Mining bacterial genomes for novel arylesterase activity.
Bottom Line: 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.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.
Affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, M5S 3E5, Canada.Show MeSH
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Mentions: While lipases and esterases are difficult to distinguish at the sequence level, differences in catalytic properties can be used to differentiate these enzymes. Therefore, each candidate gene was cloned into a pET15b‐based vector for heterologous expression in Escherichia coli. A total of 171 genes were cloned and 74 of them were expressed as soluble proteins in E. coli (Table S1). All 74 soluble proteins were purified to homogeneity and then screened using colorimetric assays; 36 proteins showed activity on pNP‐esters at pH 8 while 38 proteins did not exhibit any activity towards the test substrates (Fig. 1, Table S2). Phenyl acetate is a commonly used substrate to identify bacterial arylesterases (Shaw et al., 1994; Fenster et al., 2000). Seventeen purified enzymes demonstrated arylesterase activity; none of these hydrolysed olive oil to detectable levels (Fig. 2).
Affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, M5S 3E5, Canada.