SYSTEMS, COMPOSITIONS, AND METHODS FOR BIOCATALYZED PRODUCTION OF A GREASE THICKENER
20240368639 ยท 2024-11-07
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Inventors
Cpc classification
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Abstract
Systems, methods, and compositions for biocatalyzed conversion of oleic acid to non-racemic (R)-10-hydroxystearic acid ((R)-10-HSA) are described herein. Some systems and methods may include mixing oleic acid, a buffer, and a surfactant to produce an emulsion. The emulsion may be mixed with recombinant enzyme produced by a transformed microorganism. The recombinant enzyme may biocatalytically convert oleic acid to (R)-10-HSA, which may be extracted from the emulsion via an organic solvent.
Claims
1. A biocatalyzed method for the conversion of oleic acid to non-racemic (R)-10-hydroxystearic acid (10-HSA) comprising: mixing oleic acid, a buffer, and a surfactant to produce an emulsion; mixing the emulsion with an enzyme having at least 85% homology with any of SEQ ID. No. 19-35 to produce a catalyzed emulsion; extracting non-racemic (R)-10-HSA from the catalyzed emulsion using an organic solvent; and at least partially isolating at least a portion of the non-racemic (R)-10-HSA from the organic solvent.
2. The biocatalyzed method of claim 1, wherein the buffer includes phosphate and has a pH in a range of 4.5-7.0.
3. The method of claim 1, wherein the emulsion includes less than or equal to 25% oleic acid by volume.
4. The method of claim 1, wherein the organic solvent includes ethyl acetate.
5. The method of claim 1, wherein the surfactant comprises a monoglycerid, diglyceride, an ester of polyethylene glycol, or an ester of sorbitan.
6. The method of claim 5, wherein the surfactant comprises polysorbate-80.
7. The method of claim 1, further comprising: lysing bacterial cells that have expressed the enzyme.
8. The method of claim 7, wherein the enzyme, obtained via said lysing, is mixed with the emulsion without purification from cellular debris.
9. The method of claim 8, wherein the non-racemic (R)-10-HSA is 80% or greater in enantiomeric excess.
10. The method of claim 1, wherein the enzyme includes a proline at position 432.
11. A bacterial cell including at least one recombinant plasmid including a region having at least 85% homology with any of SEQ IDs. No. 1-17.
12. The bacterial cell of claim 11, wherein the at least one recombinant plasmid includes a promoter region that induces transcription of at least a portion of the region having at least 85% homology with any of SEQ IDs. No. 1-17.
13. The bacterial cell of claim 10, wherein the cell further includes a recombinant protein generated by transcription and translation of the region having at least 85% homology with any of SEQ IDs. No. 1-17.
14. The bacterial cell of claim 12, wherein the protein includes at least two of: a proline at position 30; an arginine at position 41; a tryptophan at position 113; an alanine at position 153; a methionine at position 230; a threonine at position 291; a leucine at position 336; a lysine at position 394; a proline at position 432; an alanine at position 501; or an alanine at position 508.
15. A composition comprising a recombinant nucleic acid having at least 85% homology with any of SEQ ID. No. 1-17.
16. The composition of claim 15, wherein the recombinant nucleic acid has at least 90% homology with any of SEQ ID. No. 1-17.
17. The composition of claim 15, wherein the recombinant nucleic acid has at least 95% homology with any of SEQ ID. No. 1-17.
18. A composition comprising recombinant protein having at least 85% homology with any of SEQ ID. No. 19-35.
19. The composition of claim 18, wherein the recombinant protein includes at least two of: a proline at position 30; an arginine at position 41; a tryptophan at position 113; an alanine at position 153; a methionine at position 230; a threonine at position 291; a leucine at position 336; a lysine at position 394; a proline at position 432; an alanine at position 501; or an alanine at position 508.
20. The composition of claim 18, wherein the recombinant protein includes at least one of: a tryptophan at position 113; a leucine at position 336; or a proline at position 432.
21. The composition of claim 20, wherein the recombinant protein also includes at least one of: an arginine at position 41; a threonine at position 291; or a lysine at position 394.
22. The composition of claim 21, wherein the recombinant protein also includes at least one of: a proline at position 30; an alanine at position 153; a methionine at position 230; an alanine at position 501; or an alanine at position 508.
Description
DESCRIPTION OF FIGURES
[0008] The present systems, methods, and recombinant DNA and proteins for biocatalyzed production of a grease thickener are described in detail below with reference to the attached drawing figures, wherein:
[0009]
[0010]
[0011]
[0012]
[0013]
[0014]
[0015]
DETAILED DESCRIPTION
[0016] Biocatalysis provides an alternative to traditional chemical manufacturing techniques. Advantageously, biocatalysis can facilitate selective partial oxidation of medium- and long-chain hydrocarbons. For example, biocatalytic oxidations can achieve region- and stereo-selectivity that are not feasible with chemical catalysis. Accordingly, biocatalysis can facilitate the development and manufacturing of non-racemic mixtures of a thickener and some of the aspects described herein are directed to methods and processes for biocatalyzed production of non-racemic mixtures of one or more thickening agents.
[0017] A common thickener that is used is Lithium (Li) (R)-12-hydroxysterate, which is manufactured from (R)-12-hydroxystearic acid (R-12-HSA) and LiOH. Although the hydroxyl group is located on the ten (10) position instead of the twelve (12) position, experiments have shown that (R)-10-HSA has comparable performance to R-12-HSA.
[0018] In various aspects, processes, methods, and compositions are provided for producing non-racemic thickening agents. In particular aspects, the thickening agent includes (R)-10-hydroxsteric acid ((R)-10-HSA). The processes and methods include biocatalysis using one or more recombinant oleic acid hydratase mutants. A generalized example of a biocatalysis process 100 is depicted in
[0019] As discussed below, a recombinant oleic acid hydratase mutant is expressed in a bacteria that includes one or more recombinant plasmids. The recombinant oleic acid hydratase mutant is mixed with oleic acid. Non-racemic (R)-10-HSA can be extracted from the reaction media using an organic solvent. The non-racemic (R)-10-HSA can be mixed with lithium or any other metal (e.g., alkali metal, alkaline earth metals, transition metals, metalloids, or post-transition metals) to produce a non-racemic thickening agent that includes R-10-HSA. In at least one aspect, the non-racemic (R)-10-HSA is mixed with lithium (Li) to produce Li(R)-10-HSA.
Definitions
[0020] In order to clarify various terms used herein, the following definitions are provided. The following terms are used in accordance with the provided definitions throughout the description unless explicitly specified. Terms that are not specifically defined are used consistent with the common meaning within the field.
[0021] As used herein the term racemic is defined as having substantially equal amounts of left-handed and right-handed enantiomers of a chiral chemical composition.
[0022] As used herein the term non-racemic is defined as having 70% or greater enantiomeric excess.
Synthesis of Recombinant Oleic Acid Hydratase Mutants
[0023] To synthesize the recombinant oleic acid hydratase mutants described herein a wild type oleic acid hydrate from bacterial strain Paracoccus aminophilus (GenBank Accession No. WP_020951013) was initially selected as the parental enzyme. The enzyme was chosen as a parental enzyme based on its relative initial performance of bioconversion of oleic acid to R-10-HSA. Sun, Q. F., Y. C. Zheng, Q. Chen, J. H. Xu, and J. Pan. 2021. Engineering of an oleic hydratase for efficient C10-Functionalization of oleic acid, Biochem Biophys Res Commun, 537:64-70. The full reference DNA and amino acid sequences were identified and are included herein as SEQ ID NO: 18 and SEQ ID NO: 36, respectively.
[0024] The amino acid sequence of SEQ ID NO: 36 was provided as input data for an artificial intelligence-guided enzyme analysis. The output of the artificial intelligence system was analyzed and twelve (12) amino acid positions (Table 1) and substitution mutations were identified as having a high probability of modifying catalytic efficiency.
TABLE-US-00001 TABLE 1 Amino Acid Position Mutation 30 V30P 41 H41R 113 F113W 153 S153A 230 T230M 245 C245A 291 S291T 336 M336L 394 T394K 432 L432P 501 S501A 508 K508A
[0025] Based on the output of the artificial intelligence system a library of mutated enzymes was prepared such that each enzyme variant in the library had a randomized combination of the substitutions identified in Table 1. For example, the DNA of SEQ ID NO: 18 may be mutated via Kunkel's targeted mutagenesis, Cassette mutagenesis, PCR site-directed mutagenesis, whole plasmid mutagenesis, CRISPR mediated mutagenesis, or any combination thereof. The mutated DNA was inserted into the plasmid. In some preferred aspects, the plasmid includes a promoter region (e.g., T7 promoter, lac operon, or any other suitable promoter). In some aspects described herein, the recombinant plasmids were transformed into competent bacterial cells (e.g. electrocompetent or chemically competent strains of E. coli, Kluyveromyces lactis, Burkholderia glumae or any other suitable bacteria).
[0026] Quality control analysis of the library was performed indicating that: 94.44% of the produced variants included a combination of the desired mutations at the 12 loci; 5.56% included insertions, deletions, or a combination thereof; and, 2.78% of the produced variants included a combination of the desired mutations at the 12 loci and included a mutation at a non-targeted location.
Evaluation of Recombinant Oleic Acid Hydratase Mutants
[0027] The library of transformed bacteria was grown in liquid growth media and screened to confirm expression of mutant oleic acid hydratase. For example, an aliquot of a particular bacterial colony in the liquid growth media was used to create 5 mL of a 10.sup.6 dilution of the aliquot. 180 L of the dilution was spiral plated onto an Agar plate containing Ampicillin. The plate was incubated overnight at 37 C.
[0028] After the overnight incubation, colonies from the plate were picked and added to individual wells of a first 48-well plate containing 500 L of LB-Ampicillin per well. The 48-well plate was incubated overnight at 37 C. with mild shaking. After the overnight incubation, 20 L of each well were added to 600 L of LB-Ampicillin in the corresponding well of a second 48-well plate. Glycerol was added to each well of the first 48-well plate and stored at 80 C.
[0029] The second 48-well plate was incubated for three (3) hours at 37 C. and shaken at 200 rpm. The second 48-well plate was then allowed to cool to 16 C. 30 L of 100 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to each well of the second 48-well plate and incubated overnight at 16 C. while being shaken at 200 rpm.
[0030] After the overnight incubation, the second plate was centrifuged at 4 C. for 10 minutes at maximum speed to pellet the cells of each well. 560 L was decanted from each well of the second plate and 200 L of a 10 concentrated tris-buffer-based mixture of non-ionic and zwitterionic detergents (e.g., BugBuster) was added to each well. The cell pellets were then broken up by shaking the second 48-well plate at 500 rpm for one (1) minute. The second 48-well plate was then incubated at room temperature (e.g., approximately 20 C.) with shaking at 200 rpm.
[0031] 500 L of a substrate were added to each well of the second 48-well plate. The substrate included 200 mL 100 mM Potassium Phosphate buffer with a pH of 6.5, 500 L Oleic Acid, and 200 L of a surfactant (e.g., polysorbate-80). To prepare the substrate the reagents were mixed and sonicated for 15 minutes on ice.
[0032] The second 48-well plate, including the 500 L of substrate, was incubated for 30 minutes at 30 C. while being shaken at 200 rpm. The second 48-well plate was then incubated for 15 minutes at 65 C. to deactivate the mutant oleic acid hydratase.
[0033] A colorimetric WST-8 assay was performed on each well of the 48-well plate. Each of the reagents of WST-8 master mix was prepared using a 100 mM Potassium Phosphate buffer at pH 7.5. For example, 70 mg NAD was added to 5 mL of buffer, 50 mg ADH010 was added to 2.5 mL of buffer, 30 mg WST-8 was added to 2.5 mL Buffer, 8.16 mg PMS was added to 10 mL buffer. Each individual reagent mix was kept on ice. Immediately prior to assessment, each reagent was mixed (all NAD, all ADH, all WST-8, and 250 L PMS) together in 89.75 mL of buffer to form a WST-8 assay master mix.
[0034] The WST-8 assay was performed by duplicative plating of 20 L of each well of the second 48-well plate to a 96-well plate. 180 L of the WST-8 assay master mix was added to each well of the 96-well plate and incubated at room temperature for 5 minutes with gentle shaking. The absorbance of each well was determined at 450 nm.
[0035] Approximately 800 mutants were screened in a similar manner. Additionally, three (3) bacterial colonies were prepared with control plasmids. The three controls were an empty plasmid (i.e., the plasmid without a mutant oleic acid hydratase inserted), a wild type plasmid (i.e., the plasmid with SEQ. ID. 18 inserted), and a triple mutant plasmid (i.e., the plasmid with the oleic acid hydratase mutant described in Sun, Q. F., Y. C. Zheng, Q. Chen, J. H. Xu, and J. Pan. 2021. Engineering of an oleic hydratase for efficient C10-Functionalization of oleic acid, Biochem Biophys Res Commun, 537:64-70).
[0036]
[0037] Enzymes that demonstrated comparatively high reaction activity were identified and selected for larger scale bioconversion testing. For example, and turning with general reference to
[0038] With specific reference to
[0039] To confirm the unanticipated outstanding performance of the recombinant enzymes described herein additional testing was performed. This additional testing included, amongst other things, a reduced enzyme load rate test. Selected results of the reduced enzyme load rate test are depicted in chart 350 of
Sequencing and Quantification of Recombinant Oleic Acid Hydratase Mutants
[0040] Traditional techniques for amino acid (AA) sequencing and deoxyribonucleic acid (DNA) sequencing were used to determine the sequence for all tested mutants. For example, the DNA sequence of SEQ. ID. Nos. 1-17 were confirmed using Sanger sequencing or Next-Generation sequencing (NGS). Similarly, the AA sequence of SEQ. ID. Nos. 19-35 were confirmed using mass spectrometry or Edman degradation. Additionally, the AA and DNA sequences for the WT enzyme and TM enzyme were determined using the same traditional techniques. The DNA sequence of each of the mutant oleic acid hydratases (i.e., SEQ. ID. Nos. 1-17) are provided in Table 2 below. The AA sequence of each of the mutant oleic acid hydratases (i.e., SEQ. ID. Nos. 19-35) are provided in Table 3 below.
TABLE-US-00002 TABLE2 SEQ ID NO: Ref Ref Name AminoAcidSequence SEQ MCC2 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCGCCCGTACCCGGGCA 1 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTTCAATTATGACAACTTGTGGGATATGT TTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTCT GGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCAA GTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCAC CTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTTG TTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTAC TTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTTC TATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACTC TACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATGT CCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTAA ACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATTT GCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAAA CGTACCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACATC GTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGCA GCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCAG TTCTGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGGGC ATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACCCG AAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCGGT ACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGACGGAG CTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCGGT ATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTGCA ACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTCTT GTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTGAA GAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAATT GTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGGCT GCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAGCA TGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCGGA GGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACGGCC AATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGCGTA TTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGACAT CAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCGCG CACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTACA CCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGCTG CCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAATTG AAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGGTG GTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCGTA AG SEQ. MCC3 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID. ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCACCGC No.2 CGGACATGATGGGTGCGTACATGCGTAATCACCCGTACCCGGGCA ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTGGAATTATGACAACTTGTGGGATATG TTTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTC TGGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCA AGGCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCA CCTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTT GTTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTA CTTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTT CTATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACT CTACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATG TCCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTA AACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATT TGCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAA ACGTAGCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACA TCGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGC AGCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCA GTTATGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGG GCATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACC CGAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCG GTACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGACGG AGCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCG GTATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTG CAACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTC TTGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTG AAGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAA TTGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGG CTGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCGC CATGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCG GAGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACG GCCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGC GTATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGA CATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCG CGCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTA CACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGC TGCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAAT TGAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGG TGGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCG TAAG SEQ MCC6 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCACCGC NO: CGGACATGATGGGTGCGTACATGCGTAATCACCCGTACCCGGGCA 3 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTTCAATTATGACAACTTGTGGGATATGT TTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTCT GGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCAA GTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCAC CTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTTG TTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTAC TTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTTC TATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACTC TACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATGT CCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTAA ACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATTT GCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAAA CGTACCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACATC GTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGCA GCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCAG TTCTGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGGGC ATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACCCG AAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCGGT ACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGACGGAG CTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCGGT ATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTGCA ACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTCTT GTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTGAA GAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAATT GTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGGCT GCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAGCA TGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCGGA GGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACGGCC AATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGCGTA TTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGACAT CAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCGCG CACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTACA CCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGCTG CCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAATTG AAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGGTG GTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCGTA AG SEQ MCC7 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCACCCGTACCCGGGCA 4 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTGGAATTATGACAACTTGTGGGATATG TTTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTC TGGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCA AGTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCA CCTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTT GTTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTA CTTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTT CTATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACT CTACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATG TCCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTA AACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATT TGCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAA ACGTACCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACA TCGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGC AGCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCA GTTCTGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGG GCATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACC CGAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCG GTACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGACGG AGCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCG GTATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTG CAACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTC TTGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTG AAGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAA TTGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGG CTGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAG CATGTTCATGCCGCGTGCGGCGGGTGACCGTCCGCACGTTGTTCCG GAGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACG GCCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGC GTATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGA CATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCG CGCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTA CACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGC TGCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAAT TGAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGG TGGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCG TAAG SEQ MCC8 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCACCGC NO: CGGACATGATGGGTGCGTACATGCGTAATCGCCCGTACCCGGGCA 5 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTTCAATTATGACAACTTGTGGGATATGT TTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTCT GGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCAA GGCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCAC CTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTTG TTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTAC TTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTTC TATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACTC TACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATGT CCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTAA ACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATTT GCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAAA CGTAGCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACAT CGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGCA GCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCAG TTCTGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGGGC ATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACCCG AAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCGGT ACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGAAGGAG CTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCGGT ATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTGCA ACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTCTT GTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTGAA GAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAATT GTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGGCT GCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCGCCA TGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCGGA GGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACGGCC AATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGCGTA TTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGACAT CAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCGCG CACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTACA CCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGCTG CCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAATTG AAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGGTG GTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCGTA AG SEQ MCC9 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCACCGC NO: CGGACATGATGGGTGCGTACATGCGTAATCACCCGTACCCGGGCA 6 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTGGAATTATGACAACTTGTGGGATATG TTTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTC TGGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCA AGTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCA CCTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTT GTTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTA CTTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTT CTATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACT CTACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATG TCCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTA AACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATT TGCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAA ACGTAGCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACA TCGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGC AGCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCA GTTATGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGG GCATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACC CGAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCG GTACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGAAGG AGCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCG GTATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTG CAACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTC TTGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTG AAGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAA TTGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGG CTGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAG CATGTTCATGCCGCGTGCGGCGGGTGACCGTCCGCACGTTGTTCCG GAGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACG GCCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGC GTATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGA CATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCG CGCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTA CACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGC TGCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAAT TGAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGG TGGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCG TAAG SEQ MCC11 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCGCCCGTACCCGGGCA 7 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTGGAATTATGACAACTTGTGGGATATG TTTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTC TGGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCA AGTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCA CCTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTT GTTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTA CTTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTT CTATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACT CTACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATG TCCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTA AACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATT TGCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAA ACGTAGCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACA TCGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGC AGCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCA GTTCTGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGG GCATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACC CGAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCG GTACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGAAGG AGCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCG GTATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTG CAACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTC TTGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTG AAGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAA TTGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGG CTGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAG CATGTTCATGCCGCGTGCGGCGGGTGACCGTCCGCACGTTGTTCCG GAGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACG GCCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGC GTATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGA CATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCG CGCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTA CACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGC TGCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAAT TGAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGG TGGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCG TAAG SEQ MCC12 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCACCCGTACCCGGGCA 8 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTGGAATTATGACAACTTGTGGGATATG TTTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTC TGGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCA AGGCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCA CCTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTT GTTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTA CTTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTT CTATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACT CTACATGCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATG TCCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTA AACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATT TGCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAA ACGTACCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACA TCGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGC AGCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCA GTTATGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGG GCATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACC CGAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCG GTACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGAAGG AGCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCG GTATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTG CAACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTC TTGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTG AAGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAA TTGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGG CTGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAG CATGTTCATGCCGCGTGCGGCGGGTGACCGTCCGCACGTTGTTCCG GAGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACG GCCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGC GTATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGA CATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCG CGCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTA CACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGC TGCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAAT TGAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGG TGGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCG TAAG SEQ MCC14 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCACCCGTACCCGGGCA 9 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTTCAATTATGACAACTTGTGGGATATGT TTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTCT GGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCAA GTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCAC CTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTTG TTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTAC TTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTTC TATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACTC TACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATGT CCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTAA ACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATTT GCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAAA CGTAGCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACAT CGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGCA GCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCAG TTATGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGGG CATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACCC GAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCGG TACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGAAGGA GCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCGG TATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTGC AACCGTCAGCCGCATTTTCTGGGCCAACCGAAGGATGTTCTGGTCT TGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTGA AGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAAT TGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGGC TGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAGC ATGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCGG AGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACGG CCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGCG TATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGAC ATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCGC GCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTAC ACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGCT GCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAATT GAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGGT GGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCGT AAG SEQ MCC15 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCACCCGTACCCGGGCA 10 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTGGAATTATGACAACTTGTGGGATATG TTTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTC TGGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCA AGTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCA CCTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTT GTTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTA CTTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTT CTATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACT CTACATGCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATG TCCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTA AACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATT TGCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAA ACGTAGCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACA TCGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGC AGCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCA GTTCTGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGG GCATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACC CGAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCG GTACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGACGG AGCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCG GTATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTG CAACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTC TTGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTG AAGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAA TTGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGG CTGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAG CATGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCG GAGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACG GCCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGC GTATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGA CATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCG CGCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTA CACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGC TGCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAAT TGAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGG TGGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCG TAAG SEQ MCC17 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTG NO: CCGGACATGATGGGTGCGTACATGCGTAATCACCCGTACCCGGGC 11 AACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATT GCAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATG AAAGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGC GGTTCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATT CGTGGTGGTCGGGAGATGAACTTCAATTATGACAACTTGTGGGAT ATGTTTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCG GTTCTGGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGG AGCAAGTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTC AGCACCTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGA TTGTTGTTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAA GATTACTTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTG GCGTTCTATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATG AAACTCTACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGG ATATGTCCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTT CGTTAAACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGT TCAATTTGCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGC AGGCAAACGTAGCGTGACCGGCATTCTCGCCAGCGTTAACGGTCA AGAACATCGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCT GACTGGCAGCATGACGGAGGGAACGGCCTACGGTGATATGGACCA CGCACCAGTTATGGAACGTGGACGTTCTGACCCGGGTCCGGACTC GGACTGGGCATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTT TGGCAACCCGAAAAAGTTTTACGGCGACATCGATAAATCCATGTG GGAAAGCGGTACACTGACGTGCAAACCGAGCCCACTGACCGATCG TCTGAAGGAGCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGT GACCGGCGGTATCATCACCTTTACCGATAGTAACTGGGTGATGAG CGTGACCTGCAACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGA TGTTCTGGTCTTGTGGGTCTATGCACTGCTTATGGACAAAGACGGA AACAAAGTGAAGAAGCCGATGCCGGCATGTACCGGCCGCGAAAT ATTGGCGGAATTGTGTCACCACCTAGGCATCCCGGACGACCAGTT CGAAGCTGTGGCTGCGAAGACCAAAGTTCGTCTCGCTCTGATGCC GTATATTACCAGCATGTTCATGCCGCGTGCGGCGGGTGACCGTCCG CACGTTGTTCCGGAGGGCTGCACTAATCTGGCGCTGATGGGTCAGT TCGTTGAGACGGCCAATGACATCGTGTTCACCATGGATTCTAGCAT TCGTACCGCGCGTATTGGTGTTTATACCCTGTTGGGCCTGCGTAAG CAGGTTCCGGACATCAGCCCGGTCCAGTATGATATCCGCACGCTG ATTAAAGCCGCGCGCACGGTTAACAACAACCAGCCGTTTCCGGGC GAGAGGCTGTTACACCGTCTGCTTGGTAAAACGTATTACGCCCATA TTCTGCCTCCGCTGCCAGATCGCACCCAGACCACCCGCGACGCGG CTGAAACCGAATTGAAGGCCTTCCTCGGCACCGGTGGCACTGCGT TAGCAGCGGTTGGTGGTTGGCTGCAGCGTGTACGTGAGGATCTGA AAGAGCGCACCCGTAAG SEQ MCC18 ATGAGTCCCAAGACATCAAAACCATTICACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCGCCCGTACCCGGGCA 12 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTGGAATTATGACAACTTGTGGGATATG TTTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTC TGGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCA AGGCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCA CCTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTT GTTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTA CTTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTT CTATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACT CTACATGCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATG TCCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTA AACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATT TGCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAA ACGTACCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACA TCGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGC AGCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCA GTTCTGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGG GCATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACC CGAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCG GTACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGACGG AGCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCG GTATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTG CAACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTC TTGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTG AAGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAA TTGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGG CTGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAG CATGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCG GAGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACG GCCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGC GTATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGA CATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCG CGCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTA CACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGC TGCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAAT TGAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGG TGGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCG TAAG SEQ MCC19 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCGCCCGTACCCGGGCA 13 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTGGAATTATGACAACTTGTGGGATATG TTTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTC TGGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCA AGTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCA CCTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTT GTTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTA CTTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTT CTATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACT CTACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATG TCCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTA AACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATT TGCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAA ACGTACCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACA TCGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGC AGCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCA GTTATGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGG GCATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACC CGAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCG GTACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGAAGG AGCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCG GTATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTG CAACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTC TTGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTG AAGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAA TTGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGG CTGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAG CATGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCG GAGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACG GCCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGC GTATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGA CATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCG CGCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTA CACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGC TGCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAAT TGAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGG TGGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCG TAAG SEQ MCC20 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCGCCCGTACCCGGGCA 14 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTTCAATTATGACAACTTGTGGGATATGT TTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTCT GGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCAA GTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCAC CTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTTG TTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTAC TTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTTC TATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACTC TACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATGT CCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTAA ACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATTT GCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAAA CGTAGCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACAT CGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGCA GCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCAG TTATGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGGG CATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACCC GAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCGG TACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGACGGA GCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCGG TATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTGC AACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTCT TGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTGA AGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAAT TGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGGC TGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAGC ATGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCGG AGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACGG CCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGCG TATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGAC ATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCGC GCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTAC ACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGCT GCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAATT GAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGGT GGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCGT AAG SEQ MCC21 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCACCCGTACCCGGGCA 15 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTTCAATTATGACAACTTGTGGGATATGT TTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTCT GGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCAA GTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCAC CTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTTG TTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTAC TTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTTC TATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACTC TACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATGT CCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTAA ACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATTT GCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAAA CGTAGCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACAT CGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGCA GCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCAG TTCTGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGGGC ATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACCCG AAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCGGT ACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGACGGAG CTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCGGT ATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTGCA ACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTCTT GTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTGAA GAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAATT GTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGGCT GCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAGCA TGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCGGA GGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACGGCC AATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGCGTA TTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGACAT CAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCGCG CACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTACA CCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGCTG CCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAATTG AAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGGTG GTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCGTA AG SEQ MCC22 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCGCCCGTACCCGGGCA 16 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTGGAATTATGACAACTTGTGGGATATG TTTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTC TGGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCA AGTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCA CCTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTT GTTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTA CTTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTT CTATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACT CTACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATG TCCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTA AACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATT TGCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAA ACGTAGCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACA TCGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGC AGCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCA GTTATGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGG GCATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACC CGAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCG GTACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGACGG AGCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCG GTATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTG CAACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTC TTGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTG AAGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAA TTGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGG CTGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCGC CATGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCG GAGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACG GCCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGC GTATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGA CATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCG CGCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTA CACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGC TGCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAAT TGAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGG TGGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCG TAAG SEQ MCC25 ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCGCCCGTACCCGGGCA 17 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTGGAATTATGACAACTTGTGGGATATG TTTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTC TGGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCA AGGCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCA CCTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTT GTTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTA CTTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTT CTATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACT CTACATGCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATG TCCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTA AACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATT TGCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAA ACGTACCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACA TCGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGC AGCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCA GTTCTGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGG GCATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACC CGAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCG GTACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGACGG AGCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCG GTATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTG CAACCGTCAGCCGCATTTTCCGGGCCAACCGAAGGATGTTCTGGTC TTGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTG AAGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAA TTGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGG CTGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAG CATGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCG GAGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACG GCCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGC GTATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGA CATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCG CGCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTA CACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGC TGCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAAT TGAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGG TGGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCG TAAG SEQ WT ATGAGTCCCAAGACATCAAAACCATTTCACGTGGAGAACGACACC ID ACCGCGGGTTATTGGTCCAACCGTCCGGAAAATACCCTGCCAGTGC NO: CGGACATGATGGGTGCGTACATGCGTAATCACCCGTACCCGGGCA 18 ACCAAGTCGAGGGCCGCAAGGCATGGATTATCGGCTCTGGTATTG CAGGCCTGGCGGCGGCGTTTTACCTGATTCGCGATGGTGGCATGAA AGGTCAAGACATCACCATTTTAGATGCATTGGACGTCACTGGCGGT TCCCTGGACGGCGCGGGTAATCCGGAAGACGGGTACATCATTCGT GGTGGTCGGGAGATGAACTTCAATTATGACAACTTGTGGGATATGT TTCAAGATGTGCAAGCGCTGGAGCTCCCGGAGGGCTACTCGGTTCT GGACGAGTATCGCCAACTGAATGACGCTGATCCGAATTGGAGCAA GTCCCGTCTGATGCACAATCAGGGTGAGATCAGAGATTTCAGCAC CTTTGGTCTGACGAAACCGCAGCAGTGGGAACTCATCAGATTGTTG TTGAAGCGCAAAGAAGACCTGGACGATCTGACCATTGAAGATTAC TTTTCGCCGGGTTTCTTACAAAGCAACTTTTGGTTTCTGTGGCGTTC TATGTTCGCCTTCGAGAACTGGCAGAGCCTGTTGGAGATGAAACTC TACACCCATCGCTTTCTGGATTCTATCGACGGTTTCGCGGATATGT CCTGCCTGGTTTTCCCGAAGTATAATCAGCATGATACCTTCGTTAA ACCTCTGGTGGATCATCTCAAGAAGCTGGGGGTGCAAGTTCAATTT GCGACCCGTGTTAGCGACCTGGAGATGACTGAAGATGCAGGCAAA CGTAGCGTGACCGGCATTCTCGCCAGCGTTAACGGTCAAGAACAT CGTATCCCGGTAGATGAAAAAGATGTGGTGTTCGCTCTGACTGGCA GCATGACGGAGGGAACGGCCTACGGTGATATGGACCACGCACCAG TTATGGAACGTGGACGTTCTGACCCGGGTCCGGACTCGGACTGGG CATTATGGCAGAACCTTGCGGCTAAGAGCCCGATCTTTGGCAACCC GAAAAAGTTTTACGGCGACATCGATAAATCCATGTGGGAAAGCGG TACACTGACGTGCAAACCGAGCCCACTGACCGATCGTCTGACGGA GCTTTCCGTGAACGACCCGTACAGCGGTAAGACCGTGACCGGCGG TATCATCACCTTTACCGATAGTAACTGGGTGATGAGCGTGACCTGC AACCGTCAGCCGCATTTTCTGGGCCAACCGAAGGATGTTCTGGTCT TGTGGGTCTATGCACTGCTTATGGACAAAGACGGAAACAAAGTGA AGAAGCCGATGCCGGCATGTACCGGCCGCGAAATATTGGCGGAAT TGTGTCACCACCTAGGCATCCCGGACGACCAGTTCGAAGCTGTGGC TGCGAAGACCAAAGTTCGTCTCGCTCTGATGCCGTATATTACCAGC ATGTTCATGCCGCGTGCGAAGGGTGACCGTCCGCACGTTGTTCCGG AGGGCTGCACTAATCTGGCGCTGATGGGTCAGTTCGTTGAGACGG CCAATGACATCGTGTTCACCATGGATTCTAGCATTCGTACCGCGCG TATTGGTGTTTATACCCTGTTGGGCCTGCGTAAGCAGGTTCCGGAC ATCAGCCCGGTCCAGTATGATATCCGCACGCTGATTAAAGCCGCGC GCACGGTTAACAACAACCAGCCGTTTCCGGGCGAGAGGCTGTTAC ACCGTCTGCTTGGTAAAACGTATTACGCCCATATTCTGCCTCCGCT GCCAGATCGCACCCAGACCACCCGCGACGCGGCTGAAACCGAATT GAAGGCCTTCCTCGGCACCGGTGGCACTGCGTTAGCAGCGGTTGGT GGTTGGCTGCAGCGTGTACGTGAGGATCTGAAAGAGCGCACCCGT AAG
TABLE-US-00003 TABLE3 SEQ IDNO: Protein Reference Name Sequence SEQ MCC2P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNRPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNFNYDNLWDMFQDVQALELP 19 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRTVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVLERGRSDPGPDSDWA LWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLTE LSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITSMFMPRAKGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC3P MSPKTSKPFHVENDTTAGYWSNRPENTLPPPDMMGAYMRNHPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNWNYDNLWDMFQDVQALELP 20 EGYSVLDEYRQLNDADPNWSKARLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRSVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVMERGRSDPGPDSDW ALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLT ELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITAMFMPRAKGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC6P MSPKTSKPFHVENDTTAGYWSNRPENTLPPPDMMGAYMRNHPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNFNYDNLWDMFQDVQALELP 21 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRTVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVLERGRSDPGPDSDWA LWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLTE LSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITSMFMPRAKGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC7P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNHPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNWNYDNLWDMFQDVQALELP 22 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRTVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVLERGRSDPGPDSDWA LWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLTE LSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITSMFMPRAAGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC8P MSPKTSKPFHVENDTTAGYWSNRPENTLPPPDMMGAYMRNRPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNFNYDNLWDMFQDVQALELP 23 EGYSVLDEYRQLNDADPNWSKARLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRSVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVLERGRSDPGPDSDWA LWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLKE LSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITAMFMPRAKGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC9P MSPKTSKPFHVENDTTAGYWSNRPENTLPPPDMMGAYMRNHPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNWNYDNLWDMFQDVQALELP 24 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRSVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVMERGRSDPGPDSDW ALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRL KELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKD VLVLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDD QFEAVAAKTKVRLALMPYITSMFMPRAAGDRPHVVPEGCTNLA LMGQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQY DIRTLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQT TRDAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC11P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNRPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNWNYDNLWDMFQDVQALELP 25 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRSVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVLERGRSDPGPDSDWA LWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLKE LSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITSMFMPRAAGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC12P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNHPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNWNYDNLWDMFQDVQALELP 26 EGYSVLDEYRQLNDADPNWSKARLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYMHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLV DHLKKLGVQVQFATRVSDLEMTEDAGKRTVTGILASVNGQEHRI PVDEKDVVFALTGSMTEGTAYGDMDHAPVMERGRSDPGPDSD WALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDR LKELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKD VLVLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDD QFEAVAAKTKVRLALMPYITSMFMPRAAGDRPHVVPEGCTNLA LMGQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQY DIRTLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQT TRDAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC14P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNHPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNFNYDNLWDMFQDVQALELP 27 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRSVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVMERGRSDPGPDSDW ALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRL KELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFLGQPKD VLVLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDD QFEAVAAKTKVRLALMPYITSMFMPRAKGDRPHVVPEGCTNLA LMGQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQY DIRTLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQT TRDAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC15P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNHPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNWNYDNLWDMFQDVQALELP 28 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYMHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLV DHLKKLGVQVQFATRVSDLEMTEDAGKRSVTGILASVNGQEHRI PVDEKDVVFALTGSMTEGTAYGDMDHAPVLERGRSDPGPDSDW ALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLT ELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITSMFMPRAKGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC17P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNHPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNFNYDNLWDMFQDVQALELP 29 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRSVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVMERGRSDPGPDSDW ALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRL KELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKD VLVLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDD QFEAVAAKTKVRLALMPYITSMFMPRAAGDRPHVVPEGCTNLA LMGQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQY DIRTLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQT TRDAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC18P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNRPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNWNYDNLWDMFQDVQALELP 30 EGYSVLDEYRQLNDADPNWSKARLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYMHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLV DHLKKLGVQVQFATRVSDLEMTEDAGKRTVTGILASVNGQEHRI PVDEKDVVFALTGSMTEGTAYGDMDHAPVLERGRSDPGPDSDW ALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLT ELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITSMFMPRAKGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC19P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNRPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNWNYDNLWDMFQDVQALELP 31 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRTVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVMERGRSDPGPDSDW ALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRL KELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKD VLVLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDD QFEAVAAKTKVRLALMPYITSMFMPRAKGDRPHVVPEGCTNLA LMGQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQY DIRTLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQT TRDAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC20P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNRPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNFNYDNLWDMFQDVQALELP 32 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRSVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVMERGRSDPGPDSDW ALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLT ELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITSMFMPRAKGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC21P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNHPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNFNYDNLWDMFQDVQALELP 33 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRSVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVLERGRSDPGPDSDWA LWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLTE LSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITSMFMPRAKGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC22P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNRPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNWNYDNLWDMFQDVQALELP 34 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRSVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVMERGRSDPGPDSDW ALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLT ELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITAMFMPRAKGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ MCC25P MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNRPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNWNYDNLWDMFQDVQALELP 35 EGYSVLDEYRQLNDADPNWSKARLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYMHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLV DHLKKLGVQVQFATRVSDLEMTEDAGKRTVTGILASVNGQEHRI PVDEKDVVFALTGSMTEGTAYGDMDHAPVLERGRSDPGPDSDW ALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLT ELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFPGQPKDVL VLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQF EAVAAKTKVRLALMPYITSMFMPRAKGDRPHVVPEGCTNLALM GQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDIR TLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTTR DAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK SEQ WTP MSPKTSKPFHVENDTTAGYWSNRPENTLPVPDMMGAYMRNHPY ID PGNQVEGRKAWIIGSGIAGLAAAFYLIRDGGMKGQDITILDALDV NO: TGGSLDGAGNPEDGYIIRGGREMNFNYDNLWDMFQDVQALELP 36 EGYSVLDEYRQLNDADPNWSKSRLMHNQGEIRDFSTFGLTKPQQ WELIRLLLKRKEDLDDLTIEDYFSPGFLQSNFWFLWRSMFAFEN WQSLLEMKLYTHRFLDSIDGFADMSCLVFPKYNQHDTFVKPLVD HLKKLGVQVQFATRVSDLEMTEDAGKRSVTGILASVNGQEHRIP VDEKDVVFALTGSMTEGTAYGDMDHAPVMERGRSDPGPDSDW ALWQNLAAKSPIFGNPKKFYGDIDKSMWESGTLTCKPSPLTDRLT ELSVNDPYSGKTVTGGIITFTDSNWVMSVTCNRQPHFLGQPKDV LVLWVYALLMDKDGNKVKKPMPACTGREILAELCHHLGIPDDQ FEAVAAKTKVRLALMPYITSMFMPRAKGDRPHVVPEGCTNLAL MGQFVETANDIVFTMDSSIRTARIGVYTLLGLRKQVPDISPVQYDI RTLIKAARTVNNNQPFPGERLLHRLLGKTYYAHILPPLPDRTQTT RDAAETELKAFLGTGGTALAAVGGWLQRVREDLKERTRK
[0041] Accordingly, some aspects include a composition of a recombinant nucleic acid having at least 65% homology with any of SEQ ID NOs: 1-17.
[0042] In some aspects, the composition of recombinant has at least 75% homology with any of SEQ. ID Nos. 1-17.
[0043] In some aspects, the composition of recombinant has at least 80% homology with any of SEQ. ID Nos. 1-17.
[0044] In some aspects, the composition of recombinant has at least 85% homology with any of SEQ. ID Nos. 1-17.
[0045] In some aspects, the composition of recombinant has at least 90% homology with any of SEQ. ID Nos. 1-17.
[0046] In some aspects, the composition of recombinant has at least 95% homology with any of SEQ. ID Nos. 1-17.
[0047] In some aspects, the composition of recombinant has at least 99% homology with any of SEQ. ID Nos. 1-17.
[0048] In some aspects, the composition of recombinant is any of SEQ. ID Nos. 1-17.
[0049] Turning to
[0050] With reference to Table 1, Table 2, and
[0051] Some aspects described herein are directed to a composition of at least one recombinant oleic acid hydratase protein. For example, at least one aspect includes recombinant protein having at least 85% homology with any of SEQ ID NOs: 18-35. In some aspects, the recombinant protein includes at least two of: a proline at position 30; an arginine at position 41; a tryptophan at position 113; an alanine at position 153; a methionine at position 230; a threonine at position 291; a leucine at position 336; a lysine at position 394; a proline at position 432; an alanine at position 501; or an alanine at position 508.
[0052] In some aspects, the recombinant protein includes at least one of a tryptophan at position 113; a leucine at position 336; or a proline at position 432. Some aspects additionally include at least one of: an arginine at position 41; a threonine at position 291; or a lysine at position 394. Some aspects additionally include at least one of: a proline at position 30; an alanine at position 153; a methionine at position 230; an alanine at position 501; or an alanine at position 508.
[0053] Turning to
[0054] The SDS-PAGE of
TABLE-US-00004 TABLE 4 PEAK AREA EXPRESSION OF TARGET LEVEL PEAK AREA PROTEIN (COMPARED OF TARGET (BACKGROUND WITH WILD VARIANT PROTEIN SUBTRACTED) TYPE ENZYME) EV 1435.196 0 WTP 3310.288 1875.092 1.0 TMP 10861.969 9426.773 5.0 MCC2P 11888.434 10453.238 5.6 MCC6P 11326.584 9891.388 5.3 MCC17P 11802.756 10367.56 5.5 MCC22P 18547.626 17112.43 9.1 MCC25P 16095.454 14660.258 7.8
Biocatalyzed Production of (R)-10-HSA
[0055] Turning to
[0056] At block 610, method 600 includes production of the enzyme. The enzyme can be any protein of SEQ ID NO: 19-35. For example, a recombinant plasmid containing any one of SEQ ID NO: 1-17 can be transferred into a competent bacterial cell. The transformation may be accomplished by electroporation or chemical stimulation of a plurality of competent bacterial cell. Chemical transformation methods typically involve the use of calcium chloride or other cationic agents to increase the permeability of the bacterial cell membrane. Electroporation involves applying an electrical field to the bacterial cell, which causes temporary pores to form in the membrane and allows for the uptake of the plasmid. Once the bacterial cell is competent, it can be mixed with the recombinant plasmid and subjected to transformation conditions. The recombinant plasmid typically contains a selectable marker, such as an antibiotic resistance gene, to allow for selection of transformed cells. The plasmid may also contain regulatory elements, such as promoters and terminators, to control the expression of the one or more mutant oleic acid hydratase enzymes.
[0057] At block 620, method 600 includes disruption of the transformed bacterial cells that express one or more mutant oleic acid hydratase enzyme. For example, the bacterial cells may be subjected to mechanical disruption, such as sonication, bead beating, or homogenization. This step involves applying mechanical force to the bacterial cells to physically break apart the cell wall and membrane, allowing the cytoplasmic proteins to be released into the surrounding solution. Said another way, a sonicator may be used to lyse the transformed bacterial cells to release one or more proteins of SEQ ID NO: 19-35.
[0058] In some embodiments, the mechanical disruption step may be followed by a chemical lysis step. This may involve the use of detergents, such as Triton X-100 or sodium dodecyl sulfate (SDS), to further disrupt the cell membrane and release the mutant oleic acid hydratase enzyme.
[0059] After lysis, the released oleic acid hydratase enzyme proteins can be separated from cellular debris using various methods, such as centrifugation or chromatography. Alternatively, some embodiments of block 604 omit separation of the mutant oleic acid hydratase enzyme from the cellular debris.
[0060] At block 630, method 600 includes mixing one or more proteins of SEQ ID NO: 19-35 with oleic acid. For example, the lysate of block 620 may be mixed with an emulsion of oleic acid, buffer, and a surfactant. The mixing may be facilitated by any suitable means. For example, mixing may be facilitated in some aspects by stirring, vibration, rocking, or any other means.
[0061] At block 640, method 600 includes extracting non-racemic (R)-10-HSA using an organic solvent. For example, an organic solvent may be added to the reaction vessel and mixed via the same or similar means as used to mix the lysate and the emulsion of oleic acid, buffer, and surfactant. In some aspects, the organic solvent is ethyl acetate.
[0062] At block 650, method 600 includes isolating at least a portion of the non-racemic (R)-10-HSA. In some aspects, the non-racemic (R)-10-HSA may be isolated from the organic solvent using crystallization. For example, the organic solvent containing (R)-10-HSA is first concentrated using various techniques such as evaporation or distillation. The concentrated solution is then allowed to cool to a temperature below the melting point of (R)-10-HSA.
[0063] As the solution cools, (R)-10-HSA begins to crystallize and precipitate out of the solution. The crystals are then separated from the remaining solution using various methods such as filtration or centrifugation. The separated crystals can be washed with a suitable solvent to remove any impurities that may be adsorbed onto the crystal surface. The washed crystals are then dried to remove any remaining solvent, producing a purified (R)-10-HSA.
[0064] In some aspects, the crystallization process may be enhanced by the use of various techniques such as seeding, where small crystals of (R)-10-HSA are introduced into the solution to promote the growth of larger crystals.
[0065] The method of the present invention for isolating (R)-10-HSA from an organic solvent can be used for a variety of applications, including the production of various industrial products such as lubricants, adhesives, and coatings.
[0066] Turning to
[0067] At block 710, method 700 includes mixing oleic acid, a buffer, and a surfactant to produce an emulsion. In some aspects, the buffer includes phosphate and has a pH in a range of 4.5-7.0. In some aspects, the organic solvent includes ethyl acetate. The surfactant may include a monoglyceride, diglyceride, an ester of polyethylene glycol, or an ester of sorbitan. For example, in some aspects, the surfactant comprises polysorbate-80. The production of the emulsion may be facilitated by any suitable means. For example, emulsification may be facilitated in some aspects by stirring, vibration (e.g., sonication), rocking, or any other means.
[0068] At block 720, method 700 includes mixing the emulsion with at least one mutant oleic acid hydratase enzyme. For example, the mutant oleic acid hydratase enzyme may have at least 65% homology with any of SEQ ID NO: 19-35.
[0069] In some aspects, the mutant oleic acid hydratase enzyme may have at least 70% homology with any of SEQ ID NO: 19-35.
[0070] In some aspects, the mutant oleic acid hydratase enzyme may have at least 75% homology with any of SEQ ID NO: 19-35.
[0071] In some aspects, the mutant oleic acid hydratase enzyme may have at least 80% homology with any of SEQ ID NO: 19-35.
[0072] In some aspects, the mutant oleic acid hydratase enzyme may have at least 85% homology with any of SEQ ID NO: 19-35.
[0073] In some aspects, the mutant oleic acid hydratase enzyme may have at least 90% homology with any of SEQ ID NO: 19-35.
[0074] In some aspects, the mutant oleic acid hydratase enzyme may have at least 95% homology with any of SEQ ID NO: 19-35.
[0075] In some aspects, the mutant oleic acid hydratase enzyme may have at least 99% homology with any of SEQ ID NO: 19-35.
[0076] In some aspects, the mutant oleic acid hydratase enzyme is homologous with any of SEQ ID NO: 19-35.
[0077] The mixing of the emulsion with the at least one mutant oleic acid hydratase enzyme produces a catalyzed emulsion. The mixing may be facilitated by any suitable means. For example, mixing may be facilitated in some aspects by stirring, vibration, rocking, or any other means.
[0078] At block 730, method 700 includes extracting non-racemic (R)-10-HSA from the catalyzed emulsion using an organic solvent. For example, an organic solvent may be added to the reaction vessel and mixed via the same or similar means as used to mix the mutant oleic acid hydratase enzyme and the emulsion of oleic acid, buffer, and surfactant. In some aspects, the organic solvent is ethyl acetate.
[0079] At block 740, method 700 includes at least partially isolating at least a portion of the non-racemic (R)-10-HSA from the organic solvent. In some aspects, the non-racemic (R)-10-HSA may be isolated from the organic solvent using crystallization. For example, the organic solvent containing (R)-10-HSA is first concentrated using various techniques such as evaporation or distillation. The concentrated solution is then allowed to cool to a temperature below the melting point of (R)-10-HSA.
[0080] As the solution cools, (R)-10-HSA begins to crystallize and precipitate out of the solution. The crystals are then separated from the remaining solution using various methods such as filtration or centrifugation. The separated crystals can be washed with a suitable solvent to remove any impurities that may be adsorbed onto the crystal surface. The washed crystals are then dried to remove any remaining solvent, producing a purified (R)-10-HSA.
[0081] In some aspects, the crystallization process may be enhanced by the use of various techniques such as seeding, where small crystals of (R)-10-HSA are introduced into the solution to promote the growth of larger crystals.
[0082] Some aspects of block 740 further include mixing (R)-10-HSA with lithium or any other metal (e.g., alkali metal, alkaline earth metals, transition metals, metalloids, or post-transition metals) to produce a non-racemic thickening agent that includes (R)-10-HSA. In at least one aspect, the non-racemic (R)-10-HSA is mixed with lithium (Li) to produce Li(R)-10-HSA.
[0083] From the foregoing, it will be seen that this invention is one well adapted to attain all the ends and objects hereinabove set forth together with other advantages which are obvious and which are inherent to the structure.
[0084] It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.
[0085] While specific elements and steps are discussed in connection to one another, it is understood that any element and/or steps provided herein is contemplated as being combinable with any other elements and/or steps regardless of explicit provision of the same while still being within the scope provided herein. Since many possible embodiments may be made of the disclosure without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
[0086] As used herein and in connection with the claims listed hereinafter, the terminology any of clauses or similar variations of said terminology is intended to be interpreted such that features of claims/clauses may be combined in any combination. For example, an exemplary clause 4 may indicate the method/apparatus of any of clauses 1 through 3, which is intended to be interpreted such that features of clause 1 and clause 4 may be combined, elements of clause 2 and clause 4 may be combined, elements of clause 3 and 4 may be combined, elements of clauses 1, 2, and 4 may be combined, elements of clauses 2, 3, and 4 may be combined, elements of clauses 1, 2, 3, and 4 may be combined, and/or other variations. Further, the terminology any of clauses or similar variations of said terminology is intended to include any one of clauses or other variations of such terminology, as indicated by some of the examples provided above.
[0087] Clause 1. A biocatalyzed method for the conversion of oleic acid to non-racemic (R)-10-hydroxystearic acid (10-HSA) comprising: mixing oleic acid, a buffer, and a surfactant to produce an emulsion; mixing the emulsion with an enzyme having at least 85% homology with any of SEQ ID. No. 19-35 to produce a catalyzed emulsion; extracting non-racemic (R)-10-HSA from the catalyzed emulsion using an organic solvent; and at least partially isolating at least a portion of the non-racemic (R)-10-HSA from the organic solvent.
[0088] Clause 2. The biocatalyzed method of clause 1, wherein the buffer includes phosphate and has a pH in a range of 4.5-7.0.
[0089] Clause 3. The biocatalyzed method of clause 1 or 2, wherein the emulsion includes less than or equal to 25% oleic acid by volume.
[0090] Clause 4. The biocatalyzed method of any of clauses 1-3, wherein the organic solvent includes ethyl acetate.
[0091] Clause 5. The biocatalyzed method of any of clauses 1-4, wherein the surfactant comprises a monoglycerid, diglyceride, an ester of polyethylene glycol, or an ester of sorbitan.
[0092] Clause 6. The biocatalyzed method of clause 5, wherein the surfactant comprises polysorbate-80.
[0093] Clause 7. The biocatalyzed method of any of clauses 1-6, further comprising lysing bacterial cells that have recombinantly expressed the enzyme.
[0094] Clause 8. The biocatalyzed method of clause 7, wherein the enzyme, obtained via the lysing, is mixed with the emulsion without purification from cellular debris.
[0095] Clause 9. The biocatalyzed method of any of clauses 1-8, wherein the non-racemic (R)-10-HSA is 80% or greater in enantiomeric excess.
[0096] Clause 10. The biocatalyzed method of any of clauses 1-9, wherein the enzyme includes a proline at position 432.
[0097] Clause 11. A bacterial cell including at least one recombinant plasmid including a region having at least 85% homology with any of SEQ IDs. No. 1-17.
[0098] Clause 12. The bacterial cell of clause 11, wherein the at least one recombinant plasmid includes a promoter region that induces transcription of at least a portion of the region having at least 85% homology with any of SEQ IDs. No. 1-17.
[0099] Clause 13. The bacterial cell of clause 11 or 12, wherein the bacterial cell further includes a recombinant protein generated by transcription and translation of the region having at least 85% homology with any of SEQ IDs. No. 1-17.
[0100] Clause 14. The bacterial cell of clause 13, wherein the protein includes at least two of: a proline at position 30; an arginine at position 41; a tryptophan at position 113; an alanine at position 153; a methionine at position 230; a threonine at position 291; a leucine at position 336; a lysine at position 394; a proline at position 432; an alanine at position 501; or an alanine at position 508.
[0101] Clause 15. A composition comprising a recombinant nucleic acid having at least 85% homology with any of SEQ ID. No. 1-17.
[0102] Clause 16. The composition of clause 15, wherein the recombinant nucleic acid has at least 90% homology with any of SEQ ID. No. 1-17.
[0103] Clause 17. The composition of clause 15, wherein the recombinant nucleic acid has at least 95% homology with any of SEQ ID. No. 1-17.
[0104] Clause 18. A composition comprising recombinant protein having at least 85% homology with any of SEQ ID. No. 19-35.
[0105] Clause 19. The composition of any of clauses 15-18, wherein the recombinant protein includes at least two of: a proline at position 30; an arginine at position 41; a tryptophan at position 113; an alanine at position 153; a methionine at position 230; a threonine at position 291; a leucine at position 336; a lysine at position 394; a proline at position 432; an alanine at position 501; or an alanine at position 508.
[0106] Clause 20. The composition of any of clauses 15-18, wherein the recombinant protein includes at least one of: a tryptophan at position 113; a leucine at position 336; or a proline at position 432.
[0107] Clause 22. The composition of any of clauses 15-18 or 20, wherein the protein also includes at least one of: an arginine at position 41; a threonine at position 291; or a lysine at position 394.
[0108] Clause 23. The composition of any of clauses 15-18, 20, or 21, wherein the protein also includes at least one of: a proline at position 30; an alanine at position 153; a methionine at position 230; an alanine at position 501; or an alanine at position 508.