ABSTRACT Beta-amylase was purified for the first time from stems of Cadaba farinosa. For this purpose, three-phase partitioning (TPP) was used to concentrate the enzyme while glycogen/amylase complex precipitation was applied to complete the purification. A Doehlert design with ratio of crude extract to t-butanol, ammonium sulphate concentration and pH as independent variables, and purification factor and activity recovery as experimental responses were used to optimize the purification process. A purification factor of 2.04 and activity recovery of 137.5% were found to be the optimum responses. The corresponding optimum parameters were 1.396, 48.2% and 5.9 for ratio of crude extract to solvent, ammonium sulphate concentration and pH, respectively. Final purification factor of 11.18 with activity recovery of 51.1% was observed after glycogen/amylase complex precipitation. High performance liquid chromatography (HPLC) analysis of products resulting from the action of purified beta-amylase on soluble starch allowed identifying the purified enzyme as beta-amylase. The purified enzyme has a molecular weight of 63 kDa and was stable at 60 °C and pH 5.0-7.0 for 4 hours. Its Michaelis constant (Km) for starch was 2.81 mg/ml with a maximum velocity (Vmax) of 10.62 U/min. Our observations suggest a potential new source for amylase production from local resources in Cameroon. The main advantages of the experimental approach are high selectivity, and reliability of results, suitable extraction time and low solvent utilization. The method is applicable with corresponding optimisation for other similar enzymes.
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