Browsing by Subject "Esterase"

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    Enzyme catalyzed perhydrolysis, molecular basis and application
    (2011-10) Yin, Delu (Tyler)
    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.
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    Evolution-guided Engineering of Alpha/Beta Hydrolases
    (2017-06) Jones, Bryan
    This work applies principles from evolution to engineering enzyme properties. Specifically, by examining the phylogeny and evolved sequence diversity in a group of α/β-hydrolase fold enzymes from plants, we are able to engineer proteins with broader chemoselectivity, altered enantioselectivity, and increased stability. A number of ancestral α/β-hydrolases fold proteins were reconstructed in one set of experiments. These were more likely than related modern proteins to have relaxed chemoselectivities and, in one case, was more useful for synthesizing medicinally important molecules. Relative to modern enzymes, ancestral enzymes near functional branch points could catalyze more esterase and hydroxynitrile lyase reactions, as well as a number of other types of reactions: decarboxylation, Michael addition, γ-lactam hydrolysis, and 1,5-diketone hydrolysis. This finding helps to demonstrate the important role that enzyme promiscuity plays in the evolution of new enzymes. Additional experiments and structural analysis on one of these reconstructed ancestral enzymes, the early hydroxynitrile lyase HNL1 found that it is both more thermostable and more promiscuous than its modern relatives, HbHNL and MeHNL. X-ray crystallographic studies revealed, counterintuitively, that larger amino acids in the active site of the ancestor actually increased the size of the substrate binding pocket relative to modern relatives. To take advantage of the promiscuity observed in HNL1, it was used in the asymmetric synthesis of a precursor for the important pharmaceutical propranolol. Another set of experiments altered enantioselectivity by making phylogenetically informed mutations. The active sites from two related hydroxynitrile lyases, HbHNL and AtHNL, were modified to resemble their last common ancestor. This resulted in altered enantioselectivity, and in the case of AtHNL, reversed enantioselectivity. Surprisingly modeling suggested that some of these mutants use a previously undescribed mechanism. This may have been the extinct ancestral mechanism that served as an evolutionary stepping stone that allowed descendant lineages to diverge to either the S-HNL mechanism used by HbHNL, or the R-HNL mechanism used by AtHNL. A final set of experiments used a variety of methods to identify stabilizing mutations in another plant α/β-hydrolase, SABP2. All of the methods were able to identify stabilizing mutations. The most stabilizing mutations were identified by methods that used no structural information. Random mutagenesis identified highly stabilizing mutations, but required screening thousands of mutants. The most efficient approaches were found to be those that used sequence information from either one stable homolog, or the consensus of many homologs, to identify potential stabilizing mutations. Residues that evolution has conserved are often important for stabilizing a protein. We created a software application, Consensus Finder, to automate the process of identifying stabilizing mutations by consensus.