On the relationship between the activity and structure of PEG-alpha-chymotrypsin conjugates in organic solvents.

Enzymes are attractive catalysts for the production of optically active compounds in organic solvents. However, their often low catalytic activity in such applications hampers their practical use. To overcome this, we investigated the effectiveness of the covalent modification of alpha-chymotrypsin with methoxy poly(ethylene glycol) (PEG) with a Mw of 5,000 to enhance its activity. The model transesterification reaction between sec-phenethyl alcohol and vinyl butyrate in various neat dry organic solvents and at a controlled water activity of 0.008 in two solvents was employed to measure the effect of PEGylation on activity and enantioselectivity. Synthesis conditions were varied to obtain various conjugates with average molar ratios of PEG-to-chymotrypsin ranging from ca. 1 to 7. While the enantioselectivity increased only modestly from ca. 4.4 to 6.1 when averaging results in all solvents, PEG was very efficient in increasing the activity of alpha-chymotrypsin up to more than 400-fold compared to that of the powder lyophilized from buffer alone. The activity increase was more pronounced in apolar than in polar organic solvents and also depended on the amount of PEG bound to the enzyme. For example, the activity of the modified enzyme towards the most reactive "S" enantiomer in octane increased 440-fold but increasing the molar ratio of PEG-to-enzyme from 1.1 to 7.1 resulted in a more than twofold decrease in enzyme activity. Controlling the water activity did not prevent the drop in activity. To investigate the possible origin of the activity changes, Fourier transform infrared (FTIR) spectroscopy experiments were conducted. It was found that PEGylation reduced lyophilization-induced structural perturbations, but exposure to the organic solvents caused structural perturbations. These perturbations were more pronounced in polar than in apolar solvents. The pronounced activity drop in polar solvents at increasing PEG-modification levels correlated with an increasing level of solvent-induced structural perturbations. This correlation was less pronounced in apolar solvents where both, activity drop and structural perturbations, were less pronounced at increasing PEGylation levels. In summary, PEG-modified alpha-chymotrypsin might be an interesting system to catalyze reactions, particularly in apolar organic solvents.

Effect of the covalent modification of horseradish peroxidase with poly(ethylene glycol) on the activity and stability upon encapsulation in polyester microspheres.

Encapsulation of proteins in polyester microspheres by coacervation methods frequently causes protein inactivation and aggregation. Furthermore, an often-substantial amount of the encapsulated proteins is released within the first 24 h from the microspheres. To overcome these problems poly(ethylene glycol) (PEG) was employed as excipient and protein-modifying agent. The model protein horseradish peroxidase (HRP) was chemically modified or co-lyophilized with PEG of differing molecular weights, namely PEG(5000), PEG(20000), and PEG(40000). The lyophilized preparations were encapsulated in poly(D,L-lactide-co-glycolic) acid (PLGA) microspheres by a coacervation method. Covalent modification of HRP with PEG increased the encapsulation efficiency (EE) from 83% to about 100% while PEG when used as an excipient reduced the EE. Encapsulation caused aggregation of ca. 5% of non-modified HRP and the residual specific activity was only 57%. Covalent modification with PEG reduced HRP aggregation to less than 1% and improved its residual activity to more than 95%. When PEG was used as excipient similar results were found with respect to a reduction in encapsulation-induced aggregation, but no more than 80% of residual activity was obtained even for the best formulation after encapsulation. It was also found that covalent modification of HRP with PEG substantially reduced the unwanted initial "burst" release observed during the initial 24 h of in vitro release from about 70% to 23%. Furthermore, HRP activity and stability were also improved during in vitro release for HRP-PEG conjugates. The data show that covalent modification of proteins with PEG might be useful to improve protein stability during coacervation encapsulation and subsequent release as well as to increase EE and reduce the burst release.