(544eu) Impact of Polymer-Based Protein Engineered ?-Chymotrypsin on Enantioselective Transesterification in Organic Media

Enzymes are able to catalyze reactions, such as transesterification or aminolysis with impressive stereo- or enantio- selectivity, in essentially organic media and ionic liquids. However, enzyme activity in organic media is often several orders of magnitude below that in aqueous conditions due mainly to lack of solubility and inefficient substrate binding. Many different strategies have been used to increase solubility and enzymatic activity in organic solvents. Specifically, conjugation of a variety of enzymes with synthetic polymers resulted in better solubility and activity increases. However, conjugates often exist as aggregates in organic media.

We have been developing next generation conjugates using polymer-based protein engineering to nano-armor proteins with densely conjugated polymers on the surface of proteins. We have previously examined the activity and stability response of nano-armor enzymes to a variety of stressors (pH and temperature) in aqueous media. Specifically, chymotrypsin-poly(2-(dimethylamino)ethyl methacrylate) (CT-pDMAEMA) prepared by atom transfer radical polymerization, showed pH-dependent activity and increased thermal stability.

Due to the amphiphilic quality of pDMAEMA and the “nano-armor” around the enzymatic core, CT-pDMAEMA conjugates can be molecularly dissolved and active in organic solvents. We first determined the effect of polymer conjugation on solubility of the conjugates in organic solvents. The hydrodynamic diameter of CT-pDMAEMA (D_h ~ 29 nm) was measured using dynamic light scattering in acetonitrile, whichwas equivalent to that in aqueous conditions (D_h ~ 34 nm), indicating that CT-pDMAEMA readily dissolves at the molecular scale in acetonitrile.

We have examined the kinetics of chymotrypsin-catalyzed transesterification and hydrolysis of N-acetyl L-phenylalanine thiophenyl ester (L-APTE). Specifically, we have investigated the effect of water content on the rate of reaction (k_cat) and substrate affinity (K_M). Since thiophenol is a byproduct of both the transesterification and hydrolysis reactions, we used 4,4’-dithiopyridine as a colorimetric indicator to quantify product formation. Thus, the resulting apparent k_cat and K_M values corresponded to the total rate of substrate removal. The conjugate exhibited good substrate binding with L-APTE (K_M ~ 17 mM). The activity (k_cat) of CT-pDMAEMA increased with increasing water concentration, while the consistent K_M values indicate that L-APTE affinity was not affected by water. The conjugate had an activity (peak activity 330 µM/min/mg enzyme) many orders of magnitude higher than that of the insoluble native chymotrypsin. In this presentation we extend our analysis to discuss the impact of molecular dissolution of nano-armored enzymes on enantioselectivity.