810re50u~e 7echn0109y 55 (1996) 167-170

• 1996 E15e~er 5~ence L1m1~d

Pr1nted 1n 6 r e ~ 8 r ~ n . A11 f19h~ re5e~ed
096~852~96 $15.00
EL5EV1ER 0960-8524(95)00194-8

0 P 7 1 M 1 2 A 7 1 0 N 0 F L1QU1D MED1A F 0 R L1PA5E

P R 0 D U C 7 1 0 N 8 Y CAND1DA R U 6 0 5 A

5af1a5 8eNam~ • A5h0k Pandey*

810techn0109y Un~ Re910na1Re5earch La60rat0ry, C0unc11 0f 5c1ent1f1c • 1ndu5tr1a1Re5earc~ 7~vandrum 695 01~ 1nd1a

(ReceNed 8 Ju~ 1995; rev15ed30 N0vem6er 1995; accepted 6 Decem6er 1995)

A65tract (Yamada et a1., 1963; V~er0 et a1., 1991). 7he fer-

5tud1e5 were carr1ed 0ut 0n 11pa5e pr0duc~0n 6y Can-
d1da ru905~ 7he yea~ wa5 9r0wn 1n Un1ver5a1 Yea5t
med1um. Add1~0n 0f 0~ve 0~ t0 th~ med1um 1nduced
11pa5e pr0duc~0n and 1n 10% (v/v) c0ncentrat10n
y1e1ded ~36 U/m1 en2ym~ Rep~cement 0f 91uc05e
w1th ma1t05e fu~her 1mpr0ved the 11pa5e y1e1d. Max#
mum en2yme ~ 2 5 5 U/m0 wa5 06ta1ned when C.
ru905a wa5 cu1t1va~d 1n Un1ver5a1 Yea5t med1um 5up-
p ~ m e n ~ d w1th urea (0.1%), ma1t05e (1%), 0~ve 011
(10%, v/v) and m1nera~ and 1ncu6a~d 0n a r0tary
5haker (180 rpm) at 30•1°C f0r 72 h. C0py~9ht •
1996 E~e~er 5c~nce Ltd.
Key w0rd5: Cand1da ru905a, 1~a5e, 01Ne 0~, 5u6-
mer9ed f e r m e n t ~ n .
mentat10n c0nd1t10n5 f0r the pr0duct10n 0f
extra-cd1u1ar 11pa5e 6y ~ee C. ru905a ce115 1n 5u5pen-
510n cu~ure have pre~0u5~ 6een 1nve5t19ated. 1t ha5
6een c0nduded that the 9r0wth 0f C. ru905a ce115
wRh 01~e 0f1 0r 01e1c ac1d appear5 t0 6e 900d. 1n
add~10n, a 519n1f1cant1nf1uence 0f aerat10n rate, w1th
a m1n1mum d~50Ned 0xY9en c0ncen~at10n ha5 a~0
6een rep0~ed (Fe~er • 501a, 1992). 1n the pre5ent
5tud~ the fermentat10n 6ehav10ur 0f C. ru905a 1n the
Un~er5~ Yea5t (UY) med1um, 5upp1emented w1th
d1fferent macr0- and m ~ r 0 - n u ~ n ~ , 1ndud1n9
1n0r9an1c n1~09en and car60n 50urce~ and the
1nduc1n9 r01e 0f 01~e 0f1, are d15cu55ed.
ME7H0D5

1N7R0DUC710N M~r00r9an15m and 9r0wth med1um

7he 11pa5e-med1ated hydr0~f15 0f ~191ycer1de5 15
c05t-effect1ve 0ver the c0nvent10na1 phy5~0-chemP
c~ mean5 (Macrae • Hamm0nd, 1985). An
en2ymat~ pr0ce55 appear5 t0 6e a~ract~e a5, wh11e
1t5 c a p ~ and ener9y re4u1rement5 are m0derate,
the un~a61e fatty add5 are m 1 n 1 m ~ de9raded 6y
~rtue 0f m11d pr0ce55-parameter5 and hence the
rec0very 0f the end pr0duct5 5h0u1d 6e h19her and
the pr0duct5 5uper10r 1n 4ua11ty (Kaur et a1., 1993).
L1pa5e5 have 6een f0und 1n var10u5 m~r00r9an15m~
and patent5 1nv0~1n9 app1~at10n5 0f the5e en2yme5
have prev10u~y 6een rep0~ed (Papapara5keva5 et
a1., 1992). Am0n9 the y e a ~ Cand1da ru905a wa5
1dent1f1ed a5 a p0tent pr0ducer 0f 11pa5e (Yamada et
a1., 1963; Ra0 et a1., 1993; V1rt0 et a1., 1994).
7he 5uper10r1ty 0f 5u6mer9ed fermentat10n (5mF)
0ver the deve10p1n9 50f1d-5tate fermentat10n (55F)
f0r cu1t~at10n 0f C. ru905a ha5 6een d15cu55ed
*Auth0r t0 wh0m c0rre5p0ndence 5h0u1d 6e addre55ed.
Pre5ent addre55: La60rat0~e de M1cr06101091eet 66n6t~
4ue, UnNerf1t6 L0u15 Pa~euL CNR5 URA n° D1481,
67083, 5~a560ur~ France.
167
Cand1da ru905a (D5M 2031) wa5 9r0wn and ma1n-
t~ned 0n the UnNer5~ Yea5t-A9ar med1um at 4°C.
7he m e ~ u m c0n515ted 0f (9/1): ~uc05e 10, pept0ne
5, yea~ extract 3, m a r extract 3 and a9ar 15 (pH
7.0) 1n ~111ed w~er.
5u6mer9ed fermentat10n and med1a
Med1a w1th d1fferent c0n5t1tuent5 were tr1ed t0 maf1-
m~e the 11pa5e pr0duct10n. Med1a f0rmu1at10n5 were
de519nated 6y 1etter5. Med1um A wa5 UY med1um
(w1th0ut a9ar) wh~h wa5 u5ed f0r 1n0cu1um pre-
parat10n. Med1um 8 had UY med1um c0n5t1tuent5
and d1fferent c0ncentrat10n5 (2, 4, 6, 8, 10, 12 and
15%, v/v) 0f 01Ne 011. F0r med1um C, 50me m0d1f1-
cat10n5 were made t0 the f0rmu1a de~5ed 6y Ferrer
and 50fa (1992): 91uc05e, 10; KH:P04, 2; urea, 3;
M9504.7H20, 1; CaC122H20, 1 (~1 9/1) and
FeC13~H20, 10; 1n051t01, 0.004; 610t1n, 0.008; th1a-
m1ne-HC1, 0.2 (a11 m9/1), and 10% (vN) 01Ne 011.
Med1um D c0mpr15ed the c0n5f1tuen~ 0f UY
med1um and KH:P0~, 0.4; K H : H P 0 ~ 0.24;
(NH~)250~ 1.6; M 9 5 0 ~ 7 H : 0 , 0.2; CaC1>2H:0, 0.5
(~1 9/1), and FeC1~6H:0, 4; 1n05k01, 0.004; 610t1n,
0.008; th1am1ne-HC1, 0.2 (H1 m9/1), and 10% (v/v)
168 ~ 8enjam1n, A. Pandey

011ve 011. Med1um E c0nt~ned UY med1um c0n5t16
uent5 w1th 01~e 011 (10%, v/v) 1n wh1ch ~uc05e wa5
rep1aced 6y 0ther car60n 50urce5 at 1% c0ncentra-
t10n (~arc~ ffu~05e, 1a~05e, 5ucr05e 0r m~t05e).
Med1um F c0nt~ned 1% urea 0r amm0n1um n~rate
and the c0n5t1tuent5 0f med1um D. Med1um 6 wa5
the f 1 n ~ ~andard~ed med1um and c0nt~ned (9/1):
ma1t05~ 10; pept0n~ 5; yea~ extracL 3;• m ~ t
extracL 3; urea, 1; 6a5~ m1nera15 a5 u5ed 1n med1um
D and 01Ne 0f1 10% (v/v).
water at 715 nm u~n9 a 5pe~r0ph0t0m~er (UV-
160A, 5h1mad2u, Japan). 0 n e un1t (U) 0f 1~a5e
a~1v1ty ~ e4u~ t0 1 m1cr0m~e 0f f1ee fat~ add
116era~d/m1n/m1 under the a55ay c0n~t10n~
50h61e 5u9ar5 and pr0te1n5 were e5t1m~ed ~ the
cu1ture 5upern~ant 6y the meth0d 0f Mf11er (1959)
and L0wry et aL (1951), re5pe~Ne1y. 7he dry wt 0f
the ce1~ wa5 determ~ed 6y keep~9 the ce11 peHet
0 6 ~ e d after centr1fu9at10n 0f ~ r m e n ~ d 6r0th (20
m1) ~ an 0ven 0vern~ht at 80°C.

1n0cn1um preparat10n
RE5UL75 AND D15CU5510N
F0r prepar1n9 the 1n0cu1um, a 100pfu1 0f ce115 ~0m
a ~e5h1y 9r0wn cu1ture (a9ar ~ant) 0f C. ru905a wa5
7he 9r0wth and fermentat10n 6ehav10ur 0f C. ru905a
~an5fe~ed t0 a 250 m1 c 0 n ~ f1a5h c0nt~n1n9 50
1n UY med1um, 1.e. med1um A, ~ 5h0wn 1n F19. 1.
m1 UY med1um w1th0ut a9ar. 7he f1a5k wa5 1ncu-
7he cu~ure wh~h a a ~ n e d max1mum ceHu1ar 9r0wth
6ated at 30 + 0n a r0tary 5haker at 180 rpm f0r
after 48 h ( 0 D 2.43 and dry ceH wt 6.5 m~m1) wa5
24 h and then the ce115were harve5ted 6y centr1fu9-
una~e t0 fu1~ ut11~e the ava11a61e 5u9ar 1n the
1n9 the 6r0th (15 m1) at 5°C f0r 10 m1n at 8000 rpm.
med1um and 0n1y a60ut 61% ~uc05e wa5 c0n5umed
7he peHet wa5 re5u5pended 1n 5 m1 5ter11e dNt11~d
after 72 h (F19. 1). 7he pH 0f the ferment1n9
water (3 m9/m1 6~ma5Q.
med1um d1d n0t chan9e 5~n1f1cant~ and wa5 7.2
after 72 h (data n0t 5h0wn). 1n th15 (UY) med1um
Fermentat10n
the yea~ cu1ture c0u1d n0t 5ynthef15e Hpa5e. Add~
Fermentat10n wa5 carr1ed w1th 50 m1 0f the med1um
t10n 0f 01~e 011, h0wever, t0 th~ (UY) med1um
1n a 250 m1 c 0 n ~ f1a5k. After aut0daf1n9 (15 p~
1nduced the ce115 f0r ~pa5e 5ynthe5~ and f19n1f1cant
f0r 15 m~), the f1a5k wa5 ~ 0 c d ~ e d w1th the ce11
am0unt5 0f ex~a-ce11u1ar 11pa5e were 5ecreted 1n the
5u5pen~0n (2%) 0f C. ru905a, prepared a5 a60ve. 1n
med1um. 7he ~e1d 0f Hpa5e 5h0wed a pr0p0~10n~
a11 the expef1ment~ the 1n1t1~ pH 0f the m e d ~ m
1ncrea5e w1th the ~crea5e 1n the 01~e 011 c0ncentra-
wa5 m ~ n t ~ n e d at 7.0. 7he f1a5k5 were ~ c u 6 ~ e d at
t ~ n up t0 10%, when max1mum 11pa5e wa5 pr0duced
30 • 1°C 0n a r0~ry 5haker (180 rpm) f0r 72-120 h.
(7a61e 1). H~her c0ncentrat10n5 0f 01~e 0f1, h0w-
5 a m N ~ 9 wa5 d0ne 6y rem0~n9 d u p 1 ~ e f1a5k5 at
eve~ d1d n0t fu~her 1ncrea5e the ~e1d. 5~n1f1cant1y,
the ~ u 1 ~ e d per10d. 7he re5uR5 rep0~ed are the
the t1me re4u1red t0 att~n the 0pt1m~ ~ d d 5 0f the
avera9e v~ue5 (5tandard de•at10n 6e~9 1e55 than
en2yme wa5 72 h 1n a11 ca5e5 (~re5pea~e 0f d1f-
5% ~ a11 ca5e~ 0f three 5et5 0f the exper1men~.
ferent c0ncen~at10n5 0f 01~e 0f1). 7he5e f1nd1n95
are 1n 900d c0rre1at10n w1th the rep0~5 0f 0ther
A55ay m~h0d5
auth0r5 (0ta et a1., 1968; De1 R10 et aL, 1990).
5amp1e5 were w1thdrawn at 12/24 h. F0r ~fferent
A5 ~ ev1dent ff0m F1~ ~ add1t10n 0f var10u5
a55ay~ an ~ u 0 t (20 m1) 0f the fermented 6r0th
m~r0- and macr0-nutr1ent~ a5 u5ed 6y Ferrer and
wa5 centr1fu9ed at 8000 rpm f0r 10 m1n at 5°C.
501a (1992), t0 med1um 8, 1.e. UY med1um w1th
5upern~ant, 50 0 6 t ~ n e ~ wa5 d ~ u ~ ff nece~ary,
10% 01~e 0ff, wa5 n0t u5efu1 and ~pa5e ~e1d wa5
t0 a 5u~a61e c 0 n c e n t r ~ n . L1pa5e a~1v1~ wa5
determ~ed 6y the m0~f1ed meth0d 0f 5atar1k
(1991). F0r th15, an ~ u 0 t 0f 01~e 011 (250 m9) wa5 20- 2.6~ ~ 6.5
~ a n 5 ~ e d 1nt0 a te5t tu6e c 0 n t ~ n 9 2 m1 0f ph0~
p h ~ e 6uffer (pH 6.5) and 1 m1 0f 5amp1e wa5 added
t0 1t. 7he m~ture wa5 v0rtexed f0r 15 5 and 1ncu- 15-
6ated at 37•1°C 1n a water 6ath under 5tat1c 1 . 8 ~ ~ 45 1
c0n~t~n5 f0r 30 m ~ . After 5t0pp~9 the react10n 6y
ad~n9 1 m1 c0nc HC1 and v0~e~n9 f0r 10 5, 3 m1 0f ~d ; 1.4~ L - --35~
6en2ene wa5 added and after v0~e~n9 f0r 90 5, the
a4ue0u5 and 0r9an~ pha5e5 were ~ w e d t0 5epa-
rate. Fr0m th15, 2 m1 0f the 6en2ene 1ayer wa5
w1thdrawn and ~an5fe~ed t0 a tu6e c0nta1n~9 1 m1 5-
0f a4ue0u5 501ut~n 0f cupr1c acetate (5% c0ncen-
~at10n, pH 6.2 a ~ u ~ e d u~n9 py~d~e) and the
m ~ t u r ~ af1er v0~e~n9 f0r 90 5, wa5 centr1fu9ed at 0 ~ ~ . 5
5000 rpm f0r 10 m1n at 5°C t0 06t~n a dear 0r9an~ 0- 0 12 24 36 48 60 72
pha5e. 7he 0r9an1c (6en2ene) 1ayer wa5 w1thdrawn ~f1~ ~ ~cu~n p
and u5ed t0 e5t1m~e the 1~era~d f1ee fat~ add5 6y F1~ 1. F e r m e m ~ n 6ehav10ur 0f C. ru905a ~ UY
mea5ur1n9 the 0 p t ~ d e n ~ ( 0 D ) a9~n~ ~5t11~d med1um.
 L~a5e pr0duct10n 6y C. ru905a 169

7a61e 1. L1pa5e p r 0 d u ~ n 6y Cand1da ru905a 1n med1um 8

1 ~ u 6 a ~ n ~r10d (N D p ~ e ~tMf1~"
C~mr~n ~ ~Ne 0~
0 2 4 6 8 10 12 15
48 0,25 1,30 1,82 2,48 3,13 4,99 4,70 4,58
72 0,33 2,58 3,15 5,81 7,58 8,36 8,18 8,00
96 0,29 2,55 3,98 5,09 7,49 8,28 8,17 7,98
120 0,25 2,52 3,85 5,67 7,47 8,03 7,95 7,97
"N0 1 ~ e w ~ ~und 1n 0 and ~ h ~ m p ~

~e 2. ~ p a ~ p ~ d u ~ ~ Cand1da ~905a 1n m ~ m E
1ncu6a~n per10d (h) ~ p ~ e aaMf1~"
5tarch Fruct05e 61uc05e Lact05e 5ucr05e Ma1t05e
48 0,35 4,87 4,92 5,16 5,19 5,40
72 0,75 8,29 8,30 8,74 9,76 10,34
96 0,75 8,27 8,28 8,74 9,75 10,20
120 0,74 8,20 8,27 8,74 8,73 9,18
"N0 1 ~ e w~ ~ d ~ 0 ~d ~ h ~m~m.

1e55 w1th th15 med1um (C) than that 06ta1ned w1th
m e ~ u m 8. H0weveC w11h med1um D, f1pa5e y1e1d
1ncrea5ed t0 8.30 U/m1 (c0mpara61e t0 med1um 8).
61uc05e wa5 the mNn car60n 50urce 1n UY
med1um. W1th 1he med1um E, the ~tent10n wa5 t0
5e1ect the m0~ 5uRa61e 5u65trate (car60n 50urce).
Am0n9 the te~ed 5u65~ate5, maR05e wa5 6e5t and
~ d d e d 10.34 U/m1 en2yme (7a61e 2). 0 n the wh01e,
d9acchaf1de5 were 6e~er than the m0n0- and p0~-
5acchaf1de5. 7he f1e1d 1n 5tarch med1um wa5
e ~ r e m e ~ p00r ( 7 a 6 ~ 2) a5 C. ru905a wa5
apparent~ una6~ t0 meta6019e 5tarch.
L1pa5e f1e1d w1th the m e N u m F wRh urea 0r
amm0n1um nRrate wa5 even 6etter and ~ the f1nN~
0pt1m~ed med1um 6 ~he 11pa5e ~ e N wa5 h19h, wRh
a m a ~ m u m 0f 12.55 U/m1 aRer 72 h fermentat10n
(F19. 2).
7he 1nduNn9 r01e 0f 11p1d 5u6~ance5 1n 11pa5e
pr0du~10n ha5 6een d15cu55ed 6y many auth0~ ( 0 t a
et a1., 1968; 0 6 r a d 0 ~ et a1., 1993). 1n aH the pre5ent
te5t5, 1nduf10n 0f 01~e 011 9ave a h19h en2yme y1e1d,
aRh0u9h the r01e 0f 0ther fact0~, car60n and nRr0-
9en 50urce5 and m1nera1 n u ~ n ~ , cann0t 6e ru1ed
0ut. 7he 11pa5e y1e1d 06ta1ned 6y u5 1n the 0pt1m~ed
med1um 6 (12.55 U/m1) wa5 6etween the rep0~ed
vNue5 0f 5.2 U/m1 1n 5mF (De1 R10 et a1., 1990) and
36.6 U/9 1n 55F (Ra0 et a1., 1993) 6y C. ru905a.
~ 0 ~ ~ ~ 5
We thank Dr A. D. Dam0daran, D ~ e ~ 0 ~ and Dr
V~ay Na1~ Deputy D ~ e ~ 0 ~ f0r the1r 1ntere5t 1n the
pre5ent w0rk. 58 9r~efu1~ ackn0w1ed9e5 the award
0f a 5en10r Re5earch Fe110w5h1p 6y 7he C0un~1 0f
5 d e n t ~ c and 1ndu~f1~ Re5earch, New Ddh1.

12 REFERENCE5
11
De1 R10, J. L., 5err~ D., V~er~ F., P0ch, M. • 5~a, C.
(1990). Rea~10n 5cheme 0f 1~a5e pr0du~10n 6y Can-
d1da ru905a 9r0w~9 0n 01~e 011. 810te~n01. Lett., 12,
335-338.
-
Ferre~ P. • 501a, C. (1992). L1pa5e pr0duct10n 6y
1mm06~ed Cand1da ru906a ce115.AppL M1cr0~01. 8~-
6- ~ ~chn01., 37, 737-741.
5- ~ KauL J., Ramamurthy, V. • K0thar1, R. M. (1993).
Chara~ef12at~n 0f 0~ 1~a5e f0r 1 ~ 0 ~ 5 0f r1ce 6ran
4- ~ 011.8~chn0L Lett., 1~ 257-263.
L0wry, 0. H., R05en6r0u9~ N. J., Fam A. L. • Randa11,
R. J. (1951). Pr0te1n mea5urement w1th the f011n phen01
rea9ent. 2 810L Chem., 193, 265-275.
Macra~ A. R. • Hamm0n~ R. C. (1985). Pre5ent and
future app11cat~n5 0f 11pa5e. 8 ~ c h n 0 L 6ene~c En9n9
12 24 36 48 60
Rev., 3, 193-217.
Per10d 0 f ~ c u 6 ~ n (h) ~ M111er, 6. L. (1959). U5e 0f ~n1tr05a11cy1~ a~d rea9ent
F19. 2. L1pa5e act1v1ty (U/m1) 0f C. ru905a 1n var10u5 f0r the determ~af10n 0f redu~n9 5u9a~. Anal Chem.,
med1~ 31, 426-428.
170 ~ 8enjam1n, A. Pandey

06md0~, ~ , M0~eNn0~ J. L, V M ~ E, L a ~ e ~ F. 5~af1L 1. (1991). A 5pe~r0ph~0m~er ~5ay ~ r f1p~e

J. • 5Na, C. (1993). E ~ c t 0f N ~ r e m ~ add5 1n a~1v1~ ut11121n9 1mm06f1~ed tr1ac~1~ycer1de5. 2 810-
~p~e ~ u ~ ~ ~n~ ~a. ~ 8~L ~ . , chem. 810phy5. Me~., 23, 249-253.
~, ~M~ V~er~ E, D~ R1~ J. L, P0c~ M. • 5Na, C. (1991).
0m, Y., ~yNf1, 5. • Yamad~ K (1968). 5tern re4u~e- Fermentat~n 6ehav~ur 0f f1pa5e p r 0 d u ~ n 6y Cand1da
ment ~ r the 1 ~ e ~ u ~ 6y ~ n ~ ~a. ~ ru905a 9r0w1n9 0n ~ f f e ~ m1xture5 0f ~uc05e and
8~L ~ . , ~, 1~1~ N~e 011.2 Fermen~ 8~en9n9, 72, 399-401.
Papapara5keva~ D., ~ ~ P., Kek0~ D. • M . V 1 ~ M. D., A9u~ 1., M0nter~ 5., 81anc~ A., 5N0-
M~f1~ 8. J. (199~. 0 N ~ 9 W~u~ 0f e9ra- 2a6a1, R., La~aray, J. M., L1ama, M. L, 5erra, J. L,
~1~ 1 ~ e ~0m ~ m ~ ~ . 8 ~ L a n d ~ L C. • de Ren6~e5 (199~. Hydr01y5~ 0f
Lea., 14, ~ M ~ a~m~ ~ 6y 1mm0~1~ed Cand1da ru905a 1 ~ e .
Ra0, P. V., J ~ a m ~ , K • Lak~manaK C. M. (1993). En2yme M1cr0~ ~chn01., 1~ 61-65.
Pr0duct10n 0f f1p~e ~ ~ n ~ ~ 1n 5 N ~ t ~ e Yamad~ K., Mac~d~ H., H19a5h1, 7., K0~e, A. • Ueda,
~rm~t~: meNum ~ 2 ~ and e ~ ~ ~- K. (1963). P r 0 d u ~ n 0f f1p~e ~0m m1~00r9an15m5
~. ~ 8 ~ h ~ . , 28, ~ 1 ~ . (111) me~um c0mp05~0n cf Cand1da cy11ndracea. A9r1c.
8101. Chem., 37, 645-649.