2007 AfrJBiotech
2007 AfrJBiotech
2007 AfrJBiotech
net/publication/224889611
CITATIONS READS
32 222
2 authors:
Some of the authors of this publication are also working on these related projects:
All content following this page was uploaded by Tarek A. A. Moussa on 15 May 2014.
The ability to produce cellulose degrading enzymes by sugarbeet pathogen Sclerotium rolfsii Sacc. in
liquid synthetic media with carboxymethy cellulose (CMC) as inducer was studied. Several cultural
conditions were examined to assess their effect in optimizing enzymes production. Shaking cultures
gave higher yields of cellulases compared with static ones. Aspargine supplement was the best
nitrogen source, especially at 3.0 g/l concentration, in promoting enzyme production. Variation of
cellulose/xylan ratio in the culture medium showed that cellulose and xylan induced both cellulases
synthesis but cellulose being the most effective specific substrate. The influence of different inhibitors
on enzymes production by S. rolfsii was also studied. Cyclohexmide and ethidium bromide inhibited
protein synthesis by S. rolfsii. Moreover, glucose repressed cellulase synthesis in S. rolfsii.
INTRODUCTION
mes (El-Abyad et al., 1996, 1997; Kurosawa et al., 1989; um was harvested and rinsed with sterile distilled water, and then
Lachke and Deshpande, 1988; Moussa, 1994). transferred to basal medium without nitrogen plus different com-
pounds: CMC, 120 mg/l + 10 g/l glucose; CMC, 120 mg/l + 0.2 mg/l
cycloheximide, CMC, 120 mg/l + 0.2 mg/l ethidium bromide; control
(glucose 10 g/l). The culture solids (mycelium and undegraded
MATERIALS AND METHODS cellulose) were separated from the culture fluids by filtration and
then centrifugation at 7000 rpm for 20 min. The culture filtrates
Micro-organism and culture conditions were dialyzed, freeze-dried and used as enzyme source.
S. rolfsii Sacc. was isolated from diseased sugarbeet roots (El-
Abyad et al., 1988) and maintained on a medium described by Enzyme assay
Johnson and Curl (1972) and composed of (g/l): dextrose, 30;
KH2PO4, 1; MgSO4.7H2O, 0.5; KCl, 0.5; KNO3, 2; agar, 20; and 1 Cellulase activity was determined at 40˚C by using carboxymethyl
ml/l of each of stock solutions (1 g/l) of FeSO4.7H2O, MnSO4.7H2O, cellulose (sodium salt, Sigma, USA) as a substrate, in 50 mM ace-
ZnSO4.7H2O and thiamine. tate buffer, pH 4.5. Reducing sugars released were assayed by the
Somogyi method (Somogyi, 1952) modified from Nelson procedure
(Nelson, 1944) with glucose as standard. One unit of enzyme
Effect of the state of culture on enzymes production activity was defined as the amount of enzyme required to liberate 1
µmol/min of reducing sugar expressed as glucose equivalents.
S. rolfsii was cultivated in 250 ml Erlenmeyer flasks with 100 ml -glucosidase was assayed at 40˚C using p-nitrophenyl- -D-
medium described by Haltrich et al. 1994 and containing the glucopyranoside as a substrate, in 50 mM acetate buffer, pH 4.5.
following (g/l): peptone, 80; NH4NO3, 2.5; MgSO4.7H2O, 1.5; One unit of enzyme activity was defined as the amount of enzyme
KH2PO4, 1.2; KCl, 0.6 and trace element solution at 0.3 ml/l. The required to liberate 1 µmol of p-nitrophenol /min.
concentration of carboxymethyl cellulose (CMC) was 200 mg/l for
production of cellulases. The flasks were inoculated with 5 mm plug
cut out from the margin of a 5 day-old culture. Incubation was Protein estimation
carried out at 25 ± 2˚C under static and shaking at 100 rpm for 11
days. The culture filtrates were dialyzed against distilled water over The protein was measured in the culture supernatant, and estima-
night at 4°C and freeze-dried. The concentrated filtrates were used ted by the Bradford method (Bradford, 1976) with bovine serum
as enzyme source. albumin as the standard.
0.9 10
[A]
Cellulase Glucosidase 9
0.8
Glucosidase activity (U ml x 10 )
-2
8
Cellulase activity (U ml )
0.7
-1
7
-1
0.6
6
0.5
5
0.4
4
0.3
3
0.2 2
0.1 1
0 0
4 5 6 7 8 9 10 11
Incubation time (days)
4.5 80
[B]
Cellulase Gulcosidase
4.0 70
-2
3.5
-1
60
3.0
-1
50
2.5
40
2.0
30
1.5
1.0 20
0.5 10
0.0 0
4 5 6 7 8 9 10 11
Incubation time (days)
1.4 [A] 70
[B]
Glucosidase activity (U ml x 10 )
-2
1.2 60
Cellulase activity (U ml )
-1
-1
1.0 50
0.8 40
0.6 30
0.4 20
0.2
10
0.0
0
Pot. Nitrate Amm. Asparagine
Pot. Nitrate Amm. Asparagine
Sulphate
Sulphate
Nitrogen source
Nitrogen source
1.4 70
[C]
1.2 60
Glucosidase activity (U ml-
Cellulase activity (U ml-1)
1.0 50
1 x 10-2)
0.8 40
0.6 30
0.4 20
0.0 0
0 1 2 3 4 5 6 7
Asparagine concentration (g/l)
Figure 2. Effect of different nitrogen sources and its concentrations on the production of cellulose
degrading enzymes by S. rolfsii. [A and B] different nitrogen sources, [C] asparagine concentrations.
Moussa and Tharwat 1051
2.5 120
[B]
[A]
0.5 20
0.0 0
C5 C3X2 C2X3 X5 C5 C3X2 C2X3 X5
Cellulose/Xylan ratio (mg/100 ml) Cellulose/Xylan ratio (mg/100 ml)
Figure 3. Effect of cellulose/xylan ratio (mg/100 ml) on the induction of cellulose degrading
enzymes by S. rolfsii. [A] cellulase, [B] -glucosidase.
control
C3X2 + glucose
Glucosidase activity (U/µg mycelial
0.6
20
0.5
0.4 15
0.3 10
0.2 5
0.1 0
0 0 1 2 3 4 5 6
0 1 2 3 4 5 6 Time (days)
Time (days)
Figure 4. Induction and repression of cellulose degrading enzymes synthesis in S. rolfsii. [A] cellulase, [B]
-glucosidase.
The data represented in Figure 3 showed that, by The inducible synthesis of the cellulase system of S.
varying the relative concentration of cellulose and xylan rolfsii was determined by adding cyclohexamide or ethi-
in the culture medium, both were able to induce and syn- dium bromide to induction media to inhibit protein synthe-
thesis of cellulose degrading enzymes. The maximum sis, although there was no growth there were very low
production of cellulose degrading enzymes was when level of enzymes produced (Figure 4). It may be conclu-
cultivated S. rolfsii on pure cellulose and decreased with ded that in S. rolfsii cellulase syntheses are repressed by
decreased cellulose concentration (Figures 3A and B). easily metabolized sugars such as glucose. It is widely
1052 Afr. J. Biotechnol.
accepted for filamentous fungi that cellulase and -glu- ner et al., 1998). There are suggestions of an interaction
cosidase production are regulated by induction and between xylanase and cellulase induction (Royer and
repression (Figures 4A and B). Nakas, 1990), although the xylanolytic and cellulolytic
systems in some filamentous fungi are likely to be under
separate regulatory control (Bajpai, 1997; Hrmovà et al.,
DISCUSSION 1991; Kulkarni et al., 1999). In S. rolfsii there was a high
Since the polymeric substrates are unable to enter the cross induction of cellulolytic and xylanolytic enzymes, in
cells by crossing the plasma membrane, the cells receive Aspergillus terreus it was mainly induced by the res-
the signal for an accelerated synthesis of secreted glyca- pective synthetic dimmers (Hrmovà et al., 1991). This un-
nases by means of low-molecular weight fragments, usu- specific effect of cellulose could be attributed to xylan
ally disaccharides, derived from the polysaccharides. The impurities found in commercially available cellulose pre-
fragments are formed by the action of small amounts of parations (Hrmovà et al., 1986; Senior et al., 1989).
the enzymes produced constitutively (Biely, 1993; Bajpai, Xylanases are generally produced together with cellula-
1997). Thus, for example, cellobiose is an inducer of ses during growth of the fungus on macromolecular subs-
cellulose-degrading enzymes (Canevascini et al., 1979; trate derived from plant polysaccharides, which inevitably
Eberhart et al., 1977; Eriksson and Hamp, 1978; Mandels always contain cellulose and xylan. The resulting xylana-
and Reese, 1957), and xylobiose is an inducer of xylan- se to cellulase ratio has been shown to be directly pro-
degrading enzymes (Biely et al., 1980; Nakanishi et al., portional to the xylan/cellulose ratio in the growth subs-
1976). trate (Senior et al., 1989). These data seem consistent
Purkarthofer et al. (1993) stated that a shaking speed with results from induction studies, which showed that
of 120 rpm provided the optimal conditions for enzyme xylanase and cellulase biosynthesis in Trichoderma ree-
formation. At a decreased shaking speed of 100 rpm, the sei is differentially regulated (Hrmovà et al., 1986). In
fungus showed poor growth, and enzyme production was contrast, the efficient xylanase induction in T. longibra-
reduced dramatically, at higher shaking speeds of 150 - chiatum required the simultaneous presence of xylo- as
250 rpm enzyme production was adversely affected. The well as cello-oligosaccharides (Royer and Nakas, 1990).
lower xylanase activity produced at the slower shaking A generally accepted view on the regulation of synthe-
speed was ascribed to poor oxygen transfer within the sis of enzymes degrading polymeric substrates is that low
medium, whereas the lower xylanase production at hig- constitutive levels of polysaccharide hydrolases interact
her shaking speeds was thought to be due to greater with the polymer and produce small soluble ‘signal’ frag-
hyphal branching, mycelial fragmentation and early spo- ments, which enter the cell and induce the synthesis of
rulation (Purkarthofer et al., 1993). Shear stress within the corresponding enzyme, thus permitting utilization of
the medium, which is directly related to the stirrer speed, polysaccharide.
has a marked influence on xylanase production by Ther- Studies using inhibitors of protein synthesis have sug-
momyces lanuginosus SSBP (Reddy et al., 2002; Singh gested that cellulase formation is regulated at the transla-
et al., 2000). tional level (Nisizawa et al., 1972). Evidence based on
The nitrogen source used in the production medium is the measurement of mRNA levels documented that the
one of the major factors affecting enzyme production and formation of cellulase occurs at the pre-translational level
level. In a study carried out with Trichoderma harzianum, (Kolbe and Kubicek, 1990; Messner et al., 1991) and the
NaNO3 and peptone were the best nitrogen sources in cellulase gene transcription occurs within 20 min, after
production medium (Abdel-Satar and El-Said, 2001), the addition of inducer (El-Gogary et al., 1989).
whilst NH4NO3 was used in a study with Schizophyllum The active growth of the fungus is crucial in cellulolysis.
commune, and (NH4)2HPO4 was found suitable in another When growth was inhibited, cellulolysis remained weak,
study with T. lanuginosus RT9 (Haltrich et al., 1993; Hoq although cellulase enzymes were present in culture broth.
et al., 1994). The effect of various organic nitrogen com- Vaheri, (1983) proposed the participation of an oxidative
pounds on the production of xylanase by T. lanuginosus reaction which is believed to disrupt the hydrogen bonds
strains showed that all sources promoted growth of the in crystalline cellulose, rendering it susceptible to attack
fungus, but yeast extract had the most pronounced effect by endoglucanase. He found that this activity was asso-
(Singh et al., 2003). ciated with cell wall in young cells of T. reesei in both
The production of both cellulases in media with xylan or induced and non-induced conditions. Thus, activities
cellulose as sole carbon source may be due to substrates associated with growing cells appear to play a crucial role
contamination or substrate cross-specificity that can ran- in the degradation of crystalline cellulose.
ge from absolute for one polymer to about the same affi- Carbon catabolite repression is another regulatory
nity for both of them (Ferreria-Filho, 1994; Hrmovà et al., mechanism known to control cellulase production in bac-
1986; Senior et al., 1989). Nevertheless, concurrent for- teria and fungi. In this case, the end product of cellulose
mation of cellulase and xylanase has been observed in hydrolysis interacts with a cellular protein and form a
several fungi using natural and synthetic substrates complex which interacts with a particular gene at the tran-
(Hrmovà et al., 1991; Royer and Nakas, 1990; Sachsleh- scription level and represses cellulase synthesis (Lewin
Moussa and Tharwat 1053
and Genes, 1987). The carbon catabolite repres-sion was herbicide pyradur on host cell wall degradation by the sugarbeet
pathogens Rhizoctona solani and Sclerotium rolfsii. Can. J. Bot. 74:
reported in Escherichia coli (Pastan and Adhya, 1976),
1407-1415.
Saccharomyces cerevisiae (Entian et al., 1985) and Clos- El-Abyad MS, Abu-Taleb AM, Abdel-Mawgoud T (1997). Response of
tridium thermocellum (Johnson et al., 1985). However, host cultivar to cell wall-degrading enzymes of the sugarbeet
Canevascini et al. (1979) reported that the cellulase syn- pathogens Rhizoctonia solani Kühn and Sclerotium rolfsii Sacc.
under salinity stress. Microbiol. Res. 152: 9-17.
thesis is regulated by both induction and catabolite repre-
El-Abyad MS, Hindorf H, Rizk MA (1988). Impact of salinity stress on
ssion in Sporotrichum thermophile. soil-borne fungi of sugarbeet. I. Pathogenicity implications. Plant Soil
The proof for carbon catabolite repression is based on 110: 27-32.
the fact that no cellulase is formed during the growth of a Entian KD, Hilbnerg F, Opitz H, Mecke D (1985). Cloning of hexokinase
structural genes from Saccharomyces cerevisiae mutants with
microorganism on glucose, glycerol and other carbon regulatory mutations responsible for glucose repression. Mol. Cell.
sources related to glycolytic metabolism. Because there Biol. 5: 3035-3040.
is no clear evidence that either glucose or a catabolite in Eriksson KE, Hamp SG (1978). Regulation of endo-1,4- -glucanase
fact controls the transcription of cellulase genes, Kubicek production in Sporotrichum pulverulentum. Eur. J. Biochem. 90: 183-
190.
recommended not to use the term "catabolite repression"
Ferreria-Filho EX (1994). The xylan degrading system. Brasilian J. Med.
(Canevascini et al., 1979). The involvement of end pro- Biol. Res. 27: 1093-1109.
duct inhibition during crystalline cellulose hydrolysis by a Haltrich D, Laussamayer B, Steiner W (1994). Xylanase formation by
rumen fungus Neocallimastix frontalis RK21 was demon- Sclerotium rolfsii: Effect of growth substrates and development of a
culture medium using statistically designed experiments. Appl.
strated (Kubicek, 1992).
Microbiol. Biotechnol. 42: 522-530.
Haltrich D, Preiss M, Steiner W (1993). Optimization of a culture
medium for increased xylanase production by a wild strain of
ACKNOWLEDGMENT Schizophyllum commune. Enz. Microb. Technol. 15: 854-860.
Hoq MM, Hempel C, Deckwer WD (1994). Cellulase-free xylanase by
Thermomyces lanuginosus RT9: Effect of agitation, aeration, and
The authors thank Prof. Dr. M. Kassas, Emeritus Profes-
medium components on production. J. Biotechnol. 37: 49-58.
sor of Botany at this department for reading the manu- Hrmová M, Biely P, Vrsanská M (1986). Specificity of cellulase and -
script. xylanase induction in Trichoderma reesei QM 9414. Arch Microbiol.
144: 307-311.
Hrmova N, Petrakova E, Biely P (1991). Induction of cellulose and xylan
degrading enzymes in Aspergillus terreus by homo and hetero-
REFERENCES
disaccharides composed of glucose and xylose. J. Gen. Microbiol.
137: 541-547.
Abdel-Satar MAM, El-Said AH (2001). Xylan-decomposing fungi and
Johnson EA, Bouehof F, Demain AL (1985). Regulation of cellulase
xylanolytic activity in agricultural and industrial wastes. Int. Biodet.
formation in Clostridium thermocellum. J. Gen. Microbiol. 131: 2303-
Biodeg. 47: 15-21.
2308.
Bajpai P (1997). Microbial xylanolytic enzyme system: properties and
Johnson LF, Curl EA (1972). Methods for research on the ecology of
applications. Adv. Appl. Microbiol. 43: 141-194.
soil-borne plant pathogens. USA: Burgess Publ Co, p. 112.
Beguin P, Anbert JP (1993). The biological degradation of cellulose.
Kolbe J, Kubicek CP (1990) Quantification and identification of the main
FEMS Microbiol. Rev. 13: 25-58.
components of the Trichoderma cellulase complex with monodonal
Bhat MK, Bhat S (1997). Cellulose degrading enzymes and their
antibodies using an enzyme-linked immunosorbent assay (ELISA).
potential industrial applications. Biotechnol. Adv. 15: 583–620.
Appl. Microbiol. Biotechnol. 34: 26-30.
Biely P (1993). Biochemical aspects of the production of microbial
Kubicek CP (1992). The cellulase proteins of T. reesei: Structure,
hemicellulases In: Coughlan MP, Hazlewood GP, eds. Hemicellulose
multiplicity, mode of action and regulation of formation. Adv.
and hemicellulases, Portland Press, London. England pp. 29-51.
Biochem. Eng. 45: 1-27.
Biely P, Krátký Z, Vršanská M, Urmani ová D (1980). Induction and
Kulkarni N, Shendye A, Rao M (1999). Molecular and biotechnological
inducers of endo-1,4- -xylanase in the yeast Cryptococcus albidus.
aspects of xylanases. FEMS Microbiol. Rev. 23: 411-456.
Eur. J. Biochem. 108: 323-329.
Kurosawa K, Hosoguchi M, Hariantono J, Sasaki H, Takao S (1989).
Bradford MM (1976). A rapid and sensitive method for the quantitation
Degradation of tough materials by cellulase from Corticium rolfsii.
of microgram quantities of protein utilizing the principle of protein-dye
Agric. Boil. Chem. 53: 931-937.
binding. Anal. Biochem. 72: 248-254. SPSS, Inc. SPSS for windows
Lachke AH, Deshpande MV (1988). Sclerotium rolfsii: status in cellulase
release 10.0.1. 1999.
research. FEMS Microbiol. Rev. 54: 177-194.
Canevascini G, Coudray MR, Rey JP, Southgate RJG (1979), Meier H.
Lewin B (1987). Genes III. An up-to-date Treatment of the Molecular
Induction and catabolic repression of cellulase synthesis in the
Genetics of both Eukaryotes and Prokaryotes, along with Related
thermophilic fungus Sporotrichum thermophile. J. Gen. Microbiol.
Cell and Molecular Biology. John Wiley, New York.
110: 291-303.
Ludwig R, Haltrich D (2002). Cellobiose dehydrogenase production by
Canevascini G, Coudray MR, Rey JP, Southgate RJG, Maier H (1979).
Sclerotium species pathogenic to plants. Lett. Appl. Microbiol. 35:
Induction and catabolic repression of cellulase synthesis in the
261-266.
thermophilic fungus Sporotrichum thermophile. J. Gen. Microbiol.
Mandels M (1985). Applications of cellulases. Biochem. Soc. Trans. 13:
110: 291-303.
414-415.
Coughlan MP (1985). Cellulases: Production properties and appli-
Mandels M, Reese ET (1957). Induction of cellulase in Trichoderma
cations. Biochem. Soc. Trans. 13: 405-406.
viride as influenced by carbon sources and metals. J. Bacteriol. 73:
Eberhart BM, Beck RS, Goolsby KM (1977). Cellulase of Neurospora
269-278.
crassa. J. Bacteriol. 130: 181-186.
Messner R, Kubicek-Pranz EM, Gsur A, Kubicek CP (1991).
Ei-Gogary S, Leite A, Crivellaro O, Eveleigh DE, Ei-Dorry H (1989).
Cellobiohydrolase II is the main conidial bound cellulase in
Mechanism by which cellulose triggers cellobiohydrolase I gene
Trichoderma reesei and other Trichoderma strains. Arch Microbiol.
expression in Trichoderma reesei. Proc. Natl. Acad. Sci. USA. 86:
155: 601-605.
6138-6141.
Moussa TAA (1994). Preliminary studies on the production of
El-Abyad MS, Abu-Taleb AM, Abdel-Mawgoud T (1996). Effect of the
pathogenicity enzymes by some sugarbeet pathogenic fungi under
1054 Afr. J. Biotechnol.
the stresses of salinity and herbicidal treatment. M.Sc. Thesis, Dept. Singh S, Madlala AM, Prior BA (2003). Thermomyces lanuginosus:
Bot. Fac. Sci. Univ. Cairo, pp. 1-132. properties of strains and their hemicellulases. FEMS Microbiol. Rev.
Nakanishi K, Yasui T, Kobayashi T. (1976). Inducers for xylanase 27: 3-16.
production by Streptomyces sp. J. Ferment. Technol. 54: 801-807. Singh S, Pillay B, Dilsook V, Prior BA (2000). Production and properties
Nelson N (1944). A photometric adaptation of the Somogyi method for of hemicellulases by Thermomyces lanuginosus strain. J. Appl.
the determination of glucose. J. Biochem. 153: 375-380. Microbiol. 88: 975-982.
Nisizawa T, Suzuki H, Nisizawa K (1972). Catabolite repression of Somogyi M (1952). Notes on sugar determination. J. Biol. Chem. 195:
cellulase formation in Trichoderma viride. J. Biochem. (Tokyo) 71: 19-23.
999-1007. Vaheri MP (1983). Formation of oxidative activity for the initial
Pastan I, Adhya S (1976). Cyclic Adenosine 5' monophosphate in degradation of crystalline cellulose by Trichoderma reesei. J. Appl.
Escherichia coil. Bacteriol. Rev. 40: 527-551. Biochem. 5: 66-74.
Purkarthofer H, Sinner M, Steiner W (1993). Effect of shear rate and
culture pH on the production of xylanase by Thermomyces
lanuginosus. Biotechnol. Lett. 15: 405-410.
Reddy V, Reddy P, Pillay B, Singh S (2002). The effect of aeration on
the production of hemicellulases by Thermomyces lanuginosus SSBP
in a 30 l bioreactor. Process Biochem. 37: 1221-1228.
Royer JC, Nakas JP (1990). Interrelationship of xylanase induction and
cellulase induction of Trichoderma longibrachiatum. Appl. Environ.
Microbiol. 56: 2535-2539.
Sachslehner A, Haltrich D, Nidetzky B, Kulbe KD (1997). Production of
hemicellulose- and cellulose-degrading enzymes by various strains of
Sclerotium rolfsii. Appl. Biochem. Biotechnol. 63(65): 189-201.
Sachslehner A, Nidetzky B, Kulbe KD, Haltrich D (1998). Induction of
mannanase, xylanase and endoglucanase activities in Sclerotium
rolfsii. Appl. Environ. Microbiol. 64: 594-600.
Senior DJ, Mayers PR, Saddler JN (1989). Xylanase production by
Trichoderma harzianum E58. Appl. Microbiol. Biotechnol. 32: 137-
142.