Discovery and design of novel glucoamylases suitable for raw starch hydrolysis at moderate temperatures via an integrated bioinformatics-assisted strategy.

Glucoamylases capable of hydrolyzing raw starch at moderate temperatures exhibit significant potential for the direct and efficient hydrolysis of raw starch. Using bioinformatics-assisted mining strategies, four candidate glucoamylases were initially identified from the GenBank database. Among these candidates, SeGA demonstrated high specific activities of 412.94 U/mg toward soluble starch and 143.51 U/mg toward raw corn starch. The half-life of SeGA was 192 h at 40 °C. Using SeGA as the initial enzyme, FuncLib design was performed to enhance its activity. The mutant SeGA-21 exhibited higher specific activity of 466.22 U/mg toward soluble starch and 173.98 U/mg toward raw corn starch, representing increases of 1.13-fold and 1.21-fold compared to SeGA, respectively. The half-life of SeGA-21 at 40 °C increased to 240 h, representing a 25% increase compared with SeGA. SeGA-21 can efficiently hydrolyze 30% raw corn starch, achieving a hydrolysis rate of approximately 31% after a 9-h reaction. When working synergistically with amylolytic enzymes, a hydrolysis rate of 71.5% was achieved for 30% raw corn starch at 40 °C within 15 h of reaction. Due to its ability to hydrolyze raw starch, moderate reaction temperature requirements, and excellent thermostability, SeGA-21 can be regarded as an efficient enzyme for the cold hydrolysis of raw starch and starch modification.