Kinetic analysis, structural studies and prediction of pKa values of Bacillus KR-8104 α-amylase: The determinants of pH-activity profile

A native α-amylase capable of activity at low pH was isolated from Bacillus sp. KR-8104 (KRA) naturally occurred in rhizoplane zone of potato cultured soils. An investigation was set out to account some evidence for its specific properties. Kinetic parameters of KRA with respect to Bacillus licheniformis α-amylase (BLA) and B. amyloliquefaciens α-amylase (BAA) showed a shift in acidic limb for KRA pH-profile. After isolation of KRA gene and determination of its nucleotide and protein sequences, the three-dimensional structure of KRA was simulated based on BLA as template. Using PROPKA program the pKa values of titrable groups in KRA model together with other few members of this family were predicted either from simulated model or available structure at protein data bank. A correlation between catalytic residue pKa values and the optimal pH-activity profile was seen. Both our experimental data and prediction studies indicated a shift in pKa value of catalytic residues to acidic pH. Moreover alignment studies raised amino acid substitutions in KRA compare to BLA which may affect the KRA putative active site leading to the formation of an extra hydrogen bond between Glu261 and Arg229 (regarding to BLA numbering) and in turn shifts the pH-activity profile to lower amount. Also a common intermediate with characteristics of molten globular state was detected at a pH value lower than other members of α-amylase family using far UV circular dichroism, intrinsic and ANS fluorescence and quenching experiments.