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United States Department of Agriculture

Agricultural Research Service

Title: Active Expression of Lipase Forms from Rhizopus Delemar in E. Coli

Authors
item Dilorenzo, M - GREIFSWALD UNIVERSITY
item Hidalgo, A - GREIFSWALD UNIVERSITY
item Pirozzi, D - FEDERICO II UNIVERSITY
item Greco, G - FEDERICO II UNIVERSITY
item Haas, Michael
item Bornscheuer, Uwe - GREIFSWALD UNIVERSITY

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 1, 2005
Publication Date: May 1, 2005
Citation: Dilorenzo, M., Hidalgo, A., Pirozzi, D., Greco, G., Haas, M.J., Bornscheuer, U. 2005. Active expression of lipase forms from rhizopus delemar in e. coli [abstract]. Bioperspective 2005. p. 7.

Technical Abstract: Lipase from the fungus Rhizopus delemar (ROL) has a strong potential in a wide range of reactions relevant to industry. In aqueous systems it has a high specificity as it hydrolyzes the ester bonds at the sn-1 and sn-3, but not the sn-2 position of triacylglycerides and are therefore, referred to as 1,3-specific lipase. This property makes this lipase useful for producing structured lipids. For large scale applications sufficient amounts of active enzyme are required. The genes encoding for the prolipase and for the mature lipase from Rhizopus delemar have been cloned into pET11-d. Unfortunately, the expression in E. coli BL 21(DE3) led to an inactive and insoluble product in the cytoplasm. Initially, strategies to facilitate the process of refolding and purification of the inclusion bodies and, on the other hand, to increase the amount of the soluble protein produced have been studied by us. To simplify the protein purification, the prolipase and the mature lipase genes have been recloned into pET28 vector, which contains a His-tag-coding sequence in the N-terminal position. The insoluble product has been denaturated with 8M urea, adsorbed onto an IMAC resin, so that the His-tagged protein could be isolated, eluted and refolded in the presence of cysteine. This process led to an active protein, but it was time consuming and the storage of the refolded protein was difficult to achieve. Alternatively, we have used the E. coli Origami strain, as it enhances the formation of disulfide bonds in the cytoplasm and allows the lipase to fold properly into a native form, instead of accumulating as inclusion bodies. Good results have been obtained and the target protein has been expressed in a soluble and active form. Further experiments have been than carried out to optimize the expression levels focusing only on the prolipase, as it has been shown to give a larger amount of soluble protein. Best results have been obtained using pET11-d as vector, carrying out the expression at 20°C and induction at OD600 = 1. The maximum activity obtained was 110 U/mg. When the vector used was pET15, in order to produce an N-terminus His-tagged product, the activity obtained under the same conditions, was 40.8 U/mg.

Last Modified: 7/31/2014
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