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Annals of Microbiology

, Volume 62, Issue 4, pp 1547–1556 | Cite as

Purification and characterization of carboxymethyl cellulase and protease by Ulocladium botrytis Preuss ATCC 18042 using water hyacinth as a substrate under solid state fermentation

  • Heba I. Abo-Elmagd
  • Manal M. Housseiny
Original Article

Abstract

The potential of 12 fungal strains to produce carboxymethyl cellulase (CMCase) and protease on Eichhornia crassipes (water hyacinth) wastes was investigated under conditions of solid state fermentation. Ulocladium botrytis (Preuss) was selected as the best fungus for the production of both enzymes. The best nitrogen sources for production of CMCase and protease were yeast extract and malt extract, respectively. CMCase and protease were purified by isopropanol (1:1) precipitation and column chromatography on Sephadex G-100 and DEAE-cellulose. Purification fold of 47.34 and 51.78, with 852.11 and 1,469.38 U/mg specific activity was achieved with 40.3 and 56.25% recovery after purification of CMCase and protease, respectively. The purified CMCase expressed its maximal activity at 60°C and pH 5.2, showed good stability in the pH range of 5.2–5.4 and its midpoint of thermal inactivation (Tm) was 60°C after 75 min exposure. The purified protease expressed its maximal activity at 35°C and pH 5.2, showed good stability in the pH range of 5.6–6.0 and its midpoint of thermal inactivation (Tm) was 35°C after 75 min exposure. The best substrate concentration for CMCase was 1.2% (w/v) Na-CMC and for protease, it was 0.8% (w/v) casein. The best enzyme concentration for the two tested enzymes was 0.4 U/ml. Ions of Ca2+, Na+ and K+ showed a stimulatory effect but sodium arsenate and iodoacetate showed an inhibitory effect. Moreover, Ag2+ and Hg2+ inhibited both enzyme activities completely. The purified enzymes from Ulocladium botrytis had molecular weights of 50 and 83 kDa for CMCase, and 47 kDa for protease on SDS-PAGE.

Keywords

Carboxymethyl cellulase Protease Ulocladium Eichhornia crassipes 

Notes

Acknowledgment

The authors wish to express their deepest gratitude to Prof. Dr. Ahmed Fouad Afifi, Professor of Microbiology and Formerly Head of Biological Sciences Department, Faculty of Education, Ain Shams University and Dr. Eman M. Fawzy associate professor of microbiology for their useful criticism and continuous encouragement.

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Copyright information

© Springer-Verlag and the University of Milan 2012

Authors and Affiliations

  1. 1.Department of Biological and Geological Sciences, Faculty of EducationAin Shams UniversityRoxyEgypt

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