Mining bacterial genomes for novel arylesterase activity.
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.
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, M5S 3E5, Canada.
Lijun
L
Wang
Valentina
V
Mavisakalyan
Elisabeth R M
ER
Tillier
Greg W
GW
Clark
Alexei V
AV
Savchenko
Alexander F
AF
Yakunin
Emma R
ER
Master
D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019295:Computational Biology; D004795:Enzyme Stability; D016680:Genome, Bacterial; D006863:Hydrogen-Ion Concentration; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases; C019734:arylesterase
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