Kinetic characterization of extracellular alpha-amylase from a derepressed mutant of Bacillus licheniformis.
Three strains of Bacillus licheniformis were isolated and screened for alpha-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-methylnN-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 (qp = 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 (DeltaH D = 94.5+/-11 kJ/mol) and entropy of activation (DeltaS = -284+/-22J/[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 endogenous metabolism of the mutant culture for alpha-amylase production.
Institute of Industrial Biotechnology, GC University Lahore, Pakistan. ikhaq@yahoo.com
Ikram
I
Ul-Haq
Hamad
H
Ashraf
Sikander
S
Ali
M A
MA
Qadeer
D001407:Bacillus / Q000235:genetics*; D018533:Biomass; D001709:Biotechnology / Q000379:methods*; D002244:Carbon / Q000737:chemistry; D003470:Culture Media / Q000494:pharmacology; D004043:Dietary Fiber / Q000378:metabolism; D005285:Fermentation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D009604:Nitrosoguanidines / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D000516:alpha-Amylases / Q000378:metabolism*
D003470:Culture Media; D004043:Dietary Fiber; D009604:Nitrosoguanidines; D011506:Proteins; D002244:Carbon; D000516:alpha-Amylases