Purification and Characterization of PrbA, a New Esterase fromEnterobacter cloacae Hydrolyzing the Esters of 4-Hydroxybenzoic Acid (Parabens)

The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 ± 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, withKm and Vmax values ranging from 0.45 to 0.88 mm and 0.031 to 0.15 mm/min, respectively. The Km and Vmax values for p-nitrophenyl acetate were 3.7 mm and 0.051 mm/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 °C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mm propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7- amino-2-heptanone (TLCK), withKi values of 0.29 and 0.20 mm, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine. The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 ± 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, withKm and Vmax values ranging from 0.45 to 0.88 mm and 0.031 to 0.15 mm/min, respectively. The Km and Vmax values for p-nitrophenyl acetate were 3.7 mm and 0.051 mm/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 °C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mm propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7- amino-2-heptanone (TLCK), withKi values of 0.29 and 0.20 mm, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine. 1-chloro-3-tosylamido-4-phenyl-2-butanone 1-chloro-3-tosylamido-7-amino-2-heptanone diisopropyl fluorophosphate 4-morpholineethanesulfonic acid high performance liquid chromatography The esters of p-hydroxybenzoic acid, commonly named parabens, are important preservative agents in the pharmaceutical, cosmetic, and food industries. Parabens are active over a wide pH range (pH 4–8), are colorless, odorless, nonvolatile, stable, and have a low acute and chronic toxicity and a broad spectrum of activity against molds, yeasts, and bacteria (1Haag T. Loncrini D.F. Cosm. Sci. Technol. Ser. 1984; 1: 63-77Google Scholar). The antimicrobial activity of the parabens increases with increasing alkyl chain length, although limitations on the use of longer chain-length parabens are imposed by their solubility in aqueous media. Hence, the methyl, ethyl, propyl, and butyl parabens are more commonly used in commercial formulations. Their anti-microbial effectiveness can be enhanced by combining two or more parabens in a formulation, with the total paraben concentration seldom exceeding 0.2–0.3% (2Gottfried N.S. Am. J. Hosp. Pharm. 1962; 19: 310-314Google Scholar, 3Rastogi S.C. Schouten A. de Kruijf N. Weijland J.W. Contact Dermatitis. 1995; 32: 28-30Crossref PubMed Scopus (177) Google Scholar). Resistance to parabens can lead to the survival and proliferation of microorganisms in commercial products that are normally well stabilized with these antimicrobial agents. One mechanism of resistance of bacteria toward parabens is hydrolysis of their ester bond. There are many such reported cases of resistance to the parabens in the literature. A strain of Cladosporium resinaeisolated from a pharmaceutical suspension containing 0.2% of methyl paraben was able to hydrolyze this paraben (4Sokoloski W.T. Chidester C.G. Honeywell G.E. Dev. Ind. Microbiol. 1962; 3: 179-187Google Scholar). A strain ofPseudomonas aeruginosa was able to grow and degrade parabens in an antimicrobial preparation used in the formulation of eye drops containing a mixture of methyl and propyl paraben at a total concentration of 0.3% (5Hugo W.B. Foster J.H.S. J. Pharm. Pharmacol. 1964; 16: 209Crossref PubMed Scopus (16) Google Scholar). The P. aeruginosa strain 396, isolated from an unpreserved oral formulation, was able to grow in the presence of 0.1–0.2% of methyl and propyl parabens and to hydrolyze propyl paraben to produce p-hydroxybenzoic acid (6Zedan H.H. Serry F.M. Egypt. J. Microbiol. 1984; 19: 41-54Google Scholar). A strain of Burkholderia cepacia isolated from an oil-in-water emulsion containing 0.1–0.2% methyl and propyl parabens was able to hydrolyze both parabens (7Close J.-A. Nielsen P.A. Appl. Environ. Microbiol. 1976; 31: 718-722Crossref PubMed Google Scholar). Although bacterial resistance to parabens has been reported in several species, there are few reports of specific enzymes with the ability to degrade parabens. The degradation of parabens by the resistant P. aeruginosa strain 396 was proposed to proceed through inducible intra- and extracellular esterases, although these enzymes were not isolated (6Zedan H.H. Serry F.M. Egypt. J. Microbiol. 1984; 19: 41-54Google Scholar). An esterase purified from Aspergillus flavuscapable to hydrolyze depside ester linkages was shown to be active against the methyl and ethyl parabens as part of its substrate specificity profile, although microorganism resistance to the parabens was not reported (8Child J.J. Oka T. Simpson F.J. Krishnamurty H.G. Can. J. Microbiol. 1971; 17: 1455-1463Crossref PubMed Scopus (17) Google Scholar). Additionally, four different carboxyl esterases capable of hydrolyzing parabens have been identified in human skin and subcutaneous fat tissues. Two such esterases from subcutaneous fat tissues were more active toward short chain parabens, whereas a third one from transformed keratinocytes was more active toward longer chain-length parabens such as butyl paraben (9Lobemeier C. Tschoetschel C. Westie S. Heymann E. Biol. Chem. Hoppe-Seyler. 1996; 377: 647-651Crossref PubMed Scopus (46) Google Scholar). A strain of Enterobacter cloacae was isolated from a contaminated mineral supplement formulation containing 1700 ppm (11.2 mm) and 180 ppm (1.0 mm) methyl and propyl paraben, respectively. The strain, named EM, was shown to be resistant to parabens through the action of an esterase that hydrolyzed the parabens to p-hydroxybenzoic acid (10Valkova N. Lépine F. Valeanu L. Dupont M. Labrie L. Bisaillon J.-G. Beaudet R. Shareck F. Villemur R. Appl. Environ. Microbiol. 2001; 67: 2404-2409Crossref PubMed Scopus (63) Google Scholar). Here, we report the purification and characterization of the PrbA esterase active against parabens and other analogues. All growth media, including tryptic soy broth and tryptic soy agar were from Difco. The CM-Sepharose Fast Flow was purchased from Amersham Biosciences, and the Avicell QMA and SP-5W columns were from Waters (Milford, MA). The methyl, ethyl, propyl, and butyl parabens and p-hydroxybenzoic acid as well as 1-chloro-3-tosylamido-4-phenyl-2-butanone (TPCK),11-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), diethyl pyrocarbonate, and hydroxylamine were purchased from Sigma. All the paraben analogues, p-nitrophenyl acetate,p-nitrophenol, and n-propanol were purchased from Aldrich. Methanol and ethanol were from EM Science (Gibbstown, NJ), and n-butanol was from A&C American Chemicals (Ville St.-Laurent, Quebec). Diisopropyl fluorophosphate (DFP) was obtained from Calbiochem-Novabiochem. Sequencing-grade modified trypsin was obtained from Promega (Madison, WI). Solid-phase extraction of tryptic peptides was carried out on C4 or C18 Ziptips from Millipore (Bedford, MA). Type D nanoflow probe tips for mass spectrometry were from Micromass Canada (Pointe-Claire, QC). A suspension of E. cloacaestrain EM was inoculated at an approximate cell density of 1 × 107 cells/ml in minimal Davis medium containing 1% glucose. The cells were incubated overnight at 37 °C, subsequently collected by centrifugation at 8000 rpm for 20 min, and resuspended in 20 mm MES-NaOH buffer, pH 5.5. The cells were lysed by a 3-fold French press treatment at a pressure of 1200 p.s.i. followed by a centrifugation step at 18,000 rpm for 45 min and 2 ultracentrifugation steps at 40,000 rpm for 90 min each. The cell-free protein suspension in 20 mm MES-NaOH buffer, pH 5.5, was filtered through a 0.2-μm membrane (Millipore) before application on the purification columns. The first purification step used a CM-Sepharose Fast Flow column with a 60:40 ratio of 20 mm MES-NaOH, pH 5.5, and 20 mm MES-NaOH containing 1 m NaCl for the first 30 min and followed by a gradient that reached 100% NaCl after 60 min, with a flow of activity was determined as previously (10Valkova N. Lépine F. Valeanu L. Dupont M. Labrie L. Bisaillon J.-G. Beaudet R. Shareck F. Villemur R. Appl. Environ. Microbiol. 2001; 67: 2404-2409Crossref PubMed Scopus (63) Google and the presence of PrbA was by of the active The active were and 20 mm buffer, pH The purification through an Avicell QMA used a gradient of to of 20 mm pH and 20 mm containing 1 m NaCl in 60 min at a flow of 2 The active were and 20 mm MES-NaOH buffer, pH 5.5, and subsequently purified through a SP-5W The gradient was to of 20 mm MES-NaOH, pH 5.5, and 20 containing 1 m NaCl within 60 min at a flow of 0.5 The active were subsequently and at All activity were in of m buffer, pH containing the paraben at of 0.5 or mm and at 30 The activity was measured after the addition of enzyme over an of The hydrolysis of the parabens and the acid was by to the previously (10Valkova N. Lépine F. Valeanu L. Dupont M. Labrie L. Bisaillon J.-G. Beaudet R. Shareck F. Villemur R. Appl. Environ. Microbiol. 2001; 67: 2404-2409Crossref PubMed Scopus (63) Google Scholar). The specific activities are in of p-hydroxybenzoic acid of activity were in and are reported with the and Vmax values for the parabens were determined using of mm of methyl and propyl paraben and of a 0.5 mm of butyl paraben in m buffer, pH All Km and Vmax values were by with the The Km and Vmax for p-nitrophenyl acetate were in of of a mm in buffer, pH containing to enzyme were The enzyme activity was measured by the of by at The was on the of a in containing The hydrolysis of p-nitrophenyl acetate was from All substrate analogues, including a of propyl paraben, were at of in m buffer, pH and to All with substrate were with enzyme as with ranging from to The enzyme activity was measured as the of substrate hydrolyzed as by as The of PrbA was obtained by the enzyme activity with paraben to the in at pH The pH was obtained by the enzyme activity at 30 °C in different A m was used at pH and at A was used at pH and a was used at pH All transesterification were to the as the substrate specificity using 0.5 mm methyl or propyl paraben in of m buffer, pH containing of or 5% of methanol or All were in The of enzyme activity after with or diethyl was to the as the substrate specificity using propyl at a concentration of was at the of the or the enzyme was with the for min at 30 °C before the addition of the of the of the diethyl by hydroxylamine was by the enzyme and for min at 30 °C followed by with hydroxylamine and the addition of the The for and TLCK was from the relative Km values for propyl paraben in the of the and in the presence of a concentration of or The stoichiometry of the addition of DFP or TLCK to PrbA was by PrbA in m buffer, pH with DFP or mm TLCK at 30 °C for 30 The modified enzyme was subsequently purified from the and by extraction with a C4 using a 60:40 with acid to acid was subsequently The molecular of the and modified enzymes was from several mass spectrometry using a mass with a in The molecular were in with a of and a of A concentration of PrbA in m buffer, pH was incubated with DFP at 30 °C for 30 this mixture was to 1 trypsin in an of buffer, pH for an overnight at 37 The tryptic peptides were purified by extraction with a C18 and with a 60:40 mixture containing the concentration of acid was to of the tryptic peptides were with at steps on a cell-free cloacae strain EM were to the esterase PrbA to The first through a CM-Sepharose in a increase in the specific activity of the The using an Avicell QMA was the in the specific activity after this step relative to the The with a SP-5W purified the enzyme to and the specific 60-fold relative to the activity of the cell-free The enzyme was purified to as determined by and its molecular mass was at The molecular of the enzyme was 54,619 ± 1 Da as measured by mass activity of PrbA after purification enzyme were with propyl protein concentration was measured by the × × × × × × × × All were with propyl The protein concentration was measured by the in a The specific activities of the purified enzyme were determined with the commonly used series of parabens, from methyl to butyl paraben, and are shown in The specific activity of PrbA is with ethyl paraben, The specific activity toward methyl paraben is as that with ethyl The activity with longer chain-length parabens, activity with propyl paraben and activity with butyl activities toward the methyl to butyl parabens and p-nitrophenyl ± ± ± ± in a The Km and Vmax values were determined for methyl paraben and for the longer chain-length propyl and butyl parabens The Km values as the alkyl chain length from 0.88 mm with methyl paraben to 0.45 mm with butyl The Vmax values with increasing chain length, from 0.15 with methyl paraben to 0.031 with butyl for p-nitrophenyl acetate is than that obtained with methyl paraben, whereas for p-nitrophenyl acetate is to the obtained with butyl The activity of PrbA toward several of the parabens was determined relative to the activity with propyl paraben The enzyme activity was with propyl paraben, although the activity with methyl was as The activity was obtained with methyl 4-aminobenzoate, at and with methyl at were obtained with methyl and ethyl The activity with the methyl methyl methyl and from to PrbA was not able to hydrolyze the ester of paraben the of activity of PrbA toward paraben The activity is as a of the activity obtained with propyl in a The activity is as a of the activity obtained with propyl The activity of PrbA at different an optimum activity at 31 °C and activity the of and The enzyme of its activity and 45 °C and more than activity at °C and The activity of PrbA at different pH values is at pH and the enzyme of its activity in the pH range The enzyme of its activity at pH values of and The activity of this at pH and at pH The enzyme is at pH PrbA was able to transesterify methyl or propyl parabens with of and n-butanol The of of propyl paraben was with methanol than in the and whereas the of acid was with methanol than and The of of methyl paraben was by in the presence of ethanol and by 60 and and n-butanol, respectively. The of of p-hydroxybenzoic acid in the presence of of these alcohols from to that The of of the paraben was that of the of of the paraben with and reached only of transesterification of methyl paraben with and n-butanol and of propyl paraben with of of of p-hydroxybenzoic paraben ± ± paraben ± ± ± ± ± ± ± ± ± ± ± ± ± ± paraben paraben in a Additionally, PrbA was able to out the transesterification of propyl paraben with 5% of methanol The of methyl paraben after 2 was 64% of the of propyl paraben in whereas at the the of the p-hydroxybenzoic acid was to of the of propyl paraben The of the paraben was as shown by the low of The transesterification with 5% ethanol was the 2 only of the propyl paraben was of ethyl paraben and of p-hydroxybenzoic acid relative to the of propyl paraben not of PrbA activity occurred DFP were at the of a hydrolysis with propyl enzyme was obtained by the addition of diethyl pyrocarbonate, and a of activity was measured after the addition of hydroxylamine to the diethyl enzyme not The addition of or TLCK in a and enzyme respectively. The and TLCK was and respectively. The addition of or TLCK the Km of PrbA toward propyl paraben by and only a on the Vmax that both of these as of PrbA not The mass spectrum of PrbA after with DFP showed only 1 at ± 1 Da not mass increase to the addition of 1 DFP molecule and the of Da from the The addition of Da a 1:1 stoichiometry of DFP addition to the The DFP-modified PrbA was The mass spectrum of TLCK-modified PrbA the of the enzyme at 54,619 ± 1 Da with at ± with of the not mass increase within the to the addition of one TLCK with the of Da from the other was to addition of of PrbA be at mm TLCK of TLCK a on the of the in a of enzyme the activity by The addition of one DFP molecule to PrbA the of the modified through of the tryptic containing the active in many The of this tryptic was with a molecular mass of An to the of this was identified and a low to the was found The was to the active by the the and the were the of the as Additionally, the and the to the were The at in the tryptic digest of the PrbA two other were identified by peptides not the active The first in a the and a molecular mass of was identified from the series of and The in a the and a molecular mass of was identified from the series of and by an at that was not in the tryptic digest of the enzyme to the molecular mass of the active with the addition of Da from the DFP to Da in the The was at a low The of the was as by the Da of its The at in the DFP-modified tryptic digest is to the presence of the two peptides The tryptic of the and DFP-modified PrbA the of a of peptides the active A total of peptides were identified from the PrbA of a total of tryptic An additional peptides were identified from tryptic that one of the the DFP-modified the total of identified peptides was with an additional peptides identified from with one of the the peptides were identified from the and DFP-modified although their relative were different of the from the DFP-modified PrbA that an active in the Although the four that DFP the of in the was the of this at to the or The spectrum of showed the to the with a mass addition of Da from the to the the addition of Da to at The is not its is by the the to the were identified as well as the and an mass of The and several other and to the of are to the series of to the were with a low at for the not The in mass at by Da the of DFP and the total of activity that is the active in the The esterase PrbA was purified from the strain of E. cloacae The molecular mass of the purified enzyme, as measured by was was by the molecular mass of the enzyme, from the of the as Da N. Lépine F. C. Dupont M. Villemur R. J. PubMed Scopus Google Scholar). The molecular mass of the enzyme as measured by mass spectrometry was 54,619 ± 1 to the addition of mass from a from the The esterase showed the specific activities toward the ethyl, methyl, and propyl parabens, indicating a for chain esters Although the Km values with increasing alkyl chain length a for the longer chain-length parabens, the Vmax values with methyl paraben that the of is with chain-length parabens. The of the enzyme acetate is as by than that for methyl paraben as well as by a low Vmax PrbA showed an activity toward methyl as for propyl paraben the activity with methyl was only that with propyl paraben, indicating that the be in the not in the for hydrolysis by The presence of two or in the in activity. The of at the or to only as shown with methyl The groups at the can be by such as an as with methyl 4-aminobenzoate, and PrbA of its activity. The methyl and methyl showed a that the presence of a at the in of activity. PrbA was capable of hydrolyzing methyl although in low that its activity was not only to ester analogues, as shown by its activity against PrbA not the ester of paraben to the at the by the of this paraben is by its solubility in The activity of PrbA to be to ester not the of the structurally It has been previously shown that a P. cepacia strain that hydrolyze low of parabens in the transesterification of ethyl paraben with alcohols T. R. J. Scopus Google Scholar). transesterification was to an the that hydrolyzed the the enzyme was not and the transesterification although for of ethyl paraben to methyl paraben with of methanol T. R. J. Scopus Google Scholar). The ability of PrbA to out paraben transesterification was with a series of alcohols of increasing chain length ranging from methanol to The and of transesterification was with methanol and ethanol and by with The of butyl paraben obtained transesterification was the only of the obtained with PrbA can out transesterification with methanol as high as 5% within a short of achieving 64% transesterification in 2 h. The in the active of PrbA were The of many esterases a of an active and The presence in the of the and was using to with these active The DFP is to with active Sci. S. A. PubMed Scopus Google Scholar). of PrbA with DFP in a with a 1:1 stoichiometry as by mass spectrometry. Analysis of the tryptic peptides of the DFP-modified PrbA showed that the modified was is part of the to the commonly found in carboxyl esterases T. M. Pharmacol. PubMed Scopus Google the of this as part of the active of An of the esterases to PrbA with the showed that this was these The TLCK with active A. E. Pharmacol. PubMed Scopus Google Scholar). one molecule of TLCK was found in the modified that the that was part of a the TLCK concentration used only of the enzyme was as by mass whereas the activity was by be to the relative of at the low pH for in The of the esterases more to PrbA showed that one was in of the was with the of the other esterases containing the is commonly found for the in the of T. M. Pharmacol. PubMed Scopus Google although was not within the of PrbA A of the in the of was in the the highly in was in of the The in PrbA was part of a modified was found in several other esterases of the A of esterases have previously been shown to have the ability to hydrolyze parabens, from P. or from human skin and subcutaneous fat as well as an esterase hydrolyzing linkages (6Zedan H.H. Serry F.M. Egypt. J. Microbiol. 1984; 19: 41-54Google Scholar, J.-A. Nielsen P.A. Appl. Environ. Microbiol. 1976; 31: 718-722Crossref PubMed Google Scholar, J.J. Oka T. Simpson F.J. Krishnamurty H.G. Can. J. Microbiol. 1971; 17: 1455-1463Crossref PubMed Scopus (17) Google Scholar, C. Tschoetschel C. Westie S. Heymann E. Biol. Chem. Hoppe-Seyler. 1996; 377: 647-651Crossref PubMed Scopus (46) Google Scholar). of these enzymes have been isolated or in their purified and their acid and other are The esterase PrbA the first carboxyl esterase for the hydrolysis of parabens, and was shown that the E. cloacae strain EM containing the was more resistant toward parabens than a E. strain that not have a activity N. Lépine F. C. Dupont M. Villemur R. J. PubMed Scopus Google Scholar). PrbA is active at pH and at a 30 °C, are that to commercial containing parabens. Two to the found in E. cloacae strain EM have been identified in isolated in N. Lépine F. C. Dupont M. Villemur R. J. PubMed Scopus Google Scholar). hydrolyzed parabens, and one of was more than a E. that of are more than Hence, the characterization of PrbA is an important step in and the growth of microorganisms in commercial formulations.

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