Atomic Force Microscopy: A Useful Tool for Evaluating Aggregationof Lipases

Extract

Lipases (glycerol ester hydrolases, E.C. 3.1.1.3) are enzymes of great industrial interest due to their ability to catalyze a broad range of hydrolytic and synthetic reactions. They find applications in the synthesis of compounds used in clinical, nutritional, environmental, pharmaceutical and chemical fields. For example, lipases are used to catalyze key intermediate steps in the synthesis of biologically active compounds such as Naproxen, Ibuprofen and Atenolol [1]. Depending on the application, lipases may need to be purified and characterized biochemically before they can be used. However, the purification of microbial lipases is often made difficult by the presence of high molecular weight aggregates. These aggregates can form due to the presence, in the fermentation medium, of lipids used to induce the production of the enzyme by the microorganism or simply due to hydrophobic interactions amongst the enzyme molecules themselves [2]. In previous work, we characterized a new lipase produced by Bacillus megaterium CCOC P2637. The enzyme eluted in the void volume during gel filtration chromatography, indicating that it was present in the form of a high molecular weight aggregate. This aggregate was dispersed when a gradient of 60% (v/v) isopropanol was used, but formed again when the enzyme was injected in a gel filtration column for further purification, even when the elution buffer contained 20% (v/v) isopropanol. Further, when the enzyme was diluted in buffer (phosphate pH 7.0 20 mM) containing 30% isopropanol, its specific activity was double the activity obtained by diluting in buffer without isopropanol [3].