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1 The Krean Jurnal f Micrbilgy, Vl. 42, N. 3, September 2006, p Cpyright 2006, The Micrbilgical Sciety f Krea Dipldia gssypina ATCC10936 ³ w (+)-Jasmnic acid y š yá½ Á½ {* w tœw tœw Dipldia gssypina ATCC 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: , Fax: kimyh@sejng.ac.kr xidatin w JAƒ (7, 10). JA 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

2 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 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 KH 2 PO K 2 HPO MgSO 4 O KCl FeSO 4 O Yeast extract Glucse ZnSO 4 O MnSO 4 O CuSO 4 O Na 2 MO 4-2H 2 O

3 212 Inh G et al. Kr. J. Micrbil š, TLC chamber ( 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, 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 ATCC (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.

4 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 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 % NaOH

5 214 Inh G et al. Kr. J. Micrbil Fig. 3. Effect f temperature n the prductin f JA by D. gssypina ATCC 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 ATCC 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 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 { »w : y ey yw w w w»w.. 30, ,», l r w wy w w. w» y,, z Methyl jasmnate w z. w ywz. 11, Aldridge, D.C., S. Galt, D. Giles, W.B. Turner Metablites f Lasipldia thebrmae. J. Chem. Sc. Chem. Cmmun Eng, F., M. Gutirrez-Rjas, and E.F. Trres Culture cnditins fr Jasmnic acid and bimass prductin by Btrydipldia thebrmae in submerged fermentatin. Prcess Bichemistry. 33, Farbd, M., R. Blcker, L. McLean, M. Sprecker. M. McLean, N. Kssiakff, A. Kim, and M. Hagedrn 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, Hamberg, M. and H.W. Gardner Oxylipin pathway t jasmnates: bichemistry and bilgical significance. Bichim. Biphysica Acta. 1165, Kim, A.Y Applicatin f bitechnlgy t the prductin f natural fla and fragrance chemicals. ACS sympsium series 908, Meyer, A., O. Miersch, C. Buttner, W. Dathe, and G. Sembdner Occurrence f the plant grwth regulatr jasmnic acid in plants. J. f plant grwth regulatin. 3, Vick, B.A. and D.C. Zimmerman Bisynthesis f jasmnic acid by several plant species. Plant Physil. 75, (Received May 22, 2006/Accepted August 24, 2006)

6 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 , 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.