Intracellular-to-extracellular localization switch of acidic lipase in Enterobacter cloacae : evaluation of production kinetics and enantioselective esterification potential for pharmaceutical applications.

Downstream processing is a significant part of a production process and accounts for 50-90% of the production cost of biotechnological products. Post-fermentation localization of a microbial metabolite contributes significantly to the recovery cost of the product. Enterobacter cloacae produced naturally, acidic lipase with a 0.023:1 extracellular localization ratio. This research aimed to re-direct the localization of lipase to the extracellular milieu to reduce recovery costs using multi-objective response surface optimization (MO-RSM). The approach resulted in a 1:0.32 extracellular: intracellular lipase ratio, with product formation kinetics of Luedeking-Piret function showing a significant switch from a completely growth-associated intracellular production to a predominantly non-growth-associated extracellular localization. The enzyme was purified by an aqueous two-phase system which extracted 95.22% lipase with 72.36 purity. Characterization of the enzyme showed a molecular weight of 55.7 kDa, kcat of 68.59 s -1 , and a K m of 0.63 mmol. Lipase activity occurred optimally at pH 2.5-3.5 and 50 °C, and was stable in most organic solvents tested. The acidic lipase demonstrated pH-dependent enantioselective esterification in resolving (R, S)-ibuprofen ( E  = 14, pH 4.5) and (R, S)-Naproxen ( E  = 13, pH 2.5), with an enantioselective preference for (S)-enantiomer in both drugs thus underpinning its potential for pharmaceutical applications.