Bioethics in India: Proceedings of the International Bioethics Workshop in Madras: Biomanagement of Biogeoresources, 16-19 Jan. 1997, University of Madras; Editors: Jayapaul Azariah, Hilda Azariah, & Darryl R.J. Macer, Copyright Eubios Ethics Institute 1997.
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83. Ecoethical Technology Using Extracellular Enzymes of Chrysosporium Species

N.S. Kavitha, A. Hilda, S.Gopinath & K. Latha
Centre for Advanced Studies in Botany, Guindy Campus, University of Madras, Chennai 600 025.


Abstract

Five species of Chrysosporium were investigated for their enzymatic potentiality. Of the five species of Chrysosporium, C. anam. Arth. cuniculi highlighted itself to be highly potent for cellulolytic (filter paper and endoglucanase), and proteolytic activity. Whereas C. pannorum projected itself to be highly potent for cellulolytic (filter paper) and lipolytic activity. While C. keratinophilum and C. tropicum were highly amylolytic fungi. To assess the amylolytic potential of the fungi 2% w/v of starch used as the substrate. Whereas to assay the cellulolytic potential, 50 mg of filter paper and 3% w/v of carboxymethylcellulose (CMC) were used as the substrate. For assessing the lipolytic and proteolytic activity p-nitrophenyl palmitate and casein respectively were used as substrates. In the present investigation, only one species of Chrysosporium recorded low enzyme activity.

Introduction

Microorganisms produce enzymes which are involved primarily in the degradation of macro-molecules to units capable of being taken into the living cell (Fogarty and Kelly, 1979). Out of the 2000 various enzymes that have been described only a few like amylolytic, cellulolytic, lipolytic and proteolytic enzymes have wide industrial and biotechnological applications.

Amylase is the enzyme which is capable of degrading starch, glycogen and related polysaccharides of ‡-1,4 and 1,6 glucosidic linkages. It is used in textile industry and also in digestive process. Cellulase has been regarded as enzymes that are capable of degrading linear glucose polymer of cellulose to give sugar as the end product. It is used for degrading cellulolytic wastes, such as, agricultural and domestic wastes, to increase the tensile strain of paper, to produce single cell protein from cellulosic waste and also in preparation of alcohol for fuel or chemical production. Lipase are glycerol ester hydrolase which catalyze the hydrolysis of triacyl glycerol into fatty acid, partial acyl glycerol and glycerol. It is used for detergent preparation, lipolyze milk fat, enhancement of dairy products, pharmaceuticals, synthesis of peptides and is also used in leather industry. Protease are capable of cleaving protein into smaller units such as, the peptides and amino acids. It is used as reagents in laboratory, food industry, dehairing of skin, feather degradation and also in pharmaceutical industry.

Organisms Chosen For The Present Study

1. Chrysosporium anamorph of Arthroderma cuniculi;2. Chrysosporium keratinophilum; 3. Chrysosporium pannorum;4. Chrysosporium species and 5. Chrysosporium tropicum. All Chrysosporium species were isolated from soil samples in school play grounds and public parks in and around Madras city by hair baiting technique (Ramesh and Hilda, 1996). The fungal species were grown and maintained on Sabouraud dextrose agar medium (Dextrose 40g; Peptone 10g; Agar 25g; Distilled water1000 ml; pH 5.6). Among these five species, Chrysosporium keratinophilum causes skin diseases.

Amylase

For amylase production, Czapek-dox medium was amended with 2% w/v concentration of starch as sole carbon source. The amylase activity was studied by the estimation of reducing sugars. The absorbance of reducing sugar was measured at 500 nm (Nelson, 1944). One unit of amylolytic activity was the amount of enzyme which liberates 1um of reducing sugar in the assay condition.

Among the five species of Chrysosporium, the following three species namely Chrysosporium keratinophilum, Chrysosporium pannorum and Chrysosporium tropicum were the highly potent fungi for amylolytic activity. However, Chrysosporium keratinophilum showed maximum activity at 72 hrs. of incubation (Fig. 1).

Cellulase

For cellulase production, Czapek-dox medium was amended with 3% w/v concentration of carboxy methyl cellulose (CMC) as sole carbon source.

Cellulase activity was monitored by the estimation of reducing sugar using dinitro salicylic method (Miller, 1959), with filter paper (50 mg) or 1% w/v of carboxy methyl cellulose as the substrate. The absorbance of reducing sugar was measured at 575 nm. One unit of filter paper activity was the amount of enzyme which liberates 1 um of reducing sugar in the assay condition. One unit of endoglucanase activity was the amount of enzyme which liberates 1um reducing sugar from 1% CMC in the assay condition.

Among the five species, Chrysosporium anamorph of Arthroderma cuniculi and Chrysosporium pannorum appeared to be highly potent fungi for cellulase activity. However, Chrysosporium ana. Arth. cuniculi and Chrysosporium pannorum showed maximum filter paper activity at 120 hrs. of incubation. Whereas, Chrysosporium ana. Arth. cuniculi showed maximum endoglucanase activity at 168 hrs. of incubation (Fig. 2a & 2b).

Lipase

For lipase production the fungi were grown in Yeast- extract - Peptone medium. Exolipase was assayed using p-nitrophenyl palmitate as the substrate. The lipase activity was assessed by measuring the increase in absorbance of p-nitrophenol at 410 nm (Thoner, 1973). One unit of lipase activity was the amount of enzyme which liberates 1 um of p-nitrophenol under the assay conditions. Species like Chrysosporium keratinophilum and Chrysosporium pannorum were the highly potent fungi for lipolytic activity. However, Chrysosporium pannorum showed maximum activity at 168 hrs. of incubation (Fig. 3).

Protease

For protease production the fungi were grown in Yeast-extract - Maltose medium. Protease activity was assayed according to the method of Kunitz (1947) using casein as the substrate. The tyrosine liberated by the hydrolysis was quantitatively estimated by measuring the absorbance at 280 nm. One unit of protease activity was the amount of enzyme which liberates 1 ug of tyrosine in the assay conditions. Species of Chrysosporium anamorph of Arthroderma cuniculi showed maximum activity at 168 hrs. of incubation and it was the highly potent fungi for proteolytic activity (Fig. 4).

Among the five species of Chrysosporium, Chrysosporium anamorph of Arthroderma cuniculi was the highly potent fungi for filter paper, endoglucanase and protease activity. While Chrysosporium keratinophilum and Chrysosporium tropicum were highly potent fungi for amylase activity. Whereas Chrysosporium pannorum for filter paper and lipase activity. While only one species of Chrysosporium recorded low enzyme activity.

Relationship between environmental incidence of fungi and human health

As these fungi were isolated from school soil environment they have a direct relevance to the health of the school going children. Many keratinophilic fungi are pathogenic and are linked with the incidence of dermatomycosis (Ajello, 1953; Mercantini et al., 1980, 1983). All these Chrysosporium species are keratinophilic fungi which could degrade keratin. Further, many Ketinophilic fungi are pathogenic and are linked with the incidence of dermatomycosis, fungal keratitis, Keratitis of cornea and allergic alveolitis. There are several reports of Chrysosporium keratinophilum causing diseases in a wide variety of animals. Their universal occurrence in our human environment was considered to be of epidemiological significance.

Biotechnological implications

The production of different enzymes by keratinophilic fungi is of immense value for their successful survival and subsequent hydrolysis of keratin. These five species of Chrysosporium have extracellular enzymes which are useful in ecoethical technology such as, for digestive processes (amylase), degradation of cellulolytic waste from pulp and paper industry (cellulase). fermentation and enhancement of dairy products (lipase), for dehairing of skin and feather degradation which remove the contaminants from soil (protease).

Furthermore, these Chrysosporium species produce large number of enzymes which have biotechnological applications. Hence if carefully manipulated they can be used for human advantage.

Acknowledgements

The authors thank Prof. A. Mahadevan, Director, Centre for Advanced Studies in Botany, University of Madras, Chennai for his encouragement. The financial assistance by U.G.C and DoEn are greatly acknowledged.

References
Ajello, 1953. The dermatophyte, Microsporum gypseum as a saprophytic parasite. Journal of Invest. Derm. 21: 157-171.
Fogarty, W. M. and Kelly, C. J. 1979. Developments in microbial extracellular enzymes. In: Alan Wiseman (ed.), Topics in Enzyme and Fermentation Biotechnology. Ellis Horwood Ltd., Publishers, England, Vol. 3: pp. 289.
Kunitz, M. 1947. Crystalline soybean trypsin inhibitor II. General properties. Journal of General Physiology. 30: 291-310.
Mercantini, R., Marsella, R., Caprilli, F. and Dovgiallo, G. 1980. Isolation of dermatophytes and correlated species from the soil of public gardens and parks in Rome. Sabouraudia 18: 123-128.
Mercantini, R., Marsella, L., Lambiase, L. and Fulvi, F. 1983. Isolation of keratinophilic fungi from floors in Roman (Italy) primary schools. Mycopathologia. 82: 115-120.
Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry. 31: 426-428.
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Ramesh, V. M. and Hilda, A. 1996. Studies on keratinophilic fungi - Characterization of keratinolytic potential and fungitoxic evaluation of some plant extracts. Ph. D thesis, University of Madras, Chennai.
Thoner, M. 1973. Diplom. Thesis. University of Bochum, Bochum, Germany.


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