Stabilization of a truncated Bacillus sp. strain TS-23 α-amylase by replacing histidine-436 with aspartate

Histidine-436 of a truncated Bacillus sp. strain TS-23 α-amylase (His6-tagged ΔNC) has been known to be responsible for thermostability of the enzyme. To understand further the structural role of this residue, site-directed mutagenesis was conducted to replace His-436 of His6-tagged ΔNC with aspartate, lysine, tyrosine or threonine. Starch-plate assay showed that all Escherichia coli M15 transformants conferring the mutated amylase genes retained the amylolytic activity. The over-expressed proteins have been purified to near homogeneity by nickel-chelate chromatography and the molecular mass of the purified enzymes was approximately 54 kDa. The specific activity for H436T was decreased by more than 56%, while H436D, H436K, and H436Y showed a higher activity to that of the wild-type enzyme. Although the mutations did not lead to a significant change in the Km value, more than 66% increase in the value of catalytic efficiency (kcat/Km) was observed in H436D, H436K, and H436Y. At 70 °C, H436D exhibited an increased half-life with respect to the wild-type enzyme.