Purification and characterization of β-Galactosidase produced by a bacterium isolated from dairy effluent.
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Date
2015
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Publisher
Microbiology,Obafemi Awolowo University
Abstract
This study purified and characterized β-galactosidase from a bacterium isolated from dairy effluent with the view to selecting a good bacterial candidate that could be useful in the hydrolysis of lactose and other industrial uses.
In this study, β-galactosidase producing organism was isolated from dairy effluent collected from Fan Milk Plc, Eleyele, Ibadan, Nigeria. The bacterium with the highest β-galactosidase activity was selected for enzyme production, purification and characterization. Ten millilitres (10 mL) of the dairy effluent sample was measured out and re-constituted in 90 mL sterile distilled water and serially diluted to 10-10. One millilitre (1 mL) of appropriately diluted sample was cultured on nutrient agar using pour plate method and incubated at 37oC for 24 h. The pure isolates was cultured on lactose broth and screened for lactose hydrolysis. The isolate with the ability to hydrolyze lactose was identified to species level by cultural characteristics, Gram’s reaction and biochemical characteristics using Bergey’s Manual of Determinative Bacteriology as a standard. The identity of the bacterium was confirmed using 16S rRNA gene sequencing technique and phylogenetic analysis. Optimal conditions for enzyme production by the bacterium was also studied which includes, incubation time, pH, temperature, carbon source, nitrogen source and percentage substrate concentration as well as inoculum size. Partial purification of the crude enzyme was carried out using ammonium sulphate precipitation and acetone precipitation. The enzyme from the method that produced the highest yield was subsequently adopted for further purification using ion-exchange chromatography on DEAE Sephadex A-50 and gel filtration on Biogel P-100. The effect of pH, temperature, metal chloride ions, inhibitors such as mercury and EDTA as well as the kinetic parameters of the partially purified enzyme were studied using standard procedures.
The bacterium isolated from the effluent was presumptively identified by morphological and biochemical characteristics as Klebsiella sp. The result of molecular analysis identified the organism as Klebsiella pneumoniae. The optimum production was obtained at 28th hour, temperature 35oC and pH of 7.0 with lactose and peptone as the best carbon and nitrogen source respectively. The specific activity of the partially purified Klebsiella pneumoniae β-galactosidase was found to be 0.892 units/mg protein with purification fold of 3.4. The optimum pH and temperature of the partially purified Klebsiella pneumoniae β-galactosidase are 7 and 35°C respectively. For the stability of the Klebsiella pneumoniae partially purified β-galactosidase, at 70ºC, there was total loss of activity after 20 min. The enzyme activity was activated by Na+ at 5 mM and 10 mM while it was inhibited at 15 mM and 20 mM, other metals used such as K+, Mn2+, Zn2+, Al3+, Hg 2+and Pb2+ inhibited the enzyme activity at all concentrations. The enzyme was inhibited by the inhibitor used. The apparent Km and Vmax was determined to be 0.63 mM and 0.16 µmol/ml/min respectively.
The study concluded that the isolated bacterium from dairy effluent was a potentially good source of β-galactosidase enzyme.
Description
xx, 126p
Keywords
β-galactosidase, Bacterium isolated, Dairy effluent, Enzymes
Citation
Popoola,O.O.(2015). Purification and characterization of β-Galactosidase produced by a bacterium isolated from dairy effluent. Obafemi Awolowo University