Applied Biochemistry and Biotechnology, Vol.141, No.2-3, 251-264, 2007
Kinetic characterization of extracellular alpha-amylase from a derepressed mutant of Bacillus licheniformis
Three strains of Bacillus licheniformis were isolated and screened for a-amylase production by solid-state fermentation. Of these, IS-2 gave relatively higher enzyme production (32 +/- 2.3 U/[g.min]) and was selected for improvement after treatment with N-methyl N-nitro N-nitroso guanidine (NG) or nitrous acid (NA) to enhance its hydrolytic potential. Among the mutant variants, NA-14 gave higher enzyme production (98 +/- 1.6 U/[g.min]), and hence, was selected for kinetic and thermal characterization. M1 as a moistening agent (pH 7.0, optimized) supported 2.65-fold improved amylolytic activity by the derepressed mutant 72 h after inoculation. The values of product yield coefficient (Y-p/x = 1833.3 U/g) and specific rate constant (q(p) = 25.46 U/[g.h]) with starch were severalfold improved over those from other carbon sources and the other cultures. The purified enzyme from NA-14 was most active at 40 degrees C; however, the activity remained almost constant up to 44 degrees C. The NA-induced random mutagenesis substantially improved the enthalpy (Delta H-D = 94.5 +/- 11 kJ/mol) and entropy of activation (Delta S = -284 +/- 22 J/[mol.K]) for alpha-amylase activity and substrate binding for starch hydrolysis. The results of this study (117.8 +/- 5.5 U/[g.min]) revealed a concomitant improvement in the enclogenous metabolism of the mutant culture for alpha-amylase production.
Keywords:Bacillus licheniformis;alpha-amylase;mutant;solid-state fermentation;wheat bran;thermal characterization