Functional expression of lipase A from Candida antarctica in Escherichia coli—A prerequisite for high-throughput screening and directed evolution

Abstract

We report for the first time the functional and heterologous expression of lipase A from Candida antarctica (CalA) in the cytoplasm of Escherichia coli Origami™ B cells. Expression under control of the lac promoter in the pUC18 vector yielded 0.7 U mg −1 lipase activity, whereas expression of a thioredoxin–CalA fusion protein using the pET-32b(+) vector yielded 1.7 U mg −1. The native enzyme was most efficiently expressed under control of the cspA promoter (9.63 U mg −1) using the pColdIII vector. Co-expression of various chaperones led to a significant increase in formation of active protein (up to 13.1 U mg −1). This expression strategy was validated in microtitre plates and therefore is suitable for high-throughput screening of large gene libraries and applications including directed evolution of CalA.