Purification and characterization of cellulase from aegerita webberi isolated from decaying orange fruits

Okodugha, Ese Louisa (2017)

xvii,122

Thesis

The objectives of the study was to isolate, screen and identify cellulase producing fungi from decaying orange fruits; determine the optimum condition of the best cellulase producing isolates to obtain crude cellulase; produce and partially purify the crude cellulase by precipitation and gel filtration; investigate the characteristics of the partially purified cellulase; and apply the partially purified cellulase to cellulose material. Six fungal strains were isolated from decaying orange fruit. They were screened for their ability to degrade cellulose by standard rapid plate screening assay. Cellulolytic fungi were evaluated after 5 days for the production of cellulolytic enzymes by staining with 1% Congo red and Aegerita webberi was selected being novel in the production of cellulase. Optimization for cellulase production was done using parameters such as carbon sources, pH, temperature, substrate concentration, nitrogen sources (inorganic and organic) and inoculum size of A. webberi.The enzyme produced was partially purified using a combination of ammonium sulphate precipitation and gel filtration on bio-gel P-100. The cellulase was characterized to determine the kinetic properties. The peak of cellulase production was on the fourth day of incubation (162.36Units/ml).The optimum temperature for the activity of cellulase produced by the fungal strain A. webberiwas 30oC with the activity of 39.2 Units/ml while the optimum pH was attained at pH 5.5 with an activity of 112.2 Units/ml. Casein was the best nitrogen source with an enzyme activity of 239.4 Units/ml while carboxyl methylcellulose was the best carbon source with an enzyme activity of 121.9 Units/ml. The partially purified cellulase specific activity on bio-gel P-100 had a specific activity of 3.06 Units/mg/ml and the Vmax and Km was 0.26 Unit/ml and 1.184 Unit/ml respectively. The optimum temperature for the partially purified enzyme was 60oC while the enzyme was stable to heat for 30 mins at 70oC before noticeable decrease in activity. Of the metal ions investigated, EDTA and HgCl2resulted in reduced activity of the purified cellulase while NaCl, KCl, CaCl2,MgCl2 andAlCl3 resulted in an increase in the activity of the enzyme. On hydrolysis of raw substrates (yam powder and maize cob), yam powder had a stable higher activity compared to corn cob. The thermostable cellulase from Aegerita webberi isolated from decaying orange fruits could be of great importance in biofuel industry for the saccharification of lignocellulolytic materials into economically useful monosaccharides.

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