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

alpha-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 alpha-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 degrees C indicating its stable storage at low temperatures. PmAmy was found to be Ca2+ ion-independent for both catalytic activity and thermostability while Mn2+ enhanced activity in a concentration-dependent manner. The optimum characteristic working conditions of PmAmy were measured as pH 7.0 and 40 degrees 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 KM and Vmax values of free PmAmy were 0.1 mg mL-1 and 556 U mg-1, respectively while immobilization resulted in an increase for both the KM and Vmax values. Kinetic analysis revealed enhanced activity for the Ca2+-independent immobilized enzyme, making it suitable for industrial applications particularly starch processing requiring moderate thermostability without the need for Ca2+ ions.