On the relationship between the activity and structure of PEG‐α‐chymotrypsin conjugates in organic solvents

Abstract

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 PEG modification on subtilisin Carlsberg activity, enantioselectivity, and structural dynamics in 1,4‐dioxane

“…Covalent modification of SBc with PEG was performed as previously described (Castellanos et al, 2005;Castillo et al, 2006). To obtain different levels of protein modification, molar ratios of 3.0, 6.0, and 9.0 of activated PEG-to-protein were used.…”

“…Determination of the average PEG molar content of the conjugates was done via the TNBSA chromogenic assay as previously described (Castellanos et al, 2005;Castillo et al, 2006;Habeeb, 1966). Protein samples of SBc and of the different PEG-SBc conjugates were dissolved in 0.1 M sodium bicarbonate buffer (pH 8.5) to achieve concentrations of 0.20, 0.10, and 0.05 mg/mL.…”

“…However, substantially reduced enzymatic activities that are generally observed in organic solvents when compared to catalysis in aqueous solution are a major drawback still diminishing their potential as useful catalysts (Castillo et al, 2006;Klibanov, 1989;Schmitke et al, 1996;Secundo et al, 2001). Reduced enzyme activities have been linked to factors, such as, diffusional limitations (Yamamoto and Kise, 1993), pronounced structural perturbations induced by the initial lyophilization process (Griebenow and Klibanov, 1995, 1997Griebenow et al, 1999b;Kendrick et al, 1997;Prestrelski et al, 1993a), unfavorable substrate desolvation energetics (Halling, 1994;Wescott and Klibanov, 1993), and reduced conformational dynamics (Burke et al, 1993;Griebenow et al, 2001;Hutcheon et al, 2000;Rupley and Carreri, 1991;Tsou, 1998).…”

“…Since one of the principal causes for the diminished activity within organic media is believed to be the heterogeneous catalytic nature of enzymes due to their lack of solubility, several strategies have been explored to ameliorate this (e.g., employment of hydrophobic ion pairing with detergents, lipid-coating of enzymes, and chemical modification of the enzyme) (Castillo et al, 2006;Inada and Matsushima, 1990;Inada et al, 1995;Koops et al, 1999;Kwon et al, 1999;Noda et al, 1997;Okahata and Mori, 1997;Paradkar and Dordick, 1994a,b).…”

“…In this way, substrate diffusional limitations were expected to be minimized since the enzyme now performed as a homogeneous catalyst. Subsequent studies employing this approach reported enhanced properties for the PEGylated enzymes within the organic media, such as, increased stability, substantially improved catalytic activities, and altered enantioselectivies (Castillo et al, 2006;Inada et al, 1995;Koops et al, 1999;Kwon et al, 1999). PEG-modified lipases showed enhanced activity and stability in organic solvents (Salleh et al, 2002).…”