【摘要】 【目的】实现β-琼胶酶的特异位点的定向固定化，以提高其热稳定性及操作稳定性。【方法】利用计算机辅助手段，通过特定位点的半胱氨酸的巯基与载体特异性结合，得到定向固定化突变型β-琼胶酶Aga50D。【结果】定向固定化的最优反应条件为：反应时间12 h、酶载量（酶与载体的质量比）1∶50、交联剂与载体质量比10∶50。不同定向固定化突变型β-琼胶酶Aga50D的固载率为40%～75%，大多数定向固定化突变型β-琼胶酶Aga50D的相对酶活力>80%。在40、45℃条件下，定向固定化突变型β-琼胶酶Aga50D比野生型游离酶具有更好的热稳定性。定向固定化突变型β-琼胶酶Aga50D在重复使用4次后的酶活力保留率仍可达30%以上。在有机试剂中，野生型游离酶丧失了全部活性，而大多数定向固定化突变型β-琼胶酶Aga50D的酶活力保留率仍可达70%以上。【结论】该研究制备的定向固定化突变型β-琼胶酶Aga50D有较高的酶活力，且热稳定性及操作稳定性较强，具有较高的工业应用潜力。更多还原

【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.更多还原