Abstract
Amylase is an enzyme that hydrolyzes starch for the production of maltose and glucose. In present study, the enzyme was isolated and partially purified to homogeneity by ammonium sulfate fractionation. The protein element drifted as a 20 kDa polypeptide on 10% SDS–PAGE in wild as well as in mutants. Kinetic evaluation of enzyme from wild and mutants revealed maximum activity at pH 4.5. The pKa1 and pKa2 of acidic and basic limbs of active site residues of native were 3.8 and 6.65 whereas 3.6 and 6.8 by An-UV-5.6 and 3.4 and 7.2 by An-Ch-4.7, respectively. The optimum temperature of enzyme was 60 C and temperature quotient was 1.0010, 1.0012 and 1.0011 of A. niger FCBP–198, AnUV-5.6 and An-Ch-4.7, respectively. Ea of An-UV-5.6 was 50.38 kJ mol-1 that was significantly higher than required by native and chemically modified α–amylases i.e., 43.14 and 44.06 kJ mol-1 , respectively. The enzyme was highly active at 50 C while the activity sharply declined after 10 min at 60 to 80 C by all the test strains. The values of Michaelis–Menten constant Km and Vmax revealed by parental, An-UV-5.6 and An-Ch-4.7 were 0.476, 0.25, 0.21 and 5.0, 5.26, 5.56, respectively
