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Bioprospecting for novel lipases from indigenous olive wastewater biofilms
Lipase-catalysed chemical transformations are today routinely considered by synthetic organic chemists as economical and competitive “green chemistry” alternatives. Although lipases can effortlessly be produced on a large-scale by fermentation, their industrial application was, until recently, limited to the detergent, oleo-chemistry and dairy industry. However, during the last few decades, the biotechnological application of lipases has expanded significantly, becoming indispensable in the manufacture of pharmaceuticals, pesticides, single cell protein production, biosensor preparations and waste management. Similarly, lipases have become a vital ingredient in the contemporary food processing industry with applications ranging from fruit juice production to baked foods, vegetable fermentations and dairy enrichment. Furthermore, lipases are routinely used as flavour development agents in cheese, butter and margarine products. Lipases are also applied in the leather industry for processing hides and skins and for treatment of activated sludge and other aerobic waste product treatments where its action enhances oxygen transfer. While lipases currently account for less than 21 % of the enzyme market, a growing interest in lipases is reflected by the publication of an average of 1000 research papers per year and the growing number of available lipases since the 1980s. There is a sustained interest to bioprospect for novel lipase enzymes from available unexplored biodiversity. This study aimed to screen for lipase-producing microorganisms resident in olive wastewater biofilms. Lipase activity of positive isolates was subsequently also quantitatively determined to select for the highest producers of true lipases. A Geotrichum candidum isolate from olive mill wastewater biofilms was selected for subsequent studies based on its superior lipase production phenotype. Using a yeast mediated ligation approach the G. candidum GCL1 lipase gene was cloned and heterologously expressed in Saccharomyces cerevisiae as an enzyme production host. The recombinant lipase was purified and analysed in terms of substrate specificity, pH optima, temperature optima and stability as well as organic solvent tolerance. The G. candidum gcl1 lipase presented enhanced thermo- and organic solvent-stability that are highly sought after traits for industrial application.