Effect of sulfur-containing compounds on Bacillus cellulosome-associated ‘CMCase’ and ‘Avicelase’ activities

E¡ectof sulfur-containing compoundsonBacillus cellulosome-associated‘CMCase’and‘Avicelase’activitiesNatasha Beukes & Brett Ivan Pletschke

Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown, South Africa

Correspondence: B.I. Pletschke, Department

of Biochemistry, Microbiology and

Biotechnology, Rhodes University, PO Box 94,

Grahamstown 6140, South Africa.

Tel.: 12746 6038081; fax: 12746 6223984;

e-mail: [email protected]

Received 11 May 2006; revised 17 August

2006; accepted 23 August 2006.

First published online 29 September 2006.

DOI:10.1111/j.1574-6968.2006.00465.x

Editor: Christiane Dahl

Keywords

avicelase; cellulase; cellulosome; CMCase;

sulfide.

Abstract

The isolation of cellulosomes from clostridial sources has been extensively studied;

however, the isolation of cellulosomes from facultative soil anaerobes of the family

Bacillaceae is not as well characterized. The Bacillus cellulosome (celluloxylano-

some) essentially consists of two complex components: C-I and C-II. This multi-

component complex enables Bacillus to degrade a variety of carbonaceous

compounds as it is composed of several enzymes, such as cellulases, xylanases and

other degradative enzymes. The cellulosomal cellulases from Bacillus megaterium

were purified using cellulose affinity chromatography, followed by Sepharose 4B

gel filtration chromatography. The objective of this investigation was to establish

the effect of sulfate and sulfide on cellulosomal ‘cellulase’ activity. An increase in

sulfide concentration led to a general enhancement of cellulosomal-associated

cellulolytic activity, whereas an increase in sulfate concentration resulted in an

inhibition of the cellulosome-associated cellulolytic activity.

Introduction

Several microorganisms that have the ability to degrade

compounds such as cellulose, xylan and pectin, produce an

array of catalytic and noncatalytic components, which act

synergistically to solubilize biomass (Davies et al., 1998).

The synergy between the various catalytic components,

which may be different enzymes or isomers of the same

group of enzymes, is established through the formation of

multi-enzyme complexes, known as cellulosomes for exam-

ple in the genus Clostridium, xylanosomes in the genus

Butyrivibrio (Kim, 1995) or celluloxylanosomes in the genus

Bacillus (Kim & Kim, 1995).

Species of the genus Bacillus are industrially important as

they have a high growth rate, are able to secrete proteins

extracellularly and are considered relatively safe to use with

regard to health and environmental aspects (Schallmey et al.,

2004). An interesting feature of the genus is its well-

characterized ability to degrade amorphous substrates, such

as carboxymethylcellulose (CMC), despite the inability to

degrade crystalline substrates (Avicels) (Okoshi et al., 1990;

Ozaki & Ito, 1991; Aa et al., 1994).

The degradation of cellulose by members of the family

Bacillaceae has not been extensively studied. The cellulo-

some of the bacterial genus Bacillus is slightly different in

structure from the well-characterized clostridial cellulosome,

as it appears to be composed of two multi-component

complexes, Complex-I and Complex-II (Kim, 1995), instead

of a single complex and a single Avicelase entity (Complex-

III), which is essentially an exoglucanase (Kim & Kim,

1995). It is widely believed that the endoglucanases are only

responsible for the degradation of amorphous regions of

cellulose, resulting in the partial degradation of cellulose.

The crystalline regions are degraded by exoglucanases/

cellobiohydrolases (CBHs) (Kim, 1995).

Anaerobic degradation occurs in a wide variety of aquatic

and terrestrial habitats (Novaes, 1986). Anaerobic digestion

is the conversion of a variety of compounds, ranging from

insoluble compounds (e.g. cellulose) to soluble monomers,

and the subsequent degradation of these compounds to

produce methane and carbon dioxide (Novaes, 1986). The

hydrolysis of complex insoluble compounds to soluble

monomers occurs predominantly through the action of

extracellular enzymes (cellulases, lipases and proteases);

thus, members of the genera Bacillus, Clostridium, Butyrivi-

brio and other facultative/obligate anaerobes are involved in

the initial step (Novaes, 1986). The second step is the

fermentation of the soluble monomers to produce fatty

acids, acetate, carbon dioxide and hydrogen, which occurs

through acetogenesis or methanogenesis. The third step is

FEMS Microbiol Lett 264 (2006) 226–231c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

the conversion of the fatty acids to acetate, hydrogen and

carbon dioxide, which are made accessible to various

methanogens to produce carbon dioxide and methane in

the final stage (Novaes, 1986).

Sulfate-reducing bacteria (SRB) are often associated with

methanogens in anaerobic environments. Sulfur and sulfur

derivatives may potentially enhance the cellulolytic activity

of cellulolytic systems, such as the cellulosome. This en-

hancement of cellulolytic activity was demonstrated by

Johnson et al. (1982) and Lamed et al. (1984), with regard

to both the crude extract and purified cellulosomal fractions.

Lamed et al. (1984) observed that a thiol-enzymatic system

produced a ninefold increase in enzymatic activity for both

crude and purified cellulosomal fractions. The increase in the

enzymatic activity under sulfate-reducing conditions is po-

tentially important with regard to achieving an accelerated

anaerobic digestion of organic wastes. The aims and objec-

tives of this investigation were therefore to isolate and purify

the cellulosome from an anaerobic microorganism and to

establish the effect of sulfate and sulfide on the ‘cellulase’

activity of cellulosomal endo- and exoglucanases.

Materials and methods

Isolation of an anaerobic isolate with ‘CMCase’and ‘Avicelase’ activity

Enrichment medium (Allen, 1949): Omeliansky’s anaerobic

media for cellulase decomposers was prepared as follows:

0.3 g (NH4)2SO4; 0.3 g K2HPO4; 0.15 g MgSO4 � 7H2O; trace

NaCl; and trace CaCl2, dissolved in 300 mL dH2O. The

medium was dispensed into 10 boiling tubes to produce a

final volume of 30 mL. MgSO4 � 7H2O and CaCl2 were

autoclaved individually, after which they were dispensed

into the 10 boiling tubes using a sterile technique. The

cellulolytic anaerobes were enriched by the addition of a

sterile piece of Whatman filter paper. The soil sample was

added and a layer of sterile liquid paraffin was added to the

top of each tube to maintain anaerobic conditions. Samples

were incubated at 30 1C for 5 weeks.

Selection medium: The broth was composed of 1 g tryp-

tone, 1 g peptone, 2 g beef extract, 0.6 g yeast extract, 1 g

CMC, 0.2 g starch, 0.1 g cysteine HCl, 0.6 g sodium acetate,

0.1 g agar and 1 g sodium chloride. The agar plates contained

an additional 1.3 g bacteriological agar. The culture media

were autoclaved, inoculated with a sterile toothpick and

covered with liquid paraffin. The agar plates were poured

and allowed to set, after which 200mL of the enrichment

medium was spread out on a plate. Anaerobic conditions

were maintained using Anaerotests (catalogue no. 1.15112)

and Anaerocults by Merck (catalogue no. 1.13807).

Scanning electron microscopy (SEM): A section of the

Whatman filter paper was placed in a phosphate-buffered

glutaraldehyde fixative overnight. The fixative was removed

with two 15 min washes with ice-cold 0.1 M phosphate

buffer (pH 7.3). The phosphate buffer was removed and

the filter paper was washed at 10-min intervals with ethanol

solutions ranging from 30% (v/v) to 100% (v/v). The

ethanol was decanted and the filter paper was washed at

20-min intervals with 75 : 25 ethanol:amyl acetate, 50 : 50

ethanol:amyl acetate, 25 : 75 ethanol:amyl acetate solutions

and pure amyl acetate. The filter paper was dried using the

critical point-drying process as described by Cross (2001).

The filter paper was then coated with a thin layer of gold.

Purification of CMCase and Avicelase enzymes

A modified purification protocol of Shoseyov & Doi (1990)

was used. A 50-mL culture was grown for 10 days at 37 1C.

The culture was centrifuged for 10 min at 8000 g. To the

supernatant, 8 g of Avicels was added and incubated at

22� 2 1C for an hour. The sample was centrifuged at

10 000 g for 10 min. The pellet was washed for 15 min and

centrifuged for 10 min sequentially at 10 000 g with 50 mL

1 M NaCl/PC (50 mM phosphate/12 mM citrate) buffer (pH

7) (twice), 50 mL PC buffer (pH 7) (twice) and 100 mL

deionized water. The fraction with the highest cellulolytic

activity was concentrated in dialysis tubing with polyethy-

lene glycol (PEG) 20 000 and applied to a Sepharose 4B size

exclusion column, as described by Morag et al. (1992). One

milliliter fractions were collected and assayed for cellulolytic

activity (Avicelase and CMCase). Samples showing activity

were pooled and used for further studies. The protein

content was determined as described by Bradford (1976),

and 10% sodium dodecyl sulfate-polyacrylamide gel elec-

trophoresis (SDS-PAGE) was performed as described by

Laemmli (1970).

Effect of sulfate and sulfide on CMCase andAvicelase activities

CMCase and Avicelase assays reactions were prepared as

follows: 0.1 mL of a fixed enzyme concentration was added

to 0.2 mL of a 2% (w/v) substrate solution. Sulfide and

sulfate stock solutions were added to the enzyme reaction to

give a concentration range of 0–12.24 mM sulfide or sulfate,

respectively. 0.05 M citrate buffer (pH 4.8) was added to a

final volume of 0.8 mL. Before the addition of the enzyme,

the reaction tubes were preincubated at 50 1C for 5 min.

Once the enzyme had been added, the reaction was allowed

to proceed over a time period of 30 min. The reactions

involving Avicels required brief shaking every 10 min. The

samples were microfuged in a Heraeus Biofuge at

13 000 r.p.m. for 1 min. To 300 mL of the test reactions,

600 mL 3,5-Dinitrosalicylic acid (DNS) was added and

incubated at 100 1C for 5 min, and then placed on ice for

10 min. Suitable controls were also performed to assess the

FEMS Microbiol Lett 264 (2006) 226–231 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

227Sulfur compounds and Bacillus cellulase activities

effect of sulfate and sulfide on the pH of the CMCase and

Avicelase assays. The reactions were performed in triplicate,

and the absorbance was read at 540 nm.

Results and discussion

Isolation of an anaerobic isolate with CMCaseand Avicelase activity

The anaerobic isolate was initially selected through the

enrichment media that made use of sterile Whatman filter

paper as the sole carbon source. The degradation of the

cellulose fibrils took place through the hydrolysis of the b-

1,4-glycosidic bonds (Schwartz, 2001) and thus the subse-

quent depolymerization of the complex fibril matrix. The

depolymerization resulted in the subsequent loss of the

cellulose. The degradation of the filter paper by cellulolytic

anaerobes was confirmed, through the comparison of a

culture containing filter paper that had undergone near-

total solubilization and a culture containing intact filter

paper (Fig. 1a). From this, it can also be concluded that the

loss in structural rigidity seen in some cultures is not due to

the influence of the culture medium, but rather the cellulo-

lytic activity of the various glucanases and b-glycosidases.

The degradation of the Whatman filter paper is illustrated in

Fig. 1a and b, indicating the presence of cellulolytic micro-

organisms. The association between the anaerobic micro-

organisms and the filter paper is illustrated in Fig. 1c and d.

Figure 1 illustrates the necessity of having cultures with

cellulolytic activities in the degradation of insoluble cellu-

lose-containing compounds.

16S rRNA gene analysis identified the isolate as Bacillus

megaterium (GenBank accession number DQ821937).

Although this species has been characterized as an aerobic

microorganism (Logan & Turnbull, 1999), it was able to

survive under anaerobic conditions. Bacillus megaterium has

the ability to hydrolyze a number of substrates such as

casein, gelatin and starch as nutrient sources (Logan &

Turnbull, 1999); the growth under anaerobic conditions

may therefore have been due to the presence of starch in

the culture media.

Purification of CMCase and Avicelase enzymes

Sepharose 4B chromatography yielded an elution profile

that revealed the presence of two peaks – peaks A (fractions

8–12) and B (fractions 18–27) (Fig. 2). The fractions that

constituted peaks A and B were assayed for CMCase and

Avicelase activity. As can be seen in Fig. 2, both CMCase and

Avicelase activities correlated with the protein elution pro-

file i.e. absorbance at 280 nm. CMCase was purified 2.89-

fold (Table 1a). The specific activity of CMCase was

0.078 mg glucose released min�1 mg�1 protein under the

assay conditions used. Avicelase was purified 8.37-fold

(Table 1b). Avicelase-specific activity was 0.209 mg glucose

released min�1 mg�1 protein. It appears, therefore, as if the

cellulosomal complex is higher in exoglucanase (Avicelase)

content in relation to the endoglucanase content, which was

Solubilized filter paper

Intact filter paper

Filter paper

Micro-organism

Solubilized region

(a)

(c) (d)

(b)

Fig. 1. Photographs taken of cellulolytic anae-

robe enrichment. (a) Degree of cellulolytic degra-

dation that occurred over a 5-week period in one

of the enrichment cultures in comparison with an

enrichment culture where no cellulolytic activity

was observed. (b) Different enrichment cultures

where cellulolytic degradation had occurred.

Scanning electron microscope photographs of

the filter paper used for the enrichment of

cellulolytic anaerobes. (c) and (d) Close associa-

tion between the anaerobes and the filter paper.


228 N. Beukes & B.I. Pletschke

unexpected, as the majority of cellulosomes that have been

studied to date are higher in endoglucanase content.

Sepharose 4B gel filtration chromatography resulted in an

overall yield of 12.9% and 37.67% for CMCase and Avice-

lase, respectively.

The results obtained with regard to the degradation of

both CMC and Avicel confirm the results previously ob-

tained by Kim & Kim (1995), in that the Bacillus cellulosome

has both CMCase and Avicelase activity; however, the

‘cellulases’ for this isolate had a higher activity with Avicels.

The more rapid rate of degradation of Avicels compared

with CMC indicates the possibility that the purified cellulo-

some isolated in this study was probably high in exogluca-

nase content.

Effect of sulfate and sulfide on CMCase andAvicelase activities

Cellulosomal ‘cellulase’ activity was enhanced with an

increase in sulfide concentration (Fig. 3) and reduced with

an increase in sulfate concentration (Fig. 4). The CMCase

and Avicelase enzyme assays were performed in order to

establish the effect of these two compounds on the cellulase-

associated hydrolysis of b-glycosidic bonds. In all cases, the

degradation of Avicels proceeded at a higher rate than that

with CMC (Fig. 3). There was a general enhancement of 1.4

fold and 1.5-fold for Avicels and CMC activity, respectively

(at a sulfide concentration of 12.47 mM). The trend seen in

the degradation of CMC and Avicels in the presence of

sulfate was similar for both substrates (see Fig. 4a and b);

however, the Avicels (as previously stated) was degraded at

a higher rate than that of CMC. The effect of sodium sulfate

on the degradation of both substrates was similar, in that

there was a general 1.5-fold decrease in the Avicelase activity,

Abs

orba

nce

at 2

80 n

m

A B

Fig. 2. Sepharose 4B gel exclusion chromatography of a cellulosome-

containing fraction using a 12.5� 1.7 cm column, flow rate-

1 mL min�1. The correlation between the cellulolytic activity (Avicelase

and CMCase) and protein content is shown.

Table 1. Purification tables for the purification of CMCase and Avicelase. (a) Purification table obtained for the optimized purification of the CMCase

activity for the purified Bacillus cellulosome. (b) Purification table obtained for the optimized purification of the Avicelase activity for the purified Bacillus

cellulosome

Volume (mL)

Total

protein (mg)

CMCase activity

(mg glucose

released min�1 mL�1)

Total CMCase

activity (mg glucose

released min�1)

Specific CMCase


released min�1 mg�1 protein) Yield (%)

Fold

purification

(a)Crude extract 46 173.88 0.102 4.69 0.027 100 1

Supernatant� 40 100.8 0.117 4.68 0.046 99.7 1.7

Sepharose 4B (peak B) 3 7.77 0.203 0.609 0.078 12.9 2.89

Volume

(mL)

Total

protein (mg)

Avicelase activity

(mg glucose

released min�1 mL�1)

Total avicelase


released min�1)

Specific avicelase


released min�1 mg�1 protein) Yield (%)

Fold

purification

(b)Crude extract 46 173.88 0.094 4.324 0.025 100 1

Supernatant� 40 100.8 0.084 3.4 0.034 78.63 1.36

Sepharose 4B (peak B) 3 7.77 0.542 1.629 0.209 37.67 8.37

�Supernatant, H2O wash of Avicel pellet.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 1.56 3.12 6.25 9.37 12.47Sulphide concentration (mM)

Fig. 3. Effect of sulfide on cellulosome-associated cellulase activity.

Values are presented as mean values� SD (n = 3).



and a general 1.4-fold decrease in the CMCase activity at a

sulfate concentration range of 12.47 mM. Ammonium sul-

fate also inhibited both enzyme activities (Fig. 4b).

In conclusion, then, sulfide enhanced the degradation of

both amorphous and crystalline substrates, and therefore

may be added to a variety of industrial applications to

enhance the rate of cellulose digestion. Sulfate had an

inhibitory affect of the degradation of amorphous and

crystalline cellulose; thus, a possible decreased rate of

hydrolysis may occur with biomass rich in sulfate. The

combined effect of both sulfide and sulfate may provide an

insight into the micro-environmental and respective com-

petitive/mutualistic associations that may occur between

various microorganisms involved in anaerobic digestion.

For example, this facultative B. megaterium may be able to

survive in a symbiotic relationship with a sulfate-reducing

organism. This form of coexistence is known to occur

between several organisms, where there is a mutualistic

relationship that involves the cycling of sulfur compounds

(Dubilier et al., 2001).

Acknowledgements

The authors would like to express their sincere gratitude to

Mrs A. Clarke, Ms M. Jiwaji and Dr B. Wilhelmi for their

technical assistance and to the National Research Founda-

tion (NRF) of South Africa and the Joint Research Commis-

sion (JRC) of Rhodes University for their financial support

during this study.

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Effect of sulfur-containing compounds on Bacillus cellulosome-associated ‘CMCase’ and ‘Avicelase’ activities