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Effect of PEG modification on subtilisin Carlsberg activity, enantioselectivity, and structural dynamics in 1,4‐dioxane
Author(s) -
Castillo Betzaida,
Solá Ricardo J.,
Ferrer Amaris,
Barletta Gabriel,
Griebenow Kai
Publication year - 2007
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21510
Subject(s) - chemistry , pegylation , catalysis , enzyme catalysis , ethylene glycol , enzyme , aqueous solution , subtilisin , organic chemistry , peg ratio , active site , combinatorial chemistry , polyethylene glycol , finance , economics
The employment of enzymes as catalysts within organic media has traditionally been hampered by the reduced enzymatic activities when compared to catalysis in aqueous solution. Although several complementary hypotheses have provided mechanistic insights into the causes of diminished activity, further development of biocatalysts would greatly benefit from effective chemical strategies (e.g., PEGylation) to ameliorate this event. Herein we explore the effects of altering the solvent composition from aqueous buffer to 1,4‐dioxane on structural, dynamical, and catalytic properties of the model enzyme subtilisin Carlsberg (SBc). Furthermore, we also investigate the effects of dissolving the enzyme in 1,4‐dioxane through chemical modification with poly(ethylene)‐glycol (PEG, M W = 20 kDa) on these enzyme properties. In 1,4‐dioxane a 10 4 ‐fold decrease in the enzyme's catalytic activity was observed for the hydrolysis reaction of vinyl butyrate with D 2 O and a 50% decrease in enzyme structural dynamics as evidenced by reduced amide H/D exchange kinetics occurred. Attaching increasing amounts of PEG to the enzyme reversed some of the activity loss. Evaluation of the structural dynamic behavior of the PEGylated enzyme within the organic solvent revealed an increase in structural dynamics at increased PEGylation. Correlation analysis between the catalytic and structural dynamic parameters revealed that the enzyme's catalytic activity and enantioselectivity depended on the changes in protein structural dynamics within 1,4‐dioxane. These results demonstrate the importance of protein structural dynamics towards regulating the catalytic behavior of enzymes within organic media. Biotechnol. Bioeng. 2008;99: 9–17. © 2007 Wiley Periodicals, Inc.