Mining bacterial genomes for novel arylesterase activity

Abstract

One hundred and seventy-one genes encoding potential esterases from 11 bacterial genomes were cloned and overexpressed in Escherichia coli; 74 of the clones produced soluble proteins. All 74 soluble proteins were purified and screened for esterase activity; 36 proteins showed carboxyl esterase activity on short-chain esters, 17 demonstrated arylesterase activity, while 38 proteins did not exhibit any activity towards the test substrates. Esterases from Rhodopseudomonas palustris (RpEST-1, RpEST-2 and RpEST-3), Pseudomonas putida (PpEST-1, PpEST-2 and PpEST-3), Pseudomonas aeruginosa (PaEST-1) and Streptomyces avermitilis (SavEST-1) were selected for detailed biochemical characterization. All of the enzymes showed optimal activity at neutral or alkaline pH, and the half-life of each enzyme at 50°C ranged from < 5 min to over 5 h. PpEST-3, RpEST-1 and RpEST-2 demonstrated the highest specific activity with pNP-esters; these enzymes were also among the most stable at 50°C and in the presence of detergents, polar and non-polar organic solvents, and imidazolium ionic liquids. Accordingly, these enzymes are particularly interesting targets for subsequent application trials. Finally, biochemical and bioinformatic analyses were compared to reveal sequence features that could be correlated to enzymes with arylesterase activity, facilitating subsequent searches for new esterases in microbial genome sequences.

Figure 1

The distribution of enzyme activities that were detected using a variety of esterase and lipase substrates. All assays were performed using standard conditions and were incubated for 30 min at 37°C. In total, 74 purified proteins were screened; the number shown in parentheses represents the number of proteins that demonstrated activity with each substrate, or that did not demonstrate activity with any of the test substrates.

Figure 2

Specific activity of 17 novel arylesterases against phenyl acetate. Reactions were performed using 30 mM of substrate and 1 µg 100 µl⁻¹ of protein. SWISSPROT accession numbers are given. n = 3; error bars correspond to standard deviation from the mean.

Figure 3

Sequence logo of active esterases. The MAFFT alignment of the 36 active enzymes isolated in this study was separated into three groups based on their specificity (see Table S2) and their sequence logos are shown aligned along with the sequence for P22862 for which a crystal structure is available. The following list indicates in parentheses, the position of amino acids in the gapped alignment of P22862 that are discussed in the text: W28 (W120), F93 (F212), M95 (M214), V121 (V254), F162 (F342), F198 (F382).