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PDFlib PLOP: PDF Linearization, Optimization, Protection Page inserted by evaluation version www.pdflib.com – [email protected]
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A Toolbox of Recombinant Lipases for Industrial Applications CLAUDIA SCHMIDT-DANNERT, JÜRGEN PLEISS, AND ROLF D. SCHMID a Institute of Technical Biochemistry University of Stuttgart 70569 Stuttgart, Germany ABSTRACT: We created a toolbox of recombinant, microbial lipases, which allows us in combination with a lipase database to choose among the overexpressed lipases the most appropriate for a specific application and to improve it further via mutage- nesis. By systematic comparison of geometry and properties of the scissile fatty acid binding site of five representative lipases of each family of structurally homologous lipases, three subgroups can be defined. Hence, efficient expression systems for the functional production of large amounts of microbial lipases, representing different lipase subgroups, were developed. In particular, recombinant lipases from Bacillus thermocatenulatus and Pseudomonas cepacia were functionally overexpressed in E. coli . The lipase genes from Geotrichum candidum CMICC 335426 and Rhizopus oryzae were overexpressed in Pichia pastoris . Due to an unusual codon usage that prevents heterologous expression, the LIP1 gene (1647 nt) of Candida rugosa was completely synthesized and overexpressed in Pichia pastoris . INTRODUCTION Lipases (triacylglycerol acylhydrolase EC, in view of their current and potential applications, are considered as being a particularly promising class of in- dustrial enzymes. Apart from their natural substrates, such as water-insoluble esters and triglycerides, lipases catalyze the enantioselective and regioselective hydrolysis and synthesis of a broad range of natural and nonnatural esters. 1,2 Thus, lipases are ideal biocatalysts for oleochemistry and organic chemistry and are currently used in the production of fine chemicals and pharmaceuticals. 3 Most commercial lipases are extracellular enzymes of microbial origin, allowing for the inexpensive manufacture of crude enzyme extracts. Such preparations, how- ever, can vary from lot to lot, especially in the case of many fungi, which may pro- duce isoforms of enzymes in varying amounts depending on cultivation conditions. 4 Modern genetic engineering techniques facilitate the economic production of pure enzymes and the preparation of tailor-made enzymes for specific applications. The genes of many lipases have been cloned, and overexpression of lipases in suitable hosts has been achieved. Moreover, distinct binding sites for the alcohol and acid moiety of esters were identified by X-ray structure analysis of various li- pases, and rational explanations for the stereoselectivity of these enzymes were pro- posed.
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