The Krean Jurnal f Micrbilgy, Vl. 42, N. 3, September 2006, p. 210-215 Cpyright 2006, The Micrbilgical Sciety f Krea Dipldia gssypina ATCC10936 ³ w (+)-Jasmnic acid y š yá½ Á½ {* w tœw tœw Dipldia gssypina ATCC 10936 w e (+)-jasmnic acid (JA) w ƒ y xk, t hrmne jasmin w w wù. (+)-JA w œ w, D. gssypina ATCC10936 w (+)-JA w w. k fructse glucse y NaNO 3 ƒ (+)-JA ƒ ww. (+)-JA z 28 C 200 rpm ùkû. ³ ³ PDMYS ƒw ù, (+)-JA SM 600 mg/lƒ. Key wrds ý bisynthesis, Dipldia gssypina, epi-jasmnic acid, ptimal cnditins ³ z(traditinal fermentatin) l y œw w, yw p y w z» p w ü. w mw Bayer-Villiger reactin, Diels-Alder reactin, Bamberger rearrangement, Strecker degradatin, Beckman rearrangement, Klbe-schmidt reactin»w» yw,»w w» w ƒ (1). ³ w ƒ ù w ³» w ƒ ƒwš (3), ³» w» w w w wš (1). ³ w w»» w, ñš t jasmnic acid (JA, 3- x-2-(2'-pentenyl)-cyclpentaneacetic acid) (8). JA, w» (10). JA,,, w l w» w (9). JA sy, linleic acidƒ ctadecanid pathway mw. Linleic acid lipxygenase, allene xide synthase allene xide cyclase w 12-x-phytdienic acid (12- x-pda) ëš, 12-x-PDA y w β- *T whm crrespndence shuld be addressed. Tel: 02-3408-3228, Fax: 02-3408-3319 E-mail: kimyh@sejng.ac.kr xidatin w JAƒ (7, 10). JA 206 105 w w (7, 10). Hrmne JA w ƒe ƒ š, ƒ ƒ w w y š w (8). w ³ $16 billin 10% $1.6 billin w x. y w wì ƒe y w y ƒ Naturalness w š. w ƒwš ù œ w w w œ w z w œ w» ƒwš (6, 8). w Jasminium grandiflrum š yw w w ƒÿ. w t yw - (cis-jasmne) p (methyl jasmnate, MJ), j rl (cyclpentenne), w e ¾» w w ƒ (2, 8). JA ù, w w ³ w (4, 5). ³ w JA y z, ³ w z mw JA w ƒ š (6, 8). p e (+)-JA w ƒ y xk, methylatiny g (+)-MJ w, jasmin w ƒeƒ j 210
Vl. 42, N. 3 Dipldia gssypina w (+)-jasmnic acid y 211 ƒw (6, 8). (+)-MJ w w e (+)- JA w œ t, Dipldia gssypina ATCC10936 w (+)-JA w w. ³ JA w œ w American Type Culture Cllectin (ATCC, Manassas, USA) ³ D. gssypina ATCC10936 w JA w x w. D. gssypina ATCC 10936 ptat dextrse agar (PDA, Difc Labratries Detrit, USA) 28 C 7 w 4C w.» D. gssypina ³ w ptat dextrse brth (PDB, Difc) w 28 C 3 w z, ³ Blender w 2ml wš w z -70 C w. x w (±)-JA mixture Sigma Chemical C. (St. Luis, USA) w Thin Layer Chrmatgraphy (TLC) High Perfrmance Liquid Chrmatgraph (HPLC) t w. x ³ w PDA sww, w PDB w, sy-peptne malt extract Difc w w. Sdium nitrate (NaNO 3 ), ptassium phsphate/mnbasic (KH 2 PO 4 ), ptassium phsphate/dibasic (K 2 HPO 4 ) Sigma w, magnesium sulfate (MgSO 4 O), ptassium chlride (KCl), irn sulfate (FeSO 4 O) ( ) y (Krea) t w w, w. w (tap water) deinizatin w k w. ³, basal minimal salt medium (b-ms, 6) x mdified basal minimal salts medium (BMS) (N), (N2) (N3)BMS salt medium (SM) (Table 1) w, ptat dextrse malt yeast salts medium (PDMYS, PDB 24 g/l, malt extract 5 g/l, sytne 5 g/l, yeast extract 10 g/l) w. 25% NaOH w ph 6.0 w z 121 C, 20 ³w w. ³ ³ w ù, ³ w. JA w» w BMS fructse, glucse, lactse, sucrse starch (stck slutin, 50% w/w slutin) ƒ 30 g/l ƒw k w xw, SM w, z w. ³ d w» w 100 ml ³ cheese clth w ³ w z, heating ven (95 C) 3 w z d w. Glucse w d ü glucse w w» w 1 10 ml w, (5,000 g, 20 min)w z d z w. z d 1/10 w, YSI 2700 SELECT Bichemistry Analyzer (YSI Life Sciences, Detrit, USA) w ü glucse w w. TLC w JA ³ 7 w 25% H 2 SO 4 w ph 4.5 z, ethyl acetate (1:1, V/V) š yww vtex z, ethyl acetated w w. ƒ silica gel TLC plate (Merck, Germany) w Table 1. Cmpnents f culture medium (disslved in deinized water) Medium (g/l) BMS (N)BMS (N2)BMS (N3)BMS b-ms SM NaNO 3 1.0 2.0 0.5 2.0 2.0 7.5 KH 2 PO 4 2.0 1.5 2.0 2.0 1.33 2.0 K 2 HPO 4 0.5 0.5 0.5 0.5 0.66 MgSO 4 O 2.0 2.0 2.0 0.5 0.5 0.6 KCl 0.5 0.5 0.2 0.5 0.5 0.3 FeSO 4 O 0.001 0.001 0.001 0.001 0.001 0.6 Yeast extract 0.5 0.5 0.5 0.5-1.0 Glucse 30 30 30 30 30 30 ZnSO 4 O - - - - 0.01 0.3 MnSO 4 O - - - - 0.001 0.003 CuSO 4 O - - - - 0.0015 0.003 Na 2 MO 4-2H 2 O - - - - 0.001 0.003
212 Inh G et al. Kr. J. Micrbil š, TLC chamber (20 20 5 cm) ww. n-hexane : ethyl acetate : acetic acid = 50 : 50 : 0.5 v/v/v), ƒ óù TLC plate k z, (H 2 SO 4 : ethanl : vanillin = 40 g : 10 g : 0.5 g) w mw JA y w. HPLC w JA 10 ml w z, w(5,000 g, 20 min) z w. z 0.25 µm PTFE syringe filter (Millipre, USA) w w w š, HPLC w (+)-JA» methanl w stck slutin (100 mg/g) w z -20 C wš, v d-h 2 O w w w. 20 µl HPLC w JA w. HPLC e P680 HPLC pump (Dinex Inc, USA) Mdel UVD 170U/340U»(Dinex) w. clumn C 18 clumn (5.0 µm, 150 4.0 mm, Bischff, Germany) w š, clumn 35 C w w.» UV q 200 nm, 210 nm, 220 nm, 230 nm ƒ» q w. 0.1% TFA w w d-h 2 O acetnitrile 6:4, 0.8 ml/min w w. š D. gssypina ATCC10936 w JA D. gssypina ATCC10936 w JA y w» w, 1 10 ml w, w z(7,000 g, 20 min) d z w JA TLC y w ( ). TLC JA y JA w HPLC w w (Fig. 1). ATCC w D. gssypina ³ ATCC œ s w PDB w JA. JA w w, D. gssypina JA s x w ƒ white yellw ƒ JA, blue dark black JA x w, Kim (8) e w. w y w x š ù, y w sprulatin x š D. gssypina x ü sprulatinx x ¾ š. w JA ³ q. wr, D. gssypina xkƒ pelletx JA x w w. D. gssypina JA s 2, D. gssypina w JA ƒ ƒ v w. ³ JA D. gssypina ATCC10936 w JA w, s ƒ ww JA yƒ v w. D. gssypina ww k wì JA w kw» w xw (Table 1). w j BMS w xw z, N-surce» y 7ƒ ³ 1% w 7 w. BMS w ƒ w N-surce kw Fig. 1. HPLC analysis f the prductin f JA by D. gssypina ATCC10936. (A) (+)-JA standard (1,000 mg/l in d-h 2 O), (B) D. gssypina cell culture brth. Cell culture was grwn in SM medium at 28 C and 200 rpm fr 7 days.
Vl. 42, N. 3 Dipldia gssypina w (+)-jasmnic acid y 213» w w N-surce ƒw ù NaNO 3ƒ JA ƒ ww N-surce y w ( ). D. gpssypina ATCC10936 nitrate reductase y m w NO 3 NH 4 + yw N-surce w. D. gssypina ATCC10936 w z 2-3 w ³ ƒ j, JA ³ ƒ ƒ j ùkû. PDMYS 7 ³ 36.8 g/l ƒ ù, JA w.» w BMS ³ ù JAƒ (Fig. 2A and B). JA w HPLC w w, SM 650 mg/l ƒ ù kû, (N)BMSƒ 500 mg/l ùkü. (N2)BMS 47.28 mg/l ƒ û (Fig. 2B). (N2)BMS NaNO 3 NaNO 3 w ph w w JA w. ³ Fig. 2. Relatinships between cell grwth and the prductin f JA by D. gssypina ATCC10936 in different media. (A) Effect f culture medium n mycelial grwth and (B) Effect f culture medium n the prductin f JA. All cell cultures was grwn at 28 C and 200 rpm fr 7days. JA ƒ ew y w (Fig. 2). w ³ ƒ wì ³ yellw dark black y JA ³ w. w JAƒ D. gssypina 2, š JA»» w ƒ ƒ v w. w D. gssypina ATCC10936 (C N-surce) w ³ JA ü sw w» w š (6, 8). p ü ++(+++) Fe w y ( ). w D. gssypina ³ ³ yƒ d. w w JA w w» w, (tap water), 18 Ω k (1 deinized water), 7 MΩ k (2 deinized water) 3 ƒ š ³ w. w D. gssypina JA ù 18 Ω k 7 MΩ k ƒƒ 200 mg/l 500 mg/l JA w y, w ³ dark black ë w. x 7 MΩ k w w yw. w w ³ j, JA ww w. k w JA sy, linleic acidƒ ctadecanid pathway m w de nv synthesis mw (7). JA w linleic acid ƒw wì ƒ y w w JA ww, JA v w C-surce w» w BMS fructse, glucse, lactse, starch, sucrse 30 g/l ƒw xw starch, fructse, lactse, glucse D. gssypina ATCC10936 ³ ƒ ( ), JA fructse ƒw glucse ƒw ùkû. ù ƒ s w» C-surce s w JA œ ww. JA œ y D. gssypina ATCC10936 w JA œ w ph, œ ³ x (dispersed grwth vs. pellet grwth) w z w d w.» ph 6.0 25% NaOH
214 Inh G et al. Kr. J. Micrbil Fig. 3. Effect f temperature n the prductin f JA by D. gssypina ATCC10936. All cultures was grwn in SM medium, and at 28 C and 200 rpm fr 7 days. w w z ph y JA y w w y w. x, 7ƒ phƒ y y w,» ph 4, ³ 6» phƒ 5-6 w w, 7ƒ ph y JA j ùkü. w JA ph y j ùkù ( ). JA w e (6), JA SM JA d w. D. gssypina w w, JA ƒƒ 24 C 250 mg/l, 28 C 500 mg/l 30 C 400 mg/l ù kù 28 Cƒ JA ƒ ww y (Fig. 3). ³ y» aeratin w, ü ³ x Fig. 4. Effect f agitatin speed n the prductin f JA by D. gssypina ATCC10936. Open bar( ý ); agitatin at 200 rpm, clsed bar ( þ ); agitatin at 150 rpm. All cultures was grwn at 28 C. aeratin sheer pressure, sheer pressureƒ pellet xk x w ƒ pellet ƒw. Pellet ƒw ³ y w pellet ³ x w. aeratin JA y w» w agitatin speed ƒƒ 150 200 rpm w w 200 rpm JA ƒ š, ³ pellet size y (Fig. 4). D. gssypina w x JA œ y w agitatin aeratin w sheer pressure y w» w 20-liter z» w x w š. w w ERC» l w wx» l 2005 w œ» f w w. š x 1. ½ {. 2004.»w : y ey yw w w w»w.. 30, 11-20. 2.,»,. 1987. l r w wy w w. w» 87-0306-06-13. 3. y,, z. 1999. Methyl jasmnate w z. w ywz. 11, 284-293. 4. Aldridge, D.C., S. Galt, D. Giles, W.B. Turner. 1971. Metablites f Lasipldia thebrmae. J. Chem. Sc. Chem. Cmmun. 1623-1627. 5. Eng, F., M. Gutirrez-Rjas, and E.F. Trres. 1998. Culture cnditins fr Jasmnic acid and bimass prductin by Btrydipldia thebrmae in submerged fermentatin. Prcess Bichemistry. 33, 715-720. 6. Farbd, M., R. Blcker, L. McLean, M. Sprecker. M. McLean, N. Kssiakff, A. Kim, and M. Hagedrn. 2004. Biprcess fr the high-yield prductin f fd flavr-acceptable jasmnic acid and methyl jasmnate, nvel jasmnic acid ismer prduced thereby and uses theref. US Patent N. 6, 458-569. 7. Hamberg, M. and H.W. Gardner. 1992. Oxylipin pathway t jasmnates: bichemistry and bilgical significance. Bichim. Biphysica Acta. 1165, 1-8. 8. Kim, A.Y. 2005. Applicatin f bitechnlgy t the prductin f natural fla and fragrance chemicals. ACS sympsium series 908, 60-74. 9. Meyer, A., O. Miersch, C. Buttner, W. Dathe, and G. Sembdner. 1984. Occurrence f the plant grwth regulatr jasmnic acid in plants. J. f plant grwth regulatin. 3, 1-8. 10. Vick, B.A. and D.C. Zimmerman. 1984. Bisynthesis f jasmnic acid by several plant species. Plant Physil. 75, 458-46. (Received May 22, 2006/Accepted August 24, 2006)
Vl. 42, N. 3 Dipldia gssypina w (+)-jasmnic acid y 215 ABSTRACT : Optimal Cnditins fr the Prductin f (+)-Jasmnic acid by Dipldia gssypina ATCC10936 Inh G, Kyungju Kim and Ynghwi Kim* (Department f Fd Science and Technlgy, Sejng University, Seul 143-747, Krea) Dipldia gssypina ATCC10936 prduced chiral specific (+)-jasmnic acid (JA) that is the mst bilgically active frm. (+)-JA is a plant grwth hrmne and als ne f the mst imprtant arma cmpunds respnsible fr jasmin-like arma nte. In rder t develp a cmmercial biprcess fr the prductin f (+)-JA, ptimal culture cnditins fr D. gssypina ATCC10936 were investigated. D. gssypina prduced (+)-JA using either fructse and glucse as a sle carbn surce. As a nitrgen surce, NaNO 3 gave relatively high (+)-JA prductin. The ptimal temperature fr the prductin f (+)-JA by D. gssypina was 28 C, and ptimal agitatin was fund t be 200 rpm. D. gssypina prduced (+)-JA upt 600 mg/l in SM medium, althugh the highest level f bimass was btained in PDMYS medium.