Method for biosynthesizing laminaribiose
阅读说明：本技术 一种生物合成昆布二糖的方法 (Method for biosynthesizing laminaribiose ) 是由 方诩 杜志强 刘正垚 于 2021-08-24 设计创作，主要内容包括：本发明涉及合成生物学领域,具体涉及一种生物合成昆布二糖的方法。本发明以非可食生物质纤维糊精为底物,经过多步级联酶催化反应合成具有药用价值的昆布二糖,本发明提供的昆布二糖合成路线拓展了纤维素生物质的应用范围,为生物质的高值化利用提供一定的基础。(The invention relates to the field of synthetic biology, in particular to a method for biosynthesizing laminaribiose. The invention takes non-edible biomass cellodextrin as a substrate, and synthesizes the laminaribiose with medicinal value through multi-step cascade enzyme catalytic reaction.)
1. A method of biosynthesizing laminaribiose, comprising:
taking cellodextrin as a substrate, and carrying out three-enzyme cascade reaction to synthesize laminaribiose;
wherein, the three-enzyme cascade reaction liquid comprises: cellodextrin phosphorylase, cellobiose phosphorylase, laminaribiose phosphorylase.
2. The method of biosynthesizing laminaribiose as defined in claim 1 wherein said cellodextrin is prepared by the process comprising: mixing microcrystalline cellulose with concentrated hydrochloric acid and concentrated sulfuric acid, reacting, adding water, performing solid-liquid separation after the reaction is finished, taking supernate, adding acetone, and precipitating soluble cellodextrin; washing with acetone for several times, and collecting precipitate; dissolving the precipitate in water, removing acetone, regulating supernatant to be neutral, and performing solid-liquid separation to obtain the cellodextrin.
3. The process for biosynthesizing laminaribiose as defined in claim 1 wherein the cellodextrin phosphorylase, cellobiose phosphorylase and laminaribiose phosphorylase are expressed using an E.coli expression system.
4. The method of biosynthesizing laminaribiose according to claim 1 wherein said cellodextrin phosphorylase is present in a concentration of 1.0-2.0 mg/ml, preferably 1.5 mg/ml.
5. The method of biosynthesizing laminaribiose according to claim 1 wherein said cellobiose phosphorylase is present in a concentration of 0.8-1.2 mg/ml, preferably 1 mg/ml.
6. The method of biosynthesizing laminaribiose according to claim 1 wherein the concentration of laminaribiose phosphorylase is 0.8-1.2 mg/mlL, preferably 1 mg/mlL.
7. The method of biosynthesizing laminaribiose as defined in claim 1 wherein said three-enzyme cascade reaction solution further comprises: KH (Perkin Elmer)2PO4、Mg2+And a citric acid buffer.
8. The method for biosynthesizing laminaribiose according to claim 1 wherein said three-enzyme cascade is carried out at 45-55 ℃ for 20-30 h, preferably 50 ℃ for 24 h.
9. Laminaribiose produced by the process of any of claims 1-8.
10. Application of cellodextrin in synthesizing laminaribiose by using three-enzyme cascade reaction is disclosed.
Technical Field
The invention belongs to the field of synthetic biology, and particularly relates to a novel biosynthesis method of laminaribiose.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Lignocellulose is currently the most abundant renewable energy source in the world. Currently, lignocellulose is produced into cellulosic ethanol or its analogs mainly by microbial or enzymatic treatment. Cellobiose is a unique structural unit of lignocellulose, and can be degraded into glucose-1-phosphate and glucose by cellobiose phosphorylase (CBP), and then converted into other compounds by enzymes or microorganisms.
Laminaribiose (3-O-beta-D-glucopyranosyl-D-glucose) is a reducing disaccharide linked by two glucose units, beta-1, 3 glycosidic linkages. It can be used as the synthesis precursor of important medicine sulfate polysaccharide ester, and also can be used as preservative and important prebiotics. Currently, laminaribiose is mainly obtained by partial acid hydrolysis of beta-1, 3-glucan, whereas chemical synthesis requires multiple steps of group protection and deprotection, leading to cumbersome purification and lower overall yields.
The inventor finds out through search that: the existing method for preparing laminaribiose by multi-enzyme cascade catalysis does not relate to the application of cellulose and cellulase. Meanwhile, reactants such as glucose and the like are added in the reaction process of the existing multi-enzyme cascade catalysis, so that the synthesis of the laminaribiose by a one-pot method cannot be realized in the reaction process under the condition of not adding other substrates.
Disclosure of Invention
In order to overcome the above problems, it is an object of the present invention to provide a method for biosynthesis of laminaribiose from cellodextrin by multi-enzyme cascade catalysis. According to the invention, the synthesis route of laminaribiose is redesigned, and the laminaribiose is synthesized from non-grain biomass through three-enzyme cascade reaction, so that the application prospect of agricultural wastes such as straws is expanded.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for biosynthesizing laminaribiose, comprising:
taking cellodextrin as a substrate, and carrying out three-enzyme cascade reaction to synthesize laminaribiose;
wherein, the three-enzyme cascade reaction liquid comprises: cellodextrin phosphorylase, cellobiose phosphorylase, laminaribiose phosphorylase.
The present invention relates to three enzymes in total, CDP (Cellodextrin phosphorylase, EC 2.4.1.49, Cellodextrin phosphorylase), CBP (cellobiose phosphorylase, EC:2.4.1.20, cellobiose phosphorylase) and LBP (laminaribiose phosphorylase, EC:2.4.1.31, laminaribiose phosphorylase). The constructed three-enzyme cascade reaction successfully synthesizes the laminaribiose by using non-edible biomass (cellodextrin) as a substrate under the condition of not additionally adding a cofactor and the substrate, and the one-pot method is a brand-new laminaribiose synthesis path and lays a foundation for high-value utilization of cellulose biomass.
In a second aspect of the present invention, there is provided laminaribiose prepared by any of the above processes.
In a third aspect of the invention, there is provided the use of cellodextrin in the synthesis of laminaribiose using a three enzyme cascade.
The invention takes non-edible cellodextrin as a substrate, constructs in-vitro multi-enzyme cascade reaction, and realizes the process for synthesizing the functional disaccharide laminabiose with high added value by cellulose biomass.
The invention has the beneficial effects that:
(1) the synthesis of laminaribiose by the method of the invention does not involve the addition of ATP and other reaction intermediates, only needs to add cellodextrin and a proper amount of phosphate ions, adds three enzymes, and directly synthesizes laminaribiose under proper buffer solution, pH and temperature. In the method provided by the invention, the reactants are easy to obtain and low in price, the problem of agricultural waste in China at present can be solved, the resource recycling is realized, and the method has good economic significance.
(2) The method has the advantages of simple operation method, low cost, universality and easy large-scale production.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
Fig. 1 is a technical route diagram of the present invention.
FIG. 2 shows the SDS-PAGE gels analyzed and purified CBP, LBP and CDP in example 1 of the present invention;
FIG. 3 is a graph showing that CDP, CBP and LBP catalyze cellodextrin production of laminaribiose in example 1 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
A method for biosynthesizing laminaribiose, comprising:
taking cellodextrin as a substrate, and carrying out three-enzyme cascade reaction to synthesize laminaribiose;
wherein, the three-enzyme cascade reaction liquid comprises: cellodextrin phosphorylase, cellobiose phosphorylase, laminaribiose phosphorylase.
In some embodiments, the cellodextrin is prepared by: mixing microcrystalline cellulose with concentrated hydrochloric acid and concentrated sulfuric acid, reacting, adding water, performing solid-liquid separation after the reaction is finished, taking supernate, adding acetone, and precipitating soluble cellodextrin; washing with acetone for several times, and collecting precipitate; dissolving the precipitate in water, removing acetone, regulating supernatant to be neutral, and performing solid-liquid separation to obtain the cellodextrin.
In some embodiments, the E.coli expression system is used to express cellodextrin phosphorylase, cellobiose phosphorylase, and laminaribiose phosphorylase.
In some embodiments, the concentration of cellodextrin phosphorylase is 1.0-2.0 mg/ml, preferably 1.5 mg/ml.
In some embodiments, the cellobiose phosphorylase is present at a concentration of 0.8-1.2 mg/ml, preferably 1 mg/ml.
In some embodiments, the laminaribiose phosphorylase is present at a concentration of 0.8-1.2 mg/mlL, preferably 1 mg/mlL.
In some embodiments, the three-enzyme cascade reaction solution further comprises: KH (Perkin Elmer)2PO4、Mg2+And a citric acid buffer.
In some embodiments, the conditions of the three-enzyme cascade reaction are 45-55 ℃ for 20-30 h, preferably 50 ℃ for 24 h.
Preparing a solution required by an experiment:
(1) high performance liquid chromatography mobile phase solution: 5mM H2SO4Solutions of ddH2And (O) preparation.
(2) 8% SDS-PAG separation gel (10 mL): Tris-HCl (1.5M, pH 8.8): 2.6mL of propyleneAmide solution (30%): 2.6mL, SDS (10%): 0.1mL, APS (10%): 0.1mL, TEMED: 6 μ L, ddH2O：4.6mL。
(3) 5% SDS-PAG concentrated gel (4 mL): Tris-HCl (1.5M, pH 8.8): 0.5mL, acrylamide solution (30%): 0.67mL, SDS (10%): 40 μ L, APS (10%): 40 μ L, TEMED: 4 μ L, ddH2O：2.7mL。
(4)5 XSDS electrophoresis buffer (1L): Tris-HCl: 15.1g, Glycine: 94g, SDS: 5g, ddH2And O is metered to 1L.
(5) Protein glue staining solution (1L): anhydrous ethanol: 450mL, glacial acetic acid: 100mL, Coomassie Brilliant blue: 5g, ddH2O：450mL。
(6) Destaining solution of protein glue (1L): anhydrous ethanol: 50mL, glacial acetic acid: 100mL, ddH2O：850mL。
(7) Protein purification PBS buffer (1L): NaH2PO4 .2H2O：2.46g，Na2HPO4 .12H2O: 12.24g, NaCl: 1.922g, adding ddH2O about 800mL, adjusting pH to 7.4 and adding ddH2And O is metered to 1L.
(8) LB culture medium: weighing 10g/L peptone (Tryptone), 5g/L Yeast powder (Yeast extract), and 10g/L sodium chloride (NaCl) using ddH2And (O) preparation. Sterilizing with high pressure steam for use. In the experiment, if necessary, to the medium adding 50mg/L kanamycin sulfate as selection resistance.
(9) Kanamycin solution (100 mg/mL): 10g kanamycin sulfate was weighed out and dissolved in 100mL ddH2And (4) in O. After complete dissolution, the solution was dispensed into 1.5mL centrifuge tubes and stored for further use.
(10) IPTG solution (1M): 28.3g of IPTG were weighed and dissolved in 100mL of ddH2And (4) in O. After complete dissolution, the solution was dispensed into 1.5mL centrifuge tubes and stored for further use.
(11)50mM Tris-HCl buffer: 6.06g Tris was weighed out using 800mL ddH2After dissolving O, adjusting the pH value to the required value by using HCl, and fixing the volume to 1L.
(12)50mM HEPES buffer: weighing 11.92g HEPES using 800mL ddH2Dissolving O, and adjustingAdjusting to the required pH value, and fixing the volume to 1L.
(13) PBS buffer (no imidazole): 2.46g NaH2PO4 .2H2O，12.24g Na2HPO4 .12H2O, 11.688g NaCl, plus ddH2O about 800mL, adjusting pH to 7.4 and adding ddH2And O is metered to 1L.
(14) PBS buffer (20mM imidazole): 2.46g NaH2PO4 .2H2O，12.24gNa2HPO4 .12H2O, 11.688g NaCl, 1.36g imidazole, plus ddH2O about 800mL, adjusting pH to 7.4 and adding ddH2And O is metered to 1L.
(15) PBS buffer (40mM imidazole): 2.46g NaH2PO4 .2H2O，12.24gNa2HPO4 .12H2O, 11.688g NaCl, 2.72g imidazole, plus ddH2O about 800mL, adjusting pH to 7.4 and adding ddH2And O is metered to 1L.
(16) PBS buffer (250mM imidazole): 2.46g NaH2PO4 .2H2O，12.24gNa2HPO4 .12H2O, 11.688g NaCl, 17g imidazole, plus ddH2O about 800mL, adjusting pH to 7.4 and adding ddH2And O is metered to 1L.
(17) Strain preservation solution: glycerol: 50% (m/v) with ddH2And O, preparing and sterilizing at high temperature for later use.
(18)10 XSDS-PAGE buffer: 30g/L Tris, 144g/L glycine, 10g/L SDS using ddH2And (O) preparation.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
In the following examples, the expression strains of CBP, LBP and CDP are Escherichia coli (E.coli), the amino acid sequence of CBP is shown as SEQ ID No:1, the amino acid sequence of LBP is shown as SEQ ID No:2, and the amino acid sequence of CDP is shown as SEQ ID No: 3.
Example 1 expression of CBP, LBP and CDP using E.coli expression system.
CBP gene and from Clostridium thermocellum, LBP gene from Paenibacillus sp, CDP gene from Clostridium thermocellum YM4(Acetivibrio thermocellum)
I. Primer design
The PET-28a plasmid was selected, and the CBP and LBP genes were inserted into the PET-28a plasmid using Snapgene software to design primers as shown in the following table.
TABLE 1 primers for amplification of CBP and LBP genes
Extraction of bacterial genomes
Taking 5mL of closterium thermocellum and Paenibacillus sp.bacterial liquid, respectively placing the closterium thermocellum and Paenibacillus sp.bacterial liquid into a 15mL centrifuge tube, centrifuging the closterium thermocellum and the Paenibacillus sp.bacterial liquid for 2min at room temperature under the centrifugal force of 13000 Xg, discarding the supernatant, resuspending the thalli by using 1mL of sterile water, transferring the thalli into a 1.5mL centrifuge tube, centrifuging the thalli at room temperature for 1min under the centrifugal force of 13000 Xg, discarding the supernatant, adding 500 mu L of extraction buffer and 1g of quartz sand, violently shaking the thalli for 3min by vortex, dispersing the thalli into the extraction buffer, carrying out water bath at 65 ℃ for 30min, and transferring the liquid into a new 1.5mL centrifuge tube. Add 500. mu.L of chloroform, vortex vigorously for 30s, and centrifuge at 13000 Xg for 10min at room temperature. Transfer 300. mu.L of the supernatant to another sterile 1.5ml centrifuge tube, add 0.1 volume of 3M NaAc (pH4.8), add 0.6 volume of isopropanol, mix by inversion, stand 20min at-20 ℃ and centrifuge at 13000 Xg for 10min at 4 ℃ to remove the supernatant. Adding 700 μ L70% ethanol, washing for 2 times (2 min under 13000 Xg centrifugal force), pouring off, centrifuging for 5min, volatilizing ethanol, and adding 20 μ L ddH2O dissolves the genome.
Amplification of CBP and LBP genes and ligation to plasmids
The high-Fidelity Phanta Max Super-Fidelity DNA Polymerase is used for amplifying a CBP fragment with 15bp PET-28a homology arms by taking CBP-F/CBP-R as a primer and a clostrium thermocellum genome as a template, and the PET28a-CBP-F/PET28a-CBP-R as a primer and the PET28a empty plasmid as a template are used for amplifying a PET-28a fragment with 15bp CBP homology arms. LBP-F/LBP-R is used as a primer, a Paenibacillus sp.genome is used as a template to amplify an LBP fragment with a 15bp PET-28a homology arm, PET28a-LBP-F/PET28a-LBP-R is used as a primer, and PET28a empty plasmid is used as a template to amplify a PET-28a fragment with a 15bp LBP homology arm. After PCR amplification is finished, each fragment is recovered by using a gel recovery kit, and the high concentration of each DNA fragment is ensured so as to ensure the success of subsequent tests. Then, ligation of the plasmid and the fragment was performed using a MultiF Seamless Assembly Mix kit to obtain a PET28a-CBP plasmid and a PET28a-CBP plasmid.
Wherein, the PCR reaction system is as follows:
transformation of PETT28a-CBP, PET28a-CBP and PET28a-CDP plasmids
The PET28a-CDP plasmid was synthesized by Kingchi corporation, and the PET28a-CBP and PET28a-CBP plasmids were constructed by the above-described procedure. Rosetta (DE3) competent cells were removed from-80 deg.C, quickly inserted into ice, 5min later when the pellet had thawed, the desired plasmid was added and gently mixed by hand-tapping the EP tube bottom (avoiding pipetting with a gun), and allowed to stand on ice for 25 min. The water bath at 42 ℃ was heat-shocked for 45s, quickly returned to ice and allowed to stand for 2min, shaking decreasing the conversion efficiency. Adding 700 μ L of antibiotic-free sterile LB medium into the centrifuge tube, mixing uniformly, and recovering at 37 deg.C and 200rpm for 60 min. The strain is collected by centrifugation at 5000rpm for 1min, about 100. mu.L of supernatant is left and the resuspended strain block is lightly blown and spread on LB medium containing kanamycin resistance. The plates were placed upside down in a 37 ℃ incubator overnight.
V. bacterial colony validation
A single colony of the overnight-cultured plate was picked up, placed in 1.5mL of an EP tube containing LB liquid medium resistant to kanamycin, cultured at 37 ℃ for 3 hours at 200rpm until turbid, and subjected to PCR. And (3) carrying out agarose gel electrophoresis by PCR (polymerase chain reaction), detecting whether a band with the same size as the CBP, LBP and CDP genes can be obtained or not, if so, indicating that the connection of the fragment and the plasmid is successful and the introduction of the plasmid is successful, and carrying out the next protein expression.
Expression and purification of CBP, LBP and CDP
(1) Preparing LB liquid culture medium, and sterilizing with high temperature steam for 30 min. The temperature was chosen to be 121 ℃.
(2) Cooling the above solution, taking out the strain stored at-20 deg.C and 100mg/mL kanamycin solution, shaking after it is completely dissolved, adding 50 μ L kanamycin solution into 300mL conical flask, and shaking; adding 50 μ L of strain preservation solution, and shaking. Activation was carried out at 37 ℃ and 200rpm overnight. After activation, the visible color obviously changes into opaque egg yolk color.
(3) Approximately 5mL of liquid was first removed from a 2L Erlenmeyer flask as a reference for subsequent absorbance measurements. 1mL of kanamycin solution was added to each 2L Erlenmeyer flask, and after shaking, 10mL of activated E.coli culture solution was added to each flask. Cultured at 37 ℃ and 200 rpm.
(4) After about 4 to 5 hours, about 3mL of the liquid was taken from one flask of the culture solution, and absorbance was measured at 600 nm. If the OD is not between 0.4 and 0.6, the incubation is continued for a further period of time, measured again until the OD reaches 0.4 to 0.6, 250. mu.L of a completely thawed 1M IPTG solution are added to each Erlenmeyer flask and the shaker temperature is lowered to 16 ℃ and incubated at 200rpm for about 16h (or overnight).
(5) The E.coli culture was poured into 1L centrifuge cups, and after balancing the four centrifuge cups, they were centrifuged at 4000 Xg for 10min (4 ℃). After centrifugation, the supernatant was directly decanted, the pellet was poured into a 50mL centrifuge tube, a small amount of 20mM imidazole in PBS (containing 10% glycerol) was added, and the cells were resuspended by vortexing, at which time they were stored at-20 ℃.
(6) Pouring the bacterial liquid into a 100mL beaker, adding one thousandth volume of PMSF solution, and uniformly mixing. Cell disruption was performed using an ultrasonicator. After disruption, ultracentrifugation, 13000 Xg, centrifugation at 4 ℃ for 50 min.
(7) And purifying the protein by using AKTA and Ni columns, and desalting after purification to obtain relatively pure CBP, LBP and CDP enzyme protein.
The purified CBP, LBP and CDP were analyzed on SDS-PAGE gels and the results are shown in FIG. 2.
Example 2 Tri-enzyme cascade catalytic Synthesis of Laminaribiose Using cellulose as substrate
I. Preparation of cellodextrins
2g of microcrystalline cellulose was weighed into a 300mL Erlenmeyer flask, 16mL of ice-cold concentrated hydrochloric acid and 4mL of ice-cold concentrated sulfuric acid were added, and a small amount of sulfuric acid was added several times to prevent the occurrence of spray. Mix at 22 ℃ for 4h on a shaker at 100rpm, then add slowly 20mL ddH 2O. After centrifugation at 10000 Xg for 10min, the precipitate was discarded and 180mL of ice-cold acetone were used to precipitate the soluble cellodextrins from the supernatant. 10000 Xg, and the precipitate was washed twice with 40mL ice-cold acetone, and the precipitate was retained. The precipitate was dissolved in 20mL ddH2O, put in a fume hood overnight to evaporate the acetone. Then use Ba (OH)2The supernatant was neutralized to pH 7.0 and the precipitate was removed by centrifugation to obtain pure cellodextrin.
One-pot synthesis of laminaribiose
Synthesizing laminaribiose by taking cellodextrin as a substrate through three-enzyme cascade reaction. The reaction conditions were as follows: 10mg/mL cellodextrin, 30mMKH2PO4，5mM Mg2+1.5mg/mL CDP, 1mg/mL CBP, 1mg/mL LBP, 1mL citric acid buffer (pH 7.0) to 1mL, 50 ℃ reaction for 24 h. At the same time, no enzyme control was performed. Detection was performed by TLC.
TABLE 2 enzymes involved in this example
We sampled the reaction solution for 2h, 4h, 6h, 8h and 10h, respectively, and the content of laminaribiose in the reaction solution was determined by HPLC. The experimental results show that the CDP, CBP and LBP mediated three-enzyme cascade can catalyze cellodextrin to produce laminaribiose, but the yield is less. The reaction reached equilibrium in 8h and 0.23g/L laminaribiose was obtained (FIG. 3).
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> university, Shandong
Shandong, university
<120> a method for biosynthesizing laminaribiose
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Asn Lys Val Thr Tyr Gly Val Phe Gly Ala Glu Ala Gly Gly Thr Ala
210 215 220
Pro Ala Gly Phe Phe Pro Val Thr Glu Asp Phe Ile Gly Glu Gly Gly
225 230 235 240
Ala Leu Asp Trp Pro Glu Ala Val Val Ala Asn Arg Glu Pro Asp Ala
245 250 255
Gln Ala Gly Thr Ala Val Glu Gly Tyr Glu Ala Val Gly Ala Leu Arg
260 265 270
Phe Ala Pro Val Glu Leu Ala Pro Gly Lys Ser Val Ser Tyr Val Val
275 280 285
Ala Met Val Ile Ser Gly Asp Arg Ile Asp Val Gly Arg Tyr Ala Ala
290 295 300
Asp Tyr Leu Ala Ala Gly Arg Phe Asp Ala Leu Leu Glu Gln Asn Arg
305 310 315 320
Ala Tyr Trp Arg Asp Lys Leu Asp Thr Val Arg Phe Ser Ser Gly Asp
325 330 335
Gly Glu Gln Asp Leu Trp Met Lys Trp Val Thr Leu Gln Pro Ile Leu
340 345 350
Arg Arg Leu Tyr Gly Asn Ser Phe Leu Pro Tyr His Asp Tyr Gly Arg
355 360 365
Gly Gly Arg Gly Trp Arg Asp Leu Trp Gln Asp Cys Leu Ala Leu Met
370 375 380
Val Met Glu Pro Ala Glu Val Arg His Leu Leu Leu Asn Asn Tyr Ala
385 390 395 400
Gly Val Arg Met Asp Gly Ser Asn Ala Thr Ile Ile Gly Ala Gly Pro
405 410 415
Gly Glu Phe Val Ala Asp Arg Asn Asn Ile Pro Arg Val Trp Met Asp
420 425 430
His Gly Ala Trp Pro Leu Met Thr Thr Leu Leu Tyr Leu His Gln Ser
435 440 445
Gly Asp Leu Asp Leu Leu Phe Gln Pro Gln Ser Tyr Phe Arg Asp Val
450 455 460
Phe Val Lys Arg Cys Arg Glu Arg Asp Ala Ser Trp Thr Pro Glu Gln
465 470 475 480
Gly Asn Lys Leu Leu Thr Ala Asp Gly Gln Ile Tyr Glu Gly Thr Ile
485 490 495
Leu Glu His Ile Leu Leu Gln Asn Ile Val Pro Phe Phe Asn Val Gly
500 505 510
Glu His Gly Asn Ile Lys Leu Glu Gly Ala Asp Trp Asn Asp Gly Leu
515 520 525
Asp Leu Ala Pro Glu Arg Gly Glu Ser Val Ala Phe Thr Ala Phe Tyr
530 535 540
Ala Ser Asn Leu Met Glu Leu Ser Glu Leu Leu Leu Glu Leu Gln Lys
545 550 555 560
Arg Thr Gly Lys Asp Ser Leu Asp Ile Ala Glu Glu Met Ala Leu Leu
565 570 575
Leu Asp Thr Leu Gly Lys Pro Ile Ser Tyr Asp Ser Ile Gln Glu Lys
580 585 590
Arg Ser Leu Leu Asp Arg Tyr Tyr Asp Ala Val Thr Pro Arg Val Ser
595 600 605
Gly Lys Lys Leu Leu Leu Asp Ile Arg Lys Val Ala Glu Asp Leu Lys
610 615 620
Arg Lys Ala Asp Trp Ala Val Ala His Leu Arg Gly Ser Glu Trp Ile
625 630 635 640
Gln Ser Lys Glu Gly Tyr Ala Trp Phe Asn Gly Tyr Tyr Asn Asn Asp
645 650 655
Gly Glu Arg Val Glu Gly Asp His Pro Asp Gly Val Arg Met Thr Leu
660 665 670
Thr Gly Gln Val Phe Ala Ile Met Gly Gly Val Ala Thr Asp Glu Gln
675 680 685
Thr Glu Lys Ile Ser Gln Ala Val Asn Arg Tyr Leu Lys Asp Glu Arg
690 695 700
Ile Gly Tyr Arg Leu Asn Ser Arg Phe Gly Gly Ile Gln Gln Asn Leu
705 710 715 720
Gly Arg Ala Phe Gly Phe Ala Phe Gly His Lys Glu Asn Gly Ala Met
725 730 735
Phe Ser His Met Thr Val Met Tyr Ala Asn Ala Leu Tyr Lys Arg Gly
740 745 750
Phe Val Gln Glu Gly Phe Glu Val Leu Asp Ser Ile Tyr Arg Leu Ser
755 760 765
Ala Asp Phe Glu Asn Ser Arg Ile Tyr Pro Gly Val Pro Glu Tyr Ile
770 775 780
Asn Glu Arg Gly Arg Gly Met Tyr Thr Tyr Leu Thr Gly Ser Ala Ser
785 790 795 800
Trp Leu Leu Leu Thr Gln Leu Thr Glu Val Tyr Gly Val Lys Gly Arg
805 810 815
Phe Gly Asp Leu Arg Leu Glu Pro Lys Leu Val Gln Ala Gln Phe Asp
820 825 830
Gly Ser Gly Glu Ala Ala Val Glu Thr Leu Phe Ala Gly Arg Met Leu
835 840 845
Arg Val Val Tyr Arg Asn Pro Gln Ala Ala Glu His Gly Gln Tyr Arg
850 855 860
Val Asp Ser Val Ser Leu Asn Gly Gln Ser Val Asp Cys Gln Asn Asp
865 870 875 880
Gly Ala Gly Cys Leu Ile Gly Arg Ser Leu Ile Glu Ala Leu Pro Ala
885 890 895
Asp Gly Val His Glu Leu Ile Val Thr Leu Gly Arg Asn Ile Ser
900 905 910
<210> 3
<211> 980
<212> PRT
<213> Artificial Synthesis
<400> 3
Met Ile Thr Lys Val Thr Ala Arg Asn Asn Lys Ile Thr Pro Val Glu
1 5 10 15
Leu Leu Asn Gln Lys Phe Gly Asn Lys Ile Asn Leu Gly Asn Phe Ala
20 25 30
Asp Arg Val Phe Thr Asp Ala Ala Phe Lys Asn Val Ala Gly Ile Ala
35 40 45
Asn Leu Pro Met Lys Ala Pro Val Met Gln Val Leu Met Glu Asn Cys
50 55 60
Ile Val Ser Lys Tyr Leu Lys Gln Phe Val Pro Asp Arg Ser Val Cys
65 70 75 80
Phe Val Glu Glu Gly Gln Lys Phe Tyr Ile Val Leu Glu Asp Gly Gln
85 90 95
Lys Ile Glu Val Pro Glu Asp Val Asn Lys Ala Leu Arg Ala Thr Val
100 105 110
Ser Asp Val Lys His Trp Ala Gly Tyr Leu Thr Glu Asp Gly Glu His
115 120 125
Val Ile Ala Leu Leu Lys Pro Ala Pro Gly Pro His Phe Tyr Val Asn
130 135 140
Leu Leu Ile Gly Asn Arg Leu Gly Phe Lys Arg Thr Leu Gln Thr Thr
145 150 155 160
Pro Lys Ser Val Val Asp Arg Phe Gly Arg Gly Ser Phe Arg Ser His
165 170 175
Ala Ala Thr Gln Val Leu Ala Thr Arg Phe Asp Met Arg Gln Glu Glu
180 185 190
Asn Gly Phe Pro Ala Asn Arg Gln Phe Tyr Leu Tyr Glu Asp Gly Lys
195 200 205
Gln Ile Phe Tyr Ser Ala Leu Ile Asp Asp Asn Ile Val Glu Ala Thr
210 215 220
Cys Lys His Ser Cys Asn Arg Thr Val Ile Lys Tyr Lys Thr Ala Cys
225 230 235 240
Asn Leu Glu Ile Thr Arg Thr Ile Phe Leu Val Pro His Lys Lys Gly
245 250 255
Phe Pro Leu Ala Thr Glu Leu Gln Arg Ile Glu Ile Lys Asn Ala Ser
260 265 270
Asp Lys Ala Arg Asn Leu Ser Ile Thr Tyr Thr Gly Met Phe Gly Thr
275 280 285
Gly Ala Val His Ala Ile Phe Glu Asp Val Thr Tyr Thr Asn Val Ile
290 295 300
Met Gln Ser Ala Ala Leu Tyr Asn Asp Lys Gly Glu Phe Ile Gly Ile
305 310 315 320
Thr Pro Asp Tyr Tyr Pro Glu Glu Phe Lys Gln Asp Thr Arg Phe Val
325 330 335
Thr Met Ile Val Arg Asn Gly Asp Glu Lys Ser Phe Pro Gln Ser Phe
340 345 350
Cys Thr Asp Tyr Asn Asp Phe Val Gly Thr Gly Thr Leu Glu His Pro
355 360 365
Ala Ala Asp Val Ile Glu Gln Gln Ala Glu Pro Gln Arg Ser Gly Ile
370 375 380
Leu Cys Pro Gly Cys Ala Val Tyr Val Glu Pro Gly Lys Thr Val Ile
385 390 395 400
Ile Asp Thr Phe Thr Gly Leu Ser Ser Ser Lys Asp Asn Glu Asn Tyr
405 410 415
Ser Asp Ala Val Met Leu Arg Glu Leu Asp Asn Leu Leu Arg Tyr Phe
420 425 430
Glu Lys Ser Glu Ser Val Glu Glu Thr Leu Asn Glu Ile Ile Asn Phe
435 440 445
His Glu Asn Tyr Gly Lys Tyr Phe Gln Phe Asn Pro Gly Asn Lys Leu
450 455 460
Phe Asp Ser Gly Phe Asn Arg Asn Leu Ala Leu Glu Val Leu Tyr Gln
465 470 475 480
Thr Phe Met Ile Cys Ser Phe Gly Gln Thr Gln Lys Gly Tyr Arg Glu
485 490 495
Ile Gly Ser Gly Asn Ser Gly Leu Phe Ala Ser Met Tyr Tyr Phe Ile
500 505 510
Asn Ile Gly Tyr Gln Asp Phe Val Lys Glu Leu Leu Phe Glu Trp Thr
515 520 525
Ala Asn Val Tyr Lys Met Gly Tyr Ala Asn His Asn Phe Tyr Leu Val
530 535 540
Gly Lys Gln Arg Asp Cys Ile Pro Met Thr Ala Cys Leu Leu Gln Ala
545 550 555 560
Tyr Tyr Arg Tyr Ile Ile Tyr Thr Lys Asp Thr Ser Val Leu Asn Glu
565 570 575
Glu Val Pro Val Ala Asp Gly Asn Asn Glu Lys Arg Ala Val Arg Glu
580 585 590
Thr Leu Lys Ala Ile Ile Gln Tyr Ser Ala Cys Ile Ser Val Gly Asp
595 600 605
His Gly Leu Pro Leu Leu Asp Leu Ala Asp Trp Asn Asp Cys Leu Lys
610 615 620
Ile Asp Ser Asn Ser Ile Asp Gly Ala Thr Lys Glu Lys Leu Tyr Tyr
625 630 635 640
Glu Gln Leu Lys Lys Thr Asn Gly Lys Tyr Gly Asp Arg Phe Met Ser
645 650 655
Asp Tyr Ser Glu Ser Val Met Asn Ala Phe Leu Leu Lys Leu Ala Ile
660 665 670
Asp His Leu Ala Glu Ile Ala Thr Leu Asp Asn Asp Thr Gln Leu Ala
675 680 685
Gln Gln Met Ser Glu Leu Ser Lys Glu Val Thr Asp Arg Ile Gln Lys
690 695 700
His Ala Trp Lys Glu Asn Phe Phe Ala Arg Val Leu Ile Asn Arg Tyr
705 710 715 720
Lys Asp Gly Ser Tyr Thr Tyr Leu Gly Ala Lys Gly Asp Lys Leu Ser
725 730 735
Ala Asp Pro Asn Ile Asp Gly Val Tyr Phe Leu Asn Ser Phe Ala Trp
740 745 750
Ser Val Leu Ser Asp Val Ala Thr Asp Glu Gln Ile Ala Ile Met Val
755 760 765
Asp Val Ile Lys Lys Tyr Leu Leu Thr Pro Tyr Gly Leu Arg Leu Val
770 775 780
Thr Pro Ala Asp Leu Asn Lys Ile Ala Asn Asp Thr Ala Thr Gly His
785 790 795 800
Tyr Phe Phe Gly Asp Arg Glu Asn Gly Ala Val Phe Lys His Ala Ser
805 810 815
Met Met Ala Val Val Ala Leu Ile Lys Ala Ala Lys Lys Val Lys Asp
820 825 830
Asn Glu Leu Ala Lys Glu Met Ala Arg Ile Ala Tyr Phe Met Ile Asp
835 840 845
Leu Val Leu Pro Tyr Lys Thr Glu Ile Arg Ser Arg Leu Gln Glu Ile
850 855 860
Gln Gly Tyr Ala Leu Asn Ile Ser Ile Leu Thr Gln Glu Lys Ile Leu
865 870 875 880
Thr Phe Val Glu Pro Gly Thr Ala Thr Trp Leu Asn Leu Asn Leu Ile
885 890 895
Ser Leu Ala Gly Ile Glu Tyr Thr Arg Asp Gly Ile Ser Phe Asn Pro
900 905 910
Ile Leu Arg Glu Glu Glu Thr Gln Leu Asn Phe Thr Leu Lys Ala Pro
915 920 925
Lys Cys Ser Tyr Lys Phe Ser Ile Thr Lys Pro Val Gly Phe Ala Arg
930 935 940
Met Glu Ser Ser Glu Tyr Glu Leu Phe Val Asp Gly Gln Lys Ile Asp
945 950 955 960
Asn Thr Val Ile Pro Met Tyr Thr Asp Glu Lys Glu His Ile Val Thr
965 970 975
Leu Lys Phe Lys
980