Stability Studies and Steady State Kinetics of Α-amylase From Vigna Radiata

Abstract

α-amylase, (endo-1,4-D glucose-D glucohydrolase; EC 3.2.1.1) is an enzyme that hydrolyses alpha bonds of large, alpha-linked polysaccharides, such as starch and glycogen, yielding glucose and maltose. It is the major form of α-amylase found in humans and other mammals. α-amylase was isolated from the germinated seeds of vigna radiata. At first, α-amylase was stored at two different temperatures for several days in order to check its storage stability with time. The plot of percent residual activity versus number of days gave a t1/2 of 50 days for α-amylase stored at 4oC and 16 days for amylase that stored at 37oC. The optimum temperature and pH was found to be 40oC and 7.0 respectively. To study the effect of substrate concentration, the experiment was carried out by measuring the amount of product formed at different substrate concentration. The Km and Vmax of α-amylase was found to be 483.091 mg ml-1 and 50.50μmole/min respectively. Further, α-amylase was investigated for the inhibitory effect of compounds such as HgCl2 and CuSO4. The effect of inhibitors on α-amylase activity from vigna radiata was studied. All the inhibitors inhibited the activity. The α-amylase activity strongly inhibited by several inhibitors since, the amino acid sequence alignment reveals that all known α-amylase, isolated from different sources, are highly similar and shares a common phylogenic relationship and proposed to have common structure and catalytic mechanism. Therefore, α-amylase from any source, may be microbes or plant, can be used as model system for studies and results would equally applicable for any system or field of application.