Bioinformatics-assisted mining and engineering of α-amylase for efficient hydrolysis of raw corn and microalgal starch

Raw starch-degrading α-amylases play a key role in the cold hydrolysis of starch granules. Using bioinformatic approaches, one α-amylase-encoding gene, AmySy , was identified in the database. AmySy exhibited high activity toward raw corn and microalgal starch, with specific activities of 7570 ± 234 and 2564 ± 33 U/mg, respectively. Through a combination of computational design strategies, the variant AmySy:G207H was obtained, which exhibited enhanced catalytic performance. The half-life of AmySy:G207H at 35 °C was extended to 113 h, representing a 3.0-fold increase over AmySy. Using 30% raw corn flour as the substrate, the hydrolysis rate of AmySy:G207H reached 34.7% after a 5-h reaction. A chemo-enzymatic saccharification process for microalgae was developed using AmySy:G207H. After a three-hour reaction, the reducing sugar yield reached 367.5 mg/g microalgal biomass, which was 2.5 times that obtained through chemical hydrolysis alone. Owing to its ability to hydrolyze raw corn and microalgal starch, as well as its excellent thermostability, AmySy:G207H can be considered an efficient enzyme for the conversion of raw starch into sugar. • Bioinformatics-assisted strategy was developed to obtain raw starch-hydrolyzing α-amylase. • Multiple computational design was employed to enhance the catalytic properties. • G207H is an efficient enzyme for converting raw corn and microalgal starch into sugar.