US008119385B2 (12) United States Patent (10) Patent No.: US 8,119,385 B2 Mathur et al. (45) Date of Patent: Feb. 21, 2012 (54) NUCLEICACIDS AND PROTEINS AND (52) U.S. Cl. ........................................ 435/212:530/350 METHODS FOR MAKING AND USING THEMI (58) Field of Classification Search ........................ None (75) Inventors: Eric J. Mathur, San Diego, CA (US); See application file for complete search history. Cathy Chang, San Diego, CA (US) (56) References Cited (73) Assignee: BP Corporation North America Inc., Houston, TX (US) OTHER PUBLICATIONS c Mount, Bioinformatics, Cold Spring Harbor Press, Cold Spring Har (*) Notice: Subject to any disclaimer, the term of this bor New York, 2001, pp. 382-393.* patent is extended or adjusted under 35 Spencer et al., “Whole-Genome Sequence Variation among Multiple U.S.C. 154(b) by 689 days. Isolates of Pseudomonas aeruginosa” J. Bacteriol. (2003) 185: 1316 1325. (21) Appl. No.: 11/817,403 Database Sequence GenBank Accession No. BZ569932 Dec. 17. 1-1. 2002. (22) PCT Fled: Mar. 3, 2006 Omiecinski et al., “Epoxide Hydrolase-Polymorphism and role in (86). PCT No.: PCT/US2OO6/OOT642 toxicology” Toxicol. Lett. (2000) 1.12: 365-370. S371 (c)(1), * cited by examiner (2), (4) Date: May 7, 2008 Primary Examiner — James Martinell (87) PCT Pub. No.: WO2006/096527 (74) Attorney, Agent, or Firm — Kalim S. Fuzail PCT Pub. Date: Sep. 14, 2006 (57) ABSTRACT (65) Prior Publication Data The invention provides polypeptides, including enzymes, structural proteins and binding proteins, polynucleotides US 201O/OO11456A1 Jan. 14, 2010 encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. Polypeptides, Related U.S. Application Data including enzymes and antibodies, and nucleic acids of the (60) Provisional application No. 60/658,984, filed on Mar. invention can be used in industrial, experimental, food and 4, 2005. feed processing, nutritional and pharmaceutical applications, e.g., for food and feed Supplements, colorants, neutraceuti (51) Int. Cl. cals, cosmetic and pharmaceutical needs. CI2N 9/48 (2006.01) CI2N 9/50 (2006.01) 4 Claims, 4 Drawing Sheets U.S. Patent Feb. 21, 2012 Sheet 1 of 4 US 8,119,385 B2 COMPUTER SYSTEM raw.she f(5 PROCESSOR / I 15 Y W. r old hadhav P www.wawr 12D DAA RETREVING DSPAY DEWCE GRE U.S. Patent Feb. 21, 2012 Sheet 2 of 4 US 8,119,385 B2 2O 2O NY STAR 22 strikt Ai SORE NEW SEQUENCE TO A MEMORY t OPEN AABASE OF SELENCES r 2O6 READ FIRST SEQUENCE IN DATABASE 2O PERFORM COMPARSON OF NEW SEQUENC STORE SENCE in YES 214 NC displaySPAYSTORE stored sequenceSELENCE nameArarNAME TO SER GO TONEX SECUENCEN AAEASE ORE SEENCES IN ---.YES OAABASE NC END GRE 2 U.S. Patent Feb. 21, 2012 Sheet 3 of 4 US 8,119,385 B2 250 254 SOREA FRST SEQUENCE TO A MEMORY 256 STOREASECOND SEQUENCE TO A MEMORY 260 READ FIRST CHARACTER OF FIRST SEQUENCE - L. 262 READ FIRST CHARACTER OF SECOND SEQUENCE 264 SAME? YES 268 READ NEXT CHARACTER OF FIRS AND SECOND YES SEQUENCES NO 276 DSPLAY HOMOOGY level BETWEEN THE FRS AND SECOND SECUENCES GTR 3 U.S. Patent Feb. 21, 2012 Sheet 4 of 4 US 8,119,385 B2 Sea N 304 STORE AFRST SEQUENCE O MEMORY - 36 OPEN CAABASE OF SEQUENCE FEATURES 3. REA) FIRST FEATURE FROM OAABASE d 3. COMPARE FEATUREATTrauTES WHE FRS SECAENCE 36 wamwr FOUN YES 38 www.aalth riottlur -- SPLAY FOUND FEATURE TO THE USER 326 NO REA NEX FEATURE EN AAEASE MORE FEATURESN Avr YES FORE US 8,119,385 B2 1. 2 NUCLECACDS AND PROTEINS AND these polypeptides, having the activities described in Table 1, METHODS FOR MAKING AND USING THEMI Table 2 or Table 3, below. The enzymes and proteins of the invention have utility in a variety of applications. REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB SUMMARY This application includes an electronically Submitted The invention provides isolated or recombinant nucleic sequence (SEQ ID) listing. The entire content of this acids comprising a nucleic acid sequence having at least sequence listing is herein incorporated by reference for all about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, purposes. The sequence listing is identified on the electroni 10 59%, 60%, 61%, 62%, 63%, 64%. 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, cally filed .txt file as follows: 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identity to an FileName Date of Creation Size (bytes) 15 exemplary nucleic acid of the invention, e.g., including SEQ ID NO:1, SEQID NO:3, SEQID NO:5, SEQID NO:7, SEQ 2009-08-24-SequenceListing Aug. 24, 2009 38,547,757 bytes ID NO:9, SEQID NO:11, SEQID NO:13, SEQID NO:15, (D2170-1N).txt SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQID NO:25, and all nucleic acids disclosed in the SEQ ID listing, which include all odd numbered SEQ ID FIELD OF THE INVENTION NOs: from SEQID NO:1 through SEQID NO:26,897, over a region of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, This invention relates to molecular and cellular biology 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, and biochemistry. In one aspect, the invention provides 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, polypeptides, including enzymes, structural proteins and 25 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, binding proteins (e.g., ligands, receptors), polynucleotides 1750, 1800, 1850, 1900, 1950, 2000, 2050,2100,2200,2250, encoding these polypeptides, and methods of making and 2300, 2350, 2400, 2450, 2500, or more residues, encodes at using these polynucleotides and polypeptides. In one aspect, least one polypeptide having an enzyme, structural or binding the invention is directed to polypeptides, e.g., enzymes, struc activity, and the sequence identities are determined by analy tural proteins and binding proteins, including thermostable 30 sis with a sequence comparison algorithm or by a visual and thermotolerant activity, and polynucleotides encoding inspection. In one aspect, the enzymes and proteins of the these enzymes, structural proteins and binding proteins and invention include, e.g. aldolases, alpha-galactosidases, ami making and using these polynucleotides and polypeptides. dases, e.g. secondary amidases, amylases, catalases, caro The polypeptides of the invention can be used in a variety of tenoid pathway enzymes, dehalogenases, endoglucanases, pharmaceutical, agricultural and industrial contexts, includ 35 epoxide hydrolases, esterases, hydrolases, glucosidases, gly ing the manufacture of cosmetics and nutraceuticals. cosidases, inteins, isomerases, laccases, lipases, monooxyge Additionally, the polypeptides of the invention can be used nases, nitroreductases, nitrilases, P450 enzymes, pectate in food processing, brewing, bath additives, alcohol produc lyases, phosphatases, phospholipases, phytases, polymerases tion, peptide synthesis, enantioselectivity, hide preparation in and Xylanases. In another aspect, the isolated and recombi the leather industry, waste management and animal degrada 40 nant polypeptides of the invention, including enzymes, struc tion, silver recovery in the photographic industry, medical tural proteins and binding proteins, and polynucleotides treatment, silk degumming, biofilm degradation, biomass encoding these polypeptides, of the invention have activity as conversion to ethanol, biodefense, antimicrobial agents and described in Table 1, Table 2 or Table 3, below. disinfectants, personal care and cosmetics, biotech reagents, In one aspect, the invention also provides isolated or in corn wet milling and pharmaceuticals such as digestive 45 recombinant nucleic acids with a common novelty in that they aids and anti-inflammatory (anti-phlogistic) agents. are all derived from a common Source, e.g., an environmental Source, mixed environmental sources or mixed cultures. The BACKGROUND invention provides isolated or recombinant nucleic acids iso lated from a common Source, e.g. an environmental source, The invention provides isolated and recombinant polypep 50 mixed environmental sources or mixed cultures comprising a tides, including enzymes, structural proteins and binding pro polynucleotide of the invention, e.g., an exemplary sequence teins, polynucleotides encoding these polypeptides, and of the invention, including SEQ ID NO:1, SEQ ID NO:3, methods of making and using these polynucleotides and SEQID NO:5, SEQID NO:7, SEQIDNO:9, SEQID NO:11, polypeptides. The polypeptides of the invention, and the SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID polynucleotides encoding the polypeptides of the invention, 55 NO:19, SEQID NO:21, SEQID NO:23, SEQID NO:25, and encompass many classes of enzymes, structural proteins and all nucleic acids disclosed in the SEQ ID listing, which binding proteins. In one aspect, the enzymes and proteins of include all odd numbered SEQID NOs: from SEQID NO:1 the invention include, e.g. aldolases, alpha-galactosidases, through SEQID NO:26,897, over a region of at least about amidases, e.g. secondary amidases, amylases, catalases, caro 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 150, 200, 250,300, tenoid pathway enzymes, dehalogenases, endoglucanases, 60 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, epoxide hydrolases, esterases, hydrolases, glucosidases, gly 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, cosidases, inteins, isomerases, laccases, lipases, monooxyge 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, nases, nitroreductases, nitrilases, P450 enzymes, pectate 1950, 2000, 2050,2100, 2200,2250,2300, 2350,2400, 2450, lyases, phosphatases, phospholipases, phytases, polymerases 2500, or more residues, encodes at least one polypeptide and Xylanases. The invention also provides isolated and 65 having an enzyme, structural or binding activity, and the recombinant polypeptides, including enzymes, structural sequence identities are determined by analysis with a proteins and binding proteins, polynucleotides encoding sequence comparison algorithm or by a visual inspection.