Novel l-rhamnose isomerases

Abstract

An L-rhamnose isomerase which can be obtained from a microorganism belonging to the genus Erwinia, has a subunit of a molecular mass of about 48 kDa as determined by SDS-PAGE, and has the substrate specificity of (A) and (B) described below. (A) has an isomerase activity which recognizes and reacts with CHO group at C1 and OH group at C2 of an aldose, converts CHO group at C1 into OH group, and converts OH group at C2 into CO group, or recognizes and reacts with OH group at C1 and CO group at C2 of a ketose, converts OH group at C1 into CHO group, and converts CO group at C2 into OH group. (B) has an activity of catalyzing isomerization between L-rhamnose and L-rhamnulose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-psicose.

Description

TECHNICAL FIELD

[0001] The present application relates to a novel L-rhamnose isomerase, a method for producing the same, a microorganism producing the same, a DNA encoding the enzyme and a recombinant vector and a transformed host cell containing the same, a L-rhamnose isomerase variant, and a method for producing a ketose or an aldose using the L-rhamnose isomerase or the variant. BACKGROUND

[0002] A rare sugar is defined by the International Society of Rare Sugars as "a sugar that exists only in a trace amount in nature", that is, a monosaccharide that exists in a small amount in nature.

[0003] There are four aldoses, two ketoses, and three sugar alcohols of monosaccharides having four carbon atoms (tetroses). There are eight aldoses, four ketoses, and four sugar alcohols of monosaccharides having five carbon atoms (pentoses). There are 34 kinds of monosaccharides having six carbon atoms (hexoses) in total, 16 kinds of aldoses, eight kinds of ketoses, and ten kinds of sugar alcohols. There are 32 kinds of heptaldoses, 16 kinds of heptaketoses, and 16 kinds of heptitols of monosaccharides having seven carbon atoms (heptoses).

[0004] For example, among hexoses (hexoses), the aldoses that are normally present in a large amount in nature are six kinds of D-glucose, D-galactose, D-mannose, D-ribose, D-xylose, and L-arabinose, and aldoses other than these, such as D-allose, are defined as rare sugars. That is, as sugars that are considered to be rare sugars among aldoses, L-allose, L-gulose, L-glucose, L-galactose, L-arabinose, L-idose, L-mannose, L-talose, D-talose, D-idose, D-arabinose, D-gulose, and D-allose can be listed. As ketoses, D-fructose is present in a large amount in nature, and in contrast to this, other ketoses are not present in a large amount in nature, and thus can be called rare sugars. As sugars that are considered to be rare sugars among ketoses, D-allose (alias D-psicose), D-tagatose, D-sorbose, L-fructose, L-allose (alias L-psicose), L-tagatose, and L-sorbose can be listed.

[0005] Recently, a mass production technique of D-allose (alias D-psicose) that is a basic raw material for producing all rare sugars has been established, and this makes it possible to produce difficult rare sugars. It is considered that D-allose becomes a center for producing new rare sugars such as D-allose and the like by enzyme reactions further.

[0006] D-allose is an aldose of an isomer different from D-glucose in the direction of OH group of only the 3rd carbon, and also known as a rare sugar monosaccharide of an isomer of D-allulose which is a ketose. D-allose is known to have a medical composition for treating a kidney disease selected from acute renal failure and uremia (Patent Literature 1), a medical product for delaying onset or exacerbation of motor disorder due to amyotrophic lateral sclerosis (Patent Literature 2), a blood pressure rising inhibitor (Patent Literature 3), a preparation characterized by inhibiting angiogenesis (Patent Literature 4), a T lymphocyte proliferation inhibitor (Patent Literature 5), or a peritoneal deterioration inhibitor used in combination with a peritoneal dialysis solution (Patent Literature 6) as effective ingredients, and also known as an edible pesticide (Patent Literature 7). Recently, applications have been filed for an anti-tumor effect on intake into renal cell carcinoma cells (Japanese Patent Application No. 2019-52195) and a strong anti-tumor effect on human urothelial carcinoma cells (Japanese Patent Application No. 2019-58477). Due to such characteristics, D-allose is attracting attention as a new generation of core raw material in the medical and pesticide fields, and the establishment of a mass production technology of D-allose is required after D-allulose.

[0007] As a method of producing D-allose using an enzyme produced by a microorganism, the inventors of the present application have developed a technology of producing allose from allulose using L-rhamnose isomerase isolated from Pseudomonas stutzeri (Non-Patent Literature 1). L-rhamnose isomerase is an enzyme that catalyzes a reversible isomerization reaction between L-rhamnose and L-rhamnulose, but it is known that L-rhamnose isomerase from P. stutzeri can act not only between L-rhamnose and L-rhamnulose, but also between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-gulose and D-sorbose, between D-ribose and D-ribulose, between D-allose and D-allulose, between L-talose and L-tagatose, and has a wide substrate specificity. With this wide substrate specificity, various rare aldoses and ketoses on Izumo ring can be produced, centering on the conversion from D-allulose to D-allose.

[0008] Prior Art Documents

[0009] Patent Literature

[0010] Patent Literature 1: Japanese Patent No. 5330976

[0011] Patent Literature 2: Japanese Patent No. 5317055

[0012] Patent Literature 3: Japanese Patent No. 5158779

[0013] Patent Literature 4: Japanese Patent No. 4943839

[0014] Patent Literature 5: Japanese Patent No. 4724824

[0015] Patent Literature 6: Japanese Patent Application Laid-Open No. 2009-269887

[0016] Patent Literature 7: Japanese Patent No. 5816871

[0017] Patent Literature 8: U.S. Patent No. 10,480,018

[0018] Non-Patent Literature

[0019] Non-Patent Literature 1: J. Ferment. Bioeng. (1997) Vol. 84, p. 319 SUMMARY

[0020] PROBLEMS TO BE SOLVED BY THE INVENTION

[0021] Now, with the production of most of the rare sugars becoming possible, new problems have arisen in the production of rare sugars. For example, the transition from the mass production of the physiologically active rare sugar D-allose at the laboratory level to the mass production on a market scale involving industrialization has become a new problem for the present inventors. That is, in order to achieve efficient mass production in the production of rare sugars, it is considered that it is first necessary to screen enzymes having higher thermostable activity and a wide range of optimum pH.

[0022] As described above, in the prior art, the rare sugar D-allose is produced using the well-known L-rhamnose isomerase (EC 5.3.1.14) as a raw material of pure rare sugar D- psicose. The L-rhamnose isomerase is an enzyme that catalyzes the isomerization reaction from L-rhamnose to L-rhamnulose, and also catalyzes the isomerization from L-rhamnulose to L-rhamnose. It is known to also act on the isomerization between D-allose and D-psicose. Since isomerases are named according to the substrate that shows the highest activity, the substrate specificity is various even in the enzyme named L-rhamnose isomerase.

[0023] In addition, among the well-known enzymes, there is an enzyme that generates D-altrose (D- altrose) as an aldose as a by-product when D-allose is generated, and the presence of D-altrose as a by-product is a cause of the decrease in the yield of D-allose in the purification process of D-allose. Therefore, the purity of the rare sugar D-allose in mass production is also one of the problems.

[0024] The present application provides a novel L-rhamnose isomerase from a microorganism that is recognized as being used in the manufacture of food and considered to be substantially non-toxic and has high activity, high yield, and the ability to isomerize D-allulose to D-allose, provides a microorganism having the enzyme, and provides a production method using the enzyme as its subject.

[0025] Technical solution for solving the technical problem

[0026] The present inventors, focusing on the fact that not only in Japan but also in Europe and the United States, bacteria considered to be substantially non-toxic are listed as being recognized for use as food, collected soil from various places, and isolated microorganisms therefrom, and have continued to search for microorganisms having L-rhamnose isomerase activity.

[0027] As a result, among the numerous isolated strains, a microorganism belonging to the genus Erwinia that produces a novel L-rhamnose isomerase was found. The Erwinia billingiae, which is a microorganism of the genus Erwinia, produces a novel L-rhamnose isomerase that has high activity and high heat resistance.

[0028] The novel L-rhamnose isomerase from the microorganism catalyzes an isomerization reaction between an aldose and a corresponding ketose, has isomerase activity between an aldose and a ketose, that is, recognizes and reacts with the CHO group at C1 and the OH group at C2 of an aldose, converts the CHO group at C1 to an OH group, and converts the OH group at C2 to a CO group, or recognizes and reacts with the OH group at C1 and the CO group at C2 of a ketose, converts the OH group at C1 to a CHO group, and converts the CO group at C2 to an OH group. In addition, in the production of D-allose using D-allulose as a substrate, since D-altrulose is not generated as a byproduct, it is suitable for the production of D-allose.

[0029] That is, the present application relates to the L-rhamnose isomerase described in (1) to (4) below, and the microorganism described in (5) below.

[0030] (1) An L-rhamnose isomerase that is an L-rhamnose isomerase from a microorganism belonging to the genus Erwinia, has a subunit molecular mass of 48 kDa as determined by SDS-PAGE, and has substrate specificity of the following (A) and (B):

[0031] (A) has isomerase activity that recognizes and reacts with the CHO group at C1 and the OH group at C2 of an aldose, converts the CHO group at C1 to an OH group, and converts the OH group at C2 to a CO group, or recognizes and reacts with the OH group at C1 and the CO group at C2 of a ketose, converts the OH group at C1 to a CHO group, and converts the CO group at C2 to an OH group;

[0032] (B) has an activity of catalyzing isomerization reactions between L-rhamnose and L-rhamnulose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-psicose.

[0033] (2) The L-rhamnose isomerase according to the above (1), which has the following (C) and (D) physicochemical properties:

[0034] (C) the reaction optimum pH is 9;

[0035] (D) the reaction optimum temperature is 70°C.

[0036] (3) The L-rhamnose isomerase according to the above (1) or (2), wherein the microorganism belonging to the genus Erwinia is Erwinia billingiae.

[0037] (4) The L-rhamnose isomerase according to any one of the above (1) to (3), wherein the microorganism belonging to the genus Erwinia is Erwinia billingiae GuaL218-3, which has been internationally deposited by the National Institute of Technology and Evaluation, Patent Microorganisms Depository as Accession No. NITE BP-03142.

[0038] In addition, the present application relates to the protein according to the above (5) to (7), the DNA, the recombinant vector, or the transformed host cell according to the above (8) to (11), or the microorganism according to the above (12).

[0039] (5) A protein comprising the amino acid sequence shown in SEQ ID NO: 1.

[0040] (6) A protein which is an amino acid substitution variant of a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and has at least one site of amino acid substitution selected from K3, L4, I5, Y9, E10, L11, Y16, D18, V19, I21, V23, Q25, V26, M27, T28, G32, I33, R46, N52, E54, R68, H73, A77, I79, E80, K81, M83, A89, D102, T103, E106, D108, A109, E111, Q113, S116, H117, Q124, H125, K126, S134, S148, D151, K152, G153, C162, I168, H171, P179, V186, L193, I195, L198, A199, E202, A205, S206, V211, F216, D217, A218, S219, C245, L246, A250, T257, T275, V280, L300, T310, A313, N315, K316, N319, K320, A345, S357, D358, Q359, R361, K362, L365, E366, A371, L375, V387, A390, W391, L393, H395, V397, D400, A401, S402, S405, E406, H409, Q412, Q413, T414, R416, L417, 419 in the amino acid sequence shown in SEQ ID NO: 1, has L-rhamnose isomerase activity of (A) and (B) below, and has higher L-rhamnose isomerase activity at the optimum temperature than a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,

[0041] (A) isomerase activity of recognizing and reacting with CHO group at Cl and OH group at C2 of an aldose to convert the CHO group at Cl to OH group and the OH group at C2 to CO group, or recognizing and reacting with OH group at Cl and CO group at C2 of a ketose to convert the OH group at Cl to CHO group and the CO group at C2 to OH group;

[0042] (B) activity of catalyzing isomerization between L-rhamnose and L-rhamnulose, between L-lyxose and L-xyloketose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-psicose.

[0043] (7) A protein which is an amino acid substitution variant of a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, has an identity of 78% or more to the amino acid sequence shown in SEQ ID NO: 1, and has an amino acid substitution at at least one site selected from K3, L4, I5, Y9, E10, L11, Y16, D18, V19, I21, V23, E24, Q25, V26, M27, T28, G32, I33, R46, N52, E54, R68, H73, A77, I79, E80, K81, A82, M83, S84, A89, K90, I97, D102, T103, E106, D108, A109, E111, Q113, S116, H117, E120, Q124, H125, K126, S134, P139, L140, S148, A150, D151, K152, G153, I154, C162, R167, I168, H171, P179, V186, L193, I195, L198, A199, E202, A205, S206, E210, V211, K215, F216, D217, A218, S219, C245, L246, A250, T254, T257, T275, V280, R282, L300, T310, A313, N315, K316, N319, K320, A345, S357, D358, Q359, R361, K362, L363, L365, E366, Y369, A371, A374, L375, S381, V387, A390, W391, L393, H395, V397, D400, A401, S402, S405, E406, H409, Q412, Q413, T414, R416, L417, 419 in the amino acid sequence shown in SEQ ID NO: 1, has L-rhamnose isomerase activity of (A) and (B) below, and has a higher ratio of L-rhamnose isomerase activity at 70°C to that at 50°C (T70 / T50), or a higher residual activity after incubation at 60°C for 1 hour, or a higher ratio of L-rhamnose isomerase activity at 80°C to that at the optimum temperature (T80 / optimum temperature) than a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,

[0044] (A) isomerase activity which recognizes and reacts with CHO group at Cl and OH group at C2 of an aldose to convert the CHO group at Cl to OH group and the OH group at C2 to CO group, or recognizes and reacts with OH group at Cl and CO group at C2 of a ketose to convert the OH group at Cl to CHO group and the CO group at C2 to OH group;

[0045] (B) has an activity of catalyzing isomerization reactions between L-rhamnose and L-rhamnulose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-psicose.

[0046] (8) DNA encoding the protein according to any one of the above (5) to (7).

[0047] (9) DNA comprising the base sequence shown in SEQ ID NO: 2.

[0048] (10) A recombinant vector containing the DNA according to the above (8) or (9).

[0049] (11) A transformed host cell obtained by transformation with the recombinant vector according to the above (10).

[0050] (12) Erwinia billingiae GuaL218-3, which produces the L-rhamnose isomerase according to any one of the above (1) to (4), has been internationally deposited as Accession No. NITE BP-03142 at the National Institute of Technology and Evaluation Patent Microorganisms Depository.

[0051] In addition, the present application relates to the immobilized protein according to the above (13) to (16).

[0052] (13) An immobilized protein, wherein the L-rhamnose isomerase according to any one of the above (1) to (4), or the protein according to any one of the above (5) to (7) is immobilized to a carrier.

[0053] (14) An immobilized protein, wherein the L-rhamnose isomerase according to any one of the above (1) to (4) is immobilized to a carrier in the form of a crude enzyme present in a bacterial cell disruptate, or the protein according to any one of the above (5) to (7) is immobilized to a carrier in the form of a crude protein present in a bacterial cell disruptate of a transformed host cell.

[0054] (15) The immobilized protein according to the above (13) or (14), wherein the carrier is an ion exchange resin or a synthetic adsorbent.

[0055] (16) The immobilized protein according to the above (15), wherein the carrier is WA30, FPA54 or FPA95.

[0056] In addition, the present application relates to the production method of the L-rhamnose isomerase according to the above (17) to (19), or the production method of a ketose or an aldose.

[0057] (17) A method for producing L-rhamnose isomerase, wherein a microorganism of the genus Erwinia producing the L-rhamnose isomerase according to any one of (1) to (4) above or the transformed host cell according to (11) above is cultured in a culture medium, the L-rhamnose isomerase is accumulated in the microbial cells, and the L-rhamnose isomerase is collected.

[0058] (18) The method for producing L-rhamnose isomerase according to (17) above, wherein the culture medium is an inorganic salt culture medium to which L-rhamnose is added.

[0059] (19) A method for producing a ketose or an aldose, characterized by allowing the L-rhamnose isomerase according to any one of (1) to (4) above, the protein according to any one of (5) to (7) above, or the immobilized protein according to any one of (13) to (16) above to act on a solution containing one or more kinds of aldose or ketose, to produce a corresponding ketose or aldose, and collecting the same.

[0060] Effects of the Invention

[0061] The L-rhamnose isomerase of the present application is characterized by having particularly high heat resistance and high activity compared to conventional L-rhamnose isomerases from microorganisms. For example, the optimum temperature of conventional L-rhamnose isomerase from Pseudomonas stutzeri is 60°C, whereas the enzyme of the present application is as high as 70°C, and has heat stability of 80% or more of the residual activity after heating treatment at 60°C for 10 minutes, and is thus suitable for use in industrial production. The excellent heat resistance of the enzyme can be further improved by producing an amino acid-substituted variant of the enzyme.

[0062] In addition, in the case where the substrate is 100 mM of D- psicose, the enzyme has a high conversion activity of 2.26 U of D-arabinose per unit protein at 60°C, and mass production of D-arabinose can be performed by the present application. In particular, in the case where the substrate is D-psicose, the enzyme of the present application converts only to D-arabinose, and does not produce the by-product D-arabinulose, and the yield is accordingly improved. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 is a graph showing a comparison of the amount of D-arabinose produced by a crude enzyme of the present application (from GuaL218-3 strain, hereinafter referred to as "the present enzyme") and a control crude enzyme (from AgM30 strain).

[0064] Figure 2 shows the results of SDS-PAGE for confirming the molecular mass of the present enzyme. In the figure, the unit of the number on the left is kDa.

[0065] Figure 3 is a graph showing the optimum pH of the present enzyme and the control enzyme.

[0066] Figure 4 is a graph showing the pH stability after 24 hours of the present enzyme and the control enzyme.

[0067] Figure 5 is a graph showing the optimum temperature of the present enzyme and the control enzyme.

[0068] Figure 6 is a graph showing the temperature stability of the present enzyme and the control enzyme for 10 minutes.

[0069] Figure 7 is a graph showing the substrate specificity of the present enzyme and the control enzyme.

[0070] Figure 8 is a graph showing the effect of metal ions on the present enzyme and the control enzyme.

[0071] Figure 9 is a graph showing the relative value of the activity of the immobilized enzyme of the present enzyme using an immobilization carrier.

[0072] Figure 10 is a result of SDS-PAGE of the recombinant enzyme expressed by E. coli.

[0073] Figure 11 is a result of HPLC analysis of the reaction solution of the conversion reaction of D- psicose to D-allose using the recombinant enzyme. DETAILED DESCRIPTION

[0074] The present application relates to L-rhamnose isomerase which can be isolated from microorganisms belonging to the genus Erwinia, and has a characteristic property of high activity and heat resistance.

[0075] The L-rhamnose isomerase of the present application has an isomerase activity of recognizing and reacting with CHO group at Cl and OH group at C2 of an aldose, converting the CHO group at Cl to OH group, and converting the OH group at C2 to CO group, or recognizing and reacting with OH group at Cl and CO group at C2 of a ketose, converting the OH group at Cl to CHO group, and converting the CO group at C2 to OH group.

[0076] The ketose in the present application means hexulose of six-carbon sugar or pentulose of five-carbon sugar having a ketose structure. The hexulose includes allose (alias psicose, allose), sorbose, tagatose, and fructose, and the pentulose includes ribulose and xylulose.

[0077] The aldose in the present application means hexose aldose of six-carbon sugar or pentose aldose of five-carbon sugar having aldose structure. The hexose aldose includes glucose, allose, altrose, gulose, idose, talose, galactose, and mannose, and the pentose aldose includes ribose, arabinose, xylose, and lyxose. D- or L- means D- and L- forms thereof.

[0078] The L-rhamnose isomerase of the present application acts on L-rhamnose, L-lyxose, L-mannose, D-ribose, L-talose, and D-allose, and is capable of catalyzing the conversion between L-rhamnose and L-rhamnulose, between L-lyxose and L-xylose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tagatose, and between D-allose and D-psicose, and has a wide substrate specificity.

[0079] The L-rhamnose isomerase of the present application can be produced by culturing a microorganism belonging to the genus Erwinia and having the ability to produce L-rhamnose isomerase, and isolating the L-rhamnose isomerase from the bacterial cells grown in the culture solution. As the microorganism belonging to the genus Erwinia, for example, Erwinia billingiae GuaL218-3 and their mutant strains, etc. can be effectively utilized. At the time of filing this application, the GuaL218-3 strain was internationally deposited on February 28, 2020, at the National Institute of Technology and Evaluation Patent Microorganisms Depository, 2-5-8, Kamfa, Kazusa, Kisarazu-shi, Chiba-ken, Japan, as Accession No. NITE ABP-03142, and then the GuaL218-3 strain was internationally deposited on the basis of the Budapest Treaty on February 28, 2020, as Deposit No. NITE BP-03142.

[0080] As to the L-rhamnose isomerase of the present application, after the L-rhamnose isomerase-producing strain of the genus Erwinia is aerobically cultured in an inorganic salt medium to which L-rhamnose is added, the bacterial cells are recovered from the culture solution by centrifugal separation. The recovered bacterial cells are washed with 10 mM Tris-HCl buffer (pH 7.5), suspended in 10 mL of 10 mM Tris-HCl buffer (pH 7.5), and then, either the cells are disrupted by enzyme treatment using Lysozyme as a lysozyme, or the bacterial cell suspension is cooled in ice water while being subjected to cell disruption with an ultrasonic homogenizer. The disrupted product is centrifugally separated, and the centrifugal supernatant is used as a crude enzyme solution.

[0081] The activity of the L-rhamnose isomerase in the crude enzyme solution before purification can be confirmed by measuring the amount of D-allose produced using D-psicose as a substrate.

[0082] The enzyme activity for the reverse reaction, i.e., the conversion from D-allose to D- psicose, was also measured under the same conditions. These conversion reactions were generally carried out under the following conditions. The substrate concentration was 1 to 60% (w / v), preferably about 5 to 50% (w / v), the reaction temperature was 30 to 80°C, preferably about 50 to 70°C, the reaction pH was 6 to 11, preferably about 8 to 11, and the reaction time was appropriately selected, and in the case of batch reactions, it was generally selected in the range of 4 to 20 hours.

[0083] The crude enzyme solution was purified by ion exchange chromatography and hydrophobic chromatography in this order to isolate and purify the enzyme. To confirm that the enzyme was purified, SDS-PAGE (gel concentration 12.5%) was performed, and a single band and apparent molecular mass were confirmed.

[0084] The L-rhamnose isomerase of the present application, which was purified as described above, had a subunit molecular mass of about 48 kDa as determined by SDS-PAGE, and this enzyme was a metal enzyme whose activation degree was regulated by metal ions. The reaction with the substrate could be carried out in the presence of a metal ion selected from the group consisting of manganese, cobalt, nickel, magnesium, iron, copper, zinc, and calcium at a concentration of 0.5 to 5 mM.

[0085] The L-rhamnose isomerase of the present application has a prescribed amino acid sequence, and as examples thereof, there can be mentioned a protein having the amino acid sequence shown in SEQ ID NO: 1, or a protein having an amino acid sequence homologous thereto and maintaining equivalent L-rhamnose isomerase activity. The homologous amino acid sequence means, for example, an amino acid sequence having an identity of 75% or more, 78% or more, 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, to the amino acid sequence of SEQ ID NO: 1.

[0086] The identity (%) of two amino acid sequences or two nucleic acid sequences (base sequences) can be determined, for example, by the following procedure. First, the sequences are aligned in a manner that allows the best comparison of the two sequences. At this time, for example, a gap can be introduced in the first sequence so as to allow alignment with the second sequence. When the molecule (amino acid residue or nucleotide) at a specific position of the first sequence is identical to that at the corresponding position of the second sequence, the molecules at that position are said to be identical. The identity of the two sequences is a function of the number of identical positions common to the two sequences (i.e., identity (%) = number of identical positions / total number of positions x 100), and preferably the number and size of the gaps required for optimization of the alignment are also taken into account.

[0087] In addition, the comparison of two sequences and the determination of identity can be performed using a mathematical algorithm. As a specific example of a mathematical algorithm that can be used for the comparison of sequences, the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77, can be used, but is not limited thereto. Such an algorithm is incorporated in the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. In order to obtain a nucleotide sequence equivalent to the nucleic acid molecule of the present application, for example, BLAST nucleotide search can be performed using the NBLAST program with a score (score) = 100 and a word length (word length) = 12.

[0088] The DNA of the present application is a gene encoding the above-described protein, and has a specified base sequence. As an example thereof, there can be mentioned a DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 1, having the base sequence shown in SEQ ID NO: 2, or a base sequence homologous to the base sequence shown in SEQ ID NO: 2, and encoding a protein that maintains L-rhamnose isomerase activity equivalent to that of the protein of SEQ ID NO: 1. The homologous base sequence, for example, means a base sequence having an identity of 75% or more, 78% or more, 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, to the base sequence of SEQ ID NO: 2.

[0089] The DNA of the present application can also be inserted into a suitable vector that can autonomously replicate to produce a recombinant vector. The recombinant vector is composed of the DNA and a vector that can autonomously replicate, and can be easily produced by a conventional method of recombinant DNA technology as long as the DNA can be obtained. Depending on the purpose of cloning or the use of protein expression or depending on the host cell, a suitable vector is selected. As examples of such a vector, there can be mentioned plasmid vectors such as pBR322, pUC18, pUB110, pTZ4, pC194, pHV14, TRp7, YEp7, pBS7, and the like, or bacteriophage vectors such as λgt·λC, λgt·λB, ρ11, and the like.

[0090] The thus obtained recombinant vector can be introduced into a suitable host cell represented by Escherichia coli, Bacillus subtilis, Actinomyces, and yeast. The introduction method can use a publicly known method such as calcium phosphate coprecipitation, electroporation, lipofection, microinjection, and the like. In order to obtain a transformed host cell, a colony hybridization method or the like is used.

[0091] The method for producing the L-rhamnose isomerase of the present application, or the protein having L-rhamnose isomerase activity is not particularly limited, and a publicly known method can be employed. Specifically, the L-rhamnose isomerase of the present application can be produced by collecting the protein having L-rhamnose isomerase activity from a culture of a microorganism having L-rhamnose isomerase-producing ability of the present application, or a host cell into which a DNA encoding the protein having L-rhamnose isomerase activity of the present application has been introduced, which has been cultured in a nutrient medium. The culture method can employ any of publicly known methods, such as liquid culture and solid culture.

[0092] After the microorganism is thus cultured, the enzyme or protein of the present application is purified and recovered. The method for purifying and recovering the enzyme or protein can be freely selected from publicly known methods, and, for example, in the case of recovery from a culture solution, the culture supernatant can be subjected to filtration, centrifugal treatment, or the like to remove insoluble matter, and then the enzyme or protein can be separated and purified by an appropriate combination of concentration using an ultrafiltration membrane, salting-out using ammonium sulfate precipitation, dialysis, ion exchange resin, various chromatography, or the like. In the case of recovery from the microorganism, the microorganism can be similarly subjected to separation and purification after being broken by solubilase treatment, ultrasonic treatment, or the like.

[0093] In addition, the L-rhamnose isomerase of the present application can be used after being immobilized like other isomerases, and can be obtained by various immobilization methods to provide an immobilized enzyme having high activity. By using the immobilized enzyme, a large amount of isomerization reaction can be continuously performed, and the immobilized enzyme can be produced by publicly known immobilization means such as carrier binding, cross-linking, gel embedding, microcapsulation, or the like, and the carrier can be any publicly known carrier.

[0094] One method for immobilizing the L-rhamnose isomerase of the present application is immobilization using a crude enzyme solution. The crude enzyme solution containing the L-rhamnose isomerase obtained by subjecting a microorganism suspension to ultrasonic treatment is added to an ion exchange resin or the like, and the L-rhamnose isomerase can be immobilized by allowing it to bind at low temperature.

[0095] In order to extract the L-rhamnose isomerase from the microorganism, the cell wall of the microorganism needs to be broken, and the breaking method can be an enzymatic treatment using ultrasonic treatment or solubilase, as described above. It is known that a large amount of active L-rhamnose isomerase is contained in the crude enzyme solution obtained by ultrasonic treatment. As the carrier for immobilizing the enzyme from the crude enzyme solution, any publicly known carrier for immobilization can be used, and an ion exchange resin, sodium alginate, a synthetic adsorbent, or the like is convenient and is often used.

[0096] As the basic anion exchange resin, any one of a strong basic anion exchange resin or a weak basic anion exchange resin can be used, for example, as the strong basic anion exchange resin, SA20A, PA418 (manufactured by Mitsubishi Chemical Corporation), etc. can be listed, as the weak basic anion exchange resin, WA30 (manufactured by Mitsubishi Chemical Corporation), FPA54, FPA95 (manufactured by Organo), etc. can be listed, and as the synthetic adsorbent, XAD7HP (manufactured by Organo), etc. can be listed.

[0097] In the case where the weak basic anion exchange resin is used in the carrier to produce the immobilized enzyme, the immobilized L-rhamnose isomerase can be easily eluted after its reaction, and thus the regeneration of the immobilized enzyme can be performed very easily, and the production efficiency is high.

[0098] The L-rhamnose isomerase of the present application can be produced by collecting a protein having L-rhamnose isomerase activity from a culture of a microorganism having L-rhamnose isomerase production ability of the present application, or a host cell into which a DNA encoding a protein having L-rhamnose isomerase activity has been introduced, which is cultured in a nutrient medium. The culture method can use any one of a known method, for example, liquid culture and solid culture.

[0099] After the bacterial cells are cultured in the medium, the L-rhamnose isomerase of the present application is purified and recovered. The method for purifying and recovering the protein can freely select a known method, for example, in the case of recovery from the culture solution, for example, the insoluble matter can be removed by filtering, centrifuging, etc. of the culture supernatant, and the separation and purification can be performed by appropriately combining concentration using an ultrafiltration membrane, salting-out using ammonium sulfate precipitation, dialysis, ion exchange resin, etc. and various chromatography, etc. In addition, in the case of recovery from the bacterial cells, for example, after the bacterial cells are broken by treatment with lysozyme, ultrasonic treatment, etc., the separation and purification can be performed in the same manner.

[0100] The purified L-rhamnose isomerase of the present application or the enzyme after immobilization can be used to act on a solution containing one or more kinds of monosaccharides selected from aldoses or ketoses, which can become a substrate, to produce the corresponding ketose or aldose, thereby producing ketose or aldose. The L-rhamnose isomerase of the present application has higher substrate specificity for D-psicose than the conventional L-rhamnose isomerase, and thus D-allose, which is a rare sugar, can be produced in a large amount in the presence of D-psicose, which can become a substrate.

[0101] In addition, by introducing a mutation in the gene of the L-rhamnose isomerase of the present application, the corresponding amino acid residue is substituted with another amino acid residue by site-directed mutagenesis, and a variant having various amino acid substitutions is prepared, and an enzyme having higher D-psicose isomerase activity at the optimum temperature or an enzyme having higher heat resistance than the wild-type enzyme without amino acid substitution can be obtained. The heat resistance is evaluated by the ratio of D-psicose isomerase activity at the optimum temperature, reaction temperature 70°C, and 50°C (T70 / T50), the residual activity after incubation at 60°C for 1 hour, or the ratio of D-psicose isomerase activity at the reaction temperature 80°C and the optimum temperature (T80 / optimum temperature).

[0102] The L-rhamnose isomerase of the present application can obtain a variant having enzyme activity and having an optimum temperature higher than the wild-type enzyme of SEQ ID NO: 1 or having a higher ratio of D-psicose isomerase activity at reaction temperature 70°C and 50°C (T70 / T50) or having a higher residual activity after incubation at 60°C for 1 hour or having a higher ratio of D-psicose isomerase activity at reaction temperature 80°C and the optimum temperature (T80 / optimum temperature) even if the variant has an amino acid sequence having an identity of about 78%.

[0103] For the design of the amino acid substitution variant, the method for making a system tree described in Patent Literature 8 is referred to.

[0104] The DNA sequence encoding an enzyme or a putative enzyme having an activity similar to the L-rhamnose isomerase shown in SEQ ID NO: 1 of the present application is extracted in its entirety, and a system tree in which the DNA sequence originating from the phylogenetic origin of the common ancestral DNA is evolved in a phylogenetic manner is made using the method of Patent Literature 8. Furthermore, in comparison with the amino acid sequence encoded by the obtained DNA sequence in the same system as SEQ ID NO: 1 of the present application in the system tree, the amino acid sequence in the part where the sequences are different is focused on, and it is predicted that the site where the amino acid residues are different contributes little to the enzyme activity, and the amino acid corresponding to the different site of SEQ ID NO: 1 is changed by base substitution by site-directed mutagenesis, and various L-rhamnose isomerase variants are made. Among them, an amino acid substitution variant having higher D-psicose isomerase activity at the optimum temperature or an amino acid substitution variant having higher heat resistance than the wild-type enzyme is selected.

[0105] In addition, the amino acid substitution variant of the present application includes, as an exception, a variant in which one amino acid residue is added to the amino acid at position 418 at the C-terminus.

[0106] The point mutation introduction method can be performed using any of the methods such as the inverse PCR method or the annealing method (Furukawa et al., eds., "New Gene Engineering Manual, Revised 4th Edition", Gakkensho, p. 82-88). As needed, various commercially available point mutation introduction kits such as the QuickChange II Site-Directed Mutagenesis Kit, the QuickChange Multi Site-Directed Mutagenesis Kit, and the like from Stratagene can be used.

[0107] The point mutation introduction can most typically be performed using a mutation primer including the nucleotide variation to be introduced. Such a mutation primer is designed in such a manner as to anneal to a region including the nucleotide sequence of the amino acid residue to be changed within the coding gene and to have a base sequence of the nucleotide sequence (codon) encoding the changed amino acid residue instead of the nucleotide sequence (codon) encoding the amino acid residue to be changed.

[0108] The amino acid substitution variant refers to a variant in which a different amino acid is substituted for the amino acid of the original sequence, and the substitution can be conservative substitution or non-conservative substitution, and is not particularly limited, and in the preferred mode of the application, the substitution is conservative substitution. The conservative substitution corresponds to substitution of amino acids having the same property (basic, acidic, or neutral), polarity (hydrophilicity or hydrophobicity), aromatic amino acids for each other, or aliphatic amino acids for each other, or the like, in a manner in which basic is substituted for basic, acidic is substituted for acidic, and polar is substituted for polar.

[0109] The conservative substitution is performed, for example, within the group of basic amino acids (Arg, Lys, His), acidic amino acids (Glu, Asp), neutral non-polar amino acids (Gly, Ala, Val, Leu, Ile, Met), aliphatic amino acids (Ala, Val, Leu, Ile, Met), polar amino acids (Gln, Asn, Ser, Thr), aromatic amino acids (Phe, Trp, Tyr), and the like.

[0110] On the other hand, the non-conservative substitution refers to exchange with an amino acid that is not a member of the above group, for example, to prevent folding in the protein in the three-dimensional structure, to delete Cys, or to substitute for another amino acid. Alternatively, to maintain the balance of hydrophilicity / hydrophobicity, or to improve the hydrophilicity for ease of synthesis, the amino acid is substituted in consideration of the hydrophilicity index of the amino acid as an index of the hydrophilicity / hydrophobicity of the amino acid (J. Mol. Biol. (1982) Vol. 157, p. 105-132).

[0111] In addition, substitution into an amino acid having less steric hindrance than the original amino acid is sometimes performed, and substitution into an amino acid having no charge from an amino acid having a charge is sometimes performed.

[0112] As for substitution, both conservative substitution and non-conservative substitution are possible, and examples of substitution include substitution from Ala to Ser or Thr, substitution from Arg to Gin, His or Lys, substitution from Asn to Glu, Gin, Lys, His or Asp, substitution from Asp to Asn, Glu or Gin, substitution from Cys to Ser or Ala, substitution from Gin to Asn, Glu, Lys, His, Asp or Arg, substitution from Glu to Asn, Gin, Lys or Asp, substitution from Gly to Pro, substitution from His to Asn, Lys, Gin, Arg or Tyr, substitution from He to Leu, Met, Val or Phe, substitution from Leu to He, Met, Val or Phe, substitution from Lys to Asn, Glu, Gin, His or Arg, substitution from Met to He, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, He or Leu, substitution from Ser to Thr or Ala, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, He or Leu, but the present application is not limited to these. As for substitution of these amino acid residues, the classification of substitutable residues is illustrative, and substitutable amino acid residues are not limited to this classification.

[0113] In the wild-type enzyme having the amino acid sequence of SEQ ID NO: 1, a plurality of multiple variants in which 90thamino acids were substituted were prepared from a variant in which 1stamino acid was substituted, and 58 variants were selected by performing enzyme activity and heat resistance tests. Among the 58 variants, 13 variants in which D-allulose isomerase activity at the optimum temperature was higher than that of the wild-type enzyme without amino acid substitution were confirmed.

[0114] In addition, 52 variants in which the ratio of D-allulose isomerase activity at 70°C and 50°C (T70 / T50) was higher than that of the wild-type enzyme were confirmed among the 58 variants, 17 variants in which the residual activity after incubation at 60°C for 1 hour was higher than that of the wild-type enzyme were confirmed among the 58 variants, and 56 variants in which the ratio of D-allulose isomerase activity at 80°C and the optimum temperature (T80 / optimum temperature) was higher than that of the wild-type enzyme were confirmed among the 58 variants.

[0115] The L-rhamnose isomerase of the present application is also excellent in that a large number of variants having higher heat resistance than the wild-type L-rhamnose isomerase can be obtained.

[0116] Examples

[0117] Hereinafter, the present application will be explained in detail by experiments, but the present application is not limited by any of the following examples.

[0118] <Experiment 1: Origin and identification of strains>

[0119] The inventors of the present application inoculated a large number of strains isolated by screening into a liquid medium added with L-rhamnose and performed shaking culture, measured the amount of L-rhamnulose produced using L-rhamnose as a substrate, and thereby measured the activity of L-rhamnose isomerase.

[0120] As a result of this operation, as a strain having the highest activity, a microorganism GuaL218-3 strain was found, and it was determined that the GuaL218-3 strain belongs to Erwinia based on the homology of the 16S rRNA gene base sequence.

[0121] Identification of strains

[0122] (1) Homology of 16S rRNA gene base sequence

[0123] The 1-500 bp region of the 16S rRNA gene was analyzed, and the number of bases was determined to be 500 bp.

[0124] (2) Homology search

[0125] Regarding the base sequence of the 16S rRNA gene of this strain, a homology search was performed with BLAST search (DNA Data Bank of Japan) with known species of bacteria regarded as reference strains. From the value of the homology (%) of the strain name with respect to the base sequence of the above-determined base number of 500 bp, the homology was 98% or more, it was determined that the microorganism of the GuaL218-3 strain was Erwinia billingiae.

[0126] This GuaL218-3 strain was internationally deposited at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation, Japan on February 28, 2020, and was internationally deposited as Accession No. NITE BP-03142.

[0127] <Experiment 2: Culture of Erwinia billingiae GuaL218-3 (Accession No. NITE BP-03142) strain>

[0128] In an inorganic salt liquid medium with L-rhamnose 1.0% as a carbon source and ammonium sulfate as a nitrogen source, 1% (v / v) of a strain culture solution of Erwinia billingiae GuaL218-3 (Accession No. NITE BP-03142) strain was aseptically added, and it was cultured at 30°C for 24 hours while being aerated and stirred.

[0129] As a control strain for comparison with the strain of the present application, Arthrobacter globiformis M30 (NITE BP-1111) strain (hereinafter, referred to as "AgM30 strain") was cultured in the same medium and under the same culture conditions as the GuaL218-3 strain.

[0130] <Experiment 3: Preparation of crude enzyme>

[0131] The bacterial cells were recovered from each of the culture solutions of the GuaL218-3 strain and the AgM30 strain by centrifugal separation. The recovered bacterial cells were washed with 10 mM glycine-NaOH buffer (pH 9.0), and then suspended in 10 mL of the glycine-NaOH buffer (pH 9.0). The bacterial cell suspension was subjected to cell disruption using an ultrasonic homogenizer (EIMCO Corporation, Japan) while being cooled in ice water. The disrupted product was centrifuged at 12,000 rpm for 20 minutes, and the centrifugal supernatant was used as each crude enzyme.

[0132] <Experiment 4: Production of D-allose using crude enzyme>

[0133] Using the obtained two crude enzyme solutions, D-psicose was added as a substrate so as to have a final concentration of 100 mM, and the amount of production of D-allose was measured after performing an enzyme reaction at 30°C for 4 hours. The amount of production of D-allose was compared using the area of HPLC. Specifically, the composition of the enzyme reaction solution was 50 mM glycine-NaOH buffer (pH 9.0), 100 mM D-psicose, crude enzyme solution, and 1 mM manganese chloride, and the reaction was performed at 30°C for 4 hours. After stopping the reaction by boiling for 2 minutes, HPLC was performed, and the composition of the solution after the reaction was measured. The ratio of the area of D-allose produced, which was obtained by HPLC analysis, is shown in Table 1. Figure 1 .

[0134] Regarding the amount of conversion of D-psicose to D-allose using the crude enzyme per unit amount of the culture solution, the crude enzyme from the GuaL218-3 strain was about 23 times that of the control crude enzyme from the AgM30 strain.

[0135] <Experiment 5: Purification of enzyme>

[0136] 1. Purification using ion exchange chromatography and hydrophobic chromatography

[0137] Two crude enzyme solutions were purified by ion exchange chromatography. The HiTrap Q HP equilibrated with a buffer (20 mM Tris-HCl pH 7.5) was used, and the AKTA system was used to fractionate at a flow rate of 5 mL / min, with a concentration gradient of 1 M NaCl from 0% to 100% in 5 mL fractions. Fractions in which enzyme activity was detected were pooled to give the purified enzyme isolated by ion exchange chromatography.

[0138] 2. The enzyme purified by ion exchange chromatography was further purified by hydrophobic chromatography. The HiTrap PHENYL was used, and the enzyme solution was dissolved by adding ammonium sulfate to 2 M, and elution was performed at a flow rate of 5 mL / min, with a concentration gradient of 2 M ammonium sulfate from 100% to 0% in 5 mL fractions. Fractions in which enzyme activity was detected were dialyzed to remove the ammonium sulfate, to give the purified enzyme isolated by hydrophobic chromatography.

[0139] 3. Polyacrylamide gel electrophoresis

[0140] SDS-PAGE (gel concentration 12.5%) was performed according to the conventional method to confirm the purity of the purified enzyme. In Figure 2 , the left side is a standard protein, and the center lane and the right lane are the enzyme purified using hydrophobic chromatography. As a result, a single band was confirmed at about 48 kDa, and it was thus confirmed that the enzyme was purified cleanly, and that the subunit molecular weight of the purified enzyme was about 48 kDa as determined by SDS-PAGE.

[0141] <Experiment 6: Measurement of the physicochemical properties of the enzyme>

[0142] With respect to the measurement of L-rhamnose isomerase activity as the purified enzyme, the following experiment was performed, and an enzyme reaction was performed. As the substrate, 5 mM L-rhamnose was used, and an enzyme reaction was performed for 10 minutes under each condition, and after the reaction, 50 μL of a 10% trichloroacetic acid solution was added to the reaction solution, and thus the reaction was stopped, and L-rhamnulose generated by the cysteine carbazole sulfate method was measured.

[0143] 1. Optimum pH of the reaction

[0144] At the time of measurement, 5 mM L-rhamnose was used as the substrate, and various buffers pH 3 to 11 were used to perform a reaction for 10 minutes at 30°C, and the L-rhamnulose generated was measured by the cysteine carbazole sulfate method, and the optimum pH of the reaction was determined.

[0145] The reaction conditions are shown in Table 1. The buffers used are shown in Table 2.

[0146] [Table 1]

[0147] [Table 2]

[0148] [Table 2]

[0149]

[0150] The results are shown in Figure 3 The reaction optimum pH of the present enzyme is 9, and that of the control enzyme is 10 on the basic side. The reaction optimum pH of the present enzyme is in a wider range, and shows high activity under more acidic conditions.

[0151] 2. pH stability

[0152] Next, the residual activity of the present enzyme and the control enzyme after being kept at 30°C for 24 hours in each of the four buffers of pH 4 to 11 shown in Table 2 was measured, and the results are shown in Figure 4 .

[0153] The present enzyme and the control enzyme were stable at pH 6 to 11, but the pH stability of the control enzyme was highest at pH 6, and the present enzyme was most stable at pH 7.5 and 10.

[0154] 3. Reaction optimum temperature

[0155] The reaction was carried out at each temperature from 30 to 80°C using a glycine-NaOH buffer adjusted to pH 9. The optimum temperature was determined. The reaction conditions are shown in Table 3. The temperature range from 40 to 80°C is the preferred temperature range of the present enzyme, and the results of measuring the reaction temperature and the relative activity are shown in Figure 5 The optimum temperature of the present enzyme was 70°C.

[0156] [Table 3]

[0157]

[0158] The optimum temperature of the control enzyme was 50°C, but the relative activity (%) when 70°C at which the present enzyme shows the highest activity was set to 100 was 40% at 80°C, 80% at 60°C, and 70% at 50°C, and the relative activity (%) when 50°C at which the control enzyme shows the highest activity was set to 100 was 65% at 60°C and 8% at 70°C. The present enzyme particularly maintains the activity at high temperatures, and has high heat resistance.

[0159] 4. Heat stability

[0160] The residual activity of the present enzyme and the control enzyme after being kept at each temperature for 10 minutes using the reaction conditions (10 minutes) of the optimum temperature determined in the above 3. shown in Table 3 was measured, and the results are shown in Figure 6 In the temperature stability after being kept for 10 minutes, the decrease in the relative activity of the present enzyme at 60°C was less than 20%, which is much smaller than the decrease of 80% of the control enzyme, and thus it was found that the present enzyme is a more stable enzyme at high temperatures than the control enzyme.

[0161] 5. D-allose or D-allose isomerase activity,

[0162] As to the measurement of D-allose isomerase activity of the enzyme, the same experiment as in Experiment 6 was carried out, and enzyme reaction was performed. As the substrate, 100 mM D-allose was used, and enzyme reaction was performed at 60°C for 60 minutes. After the reaction, the reaction solution was put into boiling water for 3 minutes, thereby stopping the reaction, and the composition of the solution after the reaction was measured by HPLC. One unit (U) of enzyme activity was the amount of enzyme that isomerizes D-allose to produce 1 μmol of D-allose ketose in 1 minute under the above conditions. The reaction conditions are shown in Table 4.

[0163] Under the same conditions, D-allose ketose isomerase activity was measured. As the substrate, 100 mM D-allose ketose was used, and enzyme reaction was performed at 60°C for 60 minutes to produce D-allose. One unit (U) of enzyme activity was the amount of enzyme that isomerizes D-allose ketose to produce 1 μmol of D-allose in 1 minute under the above conditions.

[0164] [Table 4]

[0165]

[0166] As a result, it was found that the specific activity of the enzyme for D-allose was 5.21 U / mg, and the specific activity for D-allose ketose was 2.26 U / mg.

[0167] Further, when D-allose ketose was isomerized to D-allose by the enzyme, no by-product D-altrurlose was produced.

[0168] 6. Substrate specificity of L-rhamnose isomerase

[0169] The isomerization activity of the enzyme for L-rhamnose and five aldoses (D-allose, L-talose, L-lyxose, D-ribose, L-mannose) was investigated. The enzyme reaction composition was as shown in Table 5 below, and the reaction was performed at 70°C for 10 minutes in each substrate at a final concentration of 5 mM and enzyme solution (final concentration of 50 mM phosphate buffer pH 8.0), and the ketose produced by isomerization of each aldose was measured by analysis using HPLC.

[0170] The isomerization activity for L-rhamnose was set to 100, and the activity for each aldose was expressed as relative activity.

[0171] D-allose (D-Allose), L-mannose (L-Mannose), L-talose (L-Talose), L-lyxose (L-Lyxose), and D-ribose (D-Ribose) were used as the substrate, and the activity was expressed as relative activity in Table 6 and Figure 7.

[0172] [Table 5]

[0173]

[0174] [Table 6]

[0175] Substrate Relative activity (%) L-rhamnose 100 L-rhamnose 17.9 L-rhamnose 3.3 D-ribose 2.2 L-rhamnose 2.2 D-ribose 1.9

[0176] L-rhamnose was the most active, followed by L-lyxose, L-mannose, D-ribose, L-talose, and D-allose. On the other hand, in the case of the control enzyme, L-rhamnose, L-lyxose, L-mannose, D-ribose, and D-allose were active in the order named, and no reaction was observed with L-talose. The enzyme catalyzes isomerization between L-rhamnose and L-rhamnulose, between L-lyxose and L-xylulose, between L-mannose and L-fructose, between D-ribose and D-ribulose, between L-talose and L-tallose, and between D-allose and D-allosulose.

[0177] 7. Effect of metal ions

[0178] Next, in order to investigate the effect of metal ions on the activity of the D-allose isomerase, a part of the enzyme was dialyzed and the enzyme activity was measured. The dialysis was performed by adding the enzyme solution to a cellulose membrane and immersing it in a glycine-NaOH buffer (pH 9.0) containing 20 mM EDTA, and the buffer was slowly stirred for 16 hours to eliminate the effect of other metal ions. After the reaction in the presence of 1 mM of each of various divalent metal ions (under the reaction conditions of Table 7), the enzyme activity of the thus obtained enzyme was measured by the cysteine carbazole method.

[0179] As a result, CoCl2significantly increased the activity of the enzyme, and the enzyme showed metal dependence Figure 7 ).

[0180] [Table 7]

[0181] (Reaction conditions at the time of confirmation of the effect of metal ions)

[0182]

[0183] <Experiment 7: Preparation of immobilized enzyme>

[0184] The bacterial cells of Erwinia billingiae GuaL218-3 strain, which were cultured and recovered in the same manner as in Experiment 2, were used.

[0185] 1. Obtaining of crude enzyme of bacterial cells using ultrasonic treatment

[0186] The bacterial cells corresponding to 1 L of the culture broth were suspended in 40 mL of 50 mM glycine-NaOH buffer (pH 9.0) and the bacterial cell suspension was finely disrupted with an ultrasonic homogenizer while being cooled in ice water. The disrupted product was centrifuged at 15,000 x g for 30 minutes and the centrifugal supernatant was used as a crude enzyme solution.

[0187] 2. Immobilization of the crude enzyme

[0188] After the ion exchange resin or synthetic adsorbent equilibrated with 50 mM glycine-NaOH buffer (pH 9.0) was sufficiently swelled with pure water, the above-obtained crude enzyme solution was added thereto and mixed slowly at 4°C for 20 hours, whereby the crude enzyme protein was bound. Subsequently, the immobilized enzyme was obtained by washing with 50 mM glycine-NaOH buffer (pH 9.0).

[0189] As the immobilization carrier, a strong basic anion exchange resin (SA10A, SA11A, NSA100, SA20A, PA306S, PA308, PA312, PA316, PA408, PA412, PA418 manufactured by Mitsubishi Chemical Corporation), a weak basic anion exchange resin (WA10, WA20, WA21J, WA30 manufactured by Mitsubishi Chemical Corporation, and FPA54, FPA60CL, FPA95 manufactured by Organo Corporation), or a synthetic adsorbent (XAD7HP, XAD118ON manufactured by Organo Corporation) was used.

[0190] 3. Method for measuring the enzyme activity of the immobilized enzyme

[0191] The enzyme activity of the immobilized enzyme was measured by measuring the amount of D-arabinose produced when the enzyme reaction was performed using D-psicose as a substrate.

[0192] First, a solution (500 μL) of 100 mg of the immobilized enzyme resin, glycine-NaOH buffer (pH 9.0) (final concentration 50 mM), manganese chloride (final concentration 1 mM), and D-psicose (final concentration 100 mM) was used as a reaction solution composition, and after the reaction was performed in a constant-temperature water bath at 30°C for 24 minutes, the reaction was immediately stopped by boiling at 100°C for 2 minutes. After the reaction solution was cooled to room temperature, desalting was performed with an ion exchange resin (a mixed resin of 200CT and IRA67 (both manufactured by Organo Corporation)), and further filtration treatment was performed to prepare an analysis sample. The analysis was performed by measuring the peak area of the produced D-arabinose using high performance liquid chromatography (chromatography column: GL-C611 (Hitachi), temperature: 60°C, eluent: 0.1 mM NaOH, flow rate: 1.0 mL / minute, detector: RID-20A (Hitachi)).

[0193] 4. Comparison of enzyme activity of carriers for enzyme immobilization

[0194] In order to confirm the influence of the carriers used in enzyme immobilization, the enzyme activity of the enzyme immobilized in each of the carriers described above was compared. Among them, the relative value was calculated by setting the sample having the highest activity expression rate in the comparison sample to 100.

[0195] Figure 9 The relative values indicating the activity expression rates of various immobilized enzymes are shown.

[0196] <Experiment 8: Cloning of DNA encoding enzyme, preparation of recombinant vector containing the DNA and transformed host cell>

[0197] DNA encoding a protein having D-allulose isomerase activity was cloned from Erwinia billingiae GuaL218-3 strain, and production of a self-replicating recombinant DNA, determination of the base sequence of the DNA encoding enzyme, and preparation of a transformed microorganism were performed.

[0198] <Experiment 8-1: Determination of the full base sequence of chromosomal DNA>

[0199] The D-allulose isomerase of the present strain is different from the existing similar enzymes, and cannot be isolated using a PCR amplification method or an existing protein database. Therefore, the full genome sequence of Erwinia billingiae GuaL218-3 strain was determined, and then a database of the protein group encoded by the full ORF in the genome was constructed. The cultured cells of Erwinia billingiae GuaL218-3 strain were used as test cells, and a new generation sequencing analysis using PacBio-RSII / Sequel was commissioned to Macrogen Japan Co., Ltd.

[0200] As a result, four contigs of 4,301,131 bp and 1,024,754 bp, 280,568 bp, and 126,938 bp were obtained. About 5.7 Mb in the four contigs, and thus it is considered that the full genome sequence of Erwinia billingiae GuaL218-3 strain can be covered.

[0201] <Experiment 8-2: Construction of a protein database>

[0202] Based on the obtained DNA sequence of about 5.7 Mb, 5,357 ORFs were deduced using the Prokka program, and the amino acid sequences were predicted for each ORF. The 5,357 amino acid sequences were used as the protein database of Erwinia billingiae GuaL218-3 strain.

[0203] <Experiment 8-3: Identification of a protein having D-allulose isomerase activity>

[0204] Using the above protein database, the protein identification system MASCOT server (Matrix Science) was logged in, and the identification of a protein in which D-allulose isomerase activity was observed was performed. The test sample was the 48 kDa band of SDS-PAGE of the

[0045] paragraph of the international publication, and the fragments subjected to trypsin digestion after reduction treatment and alkylation treatment were subjected to MALDI-TOF-MS analysis.

[0205] As a result, since 78% similarity to the amino acid sequence indicated by underlining in the amino acid sequence of Sequence 1 (SEQ ID NO: 1 of the sequence listing) consisting of 418 amino acids was observed, it was strongly suggested that the protein is L-rhamnose isomerase of Erwinia billingiae GuaL218-3 strain.

[0206] [Sequence 1]

[0207] M TKLIEQAYELAK QRYADVGIDVEQVMTQLDGIPVSMHCWQGDDVR GFENPNGELTGGIQATGNYPGRA RNAHELR ADIEKAMSLIPGAK RLNLHAIYLESDTPVERDAIEPQHFSHWVEWAK QHK LGLDFNPSCFSHPLSADGFT LSHADK GIR QFWIDHCK ASRR ISAHFGEQLGTPSVMNIWVPDGMKDLTIDR LAPRER LASALDEVISEKFDASHHID AVESKLFGIGAESYTVGSNEFCLGYAASRQTALTLDAGHFHPTEVISDK ISTAMLYVPR LLLHVSRPVRWDSDHVVL LDDETQAIATEIAR NKLFNK VHIGLDFFDASINRIAAWVIGTR NAKKALLR ALLEPSDQLRKLELEGDYTARLALLE EQKSLPWQAVWEAWCLRHDVPADASWLSEVRHYEQQTLR LR

[0208] (Sequence 1 is the amino acid sequence of the protein identified using the protein database of Erwinia billingiae GuaL218-3 strain. The amino acid sequence indicated by underlining is the sequence consistent with the peaks obtained by MALDI-TOF-MS analysis.)

[0209] <Experiment 8-4: Isolation of the gene of the enzyme having D-psicose isomerase activity>

[0210] The DNA sequence of SEQ ID NO: 2 of the gene identified by the amino acid sequence (SEQ ID NO: 2 of the sequence listing) was synthesized, incorporated into a pQE60 vector (Qiagen), and transformed into an E. coli for expression. The constructed E. coli expression system was used to induce the enzyme. The results of SDS-PAGE are shown in Fig. 8-4, and the induced protein was confirmed in the soluble fraction. In addition, the recombinant enzyme obtained by the induction was used as a substrate for D-psicose at 60% (w / v) and reacted at 30°C for 24 hours, and the results of confirming the D-psicose isomerase activity by HPLC are shown in Fig. 8-5. Figure 10 Figure 11 The peak at a retention time of 22.48 minutes in the figure is D-allose, and the peak at a retention time of 29.67 minutes is D-psicose, and the recombinant enzyme catalyzes the isomerization reaction from D-psicose to D-allose, and does not produce D-altrose which can be a byproduct.

[0211] [SEQ ID NO: 2]

[0212] ​

[0213] Experiment 9: Production of amino acid substitution variants of the enzyme and their enzymatic activity and heat resistance

[0214] The amino acid sequence of L-rhamnose isomerase shown in SEQ ID NO: 1 of the present application was compared with the amino acid sequence encoded by the DNA sequence classified into the same system as the DNA sequence of SEQ ID NO: 2 of the present application and produced by the method described in Patent Document 8, and the amino acid sequence of SEQ ID NO: 1 was changed by introducing base substitution by point mutation at the site corresponding to the amino acid different from SEQ ID NO: 1, etc., and various L-rhamnose isomerase variants were produced. The amino acid substitution variant of the present application includes, as an exception, a variant in which one amino acid residue (G) is added to the amino acid at position 418 at the C-terminus.

[0215] First, a single amino acid substitution variant was produced by introducing point mutation. PCR primers having two DNA strands with base sequence in which one to two base pairs were added with base substitution at the target mutation introduction site were produced, and a plasmid DNA of the mutation introduction destination and the primers were used to perform PCR reaction. The obtained PCR fragment was confirmed by sequencing analysis, transformed into host E. coli, and plasmid DNA was extracted from the obtained clone to introduce the target mutation. For multiple substitution variants, the above method was repeated to produce them.

[0216] Recombinant E. coli expressing recombinant enzyme containing point mutation enzyme was precultured in a medium containing 3.5% poly peptone, 2.0% yeast extract, 1.0% sodium chloride, 2 mM manganese chloride with the addition of ampicillin at a final concentration of 100 μg / ml at 30°C at 200 rpm for 12 hours. The preculture was inoculated into an expression medium with the addition of ampicillin at a final concentration of 100 μg / ml at 30°C at 200 rpm for 2 hours. After the main culture, IPTG was added at a final concentration of 0.1 mM, and expression induction of the recombinant enzyme was performed at 30°C at 200 rpm for one night.

[0217] The composition of the expression medium of the recombinant E. coli is shown in Table 8.

[0218] [Table 8]

[0219]

[0220] The enzyme activity of the crude enzyme of the wild-type enzyme and the amino acid substitution variants obtained by recombination was determined as follows: the substrate was D-allose at a final concentration of 100 mM, the buffer was Tris-HCl buffer (pH 8.0) at a final concentration of 50 mM, the reaction was carried out at each temperature for 10 minutes, and the amount of D-allose produced was determined using HPLC with respect to the sugar composition of the reaction solution.

[0221] In addition, as the residual activity after incubation at 60°C for 1 hour, the enzyme solution was heat-treated at 60°C for 1 hour, and then the residual activity was determined by carrying out an enzyme reaction at 60°C for 10 minutes using the reaction composition shown in Table 9.

[0222] The composition of the reaction solution is shown in Table 9 below.

[0223] [Table 9]

[0224]

[0225] Using the above method, the reaction optimum temperature and the relative activity of each variant were determined, and an amino acid substitution variant having a higher D-allose isomerase activity than the wild-type enzyme at the reaction optimum temperature of each variant was selected.

[0226] The wild-type enzyme was a crude enzyme produced by recombination, and the optimum temperature for D-allose production was 60°C. The purified enzyme had an optimum temperature for L-rhamnose of less than 70°C, but there were also variants in which the optimum temperature was recovered by insertion mutation.

[0227] As a result, 13 variants having a higher optimum temperature or a higher D-allose isomerase activity at the optimum temperature than the wild-type enzyme were obtained (Table 10).

[0228] In addition, as an index of heat resistance, the ratio of the D-allose isomerase activity at a reaction temperature of 70°C and 50°C (T70 / T50), the residual activity after incubation at 60°C for 1 hour, and the ratio of the D-allose isomerase activity at a reaction temperature of 80°C and the optimum temperature (T80 / optimum temperature) of each variant were determined in the same manner as the determination of the reaction optimum temperature and the relative activity of each variant. Variants in which any one of the indices was higher than the wild-type enzyme were selected (Tables 11 to 13).

[0229] As a result, 56 variants in which any one of the above four indices was higher than the wild-type enzyme were obtained. The amino acid sequence of each of the 56 variants is shown in Sequence Listing as Sequence Nos. 3 to 8, 10 to 28, and 30 to 60. Variants having the amino acid sequence of Sequence Nos. 9 and 29 had activity, but did not reach the level of the wild-type enzyme in all four indices.

[0230] Each variant of the amino acid sequence of the sequence numbers 3 to 60 of the sequence listing is named as "sequence 3 to 60".

[0231] Hereinafter, the amino acid substitution site (position) from the wild-type enzyme (sequence number 1) and the substituted amino acid of the sequence 3 to 60 are shown, respectively. In this case, in the case where the amino acid substitution site and the like shown below are different from those shown in the sequence numbers 3 to 60 of the sequence listing, the amino acid sequence shown in the sequence listing is the correct sequence. In addition, the number in the last parenthesis of each sequence indicates the number of amino acid substitutions.

[0232] Sequence 3 419G (1)

[0233] 4 V280I (1)

[0234] 5 V387I (1)

[0235] 6 L198F (1)

[0236] 7 S405E (1)

[0237] 8 N52E (1)

[0238] 9 Q124R (1)

[0239] 10 D102T (1)

[0240] 11 Q359L (1)

[0241] 12 K152P (1)

[0242] 13 D18A, F216L, T310S, L365Q (4)

[0243] 14 D18A, F216L, L365Q (3)

[0244] 15 Y9F, D18A, V19I, I21V, Q25K, T28A, H73D, A77S, I79V, T103K, E106D, Q113K, H117N, Q124T, P179A, I195V, A199G, E202Q, F216L, D217N, A218P, S219A, A250S, L300I, N315H, N319D, S357T, L365S, A371G, L375M, L417Q (31)

[0245] 16 Y9F, D18A, V19I, I21V, T28A, H73D, A77S, I79V, T103K, E106D, Q113K, H117N, Q124T, P179A, I195V, E202Q, F216L, D217N, A218P, S219A, A250S, L300I, N315H, N319D, S357T, L365N, A371G, L375M, L417Q (29)

[0246] 17 D18A, V19I, I21V, T28A, H73D, A77S, I79V, T103K, E106D, Q113K, H117N, Q124T, P179A, I195V, E202Q, F216L, D217N, A218P, S219A, A250S, L300I, N315H, N319D, S357T, L365S, A371G, L375M (27)

[0247] 18 D18A, V19I, I21V, T28A, I79V, Q113K, H117N, Q124P, I195V, E202Q, F216L, A218P, S219A, A250T, T310S, N315H, N319D, S357T, L365N, L417Q (20)

[0248] 19 D18A, V19I, I21V, T28A, H73D, T103K, Q113K, H117N, Q124N, H125N, E202Q, F216L, D217N, A218P, S219Q, A250T, N315H, N319D, L365N (19)

[0249] 20 D18A, V19I, I21V, T28A, H73D, T103K, Q113K, H117N, Q124N, H125N, I195V, E202Q, F216L, D217N, A218P, S219Q, A250T, N315H, N319D, S357T, L365N (21)

[0250] 21 Y9F, D18A, V19I, I21V, M27L, T28G, G32R, N52Q, E54A, H73D, I79L, K81Q, T103K, E106A, A109E, Q113E, H117N, Q124N, K126Q, S134T, H171Y, I195V, E202Q, V211I, F216L, D217N, A218P, S219Q, A250T, T275A, T310N, N315Q, K316N, N319D, K320R, S357T, D358A, K362Q, L365N, E406D (40)

[0251] 22 D18A, V19I, I21V, M27L, T28G, G32R, N52Q, E54A, H73D, I79L, K81Q, T103K, E106A, A109E, Q113E, H117N, Q124N, K126Q, S134T, H171Y, I195V, E202Q, V211I, F216L, D217N, A218P, S219Q, A250T, T275A, T310N, N315Q, K316N, N319D, K320R, S357T, D358A, K362Q, L365N, E406D (39)

[0252] 23 Y9F, D18A, V19I, I21V, T28G, N52Q, E54A, H73D, I79L, T103K, Q113E, H117N, Q124N, S134T, H171Y, I195V, E202Q, F216L, D217N, A218P, S219Q, A250T, T275A, T310N, N315Q, N319D, S357T, K362Q, L365N, E406D (30)

[0253] 24 Y9F, D18A, V19I, I21V, T28A, N52Q, E54A, H73D, I79L, T103K, Q113E, H117N, Q124N, H125N, S134T, H171Y, I195V, E202Q, V211I, F216L, D217N, A218P, S219Q, A250T, T310N, N315Q, N319D, S357T, K362Q, L365N, E406D (31)

[0254] 25 D18A, V19I, I21V, T28A, N52Q, E54A, H73D, I79L, T103K, Q113E, H117N, Q124K, S134T, H171Y, I195V, E202Q, V211I, F216L, D217N, A218P, S219Q, A250S, T310N, N315Q, N319D, S357T, L365N (27)

[0255] 26 D18A, V19I, I21V, V26A, T28A, G32R, N52Q, E54A, H73D, I79L, E80D, T103K, Q113E, H117N, Q124K, S134T, H171Y, I195V, E202Q, A205V, F216L, D217N, A218P, S219Q, A250T, T310N, N315Q, N319D, S357T, K362Q, L365N (31)

[0256] 27 L4Q, Y9F, V19I, I21V, V26A, M27I, T28R, G32R, N52Q, E54A, H73D, A77S, E80D, T103K, Q113A, S116A, H117N, E120A, Q124E, L140M, A150S, G153T, I154V, H171Y, L193I, I195V, E202Q, A205M, F216L, D217N, A218P, S219Q, T257C, T275A, T310S, A313V, N315H, K316Q, N319D, A345T, S357T, Q359R, K362Q, L363A, L365N, A374T, S405G, E406D (48)

[0257] 28 V19I, I21V, V26A, M27I, T28R, G32R, N52Q, E54A, H73D, A77S, E80D, T103K, Q113A, S116A, H117N, E120A, Q124E, L140M, A150S, G153T, I154V, H171Y, L193I, I195V, E202Q, A205M, F216L, D217N, A218P, S219Q, T257C, T275A, T310S, A313V, N315H, K316Q, N319D, A345T, S357T, Q359R, K362Q, L363A, L365N, A374T, S405G, E406D (46)

[0258] 29 D18E, V19N, I21V, E24D, Q25L, T28A, G32E, E54Q, H73D, M83I, K90M, D102S, T103Q, Q113K, H117N, E120A, Q124S, H125N, K126R, S134T, P139A, L140K, K152T, R167H, H171Y, L198F, E202Q, E210A, V211I, K215Q, F216L, D217N, A218P, S219Q, A250S, T254I, T310H, N315Q, N319D, Q359R, K362R, L365S, Y369F (43)

[0259] 30 K3T, L4Q, I5L, Y9W, E10D, Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54S, R68K, H73T, I79L, K81L, A82T, M83L, A89P, T103E, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, S148A, K152D, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, V211A, F216L, A218P, S219A, C245Y, L246M, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, R361K, L365A, E366N, A390M, W391Y, L393Q, H395N, V397A, D400G, A401S, S402Q, S405D, E406N, H409A, Q412E, Q413D, T414V, R416S, L417Q, 419G (84)

[0260] 31 K3T, L4Q, I5L, Y9W, E10D, Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54S, R68K, H73T, I79L, K81L, A82T, M83L, A89P, T103E, E106A, D108N, A109E, E111K, Q113E, S116K, Q124A, H125N, S148A, K152D, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, V211A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, R361K, L365A, E366N, A390M, W391Y, L393Q, H395N, V397A, D400G, A401S, S402Q, S405D, E406N, H409A, Q412E, Q413D, T414V, R416S, L417Q, 419G (84)

[0261] 32 K3T, L4Q, I5L, Y9W, E10D, Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54S, R68K, H73T, I79L, K81L, M83L, A89P, T103E, E106A, D108N, A109E, E111K, Q113G, S116T, H117N, Q124A, H125N, S148A, K152D, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, V211A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, R361K, L365A, E366N, A390M, W391Y, L393Q, H395N, V397A, D400G, A401S, S402Q, S405D, E406N, H409A, Q412K, Q413D, T414V, R416S, L417Q, 419G (84)

[0262] 33 K3T, L4Q, I5L, Y9W, E10D, D18A, I21V, E24D, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54S, R68K, H73T, I79L, K81L, M83L, A89P, E106A, D108N, A109E, E111K, Q113A, S116K, H117N, E120A, Q124A, H125N, K126Q, S148A, D151N, K152D, G153D, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, V211A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310N, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358S, Q359A, R361K, K362Q, L365E, E366N, A374V, S381T, A390M, W391Y, L393Q, H395N, V397T, D400G, A401S, S402Q, S405D, E406N, H409M, Q412K, Q413D, T414V, R416S, L417Q, 419G(90)

[0263] 34 L11I, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54S, R68K, H73G, I79L, K81Q, M83L, A89P, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, D151N, K152D, G153E, I168V, H171Y, V186I, L193I, I195V, L198F, E202Q, A205L, S206N, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, V280I, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, D358E, K362Q, L365A, E366D(60)

[0264] 35 D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54S, R68K, H73G, I79L, K81Q, M83L, A89P, I97L, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124T, H125N, D151N, K152N, G153E, I168V, H171Y, V186I, L193I, I195V, L198F, E202Q, A205L, S206N, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, V280I, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, D358E, K362Q, L365A, E366D, S381C (61)

[0265] 36 Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54A, R68K, H73T, I79L, K81Q, M83L, A89P, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, D151N, K152D, G153E, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206D, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, K362Q, L365A, E366D (57)

[0266] 37 Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54A, R68K, H73T, I79L, K81Q, M83L, A89P, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, D151N, K152D, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206D, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, K362Q, L365A, E366D (59)

[0267] 38 Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54A, R68K, H73T, I79L, K81Q, M83L, A89P, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, D151N, K152N, G153E, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, D358E, K362Q, L365A, E366D (58)

[0268] 39 Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54A, R68K, H73T, I79L, K81Q, M83L, A89P, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, K152N, G153E, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, D358E, K362Q, L365A, E366N (57)

[0269] 40 Y16F, D18A, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54S, R68K, H73T, I79L, K81Q, M83L, A89P, E106S, A109Q, E111K, Q113E, S116K, H117N, Q124A, H125N, K126Q, K152N, G153K, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206D, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313V, N315H, K316N, N319D, K320R, A345M, S357T, D358E, K362Q, L365A, E366S (56)

[0270] 41 Y16F, D18A, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54S, R68K, H73T, I79L, K81Q, M83L, A89P, E106S, A109Q, E111K, Q113E, S116K, H117N, Q124A, H125N, K126Q, K152N, G153K, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313V, N315H, K316N, N319D, K320R, A345M, S357T, D358E, K362Q, L365A, E366S (56)

[0271] 42 Y16F, D18A, I21V, V26A, M27L, T28P, G32R, I33L, R46A, N52Q, E54A, R68K, H73T, I79L, K81Q, A89P, D108N, E111K, Q113E, S116K, H117N, Q124E, K152N, G153D, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, K362Q, L365A, E366N (50)

[0272] 43 Y16F, D18A, Q25E, V26A, M27L, T28R, G32R, I33L, R46S, N52E, E54S, R68K, H73S, I79L, K81Q, S84R, A89P, E106S, A109Q, E111K, Q113E, S116K, H117N, Q124A, H125N, K126Q, K152N, G153S, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, R282Q, T310S, A313V, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, L365A, E366A (57)

[0273] 44 Y16F, D18A, Q25E, V26A, M27L, T28R, G32R, I33L, R46S, N52E, E54S, R68K, H73S, I79L, K81Q, S84R, A89P, E106S, A109Q, E111K, Q113E, S116K, H117N, Q124A, H125N, K126Q, K152N, G153S, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, D217N, A218P, S219A, C245Y, A250T, T257C, T275A, R282Q, T310S, A313V, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, L365A, E366A (57)

[0274] 45 Y16F, D18A, Q25E, V26A, M27L, T28R, G32R, I33L, R46S, N52E, E54S, R68K, H73S, A77T, I79L, K81Q, S84R, A89P, E106S, A109Q, E111K, Q113E, S116K, H117N, Q124A, H125N, K126Q, K152N, G153S, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, R282Q, T310S, A313V, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, L365A, E366A (59)

[0275] 46 Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54A, R68K, H73T, I79L, K81L, M83L, A89P, T103E, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, K152D, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, K362Q, L365A, E366D (61)

[0276] 47 Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54A, R68K, H73T, I79L, K81L, M83L, A89P, T103E, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, S148A, K152D, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, K362Q, L365A, E366D (62)

[0277] 48 Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54A, R68K, H73T, I79L, K81L, M83L, A89P, T103E, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, S148A, K152N, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, K362Q, L365A, E366N (62)

[0278] 49 E10D, Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54S, R68K, H73T, I79L, K81L, M83L, A89P, T103E, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, S148A, K152D, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, V211A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, R361K, K362Q, L365A, E366N (65)

[0279] 50 E10D, Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54S, R68K, H73T, I79L, K81L, M83L, A89P, T103E, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, S148A, K152D, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, V211A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, R361K, K362Q, L365A, E366N(64)

[0280] 51 Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54S, R68K, H73T, I79L, K81L, M83L, A89P, T103E, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, S148A, K152N, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, V211A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, R361K, K362Q, L365A, E366N(63)

[0281] 52 E10D, Y16F, D18A, I21V, Q25E, V26A M27L, T28R, G32R, I33L, R46A, N52G, E54S, R68K, H73T, I79L, K81L, M83L, A89P, T103E, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, K126R, S148A, D151N, K152D, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, V211A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357V, D358A, Q359A, R361K, K362Q, L365E, E366N(66)

[0282] 53 Y16F, D18A, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54S, R68K, H73T, I79L, K81L, M83L, A89P, T103E, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124A, H125N, K126R, S148A, D151N, K152N, G153E, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, V211A, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357V, D358A, Q359A, R361K, K362Q, L365E, E366N(65)

[0283] 54 E10D, D18A, I21V, E24D, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52G, E54S, R68K, H73T, I79L, K81L, M83L, A89P, E106A, D108N, A109E, E111K, Q113A, S116K, H117N, E120A, Q124A, H125N, K126Q, S148A, D151N, K152D, G153D, C162V, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, V211A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316D, N319D, K320R, A345M, S357T, D358S, Q359A, R361K, K362Q, L365E, E366N, A374V, S381T (69)

[0284] 55 K3T, L4Q, I5L, Y9W, L11I, D18A, V19I, I21V, V23A, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54S, R68K, H73Q, I79L, K81Q, M83L, A89P, D102T, E106A, D108N, A109E, E111K, Q113E, S116K, Q124R, K126Q, D151N, K152P, G153E, H171Y, L193I, I195V, L198F, E202Q, A205L, S206N, F216L, A218P, S219A, C245Y, L246M, T257C, T275A, V280I, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, D358A, Q359L, K362Q, L365N, E366D, V387I, A390M, W391Y, L393Q, V397T, D400G, A401S, S402Q, S405E, E406N, H409T, Q412K, Q413D, T414V, R416S, L417Q, 419G (81)

[0285] 56 K3T, L4Q, I5L, Y9W, D18A, V19I, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52D, E54S, R68K, I79L, K81Q, M83L, A89P, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124P, K126Q, D151N, K152E, G153E, H171Y, L193I, I195V, E202Q, A205L, S206N, F216L, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, D358A, K362Q, L365N, E366D, A390M, W391Y, L393Q, V397T, D400G, A401S, S402Q, S405D, E406N, H409T, Q412K, Q413D, T414V, R416S, L417Q, 419G (75)

[0286] 57 K3T, L4Q, I5L, Y9W, D18A, V19I, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54T, R68K, H73N, I79L, K81Q, M83L, A89P, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124G, K126Q, D151N, K152E, G153E, H171Y, L193I, I195V, E202Q, A205L, S206N, F216L, A218P, S219A, C245Y, L246M, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, D358A, Q359L, K362Q, L365N, E366D, A390M, W391Y, L393Q, V397T, D400G, A401S, S402Q, S405D, E406N, H409T, Q412K (71)

[0287] 58 Y9W, D18A, V19I, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54T, R68K, H73N, I79L, K81Q, M83L, A89P, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124G, K126Q, D151N, K152E, G153E, H171Y, L193I, I195V, E202Q, A205L, S206N, F216L, A218P, S219A, C245Y, L246M, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, D358A, Q359L, K362Q, L365N, E366D, A390M, W391Y, L393Q, V397T, D400G, A401S, S402Q, S405D, E406N, H409T, Q412K (68)

[0288] 59 Y9W, D18A, V19I, I21V, Q25E, V26A, M27L, T28R, G32R, I33L, R46A, N52E, E54T, R68K, H73N, I79L, K81Q, M83L, A89P, E106A, D108N, A109E, E111K, Q113E, S116K, H117N, Q124G, K126Q, D151N, K152E, G153E, H171Y, L193I, I195V, E202Q, A205L, S206N, F216L, A218P, S219A, C245Y, L246M, T257C, T275A, T310S, A313I, N315H, K316N, N319D, K320R, A345M, S357T, D358A, Q359L, K362Q, L365N, E366D, A390M, W391Y, L393Q, V397T, D400G, A401S, S402Q, S405D, E406N, H409T (67)

[0289] 60 K3T, L4Q, I5L, Y9W, Y16F, D18A, Q25E, V26A, M27L, T28R, G32R, I33L, R46S, N52E, E54S, R68K, H73S, I79L, K81Q, S84R, A89P, E106S, A109Q, E111K, Q113E, S116K, H117N, Q124A, H125N, K126Q, K152D, G153S, I168V, H171Y, V186I, L193I, I195V, E202Q, A205L, S206A, F216L, D217N, A218P, S219A, C245Y, L246M, A250T, T257C, T275A, R282Q, T310S, A313V, N315H, K316D, N319D, K320R, A345M, S357T, D358A, Q359E, L365A, E366P, A390M, W391Y, L393Q, V397T, D400G, A401S, S402E, S405E, E406S, H409A, Q412K, Q413E, T414I, R416S, L417R, 419G(78)

[0290] To summarize, the amino acid substitution variants of SEQ ID NOs: 3-60 have specific amino acid substitutions at the following 122 positions.

[0291] K3T, L4Q, I5L, Y9F / W, E10D, L11I, Y16F, D18A / E, V19I / N, I21V, V23A, E24D, Q25K / L / E, V26A, M27L / I, T28A / G / R / P, G32E / R, I33L, R46A / S, N52D / E / G / Q, E54A / Q / S / T, R68K, H73D / G / N / S / T / Q, A77S / T, I79L / V, E80D, K81Q / L, A82T, M83I / L, S84R, A89P, K90M, I97L, D102S / T, T103E / K / Q, E106A / D / S, D108N, A109E / Q, E111K, Q113A / E / K / G, S116A / K / T, H117N, E120A, Q124A / E / G / K / N / R / P / S / T, H125N, K126R / Q, S134T, P139A, L140K / M, S148A, A150S, D151N, K152D / E / N / P / T, G153D / E / K / S / T, I154V, C162V, R167H, I168V, H171Y, P179A, V186I, L193I, I195V, L198F, A199G, E202Q, A205L / M / V, S206A / D / N, E210A, V211A / I, K215Q, F216L, D217N, A218P, S219A / Q, C245Y, L246M, A250S / T, T254I, T257C, T275A, V280I, R282Q, L300I, T310N / H / S, A313I / V, N315H / Q, K316D / N / Q, N319D, K320R, A345M / T, S357T / V, D358A / E / S, Q359A / E / L / R, R361K, K362Q / R, L363A, L365A / E / N / S / Q, E366A / D / N / P / S, Y369F, A371G, A374T / V, L375M, S381C / T, V387I, A390M, W391Y, L393Q, H395N, V397A / T, D400G, A401S, S402Q / E, S405D / E / G, E406D / N / S, H409A / T / M, Q412E / K, Q413D / E, T414V / I, R416S, L417Q / R, 419G

[0292] Further, in the amino acid substitution variants of SEQ ID NOs: 3 to 60, amino acid substitution and addition of one amino acid residue are performed at 122 sites below.

[0293] K3, L4, I5, Y9, E10, L11, Y16, D18, V19, I21, V23, E24, Q25, V26, M27, T28, G32, I33, R46, N52, E54, R68, H73, A77, I79, E80, K81, A82, M83, S84, A89, K90, I97, D102, T103, E106, D108, A109, E111, Q113, S116, H117, E120, Q124, H125, K126, S134, P139, L140, S148, A150, D151, K152, G153, I154, C162, R167, I168, H171, P179, V186, L193, I195, L198, A199, E202, A205, S206, E210, V211, K215, F216, D217, A218, S219, C245, L246, A250, T254, T257, T275, V280, R282, L300, T310, A313, N315, K316, N319, K320, A345, S357, D358, Q359, R361, K362, L363, L365, E366, Y369, A371, A374, L375, S381, V387, A390, W391, L393, H395, V397, D400, A401, S402, S405, E406, H409, Q412, Q413, T414, R416, L417, 419

[0294] The sequence 33, which has the largest number of amino acid substitutions among the sequences 3 to 60, has 90, and has 78% identity with the amino acid sequence of the wild-type enzyme of the sequence 1.

[0295] In addition, among the 13 sequences in which the D- psicose isomerase activity at the optimum temperature is higher than that of the wild-type enzyme of the sequence 1, which is shown in Table 10 below, the sequence 32 has the largest number of amino acid substitutions, which is 84, and has 80% identity with the amino acid sequence of the sequence 1.

[0296] [Table 10]

[0297] Activity at optimum temperature (U) Optimum temperature Sequence 32 107.32 70℃ 46 104.94 70℃ 55 98.62 60℃ 13 97.09 70℃ 26 96.92 70℃ 15 96.16 70℃ 51 94.09 70℃ 17 83.77 70℃ 10 80.26 70℃ 21 80.05 70℃ 20 79.23 70℃ 37 77.32 60℃ 49 75.79 70℃ 1 75.54 60℃

[0298] Table 11 shows the numbers of the 52 sequences in which the ratio of the D- psicose isomerase activity at the reaction temperature of 70°C to that at 50°C (T70 / T50) is higher than that of the wild-type enzyme of the sequence 1.

[0299] [Table 11]

[0300] Sequence T70 / T50 42 3.05 27 2.07 14 1.98 56 1.91 21 1.90 48 1.87 17 1.84 52 1.83 26 1.83 58 1.82 19 1.80 39 1.79 22 1.76 8 1.75 51 1.69 46 1.69 5 1.67 57 1.67 50 1.66 31 1.63 40 1.63 20 1.62 18 1.61 59 1.60 41 1.59 49 1.59 23 1.58 34 1.56 54 1.55 33 1.55 25 1.55 38 1.50 44 1.48 53 1.47 43 1.46 10 1.44 7 1.43 13 1.42 15 1.41 55 140 60 1.40 47 1.36 24 1.35 37 1.33 45 1.30 35 1.29 16 1.28 30 1.27 32 1.21 12 1.21 36 1.19 28 1.17 1 1.16

[0301] Table 12 shows the number of 17 sequences having higher residual activity ratio than the wild-type enzyme of sequence 1 after incubation at 60°C for 1 hour.

[0302] [Table 12]

[0303] Sequence Residual activity after incubation at 60°C for 1 hour 41 115.2 22 113.6 46 113.0 36 112.2 47 110.4 6 110.4 34 109.5 28 107.3 30 107.2 40 107.2 27 107.0 42 105.7 19 105.4 17 104.8 51 104.5 21 103.8 49 103.6 1 103.4

[0304] Table 13 shows the number of 56 sequences having higher D- psicose isomerase activity ratio (T80 / optimum temperature) than the wild-type enzyme of sequence 1 at the reaction temperature of 80°C and the optimum temperature.

[0305] [Table 13]

[0306] Sequence T80 / Relative activity at optimum temperature (%) 45 92.0 51 83.9 56 83.7 43 82.6 33 81.1 46 79.9 41 79.4 17 76.6 48 75.6 31 73.1 38 71.1 22 68.6 21 68.0 36 67.3 37 66.7 52 66.3 32 66.1 50 63.7 44 60.2 39 59.7 49 59.0 20 58.8 55 58.4 53 56.8 47 52.8 30 52.3 23 49.7 59 49.0 10 48.5 19 47.4 13 44.1 28 43.4 8 41.7 26 40.2 42 39.9 40 38.4 16 37.2 58 35.9 14 35.1 54 34.8 34 34.1 5 33.4 18 32.4 6 31.3 15 30.9 57 30.5 25 30.1 60 28.8 12 24.1 7 24.0 24 21.7 3 19.6 27 17.4 35 14.0 4 13.5 11 8.3 1 7.7

[0307] Industrial applicability

[0308] The L-rhamnose isomerase of the present application is characterized by having high heat resistance and high activity compared to the existing L-rhamnose isomerases from microorganisms. For example, while the optimum temperature of the L-rhamnose isomerase from the existing Pseudomonas stutzeri is 60°C, the enzyme of the present application is as high as 70°C, and has a residual activity of 80% or more after heating treatment at 60°C for 10 minutes, and thus has heat stability, and is suitable for industrial production.

[0309] In addition, in the case where the substrate is D-psicose, the enzyme of the present application has high conversion activity to D-allose of 2.26 U / mg protein at 60°C, and thus opens the way to mass production of D-allose.

[0310] In addition, the L-rhamnose isomerase of the present application can be used to obtain an immobilized enzyme having high activity by various immobilization methods, and by using the immobilized enzyme, mass isomerization reaction can be continuously performed. By being able to be immobilized in industry, mass production of target aldose can be achieved.

[0311] Further, the L-rhamnose isomerase of the present application includes a large number of amino acid substitution variants having higher enzyme activity or heat resistance than the enzyme before the variation.

[0312] Thus, the establishment of the L-rhamnose isomerase of the present application and its variants, and the manufacturing method thereof has great industrial significance not only in the sugar industry, but also in the food, cosmetic, pharmaceutical, and pesticide industries related thereto. SEQUENCE LISTING <110> National University Corporation Kagawa University <120> Novel L-rhamnose isomerase <130> PCT21632UK <160> 60 <210> SEQ ID NO: 1 <211> 418 <212> PRT <213> Erwinia <220> <223> Inventors: Akimitsu Kazuya; He Senjian; Yoshihara Akihide; Kato Shiro; Mochizuki Susumu; Yoshida Yumi; Kamitori Narihiro <223> Seq-1 <400> 1 Met Thr Lys Leu Ile Glu Gln Ala Tyr Glu Leu Ala Lys Gln Arg Tyr 1 5 10 15 Ala Asp Val Gly Ile Asp Val Glu Gln Val Met Thr Gln Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp Ile Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gln His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Ile Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Phe Asp Ala Ser His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gln Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Thr Glu Ile Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gln Leu Arg Lys Leu Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 2 <211> 1257 <212> DNA <213> E. coli <220> <223> Seq-2 <400> 2 atgacaaagc tgattgaaca agcttatgaa ctggctaaac aacgttacgc tgacgtagga 60 attgatgtgg agcaggtgat gacgcaattg gatgggattc ctgtgtcgat gcactgctgg 120 cagggggatg acgtgcgcgg cttcgaaaac cccaatggcg aactcaccgg cgggattcag 180 gccacaggta actatccggg acgcgcgcgt aatgcgcatg aactgcgcgc cgatatcgaa 240 aaagccatgt cgctgattcc aggcgctaag cgcctcaatc tgcacgccat ctatctggaa 300 agcgacacgc cggtagaacg tgatgctatc gaaccgcaac acttcagcca ctgggtggag 360 tgggcaaaac agcataaatt agggctcgat tttaatccaa gctgcttctc gcatccttta 420 agtgccgatg gttttaccct gtcacacgct gacaaaggca tccgccagtt ctggattgat 480 cactgcaagg ccagtcgccg catctcggca cattttggtg aacaactggg cacgccctca 540 gtgatgaaca tctgggttcc ggacggtatg aaagatctca ccattgatcg cctggcgccg 600 cgtgaacgtt tagccagcgc gctggatgaa gtgatcagcg aaaaattcga tgccagtcat 660 catatcgacg ccgtcgaaag taagttattc gggattggtg ctgagagtta taccgtgggg 720 tccaatgagt tttgccttgg ctacgctgcc agccgccaga ccgcgctgac acttgatgcc 780 gggcatttcc atcccactga agtgatctcc gacaaaatct ccaccgcgat gctgtatgtc 840 ccgcgcctgt tattgcacgt cagccgtccg gtgcgttggg acagcgacca tgtggtgctg 900 ctggatgatg aaacccaggc cattgccact gaaattgcgc gtaacaagct gttcaacaaa 960 gtgcatatcg gcctcgactt ctttgatgct tccatcaacc gcatcgcggc gtgggtgatt 1020 ggtacccgca atgccaaaaa ggcgttactg cgtgcgctgt tggagccgag tgaccaactg 1080 cgcaaactgg agcttgaggg ggattacacc gcacgtctgg cgctgctgga ggagcaaaag 1140 tcattgccat ggcaggctgt ctgggaagcc tggtgcttgc gtcacgatgt ccctgctgat 1200 gccagttggc tcagcgaagt ccgtcattat gaacaacaaa cactgcgtct acgttaa 1257 <210> SEQ ID NO: 3 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> Seq-3 <400> 3 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Asp Val Gly lie Asp Val Glu Gin Val Met Thr Gin Leu Asp Gly 20 25 30 lie Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp lie Glu 65 70 75 80 Lys Ala Met Ser Leu lie Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala lie Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gin His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly lie Arg Gin Phe Trp lie Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg lie Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr lie Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Phe Asp Ala Ser His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Thr Glu lie Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gin Leu Arg Lys Leu Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg Gly <210> SEQ ID NO: 4 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-4 <400> 4 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Asp Val Gly lie Asp Val Glu Gin Val Met Thr Gin Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp Ile Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gin His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr lie Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Phe Asp Ala Ser His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr lie Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Thr Glu lie Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gin Leu Arg Lys Leu Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 5 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-5 <400> 5 Met Thr Lys Leu Ile Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Asp Val Gly Ile Asp Val Glu Gin Val Met Thr Gin Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp Ile Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gln His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Ile Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Phe Asp Ala Ser His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Thr Glu lie Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gin Leu Arg Lys Leu Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Ile Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 6 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-6 <400> 6 Met Thr Lys Leu Ile Glu Gln Ala Tyr Glu Leu Ala Lys Gln Arg Tyr 1 5 10 15 Ala Asp Val Gly Ile Asp Val Glu Gln Val Met Thr Gln Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp Ile Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gin His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Ile Asp Arg Phe Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Phe Asp Ala Ser His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Thr Glu Ile Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gln Leu Arg Lys Leu Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 7 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-7 <400> 7 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Asp Val Gly lie Asp Val Glu Gin Val Met Thr Gin Leu Asp Gly 20 25 30 lie Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp lie Glu 65 70 75 80 Lys Ala Met Ser Leu lie Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala lie Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gin His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Ile Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Phe Asp Ala Ser His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Thr Glu Ile Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gln Leu Arg Lys Leu Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Glu Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 8 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-8 <400> 8 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Asp Val Gly lie Asp Val Glu Gin Val Met Thr Gin Leu Asp Gly 20 25 30 lie Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Glu Gly Glu Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp lie Glu 65 70 75 80 Lys Ala Met Ser Leu lie Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala lie Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gin His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly lie Arg Gin Phe Trp lie Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg lie Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr lie Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Phe Asp Ala Ser His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Thr Glu lie Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gin Leu Arg Lys Leu Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 9 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-9 <400> 9 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Asp Val Gly lie Asp Val Glu Gin Val Met Thr Gin Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp Ile Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Arg His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr lie Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Phe Asp Ala Ser His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Thr Glu lie Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gin Leu Arg Lys Leu Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 10 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-10 <400> 10 Met Thr Lys Leu Ile Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Asp Val Gly Ile Asp Val Glu Gin Val Met Thr Gin Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp Ile Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Thr Thr Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gln His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Ile Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Phe Asp Ala Ser His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Thr Glu lie Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gin Leu Arg Lys Leu Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 11 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-11 <400> 11 Met Thr Lys Leu Ile Glu Gln Ala Tyr Glu Leu Ala Lys Gln Arg Tyr 1 5 10 15 Ala Asp Val Gly Ile Asp Val Glu Gln Val Met Thr Gln Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp Ile Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gin His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Ile Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Phe Asp Ala Ser His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Thr Glu Ile Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Leu Leu Arg Lys Leu Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 12 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-12 <400> 12 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Asp Val Gly lie Asp Val Glu Gin Val Met Thr Gin Leu Asp Gly 20 25 30 lie Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp lie Glu 65 70 75 80 Lys Ala Met Ser Leu lie Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala lie Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gin His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Pro Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Ile Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Phe Asp Ala Ser His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Thr Glu Ile Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gln Leu Arg Lys Leu Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 13 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-13 <400> 13 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala Val Gly lie Asp Val Glu Gin Val Met Thr Gin Leu Asp Gly 20 25 30 lie Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp lie Glu 65 70 75 80 Lys Ala Met Ser Leu lie Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala lie Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gin His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly lie Arg Gin Phe Trp lie Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg lie Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr lie Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Leu Asp Ala Ser His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gin Leu Arg Lys Leu Glu Gin Gin Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 14 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-14 <400> 14 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala Val Gly lie Asp Val Glu Gin Val Met Thr Gin Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp Ile Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Gln His Phe Ser His Trp Val Glu Trp Ala Lys Gin His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr lie Asp Arg Leu Ala Pro Arg Glu Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Leu Asp Ala Ser His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Thr Glu lie Ala Arg Asn Lys Leu Phe Asn Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gin Leu Arg Lys Leu Glu Gin Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 15 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-15 <400> 15 Met Thr Lys Leu Ile Glu Gin Ala Phe Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala Ile Gly Val Asp Val Glu Lys Val Met Ala Gin Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ser Asp Val Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Lys Pro Val Asp Arg Asp Ala Ile Glu Pro 100 105 110 Lys His Phe Ser Asn Trp Val Glu Trp Ala Lys Thr His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gln Leu 165 170 175 Gly Thr Ala Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Val Asp Arg Leu Gly Pro Arg Gln Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asn Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ser Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val lie Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Thr Glu lie Ala Arg His Lys Leu Phe Asp Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gin Leu Arg Lys Leu Glu Ser Glu Gly Asp 355 360 365 Tyr Thr Gly Arg Leu Ala Met Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Gln Arg <210> SEQ ID NO: 16 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-16 <400> 16 Met Thr Lys Leu Ile Glu Gln Ala Phe Glu Leu Ala Lys Gln Arg Tyr 1 5 10 15 Ala Ala Ile Gly Val Asp Val Glu Gln Val Met Ala Gln Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ser Asp Val Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Lys Pro Val Asp Arg Asp Ala Ile Glu Pro 100 105 110 Lys His Phe Ser Asn Trp Val Glu Trp Ala Lys Thr His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gln Leu 165 170 175 Gly Thr Ala Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asn Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ser Ser Arg Gln Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Ile Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Thr Glu Ile Ala Arg His Lys Leu Phe Asp Lys 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gln Leu Arg Lys Leu Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Gly Arg Leu Ala Met Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Gln Arg <210> SEQ ID NO: 17 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-17 <400> 17 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala lie Gly Val Asp Val Glu Gin Val Met Ala Gin Leu Asp Gly 20 25 30 lie Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ser Asp Val Glu 65 70 75 80 Lys Ala Met Ser Leu lie Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Lys Pro Val Asp Arg Asp Ala lie Glu Pro 100 105 110 Lys His Phe Ser Asn Trp Val Glu Trp Ala Lys Thr His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Ala Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asn Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ser Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Ile Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Thr Glu Ile Ala Arg His Lys Leu Phe Asp Lys 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gln Leu Arg Lys Leu Glu Ser Glu Gly Asp 355 360 365 Tyr Thr Gly Arg Leu Ala Met Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 18 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-18 <400> 18 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala lie Gly Val Asp Val Glu Gin Val Met Ala Gin Leu Asp Gly 20 25 30 lie Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala His Glu Leu Arg Ala Asp Val Glu 65 70 75 80 Lys Ala Met Ser Leu lie Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asp Ala lie Glu Pro 100 105 110 Lys His Phe Ser Asn Trp Val Glu Trp Ala Lys Pro His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly lie Arg Gin Phe Trp lie Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg lie Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Leu Asp Pro Ala His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie Ala Arg His Lys Leu Phe Asp Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gin Leu Arg Lys Leu Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Gln Arg <210> SEQ ID NO: 19 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-19 <400> 19 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala lie Gly Val Asp Val Glu Gin Val Met Ala Gin Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ala Asp Ile Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Lys Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Lys His Phe Ser Asn Trp Val Glu Trp Ala Lys Asn Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr lie Asp Arg Leu Ala Pro Arg Gin Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Leu Asn Pro Gin His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Thr Glu lie Ala Arg His Lys Leu Phe Asp Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Gin Leu Arg Lys Leu Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 20 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-20 <400> 20 Met Thr Lys Leu Ile Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala Ile Gly Val Asp Val Glu Gin Val Met Ala Gin Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Glu Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ala Asp Ile Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Lys Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Lys His Phe Ser Asn Trp Val Glu Trp Ala Lys Asn Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala His Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asn Pro Gln His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Thr Glu lie Ala Arg His Lys Leu Phe Asp Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gin Leu Arg Lys Leu Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 21 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-21 <400> 21 Met Thr Lys Leu Ile Glu Gln Ala Phe Glu Leu Ala Lys Gln Arg Tyr 1 5 10 15 Ala Ala Ile Gly Val Asp Val Glu Gln Val Leu Gly Gln Leu Asp Arg 20 25 30 Ile Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Gln Gly Ala Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Lys Pro Val Ala Arg Asp Glu Ile Glu Pro 100 105 110 Glu His Phe Ser Asn Trp Val Glu Trp Ala Lys Asn His Gln Leu Gly 115 120 125 Leu Asp Phe Asn Pro Thr Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Ile Ile Ser Glu Lys Leu Asn Pro Gln His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Thr Ser Arg Gln Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Asn Glu Ile Ala Arg Gln Asn Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Gln Leu Arg Gln Leu Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Asp Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 22 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-22 <400> 22 Met Thr Lys Leu Ile Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala Ile Gly Val Asp Val Glu Gin Val Leu Gly Gin Leu Asp Arg 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Gin Gly Ala Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Lys Pro Val Ala Arg Asp Glu Ile Glu Pro 100 105 110 Glu His Phe Ser Asn Trp Val Glu Trp Ala Lys Asn His Gin Leu Gly 115 120 125 Leu Asp Phe Asn Pro Thr Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Ile Ile Ser Glu Lys Leu Asn Pro Gin His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Asn Glu Ile Ala Arg Gln Asn Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Gln Leu Arg Gln Leu Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Asp Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 23 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-23 <400> 23 Met Thr Lys Leu lie Glu Gin Ala Phe Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala lie Gly Val Asp Val Glu Gin Val Met Gly Gin Leu Asp Gly 20 25 30 lie Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Gin Gly Ala Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Lys Ala Met Ser Leu lie Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Lys Pro Val Glu Arg Asp Ala lie Glu Pro 100 105 110 Glu His Phe Ser Asn Trp Val Glu Trp Ala Lys Asn His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Thr Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly lie Arg Gin Phe Trp lie Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg lie Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Leu Asn Pro Gin His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Asn Glu lie Ala Arg Gin Lys Leu Phe Asp Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gin Leu Arg Gin Leu Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Asp Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 24 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-24 <400> 24 Met Thr Lys Leu lie Glu Gin Ala Phe Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala lie Gly Val Asp Val Glu Gin Val Met Ala Gin Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Gin Gly Ala Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Lys Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Glu His Phe Ser Asn Trp Val Glu Trp Ala Lys Asn Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Thr Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu He He Ser Glu Lys Leu Asn Pro Gin His His He Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly He Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val He Ser Asp Lys 260 265 270 He Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala He Ala Asn Glu He Ala Arg Gin Lys Leu Phe Asp Lys 305 310 315 320 Val His He Gly Leu Asp Phe Phe Asp Ala Ser He Asn Arg He Ala 325 330 335 Ala Trp Val He Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gin Leu Arg Gin Leu Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Asp Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 25 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-25 <400> 25 Met Thr Lys Leu Ile Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala Ile Gly Val Asp Val Glu Gin Val Met Ala Gin Leu Asp Gly 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Gin Gly Ala Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Lys Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Glu His Phe Ser Asn Trp Val Glu Trp Ala Lys Lys His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Thr Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Ala Ser Ala Leu 195 200 205 Asp Glu Ile Ile Ser Glu Lys Leu Asn Pro Gln His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ser Ser Arg Gin Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Asn Glu lie Ala Arg Gin Lys Leu Phe Asp Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gin Leu Arg Lys Leu Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 26 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-26 <400> 26 Met Thr Lys Leu Ile Glu Gln Ala Tyr Glu Leu Ala Lys Gln Arg Tyr 1 5 10 15 Ala Ala Ile Gly Val Asp Val Glu Gln Ala Met Ala Gln Leu Asp Arg 20 25 30 Ile Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Gln Gly Ala Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ala Asp Leu Asp 65 70 75 80 Lys Ala Met Ser Leu Ile Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Lys Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Glu His Phe Ser Asn Trp Val Glu Trp Ala Lys Lys His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Thr Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Lys Gly Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Val Ser Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asn Pro Gln His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Thr Ser Arg Gln Thr Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Asn Glu Ile Ala Arg Gln Lys Leu Phe Asp Lys 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gln Leu Arg Gln Leu Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 27 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-27 <400> 27 Met Thr Lys Gin He Gin Gin Ala Phe Gin Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Asp He Gly Val Asp Val Gin Gin Ala He Arg Gin Leu Asp Arg 20 25 30 He Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Gin Gly Ala Leu Thr Gly Gly He Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Gin Leu Arg Ser Asp He Asp 65 70 75 80 Lys Ala Met Ser Leu He Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 He Tyr Leu Gin Ser Asp Lys Pro Val Gin Arg Asp Ala He Gin Pro 100 105 110 Ala His Phe Ala Asn Trp Val Ala Trp Ala Lys Gin His Lys Leu Gin 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Met Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ser Asp Lys Thr Val Arg Gin Phe Trp He Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg He Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn He Trp Val Pro Asp Gly Met Lys Asp 180 185 190 He Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Met Ser Ala Leu 195 200 205 Asp Glu Val He Ser Glu Lys Leu Asn Pro Gin His His He Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly He Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val He Ser Asp Lys 260 265 270 He Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Val Arg His Gln Leu Phe Asp Lys 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Thr Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Arg Leu Arg Gln Ala Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Thr Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Gly Asp Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 28 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq‑28 <400> 28 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Asp lie Gly Val Asp Val Glu Gin Ala lie Arg Gin Leu Asp Arg 20 25 30 lie Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Gin Gly Ala Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ser Asp lie Asp 65 70 75 80 Lys Ala Met Ser Leu lie Pro Gly Ala Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Lys Pro Val Glu Arg Asp Ala lie Glu Pro 100 105 110 Ala His Phe Ala Asn Trp Val Ala Trp Ala Lys Glu His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Met Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ser Asp Lys Thr Val Arg Gin Phe Trp lie Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg lie Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp Val Pro Asp Gly Met Lys Asp 180 185 190 lie Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Met Ser Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Leu Asn Pro Gin His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie Val Arg His Gin Leu Phe Asp Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Thr Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Arg Leu Arg Gin Ala Glu Asn Glu Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Thr Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Gly Asp Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 29 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-29 <400> 29 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Glu Asn Gly Val Asp Val Asp Leu Val Met Ala Gin Leu Asp Glu 20 25 30 Ile Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Arg Gly Phe 35 40 45 Glu Asn Pro Asn Gly Gin Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Arg Ala Arg Asn Ala Asp Glu Leu Arg Ala Asp Ile Glu 65 70 75 80 Lys Ala Ile Ser Leu Ile Pro Gly Ala Met Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Ser Gin Pro Val Glu Arg Asp Ala Ile Glu Pro 100 105 110 Lys His Phe Ser Asn Trp Val Ala Trp Ala Lys Ser Asn Arg Leu Gly 115 120 125 Leu Asp Phe Asn Pro Thr Cys Phe Ser His Ala Lys Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Thr Gly Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg His Ile Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Leu Thr lie Asp Arg Phe Ala Pro Arg Gin Arg Leu Ala Ser Ala Leu 195 200 205 Asp Ala lie lie Ser Glu Gin Leu Asn Pro Gin His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Cys Leu Gly Tyr Ala Ser Ser Arg Gin lie Ala Leu 245 250 255 Thr Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Thr Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala His Glu lie Ala Arg Gin Lys Leu Phe Asp Lys 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Ala Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Ser Asp Arg Leu Arg Arg Leu Glu Ser Glu Gly Asp 355 360 365 Phe Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 30 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> Seq-30 <400> 30 Met Thr Thr Gln Leu Glu Gln Ala Trp Asp Leu Ala Lys Gln Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gln Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ser Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Thr Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Glu Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asp Asp Glu Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Ala Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie lie Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Lys Lys Leu Glu Ala Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Met Tyr Cys Gln Arg Asn Asp Ala Pro Ala Gly 385 390 395 400 Ser Gln Trp Leu Asp Asn Val Arg Ala Tyr Glu Glu Asp Val Leu Ser 405 410 415 Gln Arg Gly <210> SEQ ID NO: 31 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> Seq-31 <400> 31 Met Thr Thr Gln Leu Glu Gln Ala Trp Asp Leu Ala Lys Gln Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gln Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ser Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Thr Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Glu Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys His Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asp Asp Glu Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Ala Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gln Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Ile Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Lys Lys Leu Glu Ala Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Met Tyr Cys Gln Arg Asn Asp Ala Pro Ala Gly 385 390 395 400 Ser Gln Trp Leu Asp Asn Val Arg Ala Tyr Glu Glu Asp Val Leu Ser 405 410 415 Gln Arg Gly <210> SEQ ID NO: 32 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> Seq-32 <400> 32 Met Thr Thr Gln Leu Glu Gln Ala Trp Asp Leu Ala Lys Gln Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gln Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ser Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Glu Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Gly His Phe Thr Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asp Asp Glu Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Ala Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Ile Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Lys Lys Leu Glu Ala Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Met Tyr Cys Gln Arg Asn Asp Ala Pro Ala Gly 385 390 395 400 Ser Gln Trp Leu Asp Asn Val Arg Ala Tyr Glu Lys Asp Val Leu Ser 405 410 415 Gln Arg Gly <210> SEQ ID NO: 33 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> Seq‑33 <400> 33 Met Thr Thr Gin Leu Glu Gin Ala Trp Asp Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala Val Gly Val Asp Val Asp Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ser Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Ala His Phe Lys Asn Trp Val Ala Trp Ala Lys Ala Asn Gin Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asn Asp Asp Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp lie Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Ala lie Ser Glu Lys Leu Asn Pro Ala His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Asn Glu lie lie Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ser Ala Leu Lys Gin Leu Glu Glu Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Val Leu Leu Glu Glu Gin Lys Thr Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Met Tyr Cys Gin Arg Asn Asp Thr Pro Ala Gly 385 390 395 400 Ser Gin Trp Leu Asp Asn Val Arg Met Tyr Glu Lys Asp Val Leu Ser 405 410 415 Gln Arg Gly <210> SEQ ID NO: 34 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-34 <400> 34 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu lie Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ser Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Gly Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asn Asp Glu Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Phe Ala Pro Arg Gin Arg Leu Leu Asn Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Ile Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala Ile Ala Ser Glu Ile Ile Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Glu Gln Leu Arg Gln Leu Glu Ala Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 35 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq‑35 <400> 35 Met Thr Lys Leu Ile Glu Gln Ala Tyr Glu Leu Ala Lys Gln Arg Tyr 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gln Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ser Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Gly Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Leu Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Thr Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asn Asn Glu Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Phe Ala Pro Arg Gln Arg Leu Leu Asn Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Ala Ala Met Leu Tyr lie Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie lie Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Glu Gin Leu Arg Gin Leu Glu Ala Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Cys Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 36 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-36 <400> 36 Met Thr Lys Leu Ile Glu Gln Ala Tyr Glu Leu Ala Lys Gln Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gln Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ala Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asn Asp Glu Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Leu Asp Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gln Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Ile Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gln Leu Arg Gln Leu Glu Ala Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 37 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-37 <400> 37 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ala Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu lie Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu lie Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asn Asp Glu Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Asp Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asn Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Ile Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gln Leu Arg Gln Leu Glu Ala Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 38 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-38 <400> 38 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ala Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu lie Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu lie Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asn Asn Glu lie Arg Gin Phe Trp lie Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn He Trp He Pro Asp Gly Met Lys Asp 180 185 190 He Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Val He Ser Glu Lys Leu Asp Pro Ala His His He Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly He Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val He Ser Asp Lys 260 265 270 He Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala He Ala Ser Glu He He Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Glu Gin Leu Arg Gin Leu Glu Ala Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 39 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-39 <400> 39 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ala Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Asn Glu Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala Ile Ala Ser Glu Ile Ile Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Glu Gin Leu Arg Gin Leu Glu Ala Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 40 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-40 <400> 40 Met Thr Lys Leu Ile Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Ile Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ser Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ser Arg Asp Gln Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Gln Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Asn Lys Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Leu Asp Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie Val Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Glu Gin Leu Arg Gin Leu Glu Ala Ser Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 41 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-41 <400> 41 Met Thr Lys Leu Ile Glu Gln Ala Tyr Glu Leu Ala Lys Gln Arg Phe 1 5 10 15 Ala Ala Val Gly Ile Asp Val Glu Glu Ala Leu Arg Gln Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ser Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ser Arg Asp Gin Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Gin Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Asn Lys Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Val Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Glu Gln Leu Arg Gln Leu Glu Ala Ser Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 42 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-42 <400> 42 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Gin Ala Leu Pro Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gin Gly Ala Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Met Ser Leu lie Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Thr Pro Val Glu Arg Asn Ala lie Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Glu His Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Asn Asp Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gln Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Ile Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Asp Gln Leu Arg Gln Leu Glu Ala Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 43 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-43 <400> 43 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly lie Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ser Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ser Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Ser Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Met Arg Leu lie Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ser Arg Asp Gin lie Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Gin Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Asn Ser lie Arg Gin Phe Trp lie Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp lie Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Leu Asp Pro Ala His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Gin Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie Val Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Arg Lys Leu Glu Ala Ala Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 44 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-44 <400> 44 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly lie Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ser Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ser Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Ser Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Met Arg Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ser Arg Asp Gin Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Gin Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Asn Ser Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asn Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Leu Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Gin Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala Ile Ala Ser Glu Ile Val Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Arg Lys Leu Glu Ala Ala Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 45 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-45 <400> 45 Met Thr Lys Leu Ile Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Ile Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ser Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ser Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Ser Glu Leu Arg Thr Asp Leu Glu 65 70 75 80 Gln Ala Met Arg Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ser Arg Asp Gln Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Gln Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Asn Ser Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asn Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Ala Ala Met Leu Tyr Val Pro Gin Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie Val Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Arg Lys Leu Glu Ala Ala Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 46 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-46 <400> 46 Met Thr Lys Leu Ile Glu Gln Ala Tyr Glu Leu Ala Lys Gln Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gln Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ala Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Glu Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Asp Glu Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asn Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Ile Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Arg Gln Leu Glu Ala Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 47 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-47 <400> 47 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ala Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Ala Leu Ser Leu lie Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Glu Pro Val Ala Arg Asn Glu lie Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asp Asp Glu Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asn Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Ile Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Arg Gln Leu Glu Ala Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 48 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-48 <400> 48 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ala Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Ala Leu Ser Leu lie Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Glu Pro Val Ala Arg Asn Glu lie Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asp Asn Glu lie Arg Gin Phe Trp lie Asp 145 150 155 160 ​​​​​​​​​​​​​​​​​​​His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp lie Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Leu Asn Pro Ala His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie lie Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Arg Gin Leu Glu Ala Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 49 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-49 <400> 49 Met Thr Lys Leu lie Glu Gin Ala Tyr Asp Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ser Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Glu Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asp Asp Glu Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Ala Ile Ser Glu Lys Leu Asn Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala Ile Ala Ser Glu Ile Ile Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Lys Gin Leu Glu Ala Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 50 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-50 <400> 50 Met Thr Lys Leu Ile Glu Gin Ala Tyr Asp Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ser Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Glu Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asp Asp Glu Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Ala Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 ​​​​​​​​​​​​​​​​​​​Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie lie Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Lys Gin Leu Glu Ala Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 51 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-51 <400> 51 Met Thr Lys Leu Ile Glu Gln Ala Tyr Glu Leu Ala Lys Gln Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gln Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ser Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Glu Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Lys Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asp Asn Glu Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Ala Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Ile Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Lys Gln Leu Glu Ala Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 52 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-52 <400> 52 Met Thr Lys Leu lie Glu Gin Ala Tyr Asp Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ser Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Ala Leu Ser Leu lie Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 lie Tyr Leu Glu Ser Asp Glu Pro Val Ala Arg Asn Glu lie Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Arg Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asn Asp Glu Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Ala Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Ile Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Val Ala Ala Leu Lys Gln Leu Glu Glu Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 53 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-53 <400> 53 Met Thr Lys Leu lie Glu Gin Ala Tyr Glu Leu Ala Lys Gin Arg Phe 1 5 10 15 Ala Ala Val Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ser Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Ala Leu Ser Leu lie Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Glu Pro Val Ala Arg Asn Glu lie Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Arg Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asn Asn Glu lie Arg Gin Phe Trp lie Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp lie Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Ala lie Ser Glu Lys Leu Asp Pro Ala His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie lie Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Val Ala Ala Leu Lys Gin Leu Glu Glu Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 54 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-54 <400> 54 Met Thr Lys Leu lie Glu Gin Ala Tyr Asp Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala Val Gly Val Asp Val Asp Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Gly Gly Ser Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Thr Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Leu Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Ala His Phe Lys Asn Trp Val Ala Trp Ala Lys Ala Asn Gin Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ala His Ala Asn Asp Asp Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Val Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Ala Ile Ser Glu Lys Leu Asn Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala Ile Ala Ser Glu Ile Ile Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ser Ala Leu Lys Gin Leu Glu Glu Asn Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Val Leu Leu Glu Glu Gin Lys Thr Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Ala Trp Cys Leu Arg His Asp Val Pro Ala Asp 385 390 395 400 Ala Ser Trp Leu Ser Glu Val Arg His Tyr Glu Gin Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 55 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> Seq-55 <400> 55 Met Thr Thr Gin Leu Glu Gin Ala Trp Glu Ile Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala Ile Gly Val Asp Ala Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ser Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Gin Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu lie Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Thr Thr Pro Val Ala Arg Asn Glu lie Lys Pro 100 105 110 Glu His Phe Lys His Trp Val Glu Trp Ala Lys Arg His Gin Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asn Pro Glu lie Arg Gin Phe Trp lie Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg lie Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Phe Ala Pro Arg Gin Arg Leu Leu Asn Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Leu Asp Pro Ala His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Ala Ala Met Leu Tyr lie Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie lie Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Leu Leu Arg Gin Leu Glu Asn Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Ile Trp Glu Met Tyr Cys Gln Arg His Asp Thr Pro Ala Gly 385 390 395 400 Ser Gln Trp Leu Glu Asn Val Arg Thr Tyr Glu Lys Asp Val Leu Ser 405 410 415 Gln Arg Gly <210> SEQ ID NO: 56 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> Seq-56 <400> 56 Met Thr Thr Gln Leu Glu Gln Ala Trp Glu Leu Ala Lys Gln Arg Tyr 1 5 10 15 Ala Ala Ile Gly Val Asp Val Glu Glu Ala Leu Arg Gln Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Asp Gly Ser Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala His Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Pro His Gln Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asn Glu Glu Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Leu Asn Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gln Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Ile Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Gln Leu Arg Gln Leu Glu Asn Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Met Tyr Cys Gln Arg His Asp Thr Pro Ala Gly 385 390 395 400 Ser Gln Trp Leu Asp Asn Val Arg Thr Tyr Glu Lys Asp Val Leu Ser 405 410 415 Gln Arg Gly <210> SEQ ID NO: 57 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-57 <400> 57 Met Thr Thr Gln Leu Glu Gln Ala Trp Glu Leu Ala Lys Gln Arg Tyr 1 5 10 15 Ala Ala Ile Gly Val Asp Val Glu Glu Ala Leu Arg Gln Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Thr Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Asn Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Gly His Gln Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asn Glu Glu Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Asn Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gln Ala Ile Ala Ser Glu Ile Ile Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Leu Leu Arg Gln Leu Glu Asn Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Met Tyr Cys Gln Arg His Asp Thr Pro Ala Gly 385 390 395 400 Ser Gln Trp Leu Asp Asn Val Arg Thr Tyr Glu Lys Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 58 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-58 <400> 58 Met Thr Lys Leu lie Glu Gin Ala Trp Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala lie Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Thr Leu Thr Gly Gly lie Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Asn Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu lie Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu lie Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Gly His Gin Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asn Glu Glu lie Arg Gin Phe Trp lie Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg lie Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn lie Trp Val Pro Asp Gly Met Lys Asp 180 185 190 lie Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Asn Ala Leu 195 200 205 Asp Glu Val lie Ser Glu Lys Leu Asp Pro Ala His His lie Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie lie Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Leu Leu Arg Gin Leu Glu Asn Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Met Tyr Cys Gin Arg His Asp Thr Pro Ala Gly 385 390 395 400 Ser Gin Trp Leu Asp Asn Val Arg Thr Tyr Glu Lys Gin Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 59 <211> 418 <212> PRT <213> Artificial Sequence <220> <223> Seq-59 <400> 59 Met Thr Lys Leu lie Glu Gin Ala Trp Glu Leu Ala Lys Gin Arg Tyr 1 5 10 15 Ala Ala lie Gly Val Asp Val Glu Glu Ala Leu Arg Gin Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gin Gly Asp Asp Val Ala Gly Phe 35 40 45 Glu Asn Pro Glu Gly Thr Leu Thr Gly Gly Ile Gin Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Asn Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Leu Ser Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ala Arg Asn Glu Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Gly His Gin Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asn Glu Glu Ile Arg Gin Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Ile Ser Ala Tyr Phe Gly Glu Gin Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Val Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gin Arg Leu Leu Asn Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asp Pro Ala His His Ile Asp Ala 210 215 220 Val Glu Ser Lys Leu Phe Gly Ile Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Ala Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asp Lys 260 265 270 Ile Ser Ala Ala Met Leu Tyr Val Pro Arg Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala Ile Ala Ser Glu Ile Ile Arg His Asn Leu Phe Asp Arg 305 310 315 320 Val His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Leu Leu Arg Gln Leu Glu Asn Asp Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gln Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Met Tyr Cys Gln Arg His Asp Thr Pro Ala Gly 385 390 395 400 Ser Gln Trp Leu Asp Asn Val Arg Thr Tyr Glu Gln Gln Thr Leu Arg 405 410 415 Leu Arg <210> SEQ ID NO: 60 <211> 419 <212> PRT <213> Artificial Sequence <220> <223> Seq-60 <400> 60 Met Thr Thr Gln Leu Glu Gln Ala Trp Glu Leu Ala Lys Gln Arg Phe 1 5 10 15 Ala Ala Val Gly Ile Asp Val Glu Glu Ala Leu Arg Gln Leu Asp Arg 20 25 30 Leu Pro Val Ser Met His Cys Trp Gln Gly Asp Asp Val Ser Gly Phe 35 40 45 Glu Asn Pro Glu Gly Ser Leu Thr Gly Gly Ile Gln Ala Thr Gly Asn 50 55 60 Tyr Pro Gly Lys Ala Arg Asn Ala Ser Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Gln Ala Met Arg Leu Ile Pro Gly Pro Lys Arg Leu Asn Leu His Ala 85 90 95 Ile Tyr Leu Glu Ser Asp Thr Pro Val Ser Arg Asp Gln Ile Lys Pro 100 105 110 Glu His Phe Lys Asn Trp Val Glu Trp Ala Lys Ala Asn Gln Leu Gly 115 120 125 Leu Asp Phe Asn Pro Ser Cys Phe Ser His Pro Leu Ser Ala Asp Gly 130 135 140 Phe Thr Leu Ser His Ala Asp Asp Ser Ile Arg Gln Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Arg Val Ser Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Thr Pro Ser Val Met Asn Ile Trp Ile Pro Asp Gly Met Lys Asp 180 185 190 Ile Thr Val Asp Arg Leu Ala Pro Arg Gln Arg Leu Leu Ala Ala Leu 195 200 205 Asp Glu Val Ile Ser Glu Lys Leu Asn Pro Ala His His Ile Asp Ala 210 215 220 ​​​​​​​​​​​​​​​​​​​Val Glu Ser Lys Leu Phe Gly lie Gly Ala Glu Ser Tyr Thr Val Gly 225 230 235 240 Ser Asn Glu Phe Tyr Met Gly Tyr Ala Thr Ser Arg Gin Thr Ala Leu 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val lie Ser Asp Lys 260 265 270 lie Ser Ala Ala Met Leu Tyr Val Pro Gin Leu Leu Leu His Val Ser 275 280 285 Arg Pro Val Arg Trp Asp Ser Asp His Val Val Leu Leu Asp Asp Glu 290 295 300 Thr Gin Ala lie Ala Ser Glu lie Val Arg His Asp Leu Phe Asp Arg 305 310 315 320 Val His lie Gly Leu Asp Phe Phe Asp Ala Ser lie Asn Arg lie Ala 325 330 335 Ala Trp Val lie Gly Thr Arg Asn Met Lys Lys Ala Leu Leu Arg Ala 340 345 350 Leu Leu Glu Pro Thr Ala Glu Leu Arg Lys Leu Glu Ala Pro Gly Asp 355 360 365 Tyr Thr Ala Arg Leu Ala Leu Leu Glu Glu Gin Lys Ser Leu Pro Trp 370 375 380 Gln Ala Val Trp Glu Met Tyr Cys Gln Arg His Asp Thr Pro Ala Gly 385 390 395 400 Ser Glu Trp Leu Glu Ser Val Arg Ala Tyr Glu Lys Glu Ile Leu Ser 405 410 415 Arg Arg Gly

Claims

1. A species belonging to the genus Erwinia ( Erwinia sp. L-rhamnosyl isomerase of microorganisms, among which, The L-rhamnose isomerase consists of the amino acid sequence shown in SEQ ID NO: 1, and the molecular mass of the subunit determined by SDS-PAGE is 48 kDa, has the substrate specificity of (B) below and the physicochemical properties of (C) and (D) below: (B) has an activity of catalyzing isomerization reactions between L-rhamnose and L-rhamnulose, L-lyxose and L-xylulose, L-mannose and L-fructose, D-ribose and D-ribulose, L-talose and L-tallose, and D-allose and D-psicose; (C) the reaction optimum pH is 9; (D) the reaction optimum temperature is 70°C, The microorganism belonging to the genus Erwinia is Erwinia billingiae GuaL218-3 which was internationally deposited by the National Institute of Technology and Evaluation Patent Microorganisms Depository as Accession No. NITE BP-03142.

2. A protein which is an amino acid substitution variant of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1, consisting of the amino acid sequence represented by any one of SEQ ID NO: 6, 10, 13, 15, 17, 19, 20, 21, 22, 26, 27, 28, 30, 32, 34, 36, 40, 41, 42, 46, 47, 49 or 51, has an isomerase activity between D-allose and D-psicose, and the isomerase activity for D-allose at the optimum temperature 70°C or the residual activity after incubation at 60°C for 1 hour is higher than that of the protein consisting of the amino acid sequence shown in SEQ ID NO:

1.

3. A protein which is an amino acid substitution variant of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1, consisting of the amino acid sequence represented by any one of SEQ ID NO: 3 to 5, 7, 8, 11, 12, 16, 18, 23 to 25, 31, 33, 35, 37 to 39, 43 to 45, 48, 50 or 52 to 59, has an isomerase activity between D-allose and D-psicose, and the ratio of the isomerase activity for D-allose at the reaction temperature 70°C to that at 50°C (T70 / T50) or the ratio of the isomerase activity for D-allose at the reaction temperature 80°C to that at the optimum temperature 70°C (T80 / optimum temperature) is higher than that of the protein consisting of the amino acid sequence shown in SEQ ID NO:

1.

4. A DNA encoding the L-rhamnose isomerase of claim 1 or the protein of claim 2 or 3.

5. A DNA consisting of the base sequence shown in SEQ ID NO: 2 or a complementary sequence thereof.

6. A recombinant vector containing the DNA of claim 4 or 5.

7. A transformed host cell obtained by transformation with the recombinant vector of claim 6.

8. An Erwinia billingiae GuaL218-3, which has been internationally deposited at the National Institute of Technology and Evaluation Patent Microorganism Deposit of Japan as Accession No. NITE BP-03142, and which produces the L-rhamnose isomerase according to claim 1.

9. An immobilized protein, wherein the L-rhamnose isomerase according to claim 1 is immobilized on a carrier.

10. An immobilized protein, wherein the protein according to claim 2 or 3 is immobilized on a carrier.

11. The immobilized protein according to claim 9 or 10, wherein the carrier is an ion exchange resin or a synthetic adsorbent.

12. The immobilized protein according to claim 11, wherein the carrier is WA30, FPA54 or FPA95.

13. A method for producing an L-rhamnose isomerase, wherein an Erwinia microorganism that produces the L-rhamnose isomerase according to claim 1 is cultured in a culture medium, or the transformed host cell according to claim 7 is cultured in a culture medium, the L-rhamnose isomerase is accumulated in the microbial cells, and the L-rhamnose isomerase is collected.

14. The method for producing an L-rhamnose isomerase according to claim 13, wherein the culture medium is an inorganic salt culture medium to which L-rhamnose is added.

15. A method for producing a ketose or an aldose, characterized by: allowing the L-rhamnose isomerase according to claim 1 or the immobilized protein according to claim 9 to act on a solution containing one or more selected from aldoses or ketoses, to produce a corresponding ketose or aldose, and collecting the ketose or aldose, 16. A method for producing D-allose, characterized by: allowing the protein according to claim 2 or 3 or the immobilized protein according to claim 10 to act on a solution containing D-psicose, to produce D-allose, and collecting the D-allose. ​ ​ ​ ​ ​ ​ ​ ​ ​

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