Applied Microbiology and Biotechnology

, Volume 72, Issue 5, pp 1024–1032

Functional expression of Candida antarctica lipase B in the Escherichia coli cytoplasm—a screening system for a frequently used biocatalyst

Applied Genetics and Molecular Biotechnology

DOI: 10.1007/s00253-006-0369-7

Cite this article as:
Liu, D., Schmid, R.D. & Rusnak, M. Appl Microbiol Biotechnol (2006) 72: 1024. doi:10.1007/s00253-006-0369-7

Abstract

In this paper, we report for the first time the functional expression of lipase B from the yeast Candida antarctica (CalB) in the Escherichia coli cytoplasm. The enzyme possessing three disulfide bonds was functionally expressed in the strain Origami B. Expression under the control of a lac promoter yielded 2 U mg−1, whereas expression of a thioredoxin–CalB fusion protein yielded 17 U mg−1. The native enzyme was most efficiently expressed under control of the cspA promoter (11 U mg−1). Coexpression of different chaperones led to a strong increase in active protein formation (up to 61 U mg−1). A codon-optimized synthetic variant of calb did not show significant effects on functional protein yield. Functional CalB expression was not only achieved in shake flasks but also in microtiter plate scale. Therefore, this CalB expression system is suitable for high-throughput applications, including the screening of large gene libraries as those derived from directed evolution experiments.

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  1. 1.Institute of Technical BiochemistryUniversity of StuttgartStuttgartGermany