Enzyme catalyzed perhydrolysis, molecular basis and application

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Enzyme catalyzed perhydrolysis, molecular basis and application

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Enzyme catalyzed perhydrolysis converts a carboxylic acid or ester to a peracid. In the former reaction, the amount of peracid generated is thermodynamically controlled (Keq = 3) – while in the latter, the reaction is kinetically controlled, thus a higher concentration of peracid can be generated. Enzymes that catalyze perhydrolysis of carboxylic acids share high sequence similarity and are thought to use an esterase-like mechanism. Alternatively, carboxylic acids can also use a non-covalent mechanism, such as those used by hydroxynitrile lyases. To test whether carboxylic acid perhydrolases use an esterase-like mechanism, we identify a key covalent intermediate by mass spectrometry that can be attributed to an esterase-like mechanism but not a non-covalent mechanism. We also find that carboxylic acid perhydrolases are good catalysts for hydrolysis of peracetic acid, suggesting that their natural role is to degrade peracids generated as by-products in a living organism. Next, we determine how perhydrolases increase the rate of perhydrolysis. Carboxylic acid perhydrolases increase the rate of perhydrolysis by either increasing the selectivity for hydrogen peroxide or lower the activation barrier towards acylenzyme formation. We measure the selectivity of hydrogen peroxide using wild-type Pseudomonas fluorescens esterase (PFE) and L29P PFE (a model carboxylic acid perhydrolase). The L29P PFE variant is less selective for hydrogen peroxide than the wild-type despite having higher perhydrolysis activity. We measure the rate of acyl-enzyme formation using isotope exchange of acetic acid in H218O/H216O. The L29P PFE variant catalyzes the isotope exchange rate faster than the wild-type. Thus, carboxylic acid perhydrolases favor the formation of acyl-enzyme from carboxylic acids. We find that carboxylic acid perhydrolase (L29P PFE) does not catalyze ester perhydrolysis for accumulating high concentrations of peracetic acid. Instead, wild-type PFE and a new variant, F162L PFE accumulate up to 130 mM of peracetic acid. We measure kinetic parameters and show that hydrolysis of peracetic acid limits maximum accumulation. The F162L PFE variant minimizes hydrolysis of peracetic acid by lower ing the Km and increasing the kcat for ethyl acetate hydrolysis. The kinetic parameters are also used to predict the maximum amount of peracetic acid that can be accumulated. The F162L PFE variant is used to improve the efficiency of lignocellulose pretreatment from a previously published result using wild-type PFE. Enzymatically generated peracetic acid reacts converts lignin into smaller and more soluble lignin pieces. The chemoenzymatic process is further improved by forming peracetic acid in a biphasic layer which allows the reuse of enzyme. The pretreatment reaction conditions were also optimized by increasing the temperature to 60 °C and reducing the reaction time to 6 hours.


University of Minnesota Ph.D. dissertation. October 2011. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Romas J. Kazlauskas. 1 computer file (PDF); xviii, 268 pages, appendix p. 219-268.

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Yin, Delu (Tyler). (2011). Enzyme catalyzed perhydrolysis, molecular basis and application. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/117998.

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