Improvement of Lipases by Medium and Biocatalyst Engineering

Lipases can be used as biocatalysts in hydrolytic and synthetic reactions. These enzymes are the most widely used biocatalysts in organic chemistry and they have 

Lipases catalyze the hydrolytic cleavage of triglycerides into fatty acids and glycerol, or into fatty acids and mono or di-glycerides, at oil interfaces in nature. However, this hydrolytic reaction can be reversed and transformed into reactions of esterification (inter or transesterification), alcoholysis or aminolysis by engineering the medium polarity or the water content of the medium. Therefore, substrates for lipases can be esters, like the natural triglyceride substrates in hydrolytic reactions or, if the reaction is reversed, carboxylic acids, alcohols, amines or esters. The reaction medium not only determines the direction of the reaction (hydrolytic or synthetic), but also determines the solubility and stability of lipase substrates. Therefore, lipase activity and selectivity are strongly influenced by reaction medium. 

Lipases are able to work in very different media. They work in biphasic systems and in monophasic (in the presence of hydrophilic or hydrophobic solvents) systems where the water content can vary significantly between aqueous and anhydrous media. They have been tested also in ionic liquid media (Lau et al. 2000 Wasserscheid and Keim 2000 Kamal and Chouhan 2004 Ha et al. 2007), in supercritical fluids (Laudani et al. 2007) and in gaseous media (Cameron et al. 2002). The different media for enzymatic catalysis has been outlined before (see section 1.6) and it will not be further discussed here. However, some examples of modulation of activity and selectivity of lipases by medium engineering will be described in this section. 

Water activity of the reaction medium plays a central role in lipase catalyzed reactions (Berglund 2001). Different authors have described and demonstrated the usefulness of controlling water activity on lipase performance. In esterification reactions on cyclohexane media, the reaction rate increased with water activity in the low activity range however, it reached a maximum at a value of 0.84 and a subsequent increase in water activity led to a decrease in the reaction rate (Mat-sumoto et al. 2001). Results on the effect of water activity on enantioselectivity of lipases are rather contradictory (Berglund 2001). However, very good papers have been published in tuning lipase enantioselectivity by reaction medium engineering (Wehtje and Adlercreutz 1997 Matsumoto et al. 2001 Bomscheuer 2002). 

Recent advances in recombinant DNA technology, high-throughput technologies, genomics and proteomics, have fuelled the development of new biocatalysts and biocatalytic processes. In particular, site directed mutagenesis, directed evolution or metagenome approach are very valuable tools to enhance lipase properties. 

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