Abstract:[Objective]This study aims to achieve site-directed immobilization of β-agarase at targeted residues, thereby enhancing its thermal stability and operational performance.[Method]Computer-aided design was employed to selectively immobilize the mutant β-agarase Aga50D via sulfhydryl groups introduced at specific cysteine residues, enabling binding to the carrier.[Result]The immobilization conditions were optimized as follows: reaction duration of 12 h, an enzyme loading level (enzyme-to-carrier mass ratio) of 1∶50, and a crosslinker-to-carrier mass ratio of 10∶50. The immobilization efficiency ranged from 40% to 75%, with most mutants retaining the relative enzyme activity >80%. Compared with the wild-type free enzyme, the site-directed immobilized mutant β-agarase Aga50D exhibited superior thermal stability at 40 ℃ and 45 ℃. After four reuse cycles, the enzyme activity retention rate of site-directed immobilized mutant β-agarase Aga50D remained above 30%. Notably, while the wild-type free enzyme completely lost its activity in organic reagents, the enzyme activity retention rate of most site-directed immobilized mutant β-agarase Aga50D remained above 70%.[Conclusion]The site-directed immobilized mutant β-agarase Aga50D demonstrated high enzyme activity and enhanced thermal/operational stability, showing significant promise for industrial applications.
