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Mining bacterial genomes for novel arylesterase activity.

Wang L, Mavisakalyan V, Tillier ER, Clark GW, Savchenko AV, Yakunin AF, Master ER - Microb Biotechnol (2010)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, M5S 3E5, Canada.

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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.
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f1: 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.

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).


Mining bacterial genomes for novel arylesterase activity.

Wang L, Mavisakalyan V, Tillier ER, Clark GW, Savchenko AV, Yakunin AF, Master ER - Microb Biotechnol (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815341&req=5

f1: 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.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON, M5S 3E5, Canada.

Show MeSH
Related in: MedlinePlus