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item Di Lorenzo, Mirella
item Hidalgo, Aurelio
item Pirozzi, Domenico
item Greco, Guido
item Haas, Michael
item Bornscheuer, Uwe

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/15/2005
Publication Date: 12/1/2005
Citation: Di Lorenzo, M., Hidalgo, A., Pirozzi, D., Greco, G., Haas, M.J., Bornscheuer, U.T. 2005. HETEROLOGOUS PRODUCTION OF FUNCTIONAL RHIZOPUS ORYZAE LIPASE FORMS IN ESCHERICHIA COLI. Applied and Environmental Microbiology. 71(12):8974-8977.

Interpretive Summary: Enzymes known as lipases are able to catalyze biochemical reactions that achieve the synthesis of valued food, pharmaceutical, industrial chemical, or fuel compounds. Full exploration of the capability of lipases as applied catalysts, and their use as such at industrial levels, is hindered by the fact that nature often produces enzymes that are less than optimal industrial catalysts, by inadequate supplies of desired enzymes and by a lack of basic knowledge of the capabilities of the enzymes. This paper describes work aimed at solving these deficiencies. Using genes previously cloned at ERRC-ARS-USDA, the authors conducted genetic engineering experiments that successfully modified the genes and identified a bacterial host species that gave a substantial increase in the production of active enzyme. This is a challenging goal and a substantial achievement that paves the way for rational gene modification to improve catalytic properties as well as for the synthesis of the large amounts of enzyme required for industrial biocatalysis.

Technical Abstract: The lipase from Rhizopus oryzae (ROL, equivalent to lipase from Rhizopus delemar, RDL) is one of the most important lipases for biotechnological applications. However, the commercial enzyme is still produced from the wild-type strain. To date recombinant expression of ROL in E. coli always led to the formation of inclusion bodies and inactive protein. In this paper, these limitations were overcome for the first time by careful choice of the expression system and further optimization of the cultivation conditions. Thus, active ROL (29 kDa) and its precursor ProROL (43 kDa) were produced in soluble form in the cytoplasm only when E.coli Origami (DE3) and pET-11d(+) was used as expression system. Expression at 30 and 37°C led to formation of inclusion bodies and only at 20°C soluble and active protein with a specific activity of up to 110.7 U/mg (proROL, with p-nitrophenyl butyrate as substrate) was formed. The highest activity (116 U/mg) was found for the C-terminal His-tagged proROL with induction at OD600 of 0.5. The majority of the enzyme is still produced as proROL and further studies are under way to increase the processing efficiency to convert proROL into mature ROL. Nevertheless, this achievement substantially facilitates production of the recombinant lipase on large scale, functional studies on ROL by site-directed mutagenesis, and especially allows directed evolution studies to further broaden the range of applications for this lipase.