World Journel
of Microbio/ogy
& Biotechnology
11, 656-660
Constitutive production of endoglucanase a Bacillus sp. isolated from a Zimbabwean hot spring
by
R. Zvauya* and C.J. Zvidzai A sporulating, aerobic Bucillus sp., isolated from Chimanimani hot springs, Zimbabwe, produced endoglucanase when cultured on medium with initial pH between 5.0 and 9.0 and at 30 to 60°C. Optimal production of endoglucanase was at pH 6.0. The enzyme was constitutively produced when the organism was cultured on starch, cellobiose, carboxymethylcellulose, sucrose, glucose, galadose, Avicel, lactose, mannose or maltose. Key words: Bacillus, constitutive,
endoglucanase.
Most research on cellulase-producing microorganisms has involved fungi (Harmova et al. 1986; Malek et al. 1988; Ali & Akhand 1992). Over the past 10 years, however, effort has been directed towards studying bacterial cellulases (Mullings & Parish 1984; Au & Chan 1986; Gilkes et al. 1991). Much of the research work on Bucilltrs cellulases has been concerned with expression of the coding gene in other, non-cellulolytic hosts (Ghanghas & Wilson 1987; Gilkes et al. 1991). As very little information is available on the physiology and biochemistry of Bacillus cellulases (Au & Chan 1986), these are currently under investigation in our laboratory. The present study was on the various factors affecting endoglucanase production by a Bacillus strain isolated from a hot spring in Chimanimani, Zimbabwe.
Materials
was deposited with the German (DSM) at Braunschweig.
Collection
of Microorganisms
Culfivafian Growth and enzyme production was studied by inoculating a IZh preculture (10 ml) into a 500-m] shake-flask with 190 ml Ml62 medium (Degryse et a/. 1978) containing (per I): yeast extract (Difco), 2.5 g; tryptone (Difco), 2.5 g; carbon source, 5 g; mineral salts, 100 ml; and phosphate buffer, 100 ml. Biomass was followed as A,,,. Similar shake-flask cultures containing Ml62 medium with different initial pH values were also incubated at 40°C. The effect of temperature was determined using Ml62 medium with initial pH 6.0 (0.05 M citrate/phosphate buffer). Enzyme
Assay
Endoglucanase activity was determined as described by Ghose (1987), using hydroxyethyl-cellulose (HEC; medium viscosity, 54290; Fluka) as substrate. The enzyme was assayed with the substrate in citric/phosphate buffer, pH 6.0, at 70°C.
and Methods
Microorganism The microorganism
used in this study was isolated from an
alkaline hot spring (pH 8.5 and 45°C) in Chimanimani, Zimbabwe, using medium with carboxymethylcellulose (CM-cellulose) as the sole carbon source (Waldron & Eveleigh 1986). Some physiological and biochemical identification tests were carried out on the microorganism, either according to Cowan & Steel (1993) or using the API-50 CHB identification kit (BioMeriaux). The strain
The authors are with the Fermentation and Food Group. Department of Biochemistry, University of Zimbabwe, Box MP 167, Mount Pleasant, Harare, Zimbabwe; fax: 263 4 333407. ‘Corresponding author.
@I 1995 Rapid Science
Publishers
Results
and Discussion
Identification of Microorganism The bacterial species is a Gram-positive rod. The microorganism forms terminal spores and is a motile, obligate aerobe. Results of the API-50 CHB identification tests and other biochemical tests are shown in Table 1. Using the identification keys from Cowan & Steel (1993) the microbe was classified as a Bacillus sp., closely related to B. subfilis. Further identification work is underway at DSM.
Consfiftrfive endoglucanase production by Bacillus Table 1. Some of the biochemical Identification results Bacillus sp. from Chlmanimanl hot springs, Zimbabwe.
for the
140 120
Result
Test Motility Catalase Oxidase Carbohydrate utilization Glucose Mannitol Sorbitol Rhamnose Sucrose Melibiose Amygdalin Arabinose Fructose lnositol Starch hydrolysis PGalactosidase production Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Citrate utilisation W Urease activity Tryptophan deaminase Indole production Acetoin production Kohn’s gelatin (gelatinase NO, production from NO, NO, reduction to N, Gram reaction
100
+ + +
80 60 40
+ + + + + + + + + + + + + + +
activity)
20 0 0
5
10
15
20
25
Time (h) Flgure 2. Endoglucanase production by the Bacillus species an initial pH of 6 (0.05 M citrate/phosphate buffer) and 30 (A), (A), 40 (W, 45 (m), 50 (0) and 55°C (0). Table 2. Effect of dlfferent production by the Bacillus Carbon source
Time
taken
carbon sources on endoglucenase sp. cultivated at 40°C and pH 6. A N.0
(h)
Mannose Sucrose Starch CM-cellulose Maltose Cellobiose Glucose Galactose
22 27 27 20 12 23 23 15
at 37
2.07 2.95 2.65 3.74 3.30 4.27 3.44 5.18
Maximum endoglucanase activity (nkat ml-‘) 123 129 108 125 123 123 97 86
pH 6 (Figure I). Optimum biomass levels were obtained on medium with an initial pH of 8. Endoglucanase was produced optimally at 50°C although high activities were obtained at 40 to 50°C. No filter-paper or cellobiase activity was detected. 60
Effect of Carbon
0
5
10
15
20
25
Time (h) Figure 1. Endoglucanase production by Bacillus species at a cultivation temperature of 45% and an initial pH of 5 (A), 6 (0) 7 (0) 8 (m). 9 (A) and 10 (0). Buffers used were 0.05~ acetate (pH 4) citrate/phosphate (pH 5 to 8) phosphate (pH 7 to 8) and Tris/HCI (pH 9 to 10).
Effect of Initial
Acknowledgements
pH and Temperature
The optimum temperature for growth was 40°C was no growth at 55°C. The Bacillm strain endoglucanase when cultured on medium with values between 5 and 9, with highest enzyme
Source
Endoglucanase was produced constitutively when the isolate was cultured on various carbon sources, as shown in Table 2. Mannose, maltose, starch and CM-cellulose gave high enzyme activity. Low endoglucanase activities were observed with galactose and glucose. Dhillon et al. (1985) and Au & Chan (1986) isolated Bacillus species that do not produce the complete cellulase system and which also produce the enzyme constitutively. The present Bacillus strain produced endoglucanase constitutively and enzyme production was maximal in a semisynthetic medium of initial pH 6.0.
and there produced initial pH activity at
The authors wish to thank the Swedish Agency for Research Co-operation with Developing Countries (SAREC) and The University of Zimbabwe Research Board for funding this
World Journal of Microbiology 6 Biotechnology, Vol 11. 1995
659
R. Zvauya and C.J. Zvidzai research. CJZ thanks the University of Zimbabwe for providing a Teaching Assistantship during the course of this work.
References Ali, S.M. & Akhand, A.A. 1992 Cellulase ]ournd
of Basic Microbiology Au, K.S. & Chan, K-Y. 1986 by Bacillus subUs. Microbios
from
Trichoderma
isolate.
32, 25%268.
Carboxymethycellulase
production
48, 93-108.
Cowan, T.S. & Steel, J.K. 1993 Characters of gram-positive bacteria. In Manual for the Iden@ution of Medical Bacteria, eds Barrow, G.I. & Feltham, R.K.A. pp. 50-93. Cambridge: Cambridge University Press. Degryse, E., Glansdorff, N. & Pierard, A. 1978 A comparative analysis of extreme fhermophilic bacteria belonging to the genus ‘I%ermus. Archives of Microbiology 117, 189-196. Dhillon, N., Chhibber, S., Saxena, M., Pajni, S. & Vadehra, D.V. 1985 A constitutive endoglucanase (CMCase) from Bacillus licheniforrnis-1. Biotechnology Letters 7, 695-697.
Ghanghas, S.G. & Wilson, D.B. 1987 Expression of a Thermomonosporu fuscu cellulase gene in Sfrepfomyces &duns and Bacillus subtilis. Applied and Environmental Microbiology 53, 1470-1475. Ghose, T.K. 1987 Measurement of cellulase activities. fire and Applied Chemistry 59, 257-268. Gilkes, N.R., Kilbum, D.G., Miller, R.C. & Warren, R.A.J. 1991 Bacterial cellulases. Bioresotlrce Technology 36, 21-35. Harmova, M., Biely, P. & Vrasnska, M. I986 Specificity of cellulase and p-xylanase induction in Trichodem reesei QM 9414. Archives of Microbiology 144, 307-311. Malek, M.A., Chowdhury, N.A., Youssouf, Q.M. & Choudhury, N. 1988 Bacterial cellulases and saccharification of lignocellulosic materials. Enzyme and Microbial Technology 10, 750-753. Mullings, R. & Parish, J.H. 1984 Mesophilic aerobic Gram negative cellulose degrading bacteria from aquatic habitats and soils. ]ournul of Applied Bacteriology 57, 455-468. Waldron, G. & Eveleigh, D.E. 1986 Saccharification of cellulosics by Microbiospora bispora. Applied Microbiology and Biotechnology
4,487~492. (Received 19 May 1995; revised and accepted 10 ]uly 1995)
of Microbio/ogy
& Biotechnology
11, 656-660
Constitutive production of endoglucanase a Bacillus sp. isolated from a Zimbabwean hot spring
by
R. Zvauya* and C.J. Zvidzai A sporulating, aerobic Bucillus sp., isolated from Chimanimani hot springs, Zimbabwe, produced endoglucanase when cultured on medium with initial pH between 5.0 and 9.0 and at 30 to 60°C. Optimal production of endoglucanase was at pH 6.0. The enzyme was constitutively produced when the organism was cultured on starch, cellobiose, carboxymethylcellulose, sucrose, glucose, galadose, Avicel, lactose, mannose or maltose. Key words: Bacillus, constitutive,
endoglucanase.
Most research on cellulase-producing microorganisms has involved fungi (Harmova et al. 1986; Malek et al. 1988; Ali & Akhand 1992). Over the past 10 years, however, effort has been directed towards studying bacterial cellulases (Mullings & Parish 1984; Au & Chan 1986; Gilkes et al. 1991). Much of the research work on Bucilltrs cellulases has been concerned with expression of the coding gene in other, non-cellulolytic hosts (Ghanghas & Wilson 1987; Gilkes et al. 1991). As very little information is available on the physiology and biochemistry of Bacillus cellulases (Au & Chan 1986), these are currently under investigation in our laboratory. The present study was on the various factors affecting endoglucanase production by a Bacillus strain isolated from a hot spring in Chimanimani, Zimbabwe.
Materials
was deposited with the German (DSM) at Braunschweig.
Collection
of Microorganisms
Culfivafian Growth and enzyme production was studied by inoculating a IZh preculture (10 ml) into a 500-m] shake-flask with 190 ml Ml62 medium (Degryse et a/. 1978) containing (per I): yeast extract (Difco), 2.5 g; tryptone (Difco), 2.5 g; carbon source, 5 g; mineral salts, 100 ml; and phosphate buffer, 100 ml. Biomass was followed as A,,,. Similar shake-flask cultures containing Ml62 medium with different initial pH values were also incubated at 40°C. The effect of temperature was determined using Ml62 medium with initial pH 6.0 (0.05 M citrate/phosphate buffer). Enzyme
Assay
Endoglucanase activity was determined as described by Ghose (1987), using hydroxyethyl-cellulose (HEC; medium viscosity, 54290; Fluka) as substrate. The enzyme was assayed with the substrate in citric/phosphate buffer, pH 6.0, at 70°C.
and Methods
Microorganism The microorganism
used in this study was isolated from an
alkaline hot spring (pH 8.5 and 45°C) in Chimanimani, Zimbabwe, using medium with carboxymethylcellulose (CM-cellulose) as the sole carbon source (Waldron & Eveleigh 1986). Some physiological and biochemical identification tests were carried out on the microorganism, either according to Cowan & Steel (1993) or using the API-50 CHB identification kit (BioMeriaux). The strain
The authors are with the Fermentation and Food Group. Department of Biochemistry, University of Zimbabwe, Box MP 167, Mount Pleasant, Harare, Zimbabwe; fax: 263 4 333407. ‘Corresponding author.
@I 1995 Rapid Science
Publishers
Results
and Discussion
Identification of Microorganism The bacterial species is a Gram-positive rod. The microorganism forms terminal spores and is a motile, obligate aerobe. Results of the API-50 CHB identification tests and other biochemical tests are shown in Table 1. Using the identification keys from Cowan & Steel (1993) the microbe was classified as a Bacillus sp., closely related to B. subfilis. Further identification work is underway at DSM.
Consfiftrfive endoglucanase production by Bacillus Table 1. Some of the biochemical Identification results Bacillus sp. from Chlmanimanl hot springs, Zimbabwe.
for the
140 120
Result
Test Motility Catalase Oxidase Carbohydrate utilization Glucose Mannitol Sorbitol Rhamnose Sucrose Melibiose Amygdalin Arabinose Fructose lnositol Starch hydrolysis PGalactosidase production Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Citrate utilisation W Urease activity Tryptophan deaminase Indole production Acetoin production Kohn’s gelatin (gelatinase NO, production from NO, NO, reduction to N, Gram reaction
100
+ + +
80 60 40
+ + + + + + + + + + + + + + +
activity)
20 0 0
5
10
15
20
25
Time (h) Flgure 2. Endoglucanase production by the Bacillus species an initial pH of 6 (0.05 M citrate/phosphate buffer) and 30 (A), (A), 40 (W, 45 (m), 50 (0) and 55°C (0). Table 2. Effect of dlfferent production by the Bacillus Carbon source
Time
taken
carbon sources on endoglucenase sp. cultivated at 40°C and pH 6. A N.0
(h)
Mannose Sucrose Starch CM-cellulose Maltose Cellobiose Glucose Galactose
22 27 27 20 12 23 23 15
at 37
2.07 2.95 2.65 3.74 3.30 4.27 3.44 5.18
Maximum endoglucanase activity (nkat ml-‘) 123 129 108 125 123 123 97 86
pH 6 (Figure I). Optimum biomass levels were obtained on medium with an initial pH of 8. Endoglucanase was produced optimally at 50°C although high activities were obtained at 40 to 50°C. No filter-paper or cellobiase activity was detected. 60
Effect of Carbon
0
5
10
15
20
25
Time (h) Figure 1. Endoglucanase production by Bacillus species at a cultivation temperature of 45% and an initial pH of 5 (A), 6 (0) 7 (0) 8 (m). 9 (A) and 10 (0). Buffers used were 0.05~ acetate (pH 4) citrate/phosphate (pH 5 to 8) phosphate (pH 7 to 8) and Tris/HCI (pH 9 to 10).
Effect of Initial
Acknowledgements
pH and Temperature
The optimum temperature for growth was 40°C was no growth at 55°C. The Bacillm strain endoglucanase when cultured on medium with values between 5 and 9, with highest enzyme
Source
Endoglucanase was produced constitutively when the isolate was cultured on various carbon sources, as shown in Table 2. Mannose, maltose, starch and CM-cellulose gave high enzyme activity. Low endoglucanase activities were observed with galactose and glucose. Dhillon et al. (1985) and Au & Chan (1986) isolated Bacillus species that do not produce the complete cellulase system and which also produce the enzyme constitutively. The present Bacillus strain produced endoglucanase constitutively and enzyme production was maximal in a semisynthetic medium of initial pH 6.0.
and there produced initial pH activity at
The authors wish to thank the Swedish Agency for Research Co-operation with Developing Countries (SAREC) and The University of Zimbabwe Research Board for funding this
World Journal of Microbiology 6 Biotechnology, Vol 11. 1995
659
R. Zvauya and C.J. Zvidzai research. CJZ thanks the University of Zimbabwe for providing a Teaching Assistantship during the course of this work.
References Ali, S.M. & Akhand, A.A. 1992 Cellulase ]ournd
of Basic Microbiology Au, K.S. & Chan, K-Y. 1986 by Bacillus subUs. Microbios
from
Trichoderma
isolate.
32, 25%268.
Carboxymethycellulase
production
48, 93-108.
Cowan, T.S. & Steel, J.K. 1993 Characters of gram-positive bacteria. In Manual for the Iden@ution of Medical Bacteria, eds Barrow, G.I. & Feltham, R.K.A. pp. 50-93. Cambridge: Cambridge University Press. Degryse, E., Glansdorff, N. & Pierard, A. 1978 A comparative analysis of extreme fhermophilic bacteria belonging to the genus ‘I%ermus. Archives of Microbiology 117, 189-196. Dhillon, N., Chhibber, S., Saxena, M., Pajni, S. & Vadehra, D.V. 1985 A constitutive endoglucanase (CMCase) from Bacillus licheniforrnis-1. Biotechnology Letters 7, 695-697.
Ghanghas, S.G. & Wilson, D.B. 1987 Expression of a Thermomonosporu fuscu cellulase gene in Sfrepfomyces &duns and Bacillus subtilis. Applied and Environmental Microbiology 53, 1470-1475. Ghose, T.K. 1987 Measurement of cellulase activities. fire and Applied Chemistry 59, 257-268. Gilkes, N.R., Kilbum, D.G., Miller, R.C. & Warren, R.A.J. 1991 Bacterial cellulases. Bioresotlrce Technology 36, 21-35. Harmova, M., Biely, P. & Vrasnska, M. I986 Specificity of cellulase and p-xylanase induction in Trichodem reesei QM 9414. Archives of Microbiology 144, 307-311. Malek, M.A., Chowdhury, N.A., Youssouf, Q.M. & Choudhury, N. 1988 Bacterial cellulases and saccharification of lignocellulosic materials. Enzyme and Microbial Technology 10, 750-753. Mullings, R. & Parish, J.H. 1984 Mesophilic aerobic Gram negative cellulose degrading bacteria from aquatic habitats and soils. ]ournul of Applied Bacteriology 57, 455-468. Waldron, G. & Eveleigh, D.E. 1986 Saccharification of cellulosics by Microbiospora bispora. Applied Microbiology and Biotechnology
4,487~492. (Received 19 May 1995; revised and accepted 10 ]uly 1995)
