Functional expression of Candida antarctica lipase B in Eschericha coli.
Candida antarctica lipase B (CalB) is an important catalyst in bio-organic synthesis. To optimize its performance, either the reaction medium is changed or the lipase itself is modified. In the latter case, mutants are generated in Eschericha coli and subsequently expressed in fungal hosts for their characterization. Here we present the functional expression of CalB in the periplasm of E. coli. By step-wise deletion of the CalB signal and propeptide we were able to express and purify two different variants of CalB (mature CalB and CalB with its propeptide). A N-terminal FLAG and a C-terminal His tag were used for the purification. For the substrates para-nitrophenol butyrate (p-NPB), para-nitrophenol laurate (p-NPL) and carboxyfluorescein diacetate (CFDA) the specific activity was shown to be similar to CalB expressed in Aspergillus oryzae. The kinetic constants k(M), v(max) and k(cat) were determined using the substrates p-NPB and p-NPL. Almost identical k(cat)/k(M) values (0.423-0.466 min(-1) microM(-1) for p-NPB and 0.068-0.071 min(-1) microM(-1) for p-NPL) were obtained for the CalB variants from E. coli and A. oryzae. The results clearly show that CalB can be functionally expressed in E. coli and that the attachment of tags does not alter the properties of the lipase.
Blank
Kerstin
K
Lehrstuhl für Angewandte Physik & Center for Nanoscience, LMU München, Amalienstrasse 54, 80799 München, Germany.
Morfill
Julia
J
Gumpp
Hermann
H
Gaub
Hermann E
HE
D020816:Amino Acid Motifs / Q000235:genetics; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D002175:Candida / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins; D015870:Gene Expression; D005800:Genes, Fungal; D008049:Lipase / Q000235:genetics* / Q000378:metabolism*; D008957:Models, Genetic; D021382:Protein Sorting Signals; D013379:Substrate Specificity; D014170:Transformation, Genetic
D005656:Fungal Proteins; D021382:Protein Sorting Signals; D008049:Lipase; C431349:lipase B, Candida antarctica