Isolation, partial purification and characterization of a cellulolytic enzymes produced by a bacterium isolated from the gut of larva of bettle, oryctes rhinoceros(L)

Sowole, Adekunle Damilola (2015)

xvi,85

Thesis

The study isolatedand optimized the production of a cellulolytic bacterial organismfrom the gut of the larva of beetleand characterize the cellulase produced. This was with a view to obtaining a bacterial candidate for cellulase production for industrial and biotechnological uses. Twenty samples of the larva of beetle were collected at Eleweran in Ile-Ife, Osun State, Nigeria. The larva of beetle were surface sterilized with 70% ethanol and dissected under aseptic condition. One gram of the gut of larva of beetlewas then macerated in 10 ml of phosphate buffered saline and ten-fold serial dilution was carried out. Serially diluted samples were plated on nutrient agar in order to isolate the bacteria which were then screened on carboxymethyl cellulose (CMC) agar for cellulase activity. The optimal conditions for maximal cellulase production by the bacterium with the highest cellulase activity were determined by varying the incubation time, inoculum size, carbon, nitrogen sources, percentage CMC, ammonium sulphate, temperature and pH. The enzyme was partially purified by ammonium sulphate precipitation, dialysis and ion-exchange chromatography on CM-Sephadex C-50. The kinetic parameters (Km and Vmax) of the purified enzyme and the application of the purified enzyme to some cellulosic materials were studied using standard procedures. Fourteen bacteria were isolated while the one with highest cellulase activitywas presumptively identified by morphological and biochemical tests as Bacillus sp. The result of 16S rRNA sequence analysis identified the isolate as Bacillus brevis. The peak of cellulase production was at 38 h of incubation (14.45 µg/ml/min). Carboxymethyl cellulose (CMC) and ammonium sulphate were found to be the best carbon and nitrogen sources for cellulase production respectively. The optimum temperature and pH for the production of cellulase was observed to be 37°C and 6.0 respectively. The specific activity of the partially purified cellulase from Bacillus brevis was determined to be 2.4595 U/mg protein with purification fold of 1.35. The cellulase had a Km of 0.18 mg/ml for CMC and Vmax of 1.67 µg/ml/min. The enzyme was also able to degrade rice husk, maize corb, micro-crystalline cellulose and sugarcane baggase with enzyme activities of 11.43 µg/ml/min, 14.89 µg/ml/min, 8.13 µg/ml/min and 16.98 µg/ml/min respectively. The study concluded that the gut of larva of beetle was a good source for cellulolytic bacteria, which could produce cellulase with desirable charactaristics for industrial uses.

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