Department: Applied Microbiology And Brewing
Cassava (Manihotesculenta Crantz) waste water is an effluent produced from cassava bio-processing. This effluent is regarded as a liquid waste and discarded into the environment where it constitutes pollution. This effluent however, is rich in starch and ligno-cellulose that can be harnessed for useful industrial purposes such as microbial enzyme production. This research aim was to produce and characterize amylolytic enzyme from Aspergillus sp. using cassava waste water. Molds were isolated from waste water samples collected from local garri processing plants in Awka. They were tentatively identified based on their phenotypic characteristics. Physico-chemical components of the waste water were also determined. Fungal isolates were screened for amylase production and the best amylase producing isolate was chosen, identified with molecular typing and used for further studies. Fungal biomass was produced from 500 ml enriched waste water in a rotary shaker. Amylase and protein were assayed using standard procedures at 540 nm and 280 nm respectively. Effect of 1% incorporation of different nitrogenous sources on amylase production using the waste water was determined. Amylase was produced in a batch fermentation using the best nitrogenous source and its time course was monitored. The produced enzyme was purified using CM-Sepharose ion exchange chromatography and Phenyl-Sepharose CL-4B column hydrophobic interaction chromatography. Purified amylase was characterized based on its temperature, pH, metal ions, inhibitors and substrate concentration reactions. Hydrolysis of gelatinized and raw starch by the enzyme was determined and the hydrolysates were separated using thin layer chromatography. Fungal isolate D gave the highest amylase activity with diameter clear zone of 11.4±0.25 mm. It became the choice isolate and was identified as Aspergillusnomius. The waste water had a pH of 3.81±1.33, starch content of 87.575±3.03 mg/L, reducing sugar of 12.93%, protein content of 11.20±2.52 mg/L, total solid of 109.34±0.69 mg/L and total dissolved solid of 42.90±1.05 mg/L. A biomass weight of 3.9 g was recovered with an amylase activity of 4.9 u/ml. Yeast extract (1% w/v) gave the highest amylase activity compared to other nitrogenous sources, however, 1.5% yeast extract was the optimum for amylase production. The enzyme production followed exponential growth of the organism with maximum enzyme occurring at 96h at pH 8.0. Ion exchange chromatography showed a major peak of amylase activity between fractions 36-42 at a flow rate of 10 ml in 5 mins. Recovery of the fractions led to a 7.25% retention of overall activity and 7.3-fold purification. Fractions 5-8 were the enzyme peaks on hydrophobic interaction chromatography. Enzyme was purified 0.88-fold to give a 1.1% yield and specific activity of 0.52 u/mg protein. The enzyme is optimally active and maximally stable at 400C and pH 7.0. Iron (II) gave mild stimulation of amylase activity while Mg2+ and EDTA exerted some levels of inhibition on amylase activity. Reaction rate of amylase at different substrate concentrations did not show hyperbolic relationship, indicating possible allosteric nature of the enzyme. Enzyme had highest amylase activity on raw millet starch and gelatinized soluble starch with 100% relative rate of hydrolysis. Thin layer chromatography of the hydrolysates showed the presence of maltodextrins at varying levels. The enzyme appears to be hydrophobic and will be good for use in detergent formulations. This research has been able to show that cassava waste water can serve as a useful industrial raw material for production of microbial amylase and should not be discarded to the environment to constitute pollution.