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

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...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2006-10, Vol.72 (5), p.1024-1032
Main Authors: Liu, D, Schmid, R. D, Rusnak, M
Format: Article
Language:English
Subjects:
Base Sequence
Biological and medical sciences
Biotechnology
Candida antarctica
Cloning, Molecular
Cytoplasm - metabolism
E coli
Enzymes
Escherichia coli
Escherichia coli - genetics
Escherichia coli - metabolism
Fundamental and applied biological sciences. Psychology
Fungal Proteins
Gene Expression Regulation, Bacterial
Lipase - metabolism
Molecular Chaperones
Molecular Sequence Data
Organisms, Genetically Modified
Proteins
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Summary: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.
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-006-0369-7