps: the method comprises the steps of sequencing the delta Delta G results of mutants from low to high, selecting a part of mutants which are sequenced at the front and a part of mutants which are sequenced at the back for carrying out frequency analysis on mutated amino acid residues, subtracting the amino acid residue type which has higher occurrence frequency in an unstable mutant from the amino acid residue type which has higher occurrence frequency in the stable mutant for a specific amino acid site to obtain a collection of theoretically mutated amino acid residue types on the site, and finally arranging and combining the mutated amino acid residues of each site as the stable mutant obtained by virtual screening and prediction of a computer.
Evaluation criteria for the results of the mutant stability calculation: delta G is less than or equal to minus 1kcal/mol, which is a stable mutant; delta G is more than or equal to 1kcal/mol and is an unstable mutant; -1kcal/mol < Δ Δ G <1kcal/mol is a null mutant. The standard is also suitable for judging stability results obtained by other calculation methods.
There are many methods available for calculating the structural stability of proteins, and therefore the use of the Rosetta program is not limited in this procedure. The creative contribution of the method in the stability calculation process is that a statistical method (3 b) is conceived and adopted to process the calculation result, the amino acid residue mutation on each site is screened by using a statistical analysis method, and then the screened mutations are combined to be used as the stable mutant obtained by computer virtual screening prediction.
The current computer virtual screening method generally stops at this point, namely, after stable mutants which are virtually screened and predicted are obtained by a simple sorting method [ 3a ], the mutants are verified by a specific experimental scheme in a laboratory, and no further calculation method is used for evaluating the catalytic activity of the stable mutants. In addition, due to the limitation of stability calculation, a part of mutants with actually high stability and activity in experimental verification can be omitted from the stable mutants judged by the simple sorting method [ 3a ]. The invention provides a statistical method (3 b) for screening the stable mutants, so that the stable mutants which cannot be predicted by the current algorithm can be obtained. Moreover, although the conventional calculation method eliminates a large number of mutants with unstable binding through virtual screening and reduces the number of mutants needing to be verified in a laboratory, the conventional calculation method cannot judge the activity of the predicted stable mutant in a further catalytic reaction through calculation and cannot evaluate whether the predicted stable mutant has activity on an unnatural substrate.
On the basis, the protease mutant design method further increases a calculation method and a calculation process of a reaction energy barrier of each mutant in a catalytic chemical reaction process (natural substrate or non-natural substrate), realizes judgment of the catalytic activity of the predicted stable mutant, and can evaluate whether the predicted stable mutant has activity on the non-natural substrate.
(4) Calculating a reaction energy barrier: the method is based on the force field description of different substrate reaction states, and simultaneously provides the quantum mechanical description of chemical reactions in a valence bond theory framework. This allows the reaction energy barrier calculation to take advantage of both the computational speed of classical methods based on force fields and to carry a large amount of chemical and thermodynamic information, thus providing a meaningful physical description of the bonding and bond breaking processes. Preparation work before calculation of the reaction energy barrier comprises selection of a simulated force field, determination of a reaction rate limiting step of a target enzyme, a reaction transition state form of combination of a substrate small molecule compound and the like. After the calculation parameters are determined, the reaction energy barrier calculation is realized in the cadee process, and the calculation result is analyzed by using Qtools. In the cadee calculation procedure, for example, a simulation calculation time of 12.6ns is set as a default, and the system is first gradually heated from 0.01K to 300K in a simulation time of 90ps, for example, 200kcal mol is applied to all protein atoms in the simulation -1 
The harmonic suppression of (2) is performed by applying 20kcal mol to all water atoms -1 
Then gradually decreasing the harmonic suppression with increasing temperature. The temperature was adjusted using a Berendsen thermostat, using a time step of 1fs, and for all simulations, the reaction coordinates were set to λ =0.5 to begin the subsequent reaction energy barrier calculation for the reaction step near the transition state. A Molecular Dynamics (MD) simulation of 8ns was performed for each copy in four parallel calculations, using the simulation results as the starting point for the empirical valence simulation. Snapshots were taken every 1ns in an 8ns MD simulation, resulting in a near-transition state structure, and from this structure an empirical valence key simulation of 520ps, distributed over 26 FEP/US windows (λ =0, 0.05, 0.075, 0.1, 0.125, 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.425, 0.45, 0.55, 0.575, 0.6, 0.65, 0.70, 0.75, 0.80, 0.85, 0.875, 0.90, 0.925, 0.95, 1) per 20 ps. If the calculation flow of the default setting of cadee is used, the calculation process is quite long, so that the MD simulation time in the calculation process is reduced from 8ns to 4ns, the number of repeated calculation cycles in each parallel calculation copy is reduced to 4, the calculation time can be reduced to less than 24 hours, and the calculation result can not be greatly changed. The cadee can realize multitask parallel computation in a multi-core computer, so that the cadee can realize the screening of the catalytic activity of large-scale mutants on a high-performance computer.
The reaction energy barrier is the minimum energy required for the reactant molecules to reach the activating molecules, and the size of the energy barrier can reflect the difficulty of the reaction. As shown in FIG. 2, the difference between the potentials of the enzyme and substrate in the free state and the potential of the activated molecule formed by the combination of the two is the reaction energy barrier, i.e., the difference from the lowest point of energy (the optimal conformation for binding of the enzyme substrate) to the highest point of energy (the optimal conformation for binding of the enzyme substrate in the transition state). The difference between the lowest energy point of the initial state and the highest energy point of the activation state is obtained in the calculation process.
Experimental validation of mutant catalytic activity: the catalytic activity mutant obtained by calculating in the figure 1 is constructed and expressed in a laboratory, and then the catalytic activity on a target substrate is detected so as to verify whether the mutant predicted by the calculation method has the expected catalytic performance.
In the traditional directed evolution process, there is usually no clear mutation site for the evolved protease, and it is impossible to predict which amino acid residues are beneficial mutations, so that usually only a large number of diversity libraries can be constructed, and the screening of the libraries takes a lot of time and material resources. After the calculation design process is implemented, the structural stability of the protease is calculated on the basis of docking protease and substrate molecules, and virtual screening is performed by using the stability result processing method disclosed by the invention, so that a large number of unstable mutations and invalid mutations can be filtered, and a mutation library required to be screened in a laboratory can be greatly reduced. Regarding the current calculation of enzyme stability, on one hand, the calculation itself is an empirical algorithm, and it is impossible to ensure complete accuracy, and many factors such as flexible changes of amino acid skeleton are not regarded as variables for calculation feasibility, and in addition, small molecules in substrate docking are often regarded as rigid bodies for docking, and surrounding environmental factors are also simplified, so that in practical application of the calculation method, the success rate of laboratory verification of the predicted mutants is not high, and many good mutants are often missed. In addition, in the current calculation methods, direct sequencing of energy is generally adopted in stability calculation result processing so as to find out mutants with the lowest energy, and the mutants are considered to be mutant sequences with the highest possibility of activity on the target reaction. In the invention, the applicant optimizes the stability result processing method again by combining years of practical experience, and particularly proposes mutation frequency analysis in the invention, namely subtracting the amino acid residue type with higher occurrence frequency in the stable mutation from the amino acid residue type with higher occurrence frequency in the unstable mutation to obtain the theoretical mutable amino acid residue type on the site, and finally combining the mutable amino acid residues of each site to obtain the stable mutant, thereby ensuring that effective mutation cannot be easily eliminated to a certain extent, and the mutant is often ignored in the virtual screening process of the existing known algorithm.
According to the analysis of each parameter of an energy function in the current algorithm, the calculation in the current algorithm design actually calculates the stability of the protein structure of the enzyme mutant, and does not relate to the activity judgment of the enzyme mutant in the specific reaction process of catalysis. The stability of the whole structure of the enzyme mutant is one aspect of enzyme engineering and also a precondition for having catalytic activity, but the discovery (especially the initiation of activity on unnatural substrates) and improvement of the enzyme catalytic activity are more concerned by the industry, because the improvement of the catalytic efficiency on the substrates by using the high-activity enzyme mutant can realize industrial practicability, but the existing calculation method cannot realize judgment on the catalytic activity after the stability screening is completed. Therefore, on the basis of optimizing the stability calculation method, the invention further applies the reaction energy barrier calculation to judge the activity of the enzyme mutant in the specific catalytic reaction process, and considers a plurality of factors such as substrate molecules, intermediates, reaction transition states and the like, thereby realizing the judgment of the predicted catalytic activity of the stable mutant. The computational design method disclosed by the invention can obtain a mutant library with a more simplified range for experimental verification, so that the accuracy and the research and development efficiency of protease mutation virtual screening are greatly improved; the calculation of the reaction energy barrier effectively screens the enzyme mutants with activity, and improves the efficiency and the effect of enzyme engineering modification.
Drawings
FIG. 1 is a scheme of the computational design of protease mutants
FIG. 2 is a schematic diagram of reaction energy barrier
FIG. 3 shows the reaction of ketoreductase catalyzing 1, 3-butanediol to produce 4-hydroxy-2-butanone
FIG. 4 is a flow chart of reaction energy barrier calculation
FIG. 5 shows the number of 1, 3-butanediol atoms
FIG. 6 shows the reaction of ketoreductase to catalyze the formation of 1, 3-butanediol from 4-hydroxy-2-butanone
FIG. 7 shows the production of 4-hydroxy-2-butanone from 1, 3-butanediol by ketoreductase
FIG. 8 is a GC spectrum of 4-hydroxy-2-butanone and 1, 3-butanediol
FIG. 9 is a GC spectrum of (R) - (-) -1, 3-butanediol and (S) - (-) -1, 3-butanediol
Detailed Description
The following examples are provided to further illustrate the present invention and to clearly and completely describe the technical solutions of the present invention, but the present invention is not limited thereto. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the apparatus and reagents used in the following examples were commercially available ones unless otherwise specified.
The embodiment is as follows: design of mutants of ketoreductase active on a non-natural substrate (enantiomer of natural substrate)
The ketoreductase disclosed in patent CN111321129A can asymmetrically convert 4-hydroxy-2-butanone into more expensive (R) - (-) -1, 3-butanediol, which is very significant and industrially valuable; meanwhile, the enzyme can catalyze the reaction of converting alcohol into ketone, but the enzyme can only catalyze the substrate of (R) - (-) -1, 3-butanediol due to the specificity of the enzyme to the substrate. If racemic 1, 3-butanediol is used as the substrate (i.e., a 1-mixture of (R) - (-) -1, 3-butanediol and (S) - (-) -1, 3-butanediol), the (R) - (-) -1, 3-butanediol therein is converted into 4-hydroxy-2-butanone, leaving (S) - (-) -1, 3-butanediol unchanged, resulting in very low overall conversion. In actual industrial production, racemic 1, 3-butanediol is very easy to obtain, the price is far lower than that of chiral pure (R) - (-) -1, 3-butanediol, and the ketoreductase which can completely convert the racemic 1, 3-butanediol into 4-hydroxy-2-butanone is developed, so that the industrial application value is very good. This example starts with the ketoreductase disclosed in CN111321129A, which has a selectivity for (R) - (-) -1, 3-butanediol of >99%, and the amino acid sequence of which is shown in SEQ ID NO:2, and the DNA sequence is shown as SEQ ID NO:1 is shown in the specification; the amino acid sequence of SEQ ID NO:2 to NO activity on (S) - (-) -1, 3-butanediol, i.e. (S) - (-) -1, 3-butanediol has NO activity on SEQ ID NO:2 is a non-natural substrate. By adopting the calculation design algorithm disclosed by the invention, 10 ketoreductase mutants which have activity on (R) - (-) -1, 3-butanediol and (S) - (-) -1, 3-butanediol at the same time are predicted by virtual screening, and the ketoreductase mutant which maintains high catalytic activity and can convert (R) - (-) -1, 3-butanediol and (S) - (-) -1, 3-butanediol into 4-hydroxy-2-butanone is successfully found through experimental verification of the 10 predicted mutants. As shown in detail in figure 3.
Calculating a design process:
(1) Structure acquisition: the YASARA software package was used to compare SEQ ID NO:2 carrying out homologous modeling to obtain a structural model of the target protein.
(2) Substrate docking analysis: the (R) - (-) -1, 3-butanediol and (S) - (-) -1, 3-butanediol are respectively butted with target proteins, and the butting process is realized by a Yasara software package. The docking results were visually analyzed in Yasara. The results of the docking of (S) - (-) -1, 3-butanediol with the target protein show that the side chains of the amino acid residues at positions I144, H145, Q150, Y188 and the like are too hindered. Therefore, these sites were selected as virtual screens for the next step in this example.
(3) And (3) mutation stability calculation: the selection of the appropriate amino acid species is based on the mutated amino acid, as shown in Table 1. Then generating a mutation combination input file required by Rosetta by using python script, wherein the possible mutation combinations comprise 400 mutants. The free energy difference (Δ Δ G) of each mutant structure from the wild-type enzyme structure was calculated using the Cartesian _ ddg algorithm.
TABLE 1 amino acid mutation species
Then, the calculation results were analyzed by the statistical analysis method described above [ 3b ], and the results are shown in table 2.
TABLE 2 computational analysis results
(4) Calculating a reaction energy barrier: the mutations obtained in the previous step were combined to obtain 36 kinds of mutants in total, and the reaction energy barrier was calculated using the cadee calculation scheme as shown in FIG. 4, and the atomic number of 1, 3-butanediol as shown in FIG. 5.
Simulation setting: firstly, dissolving the simulation system in a C3 atom with a radius of (S) - (-) -1, 3-butanediol as a substrate
In a spherical drop of water molecules of the TIP3P model, wherein all atoms are in
The inner simulation center is completely movable, using 10kcal mol -1 
Harmonic suppressor constraint simulation center located at 17 and
all atoms in between, and using a harmonic force constant constraint of 200kcal
An atom other than the above. The H atoms were constrained in the solvent using the shift algorithm. Use of
To calculate non-bonding interactions between all atoms except those in the empirical valence domain, all interactions being explicitly calculated for these atoms to
All long-range electrostatics exceeding this threshold are treated using a Local Reactive Field (LRF) method.
From the results of the calculation of the reaction energy barrier, 10 mutants having the lowest reaction energy barrier were selected, as shown in Table 3.
TABLE 3 10 ketoreductase mutants with the lowest catalytic reaction energy barrier for (S) - (-) -1, 3-butanediol
(5) And (3) experimental verification:
(5.1) verification of the modification of chiral selectivity to 1.3-butanediol of 10 mutants shown in Table 3 by the reaction shown in FIG. 6A 0.1g of 4-hydroxy-2-butanone, 0.5mL of isopropanol, 0.1g of wet cells expressing ketoreductase (the method disclosed in the recombinant expression method in patent CN 111321129A), 0.005g of cofactor NAD +, and completion of the final reaction volume in the reaction flask to 5mL with 0.1M PBS (pH 7) were added to the reaction flask. The reaction was controlled in temperature 40 ℃ by a water bath, and after stirring at 400rpm for 1h, a sample was taken and examined by HPLC, and the chiral value (ee%, enantiomeric excess) of the obtained 1, 3-butanediol product was calculated as shown in Table 4. Wherein the formula for% ee is ee% = ([ R ] - [ S ])/([ R ] + [ S ]), where [ R ] represents the concentration of (R) - (-) -1, 3-butanediol in the sample, and [ S ] represents the concentration of (S) - (-) -1, 3-butanediol in the sample.
TABLE 4
Note 1: no (S) - (-) -1, 3-butanediol was detected.
The results in% ee of Table 4 show that 10 ketoreductase mutants (i.e., SEQ ID NOS: 4,6,8,10,12,14,16,18,20, and 22) catalyzed the reaction of FIG. 6 to produce not only (R) - (-) -1, 3-butanediol but also (S) - (-) -1, 3-butanediol, indicating that the 10 ketoreductase mutants were computationally predicted to have activity on (S) - (-) -1, 3-butanediol.
(5.2) for the racemic substrate in which industrially readily available (R) - (-) -1, 3-butanediol and (S) - (-) -1, 3-butanediol were present at the same time, the applicant carried out the verification of the catalytic performance of the above 10 ketoreductase mutants by experiment, and the reaction is shown in FIG. 7.
To a reaction flask, 0.1g of racemic 1,3-butanediol (i.e., a 1. The reaction was controlled at 40 ℃ with a water bath, stirred at 400rpm, and after 1h a sample was taken and tested by HPLC, and the calculated molar conversions are shown in Table 5.
TABLE 5
Since SEQ ID NO:2 is active only on (R) - (-) -1, 3-butanediol and not on (S) - (-) -1, 3-butanediol, which catalyzes the reaction shown in FIG. 7 with a theoretical maximum conversion of 50% of the substrate (i.e. racemic 1, 3-butanediol). The results of the conversion data in Table 5 show that 10 ketoreductase mutants (i.e., SEQ ID NOS: 4,6,8,10,12,14,16,18,20, and 22) were designed to achieve >50% conversion, indicating that these mutants are capable of converting (R) - (-) -1, 3-butanediol and (S) - (-) -1, 3-butanediol to 4-hydroxy-2-butanone simultaneously.
Method for analyzing and detecting compound
GC conversion analysis method: the chromatographic column is DB-WAX 15m 0.25mm 0.25 μm, and the carrier gas is N 2 The detector is FID, the injection port temperature is 250 ℃, and the split ratio is 28:1, the detector temperature is 300 ℃, the sample introduction amount is 1 μ L, the column temperature is 130 ℃, the temperature is raised to 150 ℃ at a rate of 10 ℃/min and then raised to 160 ℃ at a rate of 20 ℃/min, wherein the retention time of 4-hydroxy-2-butanone is 1.5min, and the retention time of 1, 3-butanediol is 2.3min, as shown in figure 8.
GC chiral analysis method: the sample pretreatment method comprises the steps of taking 200 mu L of inactivation solution, adding 50 mu L of MSTFA and 30 mu L of anhydrous pyridine into a 1.5mL centrifuge tube, uniformly mixing, and oscillating for 30min. The chromatographic column is CP-Chirasil Dex CB (CP 7502) 25m 0.25mm 0.25 μm, and the carrier gas is N 2 The detector is FID, the injection port temperature is 250 ℃, and the split ratio is 28:1, the detector temperature is 300 ℃, the sample injection amount is 1 μ L, the column temperature is 105 ℃, the stop time is 9min, the retention time of (R) - (-) -1, 3-butanediol is 6.4min, and the retention time of (S) - (-) -1, 3-butanediol is 6.6min, as shown in figure 9.
It should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.
Sequence listing
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gacaagggta ataaaaccgt tgaagatatt aaagcacagg gcggtgaagc tagttttgtg 180
aaagcggaca ccagcaaccc ggaagaagtg gaagccctgg ttaaacgtac ggtcgaaatt 240
tatggtcgcc tggatgtggc atgcaacaat gctggcattg cgggtgaaaa ggcactggct 300
ggtgattacg gcctggacag ctggcgtaaa gttctgtctg tgaatctgga cggtgtcttc 360
tatggctgta aatacgaact ggaacaaatg gagaaaaacg gcggtggcgt tatcgtcaat 420
atggccagca ttcatggtat cgttgcgcag ccgctgaact ctgcatatac ctctgcgaaa 480
cacgccgtgg ttggcctgac gaaaaacatt ggtgctgatt atggccagaa aaacatccgt 540
tgcaatgcgg tgtgcccggg ctacattgaa accccgctgc tggaatcact gacgaaagaa 600
atgaaagaag ccctgatctc gaaacacccg atgggtcgcc tgggcaaacc ggaagaagtg 660
gcagaactgg ttctgtttct gagttccgaa aaatcatcgt tcatgaccgg tggctattac 720
ctggtcgatg gtggctacac ggcagtg 747
<210> 2
<211> 249
<212> PRT
<213> Artificial Sequence
<400> 2
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp Lys Gly Asn Lys Thr Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Val Ala Cys Asn Asn Ala Gly Ile Ala Gly Glu
85 90 95
Lys Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Val Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Ile
130 135 140
His Gly Ile Val Ala Gln Pro Leu Asn Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Asp Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Cys Pro Gly Tyr Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
<210> 3
<211> 747
<212> DNA
<213> Artificial Sequence
<400> 3
atgggtatcc tggacaacaa agtcgcactg gttacgggcg ctggttcggg catcggtctg 60
gcggtggcac actcctacgc taaagaaggc gctaaagtca ttgtgtcaga tatcaacgaa 120
gacaagggta ataaaaccgt tgaagatatt aaagcacagg gcggtgaagc tagttttgtg 180
aaagcggaca ccagcaaccc ggaagaagtg gaagccctgg ttaaacgtac ggtcgaaatt 240
tatggtcgcc tggatgtggc atgcaacaat gctggcattg cgggtgaaaa ggcactggct 300
ggtgattacg gcctggacag ctggcgtaaa gttctgtctg tgaatctgga cggtgtcttc 360
tatggctgta aatacgaact ggaacaaatg gagaaaaacg gcggtggcgt tatcgtcaat 420
atggccagcg gccatggtat cgttgcgcag ccgctgaact ctgcatatac ctctgcgaaa 480
cacgccgtgg ttggcctgac gaaaaacatt ggtgctgatt atggccagaa aaacatccgt 540
tgcaatgcgg tgtgcccggg ctacattgaa accccgctgc tggaatcact gacgaaagaa 600
atgaaagaag ccctgatctc gaaacacccg atgggtcgcc tgggcaaacc ggaagaagtg 660
gcagaactgg ttctgtttct gagttccgaa aaatcatcgt tcatgaccgg tggctattac 720
ctggtcgatg gtggctacac ggcagtg 747
<210> 4
<211> 249
<212> PRT
<213> Artificial Sequence
<400> 4
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp Lys Gly Asn Lys Thr Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Val Ala Cys Asn Asn Ala Gly Ile Ala Gly Glu
85 90 95
Lys Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Val Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Gly
130 135 140
His Gly Ile Val Ala Gln Pro Leu Asn Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Asp Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Cys Pro Gly Tyr Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
<210> 5
<211> 747
<212> DNA
<213> Artificial Sequence
<400> 5
atgggtatcc tggacaacaa agtcgcactg gttacgggcg ctggttcggg catcggtctg 60
gcggtggcac actcctacgc taaagaaggc gctaaagtca ttgtgtcaga tatcaacgaa 120
gacaagggta ataaaaccgt tgaagatatt aaagcacagg gcggtgaagc tagttttgtg 180
aaagcggaca ccagcaaccc ggaagaagtg gaagccctgg ttaaacgtac ggtcgaaatt 240
tatggtcgcc tggatgtggc atgcaacaat gctggcattg cgggtgaaaa ggcactggct 300
ggtgattacg gcctggacag ctggcgtaaa gttctgtctg tgaatctgga cggtgtcttc 360
tatggctgta aatacgaact ggaacaaatg gagaaaaacg gcggtggcgt tatcgtcaat 420
atggccagca ttcatggtat cgttgcgcag ccgctgaact ctgcatatac ctctgcgaaa 480
cacgccgtgg ttggcctgac gaaaaacatt ggtgctgatt atggccagaa aaacatccgt 540
tgcaatgcgg tgtgcccggg cgcgattgaa accccgctgc tggaatcact gacgaaagaa 600
atgaaagaag ccctgatctc gaaacacccg atgggtcgcc tgggcaaacc ggaagaagtg 660
gcagaactgg ttctgtttct gagttccgaa aaatcatcgt tcatgaccgg tggctattac 720
ctggtcgatg gtggctacac ggcagtg 747
<210> 6
<211> 249
<212> PRT
<213> Artificial Sequence
<400> 6
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp Lys Gly Asn Lys Thr Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Val Ala Cys Asn Asn Ala Gly Ile Ala Gly Glu
85 90 95
Lys Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Val Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Ile
130 135 140
His Gly Ile Val Ala Gln Pro Leu Asn Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Asp Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Cys Pro Gly Ala Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
<210> 7
<211> 747
<212> DNA
<213> Artificial Sequence
<400> 7
atgggtatcc tggacaacaa agtcgcactg gttacgggcg ctggttcggg catcggtctg 60
gcggtggcac actcctacgc taaagaaggc gctaaagtca ttgtgtcaga tatcaacgaa 120
gacaagggta ataaaaccgt tgaagatatt aaagcacagg gcggtgaagc tagttttgtg 180
aaagcggaca ccagcaaccc ggaagaagtg gaagccctgg ttaaacgtac ggtcgaaatt 240
tatggtcgcc tggatgtggc atgcaacaat gctggcattg cgggtgaaaa ggcactggct 300
ggtgattacg gcctggacag ctggcgtaaa gttctgtctg tgaatctgga cggtgtcttc 360
tatggctgta aatacgaact ggaacaaatg gagaaaaacg gcggtggcgt tatcgtcaat 420
atggccagca ttcatggtat cgttgcgcag ccgctgaact ctgcatatac ctctgcgaaa 480
cacgccgtgg ttggcctgac gaaaaacatt ggtgctgatt atggccagaa aaacatccgt 540
tgcaatgcgg tgtgcccggg cggcattgaa accccgctgc tggaatcact gacgaaagaa 600
atgaaagaag ccctgatctc gaaacacccg atgggtcgcc tgggcaaacc ggaagaagtg 660
gcagaactgg ttctgtttct gagttccgaa aaatcatcgt tcatgaccgg tggctattac 720
ctggtcgatg gtggctacac ggcagtg 747
<210> 8
<211> 249
<212> PRT
<213> Artificial Sequence
<400> 8
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp Lys Gly Asn Lys Thr Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Val Ala Cys Asn Asn Ala Gly Ile Ala Gly Glu
85 90 95
Lys Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Val Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Ile
130 135 140
His Gly Ile Val Ala Gln Pro Leu Asn Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Asp Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Cys Pro Gly Gly Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
<210> 9
<211> 747
<212> DNA
<213> Artificial Sequence
<400> 9
atgggtatcc tggacaacaa agtcgcactg gttacgggcg ctggttcggg catcggtctg 60
gcggtggcac actcctacgc taaagaaggc gctaaagtca ttgtgtcaga tatcaacgaa 120
gacaagggta ataaaaccgt tgaagatatt aaagcacagg gcggtgaagc tagttttgtg 180
aaagcggaca ccagcaaccc ggaagaagtg gaagccctgg ttaaacgtac ggtcgaaatt 240
tatggtcgcc tggatgtggc atgcaacaat gctggcattg cgggtgaaaa ggcactggct 300
ggtgattacg gcctggacag ctggcgtaaa gttctgtctg tgaatctgga cggtgtcttc 360
tatggctgta aatacgaact ggaacaaatg gagaaaaacg gcggtggcgt tatcgtcaat 420
atggccagca tttgcggtat cgttgcgcag ccgctgaact ctgcatatac ctctgcgaaa 480
cacgccgtgg ttggcctgac gaaaaacatt ggtgctgatt atggccagaa aaacatccgt 540
tgcaatgcgg tgtgcccggg ctacattgaa accccgctgc tggaatcact gacgaaagaa 600
atgaaagaag ccctgatctc gaaacacccg atgggtcgcc tgggcaaacc ggaagaagtg 660
gcagaactgg ttctgtttct gagttccgaa aaatcatcgt tcatgaccgg tggctattac 720
ctggtcgatg gtggctacac ggcagtg 747
<210> 10
<211> 249
<212> PRT
<213> Artificial Sequence
<400> 10
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp Lys Gly Asn Lys Thr Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Val Ala Cys Asn Asn Ala Gly Ile Ala Gly Glu
85 90 95
Lys Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Val Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Ile
130 135 140
Cys Gly Ile Val Ala Gln Pro Leu Asn Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Asp Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Cys Pro Gly Tyr Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
<210> 11
<211> 747
<212> DNA
<213> Artificial Sequence
<400> 11
atgggtatcc tggacaacaa agtcgcactg gttacgggcg ctggttcggg catcggtctg 60
gcggtggcac actcctacgc taaagaaggc gctaaagtca ttgtgtcaga tatcaacgaa 120
gacaagggta ataaaaccgt tgaagatatt aaagcacagg gcggtgaagc tagttttgtg 180
aaagcggaca ccagcaaccc ggaagaagtg gaagccctgg ttaaacgtac ggtcgaaatt 240
tatggtcgcc tggatgtggc atgcaacaat gctggcattg cgggtgaaaa ggcactggct 300
ggtgattacg gcctggacag ctggcgtaaa gttctgtctg tgaatctgga cggtgtcttc 360
tatggctgta aatacgaact ggaacaaatg gagaaaaacg gcggtggcgt tatcgtcaat 420
atggccagca tttgcggtat cgttgcgcag ccgctgaact ctgcatatac ctctgcgaaa 480
cacgccgtgg ttggcctgac gaaaaacatt ggtgctgatt atggccagaa aaacatccgt 540
tgcaatgcgg tgtgcccggg cgcgattgaa accccgctgc tggaatcact gacgaaagaa 600
atgaaagaag ccctgatctc gaaacacccg atgggtcgcc tgggcaaacc ggaagaagtg 660
gcagaactgg ttctgtttct gagttccgaa aaatcatcgt tcatgaccgg tggctattac 720
ctggtcgatg gtggctacac ggcagtg 747
<210> 12
<211> 249
<212> PRT
<213> Artificial Sequence
<400> 12
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp Lys Gly Asn Lys Thr Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Val Ala Cys Asn Asn Ala Gly Ile Ala Gly Glu
85 90 95
Lys Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Val Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Ile
130 135 140
Cys Gly Ile Val Ala Gln Pro Leu Asn Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Asp Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Cys Pro Gly Ala Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
<210> 13
<211> 747
<212> DNA
<213> Artificial Sequence
<400> 13
atgggtatcc tggacaacaa agtcgcactg gttacgggcg ctggttcggg catcggtctg 60
gcggtggcac actcctacgc taaagaaggc gctaaagtca ttgtgtcaga tatcaacgaa 120
gacaagggta ataaaaccgt tgaagatatt aaagcacagg gcggtgaagc tagttttgtg 180
aaagcggaca ccagcaaccc ggaagaagtg gaagccctgg ttaaacgtac ggtcgaaatt 240
tatggtcgcc tggatgtggc atgcaacaat gctggcattg cgggtgaaaa ggcactggct 300
ggtgattacg gcctggacag ctggcgtaaa gttctgtctg tgaatctgga cggtgtcttc 360
tatggctgta aatacgaact ggaacaaatg gagaaaaacg gcggtggcgt tatcgtcaat 420
atggccagca tttgcggtat cgttgcgcag ccgctgaact ctgcatatac ctctgcgaaa 480
cacgccgtgg ttggcctgac gaaaaacatt ggtgctgatt atggccagaa aaacatccgt 540
tgcaatgcgg tgtgcccggg cggcattgaa accccgctgc tggaatcact gacgaaagaa 600
atgaaagaag ccctgatctc gaaacacccg atgggtcgcc tgggcaaacc ggaagaagtg 660
gcagaactgg ttctgtttct gagttccgaa aaatcatcgt tcatgaccgg tggctattac 720
ctggtcgatg gtggctacac ggcagtg 747
<210> 14
<211> 249
<212> PRT
<213> Artificial Sequence
<400> 14
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp Lys Gly Asn Lys Thr Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Val Ala Cys Asn Asn Ala Gly Ile Ala Gly Glu
85 90 95
Lys Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Val Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Ile
130 135 140
Cys Gly Ile Val Ala Gln Pro Leu Asn Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Asp Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Cys Pro Gly Gly Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
<210> 15
<211> 747
<212> DNA
<213> Artificial Sequence
<400> 15
atgggtatcc tggacaacaa agtcgcactg gttacgggcg ctggttcggg catcggtctg 60
gcggtggcac actcctacgc taaagaaggc gctaaagtca ttgtgtcaga tatcaacgaa 120
gacaagggta ataaaaccgt tgaagatatt aaagcacagg gcggtgaagc tagttttgtg 180
aaagcggaca ccagcaaccc ggaagaagtg gaagccctgg ttaaacgtac ggtcgaaatt 240
tatggtcgcc tggatgtggc atgcaacaat gctggcattg cgggtgaaaa ggcactggct 300
ggtgattacg gcctggacag ctggcgtaaa gttctgtctg tgaatctgga cggtgtcttc 360
tatggctgta aatacgaact ggaacaaatg gagaaaaacg gcggtggcgt tatcgtcaat 420
atggccagcg gctgcggtat cgttgcgcag ccgctgaact ctgcatatac ctctgcgaaa 480
cacgccgtgg ttggcctgac gaaaaacatt ggtgctgatt atggccagaa aaacatccgt 540
tgcaatgcgg tgtgcccggg ctacattgaa accccgctgc tggaatcact gacgaaagaa 600
atgaaagaag ccctgatctc gaaacacccg atgggtcgcc tgggcaaacc ggaagaagtg 660
gcagaactgg ttctgtttct gagttccgaa aaatcatcgt tcatgaccgg tggctattac 720
ctggtcgatg gtggctacac ggcagtg 747
<210> 16
<211> 249
<212> PRT
<213> Artificial Sequence
<400> 16
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp Lys Gly Asn Lys Thr Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Val Ala Cys Asn Asn Ala Gly Ile Ala Gly Glu
85 90 95
Lys Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Val Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Gly
130 135 140
Cys Gly Ile Val Ala Gln Pro Leu Asn Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Asp Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Cys Pro Gly Tyr Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
<210> 17
<211> 747
<212> DNA
<213> Artificial Sequence
<400> 17
atgggtatcc tggacaacaa agtcgcactg gttacgggcg ctggttcggg catcggtctg 60
gcggtggcac actcctacgc taaagaaggc gctaaagtca ttgtgtcaga tatcaacgaa 120
gacaagggta ataaaaccgt tgaagatatt aaagcacagg gcggtgaagc tagttttgtg 180
aaagcggaca ccagcaaccc ggaagaagtg gaagccctgg ttaaacgtac ggtcgaaatt 240
tatggtcgcc tggatgtggc atgcaacaat gctggcattg cgggtgaaaa ggcactggct 300
ggtgattacg gcctggacag ctggcgtaaa gttctgtctg tgaatctgga cggtgtcttc 360
tatggctgta aatacgaact ggaacaaatg gagaaaaacg gcggtggcgt tatcgtcaat 420
atggccagcg gctgcggtat cgttgcgcag ccgctgaact ctgcatatac ctctgcgaaa 480
cacgccgtgg ttggcctgac gaaaaacatt ggtgctgatt atggccagaa aaacatccgt 540
tgcaatgcgg tgtgcccggg cgcgattgaa accccgctgc tggaatcact gacgaaagaa 600
atgaaagaag ccctgatctc gaaacacccg atgggtcgcc tgggcaaacc ggaagaagtg 660
gcagaactgg ttctgtttct gagttccgaa aaatcatcgt tcatgaccgg tggctattac 720
ctggtcgatg gtggctacac ggcagtg 747
<210> 18
<211> 249
<212> PRT
<213> Artificial Sequence
<400> 18
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp Lys Gly Asn Lys Thr Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Val Ala Cys Asn Asn Ala Gly Ile Ala Gly Glu
85 90 95
Lys Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Val Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Gly
130 135 140
Cys Gly Ile Val Ala Gln Pro Leu Asn Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Asp Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Cys Pro Gly Ala Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
<210> 19
<211> 747
<212> DNA
<213> Artificial Sequence
<400> 19
atgggtatcc tggacaacaa agtcgcactg gttacgggcg ctggttcggg catcggtctg 60
gcggtggcac actcctacgc taaagaaggc gctaaagtca ttgtgtcaga tatcaacgaa 120
gacaagggta ataaaaccgt tgaagatatt aaagcacagg gcggtgaagc tagttttgtg 180
aaagcggaca ccagcaaccc ggaagaagtg gaagccctgg ttaaacgtac ggtcgaaatt 240
tatggtcgcc tggatgtggc atgcaacaat gctggcattg cgggtgaaaa ggcactggct 300
ggtgattacg gcctggacag ctggcgtaaa gttctgtctg tgaatctgga cggtgtcttc 360
tatggctgta aatacgaact ggaacaaatg gagaaaaacg gcggtggcgt tatcgtcaat 420
atggccagcg gctgcggtat cgttgcgcag ccgctgaact ctgcatatac ctctgcgaaa 480
cacgccgtgg ttggcctgac gaaaaacatt ggtgctgatt atggccagaa aaacatccgt 540
tgcaatgcgg tgtgcccggg cggcattgaa accccgctgc tggaatcact gacgaaagaa 600
atgaaagaag ccctgatctc gaaacacccg atgggtcgcc tgggcaaacc ggaagaagtg 660
gcagaactgg ttctgtttct gagttccgaa aaatcatcgt tcatgaccgg tggctattac 720
ctggtcgatg gtggctacac ggcagtg 747
<210> 20
<211> 249
<212> PRT
<213> Artificial Sequence
<400> 20
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp Lys Gly Asn Lys Thr Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Val Ala Cys Asn Asn Ala Gly Ile Ala Gly Glu
85 90 95
Lys Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Val Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Gly
130 135 140
Cys Gly Ile Val Ala Gln Pro Leu Asn Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Asp Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Cys Pro Gly Gly Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
<210> 21
<211> 747
<212> DNA
<213> Artificial Sequence
<400> 21
atgggtatcc tggacaacaa agtcgcactg gttacgggcg ctggttcggg catcggtctg 60
gcggtggcac actcctacgc taaagaaggc gctaaagtca ttgtgtcaga tatcaacgaa 120
gacaagggta ataaaaccgt tgaagatatt aaagcacagg gcggtgaagc tagttttgtg 180
aaagcggaca ccagcaaccc ggaagaagtg gaagccctgg ttaaacgtac ggtcgaaatt 240
tatggtcgcc tggatgtggc atgcaacaat gctggcattg cgggtgaaaa ggcactggct 300
ggtgattacg gcctggacag ctggcgtaaa gttctgtctg tgaatctgga cggtgtcttc 360
tatggctgta aatacgaact ggaacaaatg gagaaaaacg gcggtggcgt tatcgtcaat 420
atggccagcg gccatggtat cgttgcgcag ccgctgaact ctgcatatac ctctgcgaaa 480
cacgccgtgg ttggcctgac gaaaaacatt ggtgctgatt atggccagaa aaacatccgt 540
tgcaatgcgg tgtgcccggg cgcgattgaa accccgctgc tggaatcact gacgaaagaa 600
atgaaagaag ccctgatctc gaaacacccg atgggtcgcc tgggcaaacc ggaagaagtg 660
gcagaactgg ttctgtttct gagttccgaa aaatcatcgt tcatgaccgg tggctattac 720
ctggtcgatg gtggctacac ggcagtg 747
<210> 22
<211> 249
<212> PRT
<213> Artificial Sequence
<400> 22
Met Gly Ile Leu Asp Asn Lys Val Ala Leu Val Thr Gly Ala Gly Ser
1 5 10 15
Gly Ile Gly Leu Ala Val Ala His Ser Tyr Ala Lys Glu Gly Ala Lys
20 25 30
Val Ile Val Ser Asp Ile Asn Glu Asp Lys Gly Asn Lys Thr Val Glu
35 40 45
Asp Ile Lys Ala Gln Gly Gly Glu Ala Ser Phe Val Lys Ala Asp Thr
50 55 60
Ser Asn Pro Glu Glu Val Glu Ala Leu Val Lys Arg Thr Val Glu Ile
65 70 75 80
Tyr Gly Arg Leu Asp Val Ala Cys Asn Asn Ala Gly Ile Ala Gly Glu
85 90 95
Lys Ala Leu Ala Gly Asp Tyr Gly Leu Asp Ser Trp Arg Lys Val Leu
100 105 110
Ser Val Asn Leu Asp Gly Val Phe Tyr Gly Cys Lys Tyr Glu Leu Glu
115 120 125
Gln Met Glu Lys Asn Gly Gly Gly Val Ile Val Asn Met Ala Ser Gly
130 135 140
His Gly Ile Val Ala Gln Pro Leu Asn Ser Ala Tyr Thr Ser Ala Lys
145 150 155 160
His Ala Val Val Gly Leu Thr Lys Asn Ile Gly Ala Asp Tyr Gly Gln
165 170 175
Lys Asn Ile Arg Cys Asn Ala Val Cys Pro Gly Ala Ile Glu Thr Pro
180 185 190
Leu Leu Glu Ser Leu Thr Lys Glu Met Lys Glu Ala Leu Ile Ser Lys
195 200 205
His Pro Met Gly Arg Leu Gly Lys Pro Glu Glu Val Ala Glu Leu Val
210 215 220
Leu Phe Leu Ser Ser Glu Lys Ser Ser Phe Met Thr Gly Gly Tyr Tyr
225 230 235 240
Leu Val Asp Gly Gly Tyr Thr Ala Val
245
Claims (8)
1. A computational method for designing ketoreductase mutants active on (S) - (-) -1, 3-butanediol, the design flow is: firstly, obtaining a three-dimensional structure model of ketoreductase; then, docking and analyzing the protein structure and the substrate molecules to determine the amino acid sites to be mutated; appointing alternative amino acid residue mutation for each amino acid site to be mutated, exhaustively exhausting all mutation combinations of all the sites to be mutated, calculating the stability of each mutant by using a protein stability calculation method, processing the stability calculation result and selecting stable amino acid residue mutation combinations to obtain a predicted stable mutant; and finally, calculating the reaction energy barrier of the obtained stable mutant aiming at the target reaction, and selecting the mutant with lower reaction energy barrier to obtain the predicted catalytic activity mutant.
2. The method of claim 1, wherein the software tool used to interface the substrate molecule with the protease is Yasara, discovery studio or Rosetta.
3. The calculation method according to claim 1, wherein the method used for calculating the structural stability of the protein mutation is a tool that can perform stability calculation, such as ddg _ monomer, cartesian _ ddg, foldX, provean, ELASPIC, amber TI, and the like.
4. The method of claim 1, wherein the results of the stability calculation are statistically processed to yield a predicted stable mutant:
the method comprises the steps of sequencing delta G results of mutants from low to high, selecting partial mutants sequenced at the front and partial mutants sequenced at the back for mutation frequency analysis, subtracting the amino acid residue type with higher occurrence frequency in an unstable mutant from the amino acid residue type with higher occurrence frequency in the stable mutant for a specific amino acid site to obtain a set of theoretically mutable amino acid residue types on the site, and finally arranging and combining the mutable amino acid residues of all the sites to serve as the stable mutant obtained by computer virtual screening prediction.
5. The method of claim 1, wherein the "reaction energy barrier" in the "calculation of reaction energy barriers" is defined as a difference from a lowest point of energy (optimal conformation for binding of enzyme substrate) to a highest point of energy (optimal conformation for binding of enzyme substrate transition state).
6. Ketoreductase mutant polypeptide obtained by the calculation method according to claim 1, capable of simultaneously catalyzing, under suitable reaction conditions, the reaction of (R) - (-) -1, 3-butanediol and (S) - (-) -1, 3-butanediol for the synthesis of 4-hydroxy-2-butanone, wherein the amino acid sequence of said polypeptide comprises the residue difference X145C, X188G or X188A compared to the sequence of SEQ ID NO. 2.
7. The polypeptide of claim 6, wherein the amino acid sequence of the polypeptide further comprises the residue difference X144G as compared to the sequence of SEQ ID NO 2.
8. The polypeptide of claims 6-7, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 4. 6,8,10,12,14,16,18,20, 22.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110486993.8A CN115295076A (en) | 2021-05-03 | 2021-05-03 | Design and application of ketoreductase mutant |
| PCT/CN2022/087782 WO2022233232A1 (en) | 2021-05-03 | 2022-04-20 | A computational methodology for designing artificial enzyme variants with activity on non-natural substrates |
| EP22798573.6A EP4334945A1 (en) | 2021-05-03 | 2022-04-20 | A computational methodology for designing artificial enzyme variants with activity on non-natural substrates |
| EP22798574.4A EP4334946A1 (en) | 2021-05-03 | 2022-04-20 | Artificial ketoreductase variants and design methodology thereof |
| PCT/CN2022/087783 WO2022233233A1 (en) | 2021-05-03 | 2022-04-20 | Artificial ketoreductase variants and design methodology thereof |
| JP2023556579A JP2024512446A (en) | 2021-05-03 | 2022-04-20 | Artificial ketoreductase mutants and their design methodology |
| JP2023556577A JP2024512445A (en) | 2021-05-03 | 2022-04-20 | Computational methodology for designing artificial enzyme variants with activity against non-natural substrates |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110486993.8A CN115295076A (en) | 2021-05-03 | 2021-05-03 | Design and application of ketoreductase mutant |
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| CN115295076A true CN115295076A (en) | 2022-11-04 |
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