US 20050003383A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0003383 A1 ROSS0n et al. (43) Pub. Date: Jan. 6, 2005 (54) LINOLEATE ISOMERASE (60) Provisional application No. 60/141,798, filed on Jun. 30, 1999. (75) Inventors: Reinhardt A. Rosson, Manitowoc, WI (US); Ming-De Deng, Manitowoc, WI Publication Classification (US); Alan D. Grund, Manitowoc, WI (US) (51) Int. Cl. ............................ C12O 1/68; CO7H 21/04; C12P 7/64; C12N 9/90 Correspondence Address: (52) U.S. Cl. ............................. 435/6; 435/69.1; 435/134; SHERIDAN ROSS PC 435/233; 435/252.3; 536/23.2 1560 BROADWAY SUTE 1200 (57) ABSTRACT DENVER, CO 80202 The present invention provides an isolated (trans,cis)-10,12 (73) Assignee: Arkion Life Sciences LLC d/b/a Bio linoleate isomerase and its nucleic acid and amino acid Technical Resources Division Sequences. The present invention also provides a method for (21) Appl. No.: 10/755,087 producing conjugated linoleic acid or conjugated linolenic acid (CLA), or derivatives thereof, from an oil using an (22) Filed: Jan. 9, 2004 immobilized cell and/or an isolated linoleate isomerase. The present invention also provides an isolated lipase-like pro Related U.S. Application Data tein and its nucleic acid and amino acid Sequences. The present invention also provides an isolated acetyltrans (63) Continuation of application No. 09/561,077, filed on ferase-like enzyme and its nucleic acid and amino acid Apr. 28, 2000, now Pat. No. 6,706,501. Sequences. Patent Application Publication Jan. 6, 2005 Sheet 1 of 59 OZZ 00Z 09|| VI(olqoree)z92.gz–Ж VTO(o?qolae)z92.gz–o– WTOsuel](O?qoJee)z92.gz–o– Patent Application Publication Jan. 6, 2005 Sheet 2 of 59 –D–O?72VT(O?qoJee)889–D–VI(O?qoJae)899 WTO(O?qoJee)899–O– viosuen(olgolee)ge9–o– Patent Application Publication Jan. 6, 2005 Sheet 3 of 59 US 2005/0003383 A1 ? CY) WTO(O?qoleeue)£1071–o– )£1071–º–GR. Patent Application Publication Jan. 6, 2005 Sheet 4 of 59 US 2005/0003383 A1 260 240 220 200 18O 160 -O-25562 (aerobic) LA 140 -o-25562 (aerobic) CLA 120 100 HOURS FIG. 4 Patent Application Publication Jan. 6, 2005 Sheet 5 of 59 US 2005/0003383 A1 2500 - HLNOLE CACID -C-CLA 2OOO 1500 1000 500 HOURS FIG. 5 Patent Application Publication Jan. 6, 2005 Sheet 6 of 59 US 2005/0003383 A1 09|| EWN?ATO/\ 00|| Lori conv olo N to Lort cov w- w v- w w r v O. O. O. O. O. O. O. O. O. O. Patent Application Publication Jan. 6, 2005 Sheet 7 of 59 US 2005/0003383 A1 (KH2PO4) 3 Q : 3 r O n CN N4, Patent Application Publication Jan. 6, 2005 Sheet 8 of 59 US 2005/0003383 A1 00’6 09'8 00'8 09”/ 00'/ 09'9 9909GGOGGy079909GZOZGL0],90 EWT\TO/\ EEEEEE?KELERI?FIE???B-OZ’0 !,FFFF\,pL0||'0 C××××××××××××××××××××××××××××00’0 Patent Application Publication Jan. 6, 2005 Sheet 9 of 59 US 2005/0003383 A1 -Ho- -> Ho- a-Ho c D ISOM -He FIG. 9 Patent Application Publication Jan. 6, 2005 Sheet 10 of 59 US 2005/0003383 A1 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 F.G. 10 Patent Application Publication Jan. 6, 2005 Sheet 11 of 59 US 2005/0003383 A1 Tested Two Constructs Flac isom . ATG #1 NeW Construct it:3: ORF HisTag New Construct #4: Partial ORF HisTag O/N/N/N/ F.G. 11 Patent Application Publication Jan. 6, 2005 Sheet 12 of 59 US 2005/0003383 A1 Expression System: Hapll promoter LAT promoter - with the secretion signal peptide - without the secretion signal peptide hapll ORF #1 Flat S.p. ORF #2 ORF HisTag 3 4. O Partial ORF ORF i5 Partial ORF 6 FIG.12 Patent Application Publication Jan. 6, 2005 Sheet 13 of 59 US 2005/0003383 A1 Total Cell Lysate: Cells lysed with lysozyme and broken by Sonication Centrifugation: 10,000 g, 30 min - TN Supernatant Pellet . (Crude Extract) (Inclusion Body) Centrifugation: Lysis buffer, Tween 20, 45k rpm, 90 min EGTA, Shaken 1 h Centrifugation: 1 N 1 s 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: 45k rpm, 90 min -1N us min Supernatant (Solubilized Unsolubilized Supernatant-2 Pellet-2 Membrane Membrane Pellet Proteins) FIG. 13 Patent Application Publication Jan. 6, 2005 Sheet 14 of 59 US 2005/0003383 A1 500 . -O-NOLEC ACID -O-(10,12-CLA) 400 3OO 200 100 O C. O 5 10 1520 25 30 3540 45 50 55 60 65 70 HOURS F.G. 14 Patent Application Publication Jan. 6, 2005 Sheet 15 of 59 US 2005/0003383 A1 P. acnes Culture Harvest, Wash, Suspend in Buffer French PreSS 15K/30 minutes. Crude Extract (A) Particulate -- 45K/90 minutes Soluble (B) Fraction Fraction Buffer Wash 45K/90 minutes --> Washed (C) N Pellet 45K Wash (D) FG.15 Patent Application Publication Jan. 6, 2005 Sheet 16 of 59 US 2005/0003383 A1 1 OO 80 60 40 20 Patent Application Publication Jan. 6, 2005 Sheet 17 of 59 US 2005/0003383 A1 140 1.20 1.00 O.80 0.60 0.40 O.20 0.00 O 10 20 30 40 50 60 MINUTES FIG. 17 Patent Application Publication Jan. 6, 2005 Sheet 18 of 59 US 2005/0003383 A1 1.00 O.80 O.60 s 0.40 0.20 0.00 O 10 20 30 40 50 60 70 80 90 MINUTES FIG. 18 Patent Application Publication Jan. 6, 2005 Sheet 19 of 59 US 2005/0003383 A1 5.00 4.50 4.00 3.50 3.00 s 2.50 2.00. 1.50 1.00 O 500 1 OOO 1500 2000 2500 LINOLEICACID (PPM) FIG. 19 Patent Application Publication Jan. 6, 2005 Sheet 22 of 59 US 2005/0003383 A1 0.60 7 0.50 - - 6.5 0.40 S. O.30 5.5 O.20 - 5 0.10 - - 4.5 O.OO 4 O 5 10 15 20 25 30 35 40 45 VOLUME FG. 22 Patent Application Publication Jan. 6, 2005 Sheet 23 of 59 US 2005/0003383 A1 O cN CN Lo L?) n O 2 & C He CD H 3 O 1S 5 H2 CN CD - 3 Wod Patent Application Publication Jan. 6, 2005 Sheet 24 of 59 US 2005/0003383 A1 S O Hg & CD L Lo CN CN O s u? 9 ve C) s V gcy Ol?) 9 n > CN gc) O H St CD s u I o 8 3 Patent Application Publication Jan. 6, 2005 Sheet 25 of 59 US 2005/0003383 A1 Harvested Cells 0.1 M Tris, pH 8.0 10 or 500 mM NaCl 10% glycerol 2 mM DTT 10,000 xg, 30 min Crude Extract 10K pellet 45,000 xg, 90 minutes 45K supernatant 45Kpellet 0.1 M Tris, pH 8.0 10 Or 500 mM. NaC 10% glycerol 2 mM DTT 0.3% OTGP 45,000 xg, 90 min 45K detergent Sol. 10K detergent Sol. FIG. 24 Patent Application Publication Jan. 6, 2005 Sheet 26 of 59 US 2005/0003383 A1 O.4 0.3 0.2 0.1 Patent Application Publication Jan. 6, 2005 Sheet 27 of 59 US 2005/0003383 A1 9z914 00:00!00:0800:0900:0; (uudd)[GIOVOBTONIT] 00’02 Patent Application Publication Jan. 6, 2005 Sheet 28 of 59 US 2005/0003383 A1 0.3 O.25 O.05 O O O ve . CN S 3 TIME (min) FIG. 27 Patent Application Publication Jan. 6, 2005 Sheet 29 of 59 US 2005/0003383 A1 OTriS Phosphate ONCD LO N S? 07N 09 09 0Z). TIME (min) N FIG. 28 Patent Application Publication Jan. 6, 2005 Sheet 30 of 59 US 2005/0003383 A1 09 ?:CCCCCCCCF0 0 0 0 0 ?IIIIIIIIIIIIIIIIIIIIIIIIIIIIIJOEXXTTTTTTTTTT 8 Patent Application Publication Jan. 6, 2005 Sheet 31 of 59 US 2005/0003383 A1 O.8 0.6 0.4 [] ! [] O.2 ) MEDIUM FIG. 30 Patent Application Publication Jan. 6, 2005 Sheet 32 of 59 US 2005/0003383 A1 —) ...N 8£ —Ñ |SŠ |OBO+ – TRIS, pH 7.5 PHOSPHATE, pH 7.5 F.G. 31 Patent Application Publication Jan. 6, 2005 Sheet 33 of 59 US 2005/0003383 A1 0.4 O. 3 O. 2 ; O. 1 FG. 32 Patent Application Publication Jan. 6, 2005 Sheet 34 of 59 US 2005/0003383 A1 125 fresh Z o?n 24h ", FIG. 33 Patent Application Publication Jan. 6, 2005 Sheet 35 of 59 US 2005/0003383 A1 pH Effect on Extraction Efficiency of lsomerase 10 100 7.5 75 s e R 2 S. 5 5l sO Ol He CO 2.5 25 -H Specific Activity -o- Total Activity O O Patent Application Publication Jan. 6, 2005 Sheet 36 of 59 US 2005/0003383 A1 Half Lives of isomerase vs pH 125 100 75 SS 50 25 O N1 s T s i 3. FIG. 35 Patent Application Publication Jan. 6, 2005 Sheet 37 of 59 US 2005/0003383 A1 NIE.LOÀIc]No.tti/WCZ\/ .500d){ CRUDE EXTRACT DE TERGGENT SOL. FRA CTION FG. 36. Patent Application Publication Jan. 6, 2005 Sheet 38 of 59 US 2005/0003383 A1 Effect of Glycerol and Salt Concentration on isomerase Stability 110 1 OO & 90 80 . -A-TriS -o-Tris +20% glycerol -O-ris + 0.5M NaCl 70 .