Heterologous expression of calcium-independent mesophilic α-amylase from Priestia megaterium: Immobilization on genipin-modified multi-walled carbon nanotubes and silica supports to enhance thermostability and catalytic activity.

α-Amylases, constituting a significant share of the enzyme market, are mainly synthesized by the genus Bacillus. Enzymes tailored for specific industrial applications are needed to meet the growing demand across a range of industries, and thus finding new amylases and optimizing the ones that already exist are extremely important. This study reports the successful expression, characterization and immobilization of P. megaterium α-amylase (PmAmy) in E. coli protein expression systems. The recombinant PmAmy has a molecular weight of 56 kDa and its in silico predicted model structure presents a monomer composed of three domains, like most amylases. Regarding long-term storage, PmAmy remained 60 % active after 6 weeks of storage at -20 and -80 °C indicating its stable storage at low temperatures. PmAmy was found to be Ca ion-independent for both catalytic activity and thermostability while Mn enhanced activity in a concentration-dependent manner. The optimum characteristic working conditions of PmAmy were measured as pH 7.0 and 40 °C. Immobilizing PmAmy significantly improved its thermal stability, increasing its resistance to thermal denaturation by at least 4.1-fold. Kinetic analyses revealed that the K and V values of free PmAmy were 0.1 mg mL and 556 U mg, respectively while immobilization resulted in an increase for both the K and V values. Kinetic analysis revealed enhanced activity for the Ca-independent immobilized enzyme, making it suitable for industrial applications particularly starch processing requiring moderate thermostability without the need for Ca ions.