Glutamic acid 219 is critical for the thermostability of a truncated α-amylase from alkaliphilic and thermophilic Bacillus sp. strain TS-23

Abstract

The functional and structural significance of glutamic acid 219 of a N- and C-terminally truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was explored by the approach of site-directed saturation mutagenesis. The expressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular mass was determined to be approximately 54 kDa by SDS/PAGE. Except E219F, E219P, and E219W, all other mutant enzymes exhibited a lower shift in their optimum temperatures with respect to the wild-type enzyme. A decreased thermostability was also found in all of the mutant enzymes when compared with the wild-type form of BACΔNC. Except E219F, E219P, and E219W mutant enzymes, greater than 2-fold decrease in k cat and a similar substrate affinity relative to the wild-type BACΔNC were observed for the rest mutant enzymes. Based on these observations, it is suggested that Glu-219 apparently plays an important role in the thermostability of BACΔNC.