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Short communication
studied
the
diauxic
of C e l l u l o m o n a s sugar
lulases
in recent
years
from
use f o r
the i n d u s t -
growth
of c e l l u l o s i c In
direction, undertaken
studies
the s e c o n d
phase.
of all o e l l u l o l y t i c ones, At
increased the
1981). cane
In c o u n t r i e s
of c e l l s . growth
industry,
phase
almost
known
as b a g a s s e , suitable Before
cellulolytic -bound
enzymes,
except
te p a r t i c u l a r l y for this
aryl-~-glucosidase,
are r e l e a s -
process.
ed to the m e d i u m complex.
as a n e x t r a c e l l u l a r level of the
application of
of the p r o c e s s , mechanism
A considerable
the e n z y m a t i c
intraeellular activity
a n d its b e h a v i o u r growth
is s t i l l p r e s e n t
on the c e l l u l o s i c It w a s
of the f e r m e n t a t i o n .
required. sent w o r k
to c h a r a c t e r i z e
the f o r m a t i o n i p r e s e n d a d d r e s s : D e p a r t m e n t of Industrial Microbiology, National C e n t e r of S c i e n t i f i c R e s e a r c h , A p a r t a d o 6990, H a v a n a , C u b a Offprint request to: O.VolfovA cellulolytic growth sugar
during
on pretreated in the S C P
production
135
Material
Strain. 1978).
M e d i a . T h e s t r a i n was m a i n t a i n e d on CMC-agar and cultivated on optimized c u l t i v a t i o n m e d i u m ( R o d r i g u e z et al. 1983) of the f o l l o w i n ~ c o m p o s i t i o n (g/l); K H o P 0 h 0.6; N H A C I 2.0; N a C I 0.25; M g S O L . T H o 0 0 . 2 5 ~ T h i a m i n e 0.01; alkaline p~etr~ated bagasse (Dunlap
15 OOOxg for 15 rain. The sedimen~ was washed and resuspended in phosphate burrer pii 7.0 (0.6 g/ml)~ disrupted in a Braun disintegrator and centrifuged at 26 O00xg for 30 min. The supernatant w a s a s s a y e d f o r the i n t r a e e l l u l a r a c t i v i t y . The sediment was made up to the initial volume with buffer and assayed for the cell debris bound activity. Reducing sugars were determined by the Somo~yi-Nelson method (Somogyi 1952). T h e g r o w t h of c e l l s w a s f o l l o w e d t u r b i d i m e t r i c a l l y a f t e r f i l t r a t i o n of the s a m p l e t h r o u g h the s i n t e r e d g l a s s f i l t e r N o . l p o r o s i t y at 600 nm. The extraoellular p r o t e i n s w e r e det e r m i n e d a c c o r d i n g to L o w r y eL al.
(1951)
Results The and discussion sp. diau_xie ~rowtl~ of C e l l u l o m o n a s detected in m o s t
Ilbc w a s tures
on
alkali-pretreated
bagasse 1981)
pith.
The
prediction grows
(Enriquez first on
that
this
strain
the r e s i d u a l an a d a p t a t i o n
and~
after of
on e e ! l u l o s e pith was
the p r e t r e a t e d confirmed
fully of
by our
detailed eomp!ex
studies
in the C e l l u cultiof
cu%der c o n s t a n t At
conditions.
the b e g i n n i n g the f i r s t
growth
and during
growth
no e e l l u l a s e s in the
detected
of c e l l - b o u n d introduced prowere
the o r i g i n a l i).
inoeulum~
detected
(Fig.
During
of r e d u c i n g
sugars (Fig.
rapidly
decreased
solid
sediment
of the c u l t u r e the c o n t e n t of at
served free
as i n o e u l u m , sugars
reducing
of c e l l to
the r a p i d
of b a g a s s e xylanase cellulose
hemicellulose.
aetivity
content
aa~d d e c r e a s i n g
21
the s y s t e m
this
(]{odricuez 1983)
supported
as-
136
sumption.
The at
activity
is
derepressed tween
the l a g begrowth
O6 3 05 ! <
the f i r s t
and s e c o n d
1
Z.O
04
'0~
o 3I
zO
1.0 @5
03
02
//
02
Jo
0~
O.J
0.2
Fig. i. T h e l e v e l s of a r y l - ~ - g l u e o s i dase a c t i v i t i e s ( I U / m l x i0 "~) in diff e r e n t c u l t u r e f r a c t i o n s d u r i n g the g r o w t h of C e l l u l o m o n a s sp. I I b c on p r e t r e a t e d b a g a s s e pith. i, c e l l g r o w t h ( a b s o r b a n o e at 600 nm); 2, e x t r a e e l l u l a r enzyme a c t i v i t y ; 3, c e l l - b o u n d enzyme a c t i v i t y ; 4, b a g a s s e - b o u n d enzyme a c t i v i t y
Fig. 2. T h e p r o d u c t i o n of e x t r a e e l l u l a r proteins and reducing sugars during the g r o w t h of C e l l u l o m o n a s sp. I I b c on p r e t r e a t e d b a g a s s e pish. l, c e l l g r o w t h ( a b s o r b a n c e at 600 ram); 2, e x t r a c e l l u lar p r o t e i n s (mg/ml); 3, f r e e r e d u c i n g s u g a r s (mg/ml x 3.4)
whole
fermentation mainly
remains
cell-bound
exhibiting vity phases; simultaneously the c e l l - b o u n d , enzyextraeellular mes b e c o m e of e n z y m e with and bagasse-bound (Fig. until l).
intracellular
(Table -bound
i)
is
active
other the b a c -
activities cell
then
and o r i g i n a t e s attached
growth
cells
of e x t r a c e l l u l a r Similarly, but
particles. decrease
(Fig. later,
of c e l l -
FP-eellulase first
becomes
the s e c o n d
phase
is l a t e r
growth
on b a g a s s e
a n d 4) a n d lular
the l o w l e v e l at
of i n t r a c e l -
(Fig. cell
FP-cellulase (Table i)
growth
on b a g a s s e unlike
mentation
limitation
are not
and FP-cellulases,
exist
by bagasse. sugars
It is p o s s i b l e synthe(Fig. 2) m i ~ i t
as e x t r a c e l During the
that
a repression
of o e l l u l a s e
enzymes.
sis by r e d u c i n g
137
J
2 /
4.0
30
ZO
20 10
2O
05 1.0
4 02
0I 010 20 40 h 0
Jo
20
40
60
Fig. 3- T h e l e v e l s of ~ M - c e l l u l a s e activities (IU/ml x i0 ) in d i f f e r e n t c u l t u r e f r a c t i o n s d u r i n g the g r o w t h of C e l l u l o m o n a s sp. Ilbc on p r e t r e a t e d b a g a s s e pith. l~ c e l l g r o w t h (absorbance at 600 nm); 2, e x t r a c e l l u l a r e n z y m e a c t i v i t y } 3~ c e l l - b o u n d e n z y m e a c t i v i t y ; 4, b a g a s s e - b o u n d enzyme activity
Fig. 4. T h e l e v e l s of ~ P - e e l l u l a s e activities (IU/ml x i0 ~) in d i f f e r e n t c u l t u r e f r a c t i o n s d u r i n g ~he g r o w t h of C e l l u l o m o n a s sp. IIbe on p r e t r e a t e d b a g a s s e pith. l, c e l l g r o w t h (absorbanee at 600 nan); 2~ e x t r a c e l l u l a r e n z y m e a c t i v i t y ; 3~ c e l l - b o u n d e n z y m e a c t i v i t y } 4, b a g a s s e - b o u n d e n z y m e activity
be i n v o l v e d .
Relatively bagasse
high pith
amounts and h i g h
of n o n - d e g r a d e d levels
T a b l e i. L ~ t r a c e l l u l a r e n z y m e a c t i v i t y of C e l l u l o m o n a s sp. IIbc grown on pretreated bagasse pith
of the a c t i v e complex
eellulolytie the
(Figs.
Fraction a
Enzyme activity FPA CMCA 1.51 1.63 0.65 0.76 0.68 0,40
that
interfere
of the r e s i d u a l enzymes
to the b a c t e r i a l of b a g a s s e from
It f o l l o w s
aObtained
as d e s c r i b e d i n M e t h o d s bNot determined
that
the s y n t h e s i s
of e e l l u l a s e s and
is i n d u e i b l e
that could
of a n a with
cellulase
Compared
cellulases,
the e n z y m e s
refer-
138
red
to h e r e
are m a i n l y
and
bound
i.e. parpre-
bound
to b a g a s s e forms
titles. dominate
extraeellular at the
e n d of the f e r -
mentation.
References D u n l a p CE (1969) P r o t e i n f r o m w a s t e cellulose by chemical-microbial processing. PhD Thesis, Dept Chem Eng, L o u i s i a n a S t a t e U n i v E n r i q u e z i (1978) T h e o b t a i n e d SCP f r o m c e l l u l o s i c w a s t e s b y the f e r m e n t a t i o n p r o c e s s . P h D T h e s i s , Inst M i e r o b i o l . C z e c h k o a d Sei~ P r a g u e E n r i q u e z A (i981) Growth of eellulolytie b a c t e r i a o n s u g a r c a n e b a g a s s e . Bioteehnol Bioeng 23:1423-1429
H a n z YW, S r i n i v a s s a n V R (1968) I s o l a t i o n zLnd c h a r a c t e r i z a t i o n of a cellulose utilizing bacterium. Appl Miorobiol 16:1140-1145 L o w r y 01{, R o s e b r o u g h NJ, F a r m AL~ R a n d a l l R J (1951) P r o t e i n m e a s u r e m e n t w i t h the F o l i n p h e n o l r e a g e n t . J Biol Chem 193:265-275 R o d r i g u e z H~ E n r i q u e z A, V o l f o v ~ 0 (1983) O p t i m i z a t i o n of c u l t u r e m e d i u m c o m p o s i t i o n for e e l l u l o l y t i e bacteria by mathematical methods. Folia Microbiol 28:163-171 R o d r i g u e z tt (1983) G r o w t h of c e l l u l o l y r i c b a c t e r i a on s u g a r c a n e w a s t e s . P h D T h e s i s , Inst M i e r o b i o l , C z e c h A e a d Sci, P r a g u e S o m o g y i M (19.52) N o t e s on s u g a r d e t e r mination. J B i o l C h e m 1 9 5 : 1 9 - 2 3