CHARACTERIZATION OF PURIFIED FREE AND IMMOBILIZED ?- AMYLASE ENZYME PRODUCED BY BACILLUS MEGATERIUM KLMI 4
Masarath Irfana and Lingappa K.*
ABSTRACT
The present microbe is also a mesophilic as indicated by its activities based on temperature levels. The ambient temperature plays a great role in the growth and any of the activities of any microbe. Lower temperature levels (21 to 29 0C) did not support the enzyme activity. Slightly lower activity was observed in case of immobilized form at 21 and 370 C than that in free form of the enzyme. While peak activity was noticed at 370 C in free form, peak activity occurred at 410 C in case of immobilized form. Thus, a shift in temperature-related enzyme activity appears to have set in due to immobilization. Both forms of the enzyme exhibited nil activity at pH 5.0. From pH 6.0 onwards, the relative activity of immobilized form was certainly higher than that of the free form and in both forms peak relative activity of 100% was recorded at pH 8.0. The relative activity decreased sharply to 25% in case of free form of the enzyme, while it was very near to 100% at pH 9.0 and decreased to only 65% at pH 10.0, thus indicating an improved tolerance to higher pH levels due to immobilization. Reusability of enzyme in repeated batch fermentations: > 60% activity retained in the 7th cycle and Storage stability ~ 70% residual activity on 25th day of storage. The optimum substrate concentration for purified and free form of enzyme was found to be 1.0%. While, purified and immobilized form of enzyme utilizes 1.2% of substrate concentration with 100% its relative activity. Km =0.66, Vmax= 166.66, values of purified free α-amylase and Km =2.7, V max =142.85, values of purified immobilised α-amylase were calculated from Lineweaver- Burke plot.
Keywords: Alpha-amylase, Immobilization, Characterization.
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