PURIFICATION AND CHARACTERISATION OF BETA-AMYLASE FROM Bacillus subtilis ISOLATED FROM FERMENTED AFRICAN LOCUST BEAN (Parkia biglobosa) SEEDS

Abstract

β-amylase was purified and characterized from Bacillus subtilis isolated from fermented Parkia biglobosa seeds. Purification was achieved using ion exchange DEAE column and gel filtration (Sephadex G-200) chromatography. Effects of temperature; pH and production time on β-amylase production were investigated, while physicochemical characteristics of the purified enzyme were in investigated. The optimum production of β-amylase was obtained at temperature, pH and time of 37oC, 7.0 and 24 h respectively. The results showed that purified β-amylase had more enzymatic activity than crude samples from Bacillus subtilis whereby the activity of crude enzyme was 3.21 mM/min/ml while the purified enzyme had an improved activity of 21.46 mM/min/ml. Optimum temperature and pH values of the purified amylase were found to be 50°C and pH 5.0, respectively. pH stability of the enzyme ranged from pH 4.0- 9.0. At pH 5.0 and 7.0 it retained 70% and 60% of its activity respectively after 5 h of incubation. Temperature stability ranged between 40oC and 70oC but most stable at 50oC retaining 64% of its activity after 1 h of incubation. The enzyme exhibited maximum activity on soluble starch and sucrose, among other carbohydrate substrates. EDTA, Cu2+ and Fe2+ inhibited its activity while Ca2+ and K+ enhanced it. The Lineweaver-Burke plot of the purified β-amylase activity of B. subtilis indicates that the β-amylase enzyme has apparent Km and Vmax values for the hydrolysis of soluble starch of 17.74 mg ml-1and 14.09U respectively. The enzyme was purified 18.76 fold and the molecular weight was 42.2 kDa. The study revealed that β-amylase from B. subtilis can be exploited for starch conversion biotechnologies.