One-step purification and immobilization of his-tagged GL-7-ACA acylase

Abstract

GL-7-ACA acylase (GLA) is an enzyme that converts GL-7-ACA to 7-ACA, a starting material for semi-synthetic cephalosporin antibiotics. Four his-tagged GLAs were constructed in this work different in their N-terminal amino acid residues. Among them the highest activity of GLA reached 3800 U/l in 19 h for the recombinant strain BL21 (DE3)/pET28GA01. The immobilized GLA on TALON affinity support, agar-EPI-IDA-Co 2+ and FP-IDA-Ni 2+ was evaluated by one-step purification and immobilization, and the highest immobilized activity was 163 U/g for TALON affinity support. But more robust matrix made FP-IDA-Ni 2+ suitable for the industrial application. The activities of four immobilized GLAs on FP-IDA-Ni 2+ ranged from 66.7 to 80 U/g. Immobilized GLA showed good stability at pH value below 11.0 and at temperature up to 30 °C. To the consecutive conversion of GL-7-ACA, immobilized GLA was recycled 21 times without significant loss of activity, and the average yield rate of 7-ACA reached 90%. These results indicated that intracellular production of his-tagged GLA in E. coli, followed by one-step affinity purification and immobilization on FP-IDA-Ni 2+ resins, might serve as an effective process for the industrial application.