Appl Microbiot Biotechnol (1984) 19 : 134-138

Applied Microbiology Biotechnology

Springer-Verlag 1984

Short communication

Formation and localization of cellulases in Cellulomonas culture on bagasse

Hiida Rodriguez ~ and OIga Volfofi Institute of Microbiology, Czechoslovak Academy of Sciences, 142 20 Prague 4. Czechoslovakia
Summary. plex was growth The eellulolytic during enzyme comIntroduction The growing interest in m i c r o b i a l arises eel-

studied

the

diauxic

of C e l l u l o m o n a s sugar

sp. Il-be on cane bagasse phase only a

lulases

in recent

years

from

alkali-pretreated pith. I n the f i r s t

their potentional rial

use f o r

the i n d u s t -

growth

sacoharification and for

of c e l l u l o s i c In

low cell-bound activity was

aryl-~-glucosidase Formation (cell-, of ba-

substrates the l a t t e r have been

SCP production. numerous

detected. and bound

direction, undertaken

studies

extraeellular gasse-) later, during levels CMi.e.

o n the c u l t i v a bacteria wastes on p r e for

and FP-eellulases at the b e g i n n i n g growth

occurred and The

t i o n of c e l l u l o l y t i c treated protein vassan 1978; sugar wastes

lignocellulosic production 1968; Dunlap

the s e c o n d

phase.

(Hanz a n d S r i n i 1969; Enriquez with a large

of all o e l l u l o l y t i c ones, At

enzymes, with the

mainly bound growth linear

increased the

1981). cane

In c o u n t r i e s

of c e l l s . growth

e n d of the all bound for cell-

industry,

the i n d u s t r i a l can constitusubstrates a practical knowledge involved

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

aryl-p-glucosidase at the end

a n d its b e h a v i o u r growth

during bacterial substrate is

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

the a i m of the p r e precisely of the the

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

and localization enzyme complex

during

of C e l l u l o m o n a s cane bagasse system. pith

on pretreated in the S C P

production

H. Rodriguez and O. Volfofi: Cellulases in Celhdomonas culture

135

Material

~nd methods Cellulomonas sp l l b e (Enriquez

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

1969) pith lo.

Cultivation conditions. Cultivation w a s in a 3 - 1 i t e r f e r m e n t e r ( i m p e l l e r s p e e d i0 Hz; a e r a t i o n r a t e 1.5 i/min9 t e m p e r a t u r e 32C; c o n s t a n t p H 6.5, regulated automatically b y a d d i t i o n of 10% sodium hydroxide). ~oculum (lO (v/v)) w a s p r e p a r e d in s h a k e n f l a s k s , c e n t r i f u g e d and the s e d i m e n t w a s res u s p e n d e d in the s a m e v o l u m e of the sterile medium. enzyme activities. Enzyme activities were determined under the o p t i m u m a s s a y c o n d i t i o n s (Rodriguez 1983). Carboxymethyl-(CM-)cellulase activ i t y w a s a s s a y e d as the a m o u n t of reducing sugars released from carboxy~ methylcellulose after a ~O-min incubat i o n at 5 0 C a n d p H 6.5. F i l t e r p a p e r -(FP-)cellulase activity was determined as the a m o u n t of r e d u e i n g s u g a r s r e l e a s e d f r o m a s t r i p of W h a t m a n N o . l filter paper after a 60-min incubation at 5 0 C a n d p H 7.0. Aryl-~-Glucosidase activity was a s s a y e d a c e o r d i n g to the m o d i f i e d m e t h o d of O k a d a et al. (1968) as the a m o u n t of 4 - n i t r o p h e n o l r e l e a s e d f r o m 4-nitrophenyl-~-glucoside a f t e r a 60- m i n i n c u b a t i o n at 45C a n d p H 7.0. T h e e x t r a o e l l u l a r a c t i v i t y w a s det e r m i n e d in the s u p e r n a t a n t a f t e r centrifugation of the c u l t u r e at 15 0 0 0 x g f o r i0 min. The cell bound activity was ealcuf a t e d f r o m the d i f f e r e n c e b e t w e e n the a c t i v i t y of i n t a c t c e l l s u s p e n s i o n a n d the a c t i v i t y of the s u p e r n a t a n t . F o r the d e t e r m i n a t i o n of the b a g a s s e b o u n d a c t i v i t y , the s a m p l e w a s f i l t e r e d through a sintered glass filter No.l p o r o s i t y a n d the b a g a s s e w a s r e s u s p e n d ed in the s a m e v o l u m e of the m e d i u m . The intraeellular enzyme activity was d e t e r m i n e d in a c e l l f r e e e x t r a c t f r o m w a s h e d cells. T h e s u s p e n s i o n of b a c t e ria and residual bagasse were filtered t h r o u g h s i n t e r e d g l a s s f i l t e r N o . l por o s i t y (in o r d e r to r e m o v e b a g a s s e ) and the f i l t r a t e w a s e e n t r i f u g e d at
Assessment of

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

of the e u l sugar cane

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

hemicellulose period, bagasse

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

the c e l l u l o l y t i o lomonas vation cell culture

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

phase, were time

no e e l l u l a s e s in the

(FP- a n d CM-) system. At this

detected

only low levels

of c e l l - b o u n d introduced prowere

aryl-p-glucosidase, bably with

the o r i g i n a l i).

inoeulum~

detected

(Fig.

During

this p h a s e , in the 2). As

the a m o u n t medium only

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

i n the m e d i u m growth should

the b e g i n n i n g increase due

of c e l l to

the r a p i d

degradation The high hemi-

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

H. Rodriguez and O. Volfofi: Cellulases in Celhdomonas culture

sumption.

The at

CM-cellulase the end of

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

aryl-~-glucosidase (Fig. l) acti-

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).

a high the The

intracellular

end of the f e r m e n t a t i o n low level of b a g a s s e (Fig. its

from

(Table -bound

aryl-~-glueosidase associated with

i)

is

active

3). Tile l e v e l s increase and inprosomeacti-

probably functions terial

other the b a c -

activities cell

then

and o r i g i n a t e s attached

increasing level 2).

growth

cells

to the b a g a s s e and rapid and

creasing teins what

of e x t r a c e l l u l a r Similarly, but

particles. decrease

A significant in the l e v e l FPand

(Fig. later,

of c e l l -

FP-eellulase first

becomes

bagasse-bound during cell

CM-eellulase of the a c t i v e pith (Figs. 3

ve in the system, -bound creted bagasse enzyme but

as the cellalso exto the

the s e c o n d

phase

is l a t e r

growth

on b a g a s s e

to the m e d i u m particles active

and binds 4).

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)

the end of f e r due

to a

During pith, CM-

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

aryl-~-glucosidase, lular and b o u n d

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

H. Rodriguez and O. Volfo~i: Cellulases in Cellulomonas culture

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

extracellular in the system l, 3 at

eellulolytie the

end of f e r m e n t a t i o n indicate probably

(Figs.

G~owth Pha~e Exp~nentlenal Early staticnaz~ Late static~Dy

Fraction a

Enzyme activity FPA CMCA 1.51 1.63 0.65 0.76 0.68 0,40

(IU/ml x i0) ~-GA 0,40 0.52 b _b 1.7 0.47

a n d 4) also lignin might

that

residual with celin the

Intracellular Cell debris Intracellular Cell debris I~tracellular Cell debris

0 0 0.24 0.03 0.09 O.O1

interfere

the a v a i l a b i l i t y lulose complex

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

structure. the above results in the

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

Cellulomonas cell-bound play tural fungal a role

is i n d u e i b l e

that could

aryl-~-glucosidase in the f o r m a t i o n inducer.

of a n a with

cellulase

Compared

cellulases,

the e n z y m e s

refer-

138

H. Rodriguez and O. Volfod: Cellulases in Cellulomonas culture

red

to h e r e

are m a i n l y
and

bound

i.e. parpre-

bound

to the c e l l s Their only

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

Received October 25, 1983