Surface packing characterization of Langmuir monolayer-anchored enzyme.

We have synthesized a novel interface-anchoring alcohol dehydrogenase by covalent attachment of a hydrophobic polymer tail to the hydrophilic protein head. Analogous to a protein-based surfactant, this polymer-enzyme conjugate self-assembled at liquid-liquid or liquid-air interfaces to form a membrane similar to other surfactant monolayers. The packing and morphology of the interface-anchored enzymes play an important role in regulating the membrane behaviors including enzyme mobility and interfacial interactions of enzymes with reactant and product molecules. To characterize the surface assembly morphology of the interface-anchored enzymes, Langmuir film balance and fluorescence microscopy techniques were used. The Langmuir isotherm of the interface-anchored enzyme demonstrated a pronounced molecular rearrangement upon compression of the isotherm. This corresponded to changes in membrane morphology and state observed using fluorescence microscopy. The molecular diffusion within the novel interface-anchored enzymes was further evaluated by using a fluorescence recovery after photobleaching technique. We report a diffusion coefficient of 6.7x10(-10) cm2/s. The study represents the first in-depth analysis of surface packing and interfacial mobility of such interface-anchored enzymes.

Stabilization of interface-binding chloroperoxidase for interfacial biotransformation.

The stability of an interface-binding chloroperoxidase (CPO) against the deactivation effect of H(2)O(2) was examined. Native CPO was conjugated with polystyrene and thus self-assembled at the water-oil interface. Although the interface-assembled CPO showed improved stability as compared to native CPO, enzyme deactivation as a result of the side effect of H(2)O(2), still limits the overall productivity of the enzyme. Two approaches to further improve the stability of CPO were examined in this work. In one approach, several stabilizers including poly(ethylene glycol) (PEG), PEI, glycerol, sugars and sucrose monododecanoate were used; while in a second approach, in situ generation of hydrogen peroxide (H(2)O(2)) by using glucose oxidase (GOx) was applied. PEG was found exceptional in that it increased both the operational and storage stability of CPO. The best improvement of enzyme productivity was obtained with addition of PEG which led to an increase of 57% for interface-bound CPO and 33% for native CPO. One interesting observation with PEI is that it enhanced the storage stability against H(2)O(2) deactivation, but did not affect the enzyme's operational stability. On the other hand, glucose enhanced the operational stability by two folds, but exhibited no significant effect on storage stability. It was also found that the extended operational lifetime of CPO with in situ generation of H(2)O(2) by GOx was a result that combines the stabilizing effect of glucose and lowered concentration of H(2)O(2). Interestingly, the addition of stabilizers could improve the enantioselectivity of CPO by as much as 10%.