USOO6743609B1 (12) United States Patent (10) Patent No.: US 6,743,609 B1 ROSS0n et al. (45) Date of Patent: Jun. 1, 2004 (54) LINOLEATE ISOMERASE Hunter et al., J. Biological Chemistry, 251 (8):2241-2247 (1976). (75) Inventors: Reinhardt A. Rosson, Manitowoc, WI Jack et al., Clinica Chimica Acta, 224:139-146 (1994). (US); Alan D. Grund, Manitowoc, WI Jiang et al., J. Applied Microbiology, 85:95-102 (1998). (US); Ming-De Deng, Manitowoc, WI Kemp et al., J. General Microbiology, 130:527–533 (1984). (US); Fernando Sanchez-Riera, Kemp et al., British J. Nutrition, 52:165–170 (1984). Manitowoc, WI (US) Kemp et al., British J. Nutrition, 52:171-177 (1984). Kemp et al., J. General Microbiology, 90:100-114 (1975). (73) Assignee: Arkion Life Sciences LLC, Kepler et al., J. Biological Chemistry, 242(24):5686-5692 Wilmington, DE (US) (1967). Kepler et al., Methods in Enzymology, 14:105-110 (1969). (*) Notice: Subject to any disclaimer, the term of this Kepler et al., J. Biological Chemistry, 241(6):1350-1354 patent is extended or adjusted under 35 (1966). U.S.C. 154(b) by 0 days. Kepler et al., J. Biological Chemistry, 245(14):3612-3620 (1970). (21) Appl. No.: 09/221,014 Kepler et al., J. Biological Chemistry, 246(9):2765-2771 (22) Filed: Dec. 23, 1998 (1971). Kil et al., Infect. Immun., 62:2440-2449 (sequence search) Related U.S. Application Data (1994). (60) Provisional application No. 60/068,617, filed on Dec. 23, Klein et al., 1983, Immobilized Microbial Cells, vol. 4, pp. 1997, and provisional application No. 60/089,560, filed on 11-51, "Methods for the Immobilization of Microbial Jun. 17, 1998. Cells”, In Wingard & Katchalski-Katzir (eds.) Academic (51) Int. Cl." ........................... C12P 7/64; C12N 11/02; Press. C12N 11/14; C12N 9/58 Koritala et al., Applied Microbiology and Biotechnology, (52) U.S. Cl. ....................... 435/134; 435/176; 435/177; 32:299-304 (1989). 435/233 Lanser, J. American Oil Chemists Society, (58) Field of Search ................................. 435/134, 232, 75(12):1809–1813 (1998). 435/233, 176, 177 Lilly, Chemical Engineering Science, 49(2):151–159 (1994). (56) References Cited Mills et al., Australian J. of Biological Sciences, U.S. PATENT DOCUMENTS 23:1109-1113 (1970). Mortimer et al., J. Biological Chemistry, 249(9):2833–2842 5,856,149 A * 1/1999 Pariza et al................. 435/134 (1974). 6,015,833 A 1/2000 Saeb) ........................ 514/558 Niehaus, Mechanisms of cis-trans isomerization of unstatu FOREIGN PATENT DOCUMENTS rated fatty acids, pp. 229-245, In E.E. van Tamelen (ed.) WO WO 96/38137 12/1996 Bioorganic Chemistry, Academic Press, New York, 1989. WO WO 99/29886 6/1999 Niehaus et al., J. Bacteriology, 134(1):177-183 (1978). Niehaus et al., J. Biological Chemistry, 245(15):3790-3797 OTHER PUBLICATIONS (1970). Hart et al., “Complete nucleotide Sequence and transcrip Niehaus et al., J. Biological Chemistry, 245(15):3802–3809 tional analysis of shakehead fish retrovirus,” J. Virol. vol. 70 (1970). pp. 3606–3616, Nov. 1996.* Park et al., J. Food Sci. and Nutri, 1(2):244-251 (1996). Burgess et al., Lipids, 26(2):162-165 (1991). Polan et al., J. Bacteriology, 88(4): 1056–1064 (1964). Chibata, 1978, Immobilized Enzymes, pp. 73–81, Halsted Powell, 1996, Chapter 2.14 -Immobilized Enzymes, pp. PreSS, Japan. 267-272, In T. Godfrey and S. West (ed.), Industrial Enzy Eyssen et al., Amer. J. Clin. Nutrition, 27:1329-1340 (1974). mology. The Nature Press, New York. Eyssen et al., Applied and Environmental Microbiol., Sako et al., Nucleic Acids Res., 11:7679-7693 (sequence 47(1):39–43 (1984). search) (1983). Faber, Ch 3. Special Techniques, pp. 270–340. In (ed.), Biotransformations in organic chemistry (1995). (List continued on next page.) Fujimoto et al., BioScience and Biotechnology Biochemistry, 57(6):1026–1027 (1993). Primary Examiner Rebecca E. Prouty Fukui et al., Endeavour, New Series, 9(1):10–17 (1985). ASSistant Examiner David J Steadman Garcia et al., Biochimica et Biophysica Acta, 424:296-302 (74) Attorney, Agent, or Firm-Sheridan Ross P.C. (1976). (57) ABSTRACT Giesel-Bihler et al., 1986, The anearobic transformation of linoleic acid by Acetobacterium woodii. International Sym The present invention provides an isolated linoleate posium-Biocatalysis in organic media. Wageningen, The isomerase and its nucleic acid and amino acid Sequence. The Netherlands. Elsevier Science Publishers B.V. present invention also provides a method for producing CLA Hamberg, Biochem. and BiophyS. Res. Comm., from an oil using an immobilized bacterial cell or an isolated 188(3):1220–1227 (1992). linoleate isomerase. Hughes et al., J. Biological Chemistry, 257(7):3643-3649 (1982). 47 Claims, 42 Drawing Sheets US 6,743,609 B1 Page 2 OTHER PUBLICATIONS Van Sonsbeek et al., Enzyme and Microbial Technology, 15:722-729 (1993). Schwab et al., Chemical Reviews, 90:1203–1245 (1990). Verhulst et al., Systematic and Applied Microbiology, Seltzer, 1972, Cis-Trans Isomerization, pp. 381–406, In P.D. Boyer (ed.), The Enzymes, Academic Press, New York. 9:12–15 (1987). Seo et al., Agricultural Biological Chemistry, Verhulst et al., Microbiology, 51(3):532–538 (1986). 45(9):2025-2030 (1981). Verhulst et al., FEMS Microbiology Ecology, 31:255-259 Swaisgood, 1985, Chapter 1-Enzymes and Immobilized (1985). Cells in Biotechnology, pp. 1-24, In Allen I. Laskin (ed.), Wise et al., Biochemistry, 36(10):2985-2992 (1998). New Jersey Center for Advanced Biotechnology and Medi Wise et al., Experientia, 52:88-92 (1996). cine, New Jersey. Yamazaki et al., J. Biological Chemistry, Tulloch, Lipids, 17:544–550 (1982). 254(10):3812–3817 (1979). Uchida, Biochimica et Biophysica Acta, 348:86-93 (1974). Miskin et al., Microbiology, 143:1745–1755 (1997). Uchida, Agricultural and Biological Chemistry, Steiner et al., Can. J. Microbiol., 43:315-321 (1997). 39(2):561-563 (1975). Yang, “Isolation, identification and characterization of Uchida et al., J. General and Applied Microbiology, linoleate isomerase from Lactobacillus reuteri” (1997) 180 18:109–129 (1972). pp., UMI Order No. DA9823268. Uchida et al., J. General and Applied Microbiology, 19:233-249 (1973). * cited by examiner U.S. Patent US 6,743,609 B1 OZZ 00Z U.S. Patent Jun. 1, 2004 Sheet 3 of 42 US 6,743,609 B1 U.S. Patent Jun. 1, 2004 Sheet 4 of 42 US 6,743,609 B1 - H25562 (aerobic) LA -(-25562 (aerobic) CLA HOURS FG. 4 U.S. Patent Jun. 1, 2004 Sheet S of 42 US 6,743,609 B1 -OHLNOLECACD -KX- CLA 15OO 1 OOO 500 - HOURS FIG. 5 U.S. Patent US 6,743,609 B1 00|| Lori cacyw Ooo N to Lo Nico Ci Y v- w w a v- w - O O O. O. O. O. O. O. O. O. U.S. Patent Jun. 1, 2004 Sheet 7 of 42 US 6,743,609 B1 (KH2PO4) U.S. Patent Jun. 1, 2004 Sheet 8 of 42 US 6,743,609 B1 G90999 09GV0W98GZOz9l EWTTO/\ ?××××××××××××××××××××××××××× G0 -01°0 00°0 U.S. Patent Jun. 1, 2004 Sheet 9 of 42 US 6,743,609 B1 i-e- also arre C D ISOM E --- FG. 9 U.S. Patent Jun. 1, 2004 Sheet 10 Of 42 US 6,743,609 B1 A - T A - T A - T G - C T G C - G G - C A - T A - T G - C A - T A - T 5-TTA CTA TAA AGA TGA - TTTTTATA-3' L L STOP FIG 10 U.S. Patent Jun. 1, 2004 Sheet 11 of 42 US 6,743,609 B1 Tested TWO Constructs Flac isom ATG ii.1 ORF T -am-Ha-m-o- D ro ATG i2 ORF -Him-Ho -o- New Construct #3: ORF HisTag OCCT/N/N/N/ New ConstructiFA: Partial ORF HisTag O - T/N/N/N/ FIG 11 U.S. Patent Jun. 1, 2004 Sheet 12 of 42 US 6,743,609 B1 Expression System: Hapll promoter LAT promoter - with the Secretion signal peptide - without the secretion signal peptide hapll ORF fi1 Flat S.p. ORF #2 ORF HisTag 3 #4 ( ) Partial ORF ORF i5 Partial ORF #6 FIG. 12 U.S. Patent Jun. 1, 2004 Sheet 13 of 42 US 6,743,609 B1 Total Cell lysate: Cells lysed with lysozyme and broken by sonication Centrifugation: 10,000 g, 30 min - " - Supernatant Pelet (Crude Extract) (Inclusion Body) Centrifugation: Lysis buffer, Tween 20, 45 k rpm, 90 min EGTA, shaken 1 h Centrifugation: 1 N 1 . g, 30 min Supernatant Membrane (Soluble Protein) Pellet Supernatant-1 Pellet-1 Lysis buffer, Lysis buffer, Tween 20, Tween 20, EGTA, shaken EGTA, shaken overnight overnight Centrifugation: Centrifugation: 45 k rpm, 90 min 10,000 g, 30 min Supernatant (Solubilized Unsolubilized Supernatant-2 Pelet-2 Membrane Membrane Pellet Proteins) F.G. 13 U.S. Patent Jun. 1, 2004 Sheet 14 of 42 US 6,743,609 B1 5OO -O-LINOLEIC ACID -0-10,12-CLA) 400 300 - 2OO 1 OO O C3 O 5 10 15 20 25 30 35 40 45 50 55 60 65 70 HOURS FIG. 14 U.S. Patent Jun. 1, 2004 Sheet 15 of 42 US 6,743,609 B1 P. acnes Culture Harvest, wash, Suspend in Buffer French Press 15K/30 minutes l Crude Extract (A) Particulate -- 45K/90 minutes Soluble (B) Fraction Fraction Buffer Wash 45K/90 minutes - - - Washed (C) N Pelet 45K Wash (D) FIG.15 U.S. Patent Jun. 1, 2004 Sheet 16 of 42 US 6,743,609 B1 1 OO - 80 60 40 U.S. Patent Jun. 1, 2004 Sheet 17 of 42 US 6,743,609 B1 1.40 1.20 1.00 O.80 0.60 0.40 0.20 O.OO O 10 20 30 40 50 60 MINUTES F.G.