Applied Genetics and Molecular Biotechnology

Applied Microbiology and Biotechnology

, Volume 72, Issue 5, pp 1024-1032

First online:

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

  • D. LiuAffiliated withInstitute of Technical Biochemistry, University of Stuttgart
  • , R. D. SchmidAffiliated withInstitute of Technical Biochemistry, University of Stuttgart
  • , M. RusnakAffiliated withInstitute of Technical Biochemistry, University of Stuttgart Email author 

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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.