STUDIES ON ISOLATION, PURIFICATION, KINETIC CHARACTERIZATION AND IMMOBILIZATION OF SOME LEGUMES FROM GARHWAL HIMALAYAN REGION.
The present study was performed on two legumes: Phaseolus vulgaris HUR15 (vern. white rajma) and Dolichos biflorus (vern. Kulathee). Both these legumes are abundant in Garhwal Himalayan Region. These had been purchased from local markets of Uttarakhand, Pauri District, as the initial sample. After initial sterilization techniques, the legumes were made to sprout for 5-6 days under controlled conditions in incubator; for optimum yield of the enzyme. Then these sprouted legumes were crushed, and filtered through four layers of cheese cloth. Then, centrifugation was performed on the filtrate at 14000rpm for 10 min to obtain a pellet and a supernatant. The supernatant contained the enzyme. This part completes the first part of the study, i.e. isolation of the enzyme. Further purification of the crude enzyme was done, via ion-exchange chromatography.
Then kinetic characteristics of this purified enzyme were calculated. Kinetic characteristics were based on the following parameters: optimum pH, optimum temperature, optimum substrate concentration, optimum time, thermo-stability, Km, and Vmax of the enzyme. The enzyme was immobilized on calcium alginate matrix, and the same kinetic characteristics were determined on the immobilized enzyme as well.
Lastly, a comparison was made between purified enzyme, crude enzyme and the immobilized enzyme. Also a comparison was made between alpha-amylases obtained from bacteria and these plant sources and it was concluded that these isolated enzymes were as good as the bacterial enzymes.
This research opened a new are of study i.e. preparation of biosensor based on this enzyme, for vital functions, is the next area of study which is yet to be explored.
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