聚糖
内糖苷酶
纤维素
化学
突变体
生物化学
大肠杆菌
融合蛋白
酶
糖蛋白
重组DNA
基因
作者
Kai Zhao,Feng Tang,Wei Shi,Haofei Hong,Zhifang Zhou,Wei Huang,Wei Huang
标识
DOI:10.1016/j.bioorg.2019.103114
摘要
The endoglycosidase (EndoS and its glycosynthase mutants D233A, D233Q) gene was fused with cellulose binding domain (CBD) using pET-35b vector and the fusion enzymes were successfully expressed in Escherichia coli. Then a simplified approach for one-step immobilization and purification of EndoS enzymes using cellulose as matrices were developed and excellent loading efficiency (81-90%) was achieved in optimal condition. The cellulose immobilized CBD-EndoS and the glycosynthase mutants presented high catalytic activity and were successfully applied in a two-step antibody Fc N-glycan remodeling, generating a therapeutic antibody with homogeneous glycoform in high efficiency. The cellulose immobilized CBD-EndoS and its mutants (D233A and D233Q) displayed excellent storage stability when stored at 4 degrees for one month. Reusability studies demonstrated that the cellulose immobilized CBD-EndoS and its mutants could be recycled for five times without obvious activity loss.
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