Isolation, Purification and Characterization of Cellulase Produced by Aspergillus niger Cultured on Arachis hypogaea Shells
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Date
2018-06
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UNIVERSITY OF ILORIN
Abstract
The substantial amounts of waste materials such as shells, tatters, trunks, peels and seeds are produced as a result of agricultural practice. Large quantities of these agrowastes obtained are from heavy consumptions of agricultural products. These wastes particularly groundnut shells are abundant because once the nuts have been removed, the shells are always discarded. Accumulation of these shells constitutes what we called “wastes” and consequently lead to environmental pollution. Transformation of these wastes to expedient products will not only combat environmental pollution arising from unnecessary discarding of shell but also boast the economy of our country. The uses of different agricultural wastes such as corn cob, rice bran, bagasses, wheat bran, banana trunk for production and characterization of cellulase have been studied in the past. Therefore, this study addressed the possible use of Arachis hypogaea shells as a substrate for cellulase production from Aspergillus niger. The A. hypogaea shells were dried and subjected to acid and alkali pretreatment. Proximate analysis was carried out on the treated and untreated substrates. The remains of pretreated substrates were then used as substrates in a shake-flask containing enriched media preparation and A. niger was inoculated. Fermentations were carried out in flasks containing the enriched media inoculated with A. niger for 168 hours. Optimization parameters for maximum cellulase production were determined by varying the fermentation conditions. The crude cellulase was precipitated at 80% ammonium sulphate saturation, followed by dialysis and gel filtration using Sephadex G-100 as stationary phase in chromatographic column. The molecular weight was estimated using sodium dodecyl sulphate polyacrylamide gel electrophoresis. Cellulase activity was assayed by determining the concentration of glucose produced from the hydrolysis of carboxymethylcellulose catalysed by cellulase. The effects of pH and temperature on the activity of purified cellulase were determined. The kinetic parameters (Km and Vmax) were also evaluated. The effects of some cations (Na+, K+, Mg2+, Cu2+, Zn2+, Ca2+, Fe2+, Mn2+, Co2+) and anions (CO32-, Cl-, and SO42-) on the activity of purified cellulase were investigated. Also, the effects of some surfactants (DMSO, Triton X, Tween 20 and Mercaptoethanol) on the activity of purified cellulase as well as detergent compatibility of purified cellulase were determined. The results obtained from proximate analysis of pretreated A. hypogaea shells showed that crude fiber content, alkaline pretreated substrates had the highest percentage of crude fibre with 87.0% compared to acid treated and untreated with 85.5 and 82.0% respectively. There was a significant increase (p ˂ 0.05) in the content of crude protein following the treatment of A. hypogaea shell with alkali compared to acid treated and untreated shells. The alkaline pretreated substrates had the highest percentage of cellulose with 2.2 folds increase when compared to untreated substrate. Also, the percentage of cellulose in the acid treated substrate was higher with a 1.7-fold increased, compared to untreated A. hypogaea. This study also revealed that the: optimal production of crude cellulase was achieved at incubation period of 120 hours, pH of 4, temperature of 40 oC, and inoculum size of 13 cfu/ml; enzyme was purified to 68.12 fold with a yield and specific activity of 3.87% and 484.3 U/mg respectively; Vmax for the cellulase was 9.26 U/ml while the Km was 0.23 mg/ml; molecular weight of the cellulase was 13.5 kDa; optimum pH and temperature for the cellulase activity were 4 and 40 oC respectively; CO32-, Cl- and SO42- decreased the activity of cellulase. Na+ activated the cellulase activity at 1 to 5mM while K+ did not affect the cellulase activity; Mg2+ competitively inhibited cellulase activity while Zn2+, Cu2+, Ca2+ and Fe2+ non-competitively inhibited the activity of the enzyme. Mn2+ and Co2+ enhanced the cellulase activity; and cellulase activity was inhibited by dimethyl sulfoxide, triton X, tween 20 and mercaptoethanol. The study concluded that A. hypogaea shells can be used as a source of carbon by A. niger for the production of cellulase. Therefore, the purified cellulase produced may be explored for the various industrial applications of the enzyme.
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Isolation, Purification, Characterization, Cellulase, Aspergillus niger, Arachis hypogaea Shells