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Functional Expression of Candida Antarctica Lipase B in Eschericha Coli

Functional Expression of Candida Antarctica Lipase B in Eschericha Coli

Kerstin Blank, Julia Morfill, Hermann Gumpp, Hermann E Gaub

J Biotechnol. 2006 Oct 1;125(4):474-83.

PMID: 16713003

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP9001621-E	Lipase B Candida antarctica, recombinant from Aspergillus oryzae		9001-62-1	Price

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