JOURNAL OF BACTERIOLOGY, June 1974, p. 880-889Copyright © 1974 American Society for Microbiology

Vol. 118, No. 3Printed in U.S.A.

Purification and Properties of Undecyl Acetate Esterase fromPseudomonas cepacia Grown on 2-Tridecanone

A. C. SHUM AND A. J. MARKOVETZDepartment of Microbiology, University of Iowa, Iowa City, Iowa 52242

Received for publication 14 January 1974

Undecyl acetate esterase has been purified from Pseudomonas cepacia grownon the methyl ketone, 2-tridecanone. The Km for undecyl acetate was 2.3 x 10-2M. Polyacrylamide gel electrophoresis indicated that two esterase bands werebeing recovered during purification. These bands were separated by preparativepolyacrylamide gel electrophoresis. Molecular weights were estimated to beapproximately 34,500 by several methods. Molecular sieve polyacrylamide gelelectrophoresis indicated that the two esterases had the same molecular weightbut different charge, which is indicative of isoenzymes.

The natural origin and distribution of methylketones has recently been reviewed (15). Lack ofextensive accumulation of these compoundsindicates that an efficient recycling process isoperative. The first report on the isolation andcharacterization of an intermediate from thecatabolism of any methyl ketone, other thanacetone, was the formation of 1-undecanol from2-tridecanone by a pseudomonad (16), lateridentified as Pseudomonas multivorans (12).Formation of 1-undecanol in large quantitiesindicated that a mechanism other than methylgroup oxidation was occurring whereby the C13ketone was split to a C,1 alcohol. Subsequently,an intermediate, undecyl acetate, was isolatedthat would account for this result (12). Thisacetate ester was cleaved to form 1-undecanolplus acetate. These reactions were also carriedout by P. aeruginosa, and this organism wasused for preliminary cell-free enzymatic studieswith [3- "4C ]2-tridecanone (13). Unfractionatedextracts, in the presence of oxygen and reducedpyridine nucleotide, formed labeled undecylacetate. Hydrolysis of the recovered esteryielded radioactive 1-undecanol. When undecyl[2- "C ]acetate was employed, labeled acetatewas detected. Based on the evidence providedby metabolic products isolated and identifiedfrom studies using whole cells and cell-freeextracts, it was proposed that the biodegrada-tion of the long-chain methyl ketone, 2-trideca-none, by pseudomonads proceeds through anester intermediate, undecyl acetate, that ishydrolyzed to 1-undecanol and acetate. Al-though both oxygenase and esterase activitiesresponsible for the catabolism of 2-tridecanonehave been demonstrated in unfractionated ex-

tracts from cells grown on the methyl ketone,these enzymes have not been purified. Thisstudy was undertaken in an attempt to purifyundecyl acetate esterase and to investigate itsproperties.

MATERIALS AND METHODSOrganism and cultural methods. P. multivorans

strain 4G9, isolated by Forney, Markovetz, and Kallio(16) and characterized by Forney and Markovetz (12)was used. This organism has now been designated asP. cepacia according to the report that the newspecies, P. multiuorans, proposed by Stanier, Pal-leroni, and Doudoroff (37), is so similar to P. cepaciathat the former name should be regarded as a syn-onym (4). Media and cultural methods were describedpreviously (12). Cells used for enzyme purificationwere cultured in a Micro-Ferm fermentor (NewBrunswick Scientific Co., New Brunswick, N.J.) at 30C, 400 rpm stirrer speed, and aeration at 12 liters/min. Usually 2 liters of log-phase cells were inoculatedinto 10 liters of basal-salts medium supplementedwith 0.4% 2-tridecanone.

Esterase and protein assays. Three methods forquantitative esterase activity were employed. Oneenzyme unit is defined as 1 gmol of substrate hydro-lyzed per min.

(i) Colorimetric assay. Hydrolysis of p-nitrophe-nyl acetate (PNPA) was used and the quantitativeassay described by Bier (6) was followed. The reactionmixture contained 200 nmol of PNPA in 3.0 ml of 0.05M potassium phosphate buffer (pH 7.5). The concen-tration of enzyme in the mixture was such thatabsorbance of 0.30 would not be exceeded after 3 min.Change of absorbance was monitored at 400 nmduring assay at 25 C. This method was rapid butrelatively nonspecific (1, 19, 20, 38). Therefore, it wasused only to locate enzymatic activity in eluatesduring fractionation.

(ii) Radioactivity assay. The technique for mea-880

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suring hydrolysis of undecyl [2- 4C ]acetate into 1-undecanol and [1-_4C ]acetate as described by Forney(Ph.D. thesis, University of Iowa, Iowa City, 1969) wasmodified. Undecyl [2- 4C ]acetate was homogenizedfor 1 min in 3% Triton X-155 (wt/vol) and 0.1% gumarabic (wt/vol) by means of a Virtis homogenizer. Thereaction mixture contained 205 gmol of undecyl[2-1"C lacetate (2,177 disintegrations per min perumol), 50 to 100 Amol of potassium phosphate buffer(pH 7.5), and the designated amount of enzyme to afinal volume of 1.5 ml. For each assay, the reactionwas carried out in a polyethylene hollow stopper (size6) which was placed in a Dubnoff metabolic shakingincubator at 30 C. The assay was initiated by addingthe radioactive substrate to the preincubated enzyme-buffer mixture, and the reaction time varied from 5 to30 min. The reaction was stopped by the addition of0.5 ml of 1 M zinc acetate, the mixture was shaken foranother 5 min, and 1.0 ml of tritiated acetate ofknown specific radioactivity was added to serve as aninternal standard, thereby permitting the monitoringof recovered [14C Jacetate. The mixture was shaken for10 min to insure mixing, was transferred with aPasteur pipette to a test tube (16 by 100 mm), andwas centrifuged at 2,000 x g for 10 min to removeprecipitated protein. After centrifugation, the entiresupernatant fluid was transferred with a Pasteurpipette from the tube to a column containing 1.5 g ofDowex 21K (pretreated with 2 M NaOH) and 1.5 gChelex-100 in the sodium form. After the radioactivematerials were washed completely into the ion-exchange column, the column was washed with an-other 100 ml of deionized water. Elution of radioactiveacetate was achieved by running 1.0 M NH4NOSthrough the column until 25 ml of eluate had beencollected in a 25-ml volumetric flask. One milliliter ofthe eluate was pipetted into a scintillation vialcontaining 15 ml of scintillation cocktail (Insta-Gel,Packard Instrument Co.), and the vial was counted ina model 2420 Tri-Carb liquid scintillation spectrome-ter (Packard Instrument Co.). The efficiency correla-tion curves for 3H and "4C were achieved by thescreening method as described by Okita et al. (33).

(iii) Manometric assay. The conventional ma-nometric determination for CO, evolved fromNaHCOs as acetic acid or other aliphatic acids arewhich liberated from acetate or aliphatic esterswas employed at 30 C and pH 7.5 (41). In the maincompartment of the Warburg flask, 63 umol ofNaHCOs and the enzyme preparation were mixed.The side arm contained 82 ,umol of undecyl acetatehomogenized in 3% Triton X-155 and 0.1% gumarabic. The final reaction volume was 3.0 ml. Whenlarge amounts of substrate were used, the maincompartment contained 63 Amol of NaHCO, and 770Amol of ester, and the enzyme preparation was placedin the side arm. Flasks were attached to the manome-ter and the system was gassed with a mixture of 5%CO, and 95% N, with the side-arm vents open. Aftergassing for 10 min, side-arm vents and stopcocks wereclosed, the pressure was adjusted such that the fluidin the open arm of the manometer read between 60and 80 gliters, and the flasks were equilibrated for 10min. Enzyme reactions were initiated by tipping the

contents of the side arm into the main compartment.Determination of protein was by the method of

Lowry et al. (28). Bovine serum albumin was used toprepare a standard curve.Enzyme purification. A cell suspension (approxi-

mately 25% [wt/vol] in 0.01 M phosphate buffer, pH7.5) was placed in an ice bath and subjected to sonicdisruption (Branson model W-185C) at 120 W by asuccession of 1-min exposures, totaling 30 in number.Cellular debris was removed by centrifugation at20,000 x g for 30 min at 4 C. The supernatant fluid,recovered by aspiration to avoid collecting unusedsubstrate and other fatty materials floating on thesurface, was extensively dialyzed against 0.01 Mpotassium phosphate buffer (pH 7.5), and was recen-trifuged.Ammonium sulfate (521.7 g) was added slowly with

stirring to 1,300 ml of the supernatant fluid. Thepellet obtained by centrifugation at 15,000 x g for 30min was suspended in 250 ml of buffer, dialyzedagainst three 14-liter portions of the same buffer, andconcentrated with an Amicon ultrafiltration cell usinga PM-10 membrane to 210 ml. This 210-ml samplewas applied to a column (5.0 by 60 cm) containing 760g of diethylaminoethyl (DEAE)-cellulose (WhatmanDE-52), and 1,200 ml of 0.01 M potassium phosphatebuffer (pH 7.5) was passed through the column.Elution of undecyl acetate esterase was carried outwith a KCl gradient between 1 liter of 0.01 Mphosphate buffer and 1 liter of the same buffercontaining 1.0 M KCl. Pooled fractions containingactivity were dialyzed extensively, and the samplewas concentrated to 72 ml. Seventy milliliters of thesample was applied to a column (2.5 by 90 cm) ofSephadex G200-120 that was linked to a secondcolumn (2.0 by 95 cm) of Sephadex G200-120. Frac-tions containing high enzyme activity were pooled,concentrated to 14 ml, and 13 ml was chromato-graphed on a column (2.5 by 90 cm) of SephadexG200-120. Pooled fractions containing enzyme activ-ity were concentrated to 17 ml, and 16 ml was appliedto a DEAE-cellulose (DE-52) column (1.5 by 55 cm).Undecyl acetate esterase was eluted with a KClgradient between 500 ml of 0.01 M potassium phos-phate buffer (pH 7.5) and 500 ml of 0.8 M KCl in thesame buffer. The pool of enzyme fractions wasdialyzed against three changes of 7 liters of buffer andconcentrated to 11 ml.

Further purification was achieved by preparativepolyacrylamide gel electrophoresis using a Poly-Prep200 apparatus (Buchler Instrument Co., Fort Lee,N.J.). The method of Davis (8), as modified by Jovin,Chrambach, and Naughton (23), was employed forthe gel system. A 9-cm resolving gel of 10% acrylamideand 0.26% N, N'-methylenebisacrylamide was polym-erized at room temperature for 2 h. The total mixturefor polymerization was 160 ml. Spacer gel at 20% theamount of the resolving gel was added and polymer-ized at 4 C overnight. The lower chamber buffer was0.1 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 8.1), and the upper chamber bufferwas 0.025 M Tris-0.19 glycine (pH 8.3) (23). Elutionbuffer was the same as the lower chamber buffer.After polymerization of the spacer gel, 32 ml of

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enzyme solution (30 mg of protein, 10% sucrose, and0.00025% bromophenol blue in 0.03 M Tris-PO4buffer, pH 6.9) was overlaid onto the spacer gel belowthe upper chamber buffer by means of a New Bruns-wick peristaltic pump. A constant current of 50 mAwas maintained through the spacer gel until thetracking dye, bromophenol blue, migrated into theresolving gel. At this point the current was increasedto 100 mA. The elution buffer was maintained at a

flow rate of 1.0 ml/min by a Multi-Staltic pump(Buchler Instruments), and 5-ml fractions were col-lected. Protein concentrations could not be deter-mined spectrophotometrically on the fractions be-cause of continuous elution of ultraviolet (UV)-absorbing material. Therefore, fractions were moni-tored for esterase activity, and a major and a minorpeak were detected. Fractions corresponding to thesepeaks were pooled, concentrated by ultrafiltration,and dialyzed against 0.01 M potassium phosphatebuffer (pH 7.5). Analyses of the two pooled peakfractions with analytical polyacrylamide gel electro-phoresis showed that the major fast-moving peakcontained two esterase bands, and the minor slow-moving peak had only one esterase band. To separatethe two esterase bands in the major peak, this samplewas subjected to a second preparative electrophoresisrun with a 9-cm resolving gel of 15% acrylamide and0.4% N,N'-methylenebisacrylamide.

Analytical electrophoresis. Polyacrylamide gelelectrophoresis was carried out by the method ofDavis (8) with a Polyanalyzer (Buchler InstrumentsCo.). Gels containing 7% acrylamide were used unlessspecified. Protein samples were added as 10% glycerolsolutions. A constant current of 1.5 mA/gel was

maintained for migration through the spacer gel. Assoon as the tracking dye, bromophenol blue, migratedinto the resolving gel, the current was increased to 3mA/gel. After each run, the position of the trackingdye was marked by insertion of a fine-gauge steel wire.Esterases were located using the method of Dulaneyand Touster (9). Approximately 100 mg of Fast BlueRR salt was added to an ice-cold solution of 100 ml of0.1 M potassium phosphate buffer (pH 7.5), and themixture was rapidly and thoroughly mixed with a

magnetic stirrer, followed by suction filtration of thesolution through Whatman no. 1 filter paper. About1.0 ml of a solution of 10 mg of a-naphthyl acetate in1.0 ml of methanol was added dropwise to thediazonium salt solution with vigorous mixing toprevent precipitation. The gel was placed in thesolution and incubated in ice until brown or purplishbands of a-naphthyl diazotate appeared (1 to 30 min).If cloudiness occurred, the gel was transferred to a

fresh solution. The reaction was stopped by fixing thegel in 7% acetic acid.

Protein was stained by immersing gels for 1 h in a

filtered solution of 7% acetic acid containing 1%amido schwarz. Gels were destained with severalchanges of 7% acetic acid over a period of 1 to 2 days.When Coomassie brilliant blue was used for proteinstaining, the method of Weber and Osborn (42) was

followed.Gel filtration. A column of Sephadex G150 (2.5 by

50 cm) was equilibrated at 4 C with 0.01 M phosphate

buffer at pH 7.0, and the void volume of the columnwas determined with 1.0 ml of freshly prepared bluedextran 2000 (1 mg/ml). The calibration curve wasestablished with the purified protein standards. Twoseparate sample runs were employed, with aldolaseand chymotrypsinogen A being applied in one run,followed by ovalbumin and ribonuclease A in thesecond run. After standardization, a partially-purifiedesterase was filtered through the column, the elutionvolume was monitored by assaying for activity, andthe molecular weight was estimated by the usual plot(43).

SDS-polyacrylamide gel electrophoresis. Theelectrophoresis of dissociated undecyl acetate esterasein 0.1% sodium dodecyl sulfate (SDS) was performedusing the method described by Weber and Osborn(42). Samples (50 to 100 vg of protein) were added to1% SDS and 1% 2-mercaptoethanol in 0.01 M phos-phate buffer (pH 7.0), and were incubated for 3 h at 37C. Electrophoresis in gels containing SDS was per-formed at a constant current of 8 mA/gel at room

temperature, and gels were stained with Coomassiebrilliant blue. Relative electrophoretic mobilities were

calculated according to Weber and Osborn (42).Ribonuclease A, chymotrypsinogen A, ovalbumin,and bovine serum albumin were used as markers. Aplot of log molecular weight of markers versus theirmobilities provided a standard curve.

Molecular sieving. The molecular sieving effect inpolyacrylamide gel, as described by Hedrick andSmith (21), was also used for molecular weightdeterminations. Gels were prepared by the method ofDavis (8) with gel concentrations of 6.75, 7.50, 8.25,9.75, 10.50, and 11.25%. Electrophoresis was carriedout at 4 C using 5.0-cm resolving gels and 1.0-cmstacking gels. The gels were stained for protein withCoomassie brilliant blue, or for esterase activity as

described above. A plot of log mobility versus percentof gel concentration gave a negative slope for eachprotein marker used. When negative slope values were

plotted against molecular weight, a calibration curve

was established.Sedimentation studies. Sedimentation equilib-

rium was carried out at 20 C using a 12-mm double-sector cell (aluminum-filled Epon), sapphire win-dows, and a Spinco model E analytical ultracentri-fuge equipped with a photoelectric scanner. Themethod used is described by Chervenka (7), and hasbeen used extensively by Schachman (35). The puri-fied enzyme was dialyzed overnight against 0.2 MNaCl in 0.01 M phosphate buffer (pH 7.5). The An-Drotor was used for the operation with rotor speed at12,000 rpm for 24 h. Base-line corrections for the UVscan were made at 12,000 rpm after the experimentalrun using the solvent instead of the protein solution inthe measuring sector. Log absorbance at 280 nm

versus the square of the distance (r) from the axis ofthe rotor was plotted, and the slope of the graph was

used for molecular weight calculation (40).Chemicals. Materials were obtained as follows.

Triton X-100 and X-155, streptomycin sulfate, p-

nitrophenylacetate, a-naphthylacetate, Fast Blue RRsalt, Coomassie brilliant blue R, and Trizma Basewere from Sigma Chemical Co., St. Louis, Mo. SDS,

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2- mercaptoethanol, acrylamide (electrophoresisgrade), N, N, N', N'-tetramethylethylenediamine,N,N'-methylenebisacrylamide, and riboflavine were

from Eastman Organic Chemicals Co., Rochester, N.Y. Amido schwarz was from Allied Chemicals, Morris-town, N. J. Crystallized bovine serum albumin andammonium sulfate, enzyme grade, were from MannResearch Laboratories, New York, N. Y. 2-Tridecanolwas from K and K Laboratories, Inc., Plainview, N.Y. 2-Tridecanone (97%) and 1-undecanol (99%) were

from Chemical Samples Co., Cleveland, Ohio. Dowex21K (50 to 100 mesh, Cl- form) and Chelex 100 (100to 200 mesh, Na+ form) were from Bio-Rad Laborato-ries, Richmond, Calif. A gas mixture, 5% CO2 and 95%N2, was from Matheson Gas Products, Joliet, Ill., andSephadex G200-120 and G150, aldolase, chymotryp-sinogen A, ovalbumin, and ribonuclease A were fromPharmacia Chemicals, Inc.

Undecyl acetate was synthesized by refluxing equi-molar amounts of 1-undecanol and acetic anhydridefor 5 h in dry, redistilled pyridine. The ester was

purified on a column of Adsorbosil-CAB (1.5 by 100cm) (Applied Science Labs, State College, Pa.)packed from a slurry in redistilled hexane. Hexane-diethyl ether (90: 10 vol/vol) was used as the elutingsolvent. Fractions were collected and analyzed bygas-liquid chromatography, and those fractions con-taining only undecyl acetate were pooled and thesolvent was removed. Radioactive undecyl [2- 4C ]ace-tate was synthesized as above except [1-_4C]aceticanhydride was used. The ester had a specific activityof 2.2 x 101 disintegrations per min per mmol.

[1- 4C ]acetic anhydride, specific activity 122 mCi/mmol, and sodium [3H ]acetate, specific activity 500mCi/mmol, were obtained from Amersham/SearleCorp., Des Plaines, Ill. Sodium [1-_4C]acetate, spe-cific activity of 2.2 mCi/mmol, was obtained fromNew England Nuclear Corp., Boston, Mass.

Thin-layer radiochromatography. To check thereversibility of the esterase, 500 umol of sodium[1-"1C]acetate (2.5 x 101 dpm), 500 Mmol of 1-undecanol, and purified esterase were shaken for 1 hat 30 C. The reaction mixture was extracted withhexane. The hexane solution was reduced in volume,was spotted on a strip of Baker-Flex Silica Gel 1B (5by 20 cm) (Baker Chem. Co., Phillipsburg, N.J.) andwas developed in n-butanol saturated with NH. for 5to 6 h. Radioactive spots were detected by scanningwith a Radiochromatogram Scanner, model 7201(Packard Instrument Co.), using a gas mixture of 1.3%butane and 98.7% helium. R, values for known unde-cyl [2-"4C]acetate and [1-_4C]acetate were 0.80 and0.13, respectively.

RESULTS

Purification of undecyl acetate esterase. Asummary of the data from the various purifica-tion steps is presented in Table 1. Spectralanalysis of the crude extract (280/260 = 0.64)revealed the presence of approximately 15%nucleic acids. Treatment of the extract withstreptomycin sulfate effectively precipitated a

large amount of nucleic acid, with a 280/260ratio of 1.20 indicating the presence of less than2%. Most of the enzymatic activity was re-

covered in the precipitate formed in the 60%(NH4)IS04 saturation step. DEAE-cellulosechromatography resolved three peaks that hy-drolyzed undecyl acetate. However, only themajor peak showed good specific activity. Frac-tions corresponding to this peak were pooledand subjected to further purification by Sepha-dex G200 gel filtration. A single enzyme activity

TABLE 1. Purification of undecyl acetate esterasea

TotalTotal ~~~~~~~~~Purifi-Fraction : Vol Tprotaln Totativityo Sp act Yield cPFraction ~~(Ml) protein activity cUIg)Mation(mg) (U)' Umg % (fold)

1. Extract 1,150 10,730 31,223 2.9 100 12. Streptomycin sulfate 1,300 6,363 31,560 5.0 101 2

supernatant3. (NH4),SO4 (60% saturation) 210 3,986 28,618 7.2 92 34. DEAE-cellulose 72 1,866 27,386 15 88 55. 1st Sephadex G200 14 323 10,497 33 34 116. 2nd Sephadex G200 17 170 10,572 62 34 217. DEAE-cellulose 11 34 5,417 159 17 558. 1st polyacrylamide gel 25 11 1,846 168 6 58

electrophoresis9. 2nd polyacrylamide gel

electrophoresisFraction A 6.7 0.7 134 191 0.4 66Fraction B 8.4 5.2 1,217 234 3.9 80Fraction C 10.8 0.2 45 223 0.2 77

a Enzyme activities were determined by the radioactive assay. Undecyl [2- 'C ]acetate at a final concentra-tion of 0.2 M was employed throughout with protein concentrations ranging from 0.01 to 0.1 mg/1.5 ml.

b U = micromoles of undecyl acetate hydrolyzed per min.

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peak was detected that eluted from the columnafter most of the 280 nm-absorbing material waseluted. When fractions corresponding to thisactivity peak were pooled and chromatographedon a second column of Sephadex G200, a singleenzymatic peak that corresponded to the majorprotein peak was eluted from the column (Fig.1). However, polyacrylamide gel electrophoresicanalysis of the pooled peak fractions showedseveral protein bands and three esterase bands.Hence, the pooled peak fractions were furtherpurified by DEAE-cellulose chromatography(Fig. 2). The enzyme activity peak coincidedwith the major protein peak, and three esterasebands were still present in the pooled peakfractions when analyzed with polyacrylamidegel electrophoresis.To separate the three esterase bands, the

pooled peak fraction was subjected to prepara-

0 30 4%0 50 60 70 80 90 100 110Froction Nurnber

FIG. 1. Second Sephadex G200 gel filtration ofundecyl acetate esterase. Five-milliliter fractions werecollected and assayed for protein (0). Enzyme activ-ity (A) was determined by the manometric assayusing 27 mM undecyl acetate as a substrate.

CFroct,onNum,ber

FIG. 2. DEAE-cellulose column chromatographyof undecyl acetate esterase collected from the secondSephadex G200 column. Linear gradient of 0 to 0.8 MKCI in 0.01 M phosphate buffer was used to elute theesterase. Each fraction contained 2.5 ml and wasassayed for protein (0) and enzyme activity (A) (seeFig. 1).

tive polyacrylamide gel electrophoresis. Twoenzyme activity peaks were resolved with themajor peak having a faster electrophoretic mo-bility towards the anode. These two peak frac-tions were pooled separately and tested forenzyme specificity. The major peak fractionhad a specific activity of 168 U/mg of protein,whereas the minor peak fraction had a specificactivity of 21 U/mg of protein. Analytical poly-acrylamide gel electrophoresis revealed that themajor peak fraction contained two closely mi-grating esterase bands (R, = 0.81 and 0.83,respectively, at 7.5% gel concentration), and theminor peak fraction contained three esterasebands. Two of these bands were analogous tothose in the major peak fraction, and the thirdwas the predominant band having a slowermobility (Rr = 0.75).An attempt to separate the two closely-mov-

ing esterase bands was made by a secondpreparative polyacrylamide gel run. Each frac-tion collected having enzyme activity waschecked for esterase bands by analytical poly-acrylamide gel electrophoresis. A single enzymeactivity peak was obtained. Analytical poly-acrylamide gel electrophoresis demonstratedthat fractions from the leading (Fig. 3A) andtrailing (Fig. 3B) portions of the peak containedonly one esterase band, whereas fractions fromthe middle portions of the peak contained bothesterase bands (Fig. 3).

Further studies on both esterase bands werecarried out by polyacrylamide gel electrophore-sis using different gel concentrations. A plot oflog mobility versus percent of gel concentrationrevealed parallel lines for the two esterase bands

A B C

300

200 )

1k ::.::--::z tF"l'-----,-----'l---:::::.:: .:.:::: ..::..

a100 .

FIG. 3. Analytical polyacrylamide gel electropho-resis of undecyl acetate esterase collected from secondpreparative polvacrylamide gel electrophoresis. A7.5% resolving gel was used. Esterase bands weredeveloped with a-naphthyl acetate and Fast Blue RRsalt. (A) RB = 0.81; (B) Rt = 0.81 and 0.83; (C) R, =0.83.

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(Fig. 4). According to Hedrick and Smith (21),this indicates the occurrence of proteins ofidentical size but different charge. When frac-tion B, which contained both esterase bands,was analyzed by SDS-polyacrylamide gel elec-trophoresis, a single protein band was obtained(Fig. 5), whereas two protein bands were formedwhen the same fraction was analyzed in polya-cryalmide gel electrophoresis without SDS. Thespecific activities of fractions A, B, and C weredetermined (Table 1). Fraction B, which con-tained both bands, had a slightly higher specificactivity than either A or B, each of whichcontained only one band. Judging from the factthat both esterases had approximately the samespecific activity and the same molecular sizebut different charge, they are isoenzymes (21).Molecular weight determination. Partially

purified undecyl acetate esterase was used formolecular weight estimation with a precali-brated Sephadex G150 column. A calibrationcurve was drawn (Fig. 6), and the resultingenzyme activity peak gave a Kavg value corre-sponding to a molecular weight of approxi-mately 34,500 on the curve.SDS-polyacrylamide gel electrophoresis was

used for determining the molecular weight ofthe esterase. Figure 7 shows the calibrationcurve with standard proteins. The mobility ofundecyl acetate esterase corresponded to a mo-lecular weight of 34,800.

Molecular weight of undecyl acetate esterasewas also estimated by the molecular sievingeffect in polyacryalmide gel electrophoresis(21). After electrophoresis in increasing gelconcentration, each standard protein used forcalibration formed a straight line having anegative slope when log mobilities were plotted

8

0

K

Gel Concentration %

FIG. 4. Effect of different gel concentrations onmobility of undecyl acetate esterase. Rm = distanceof migration relative to the distance of the brom-phenol blue front. Gel concentration refers to acryl-amide content (constant bis/acrylamide ratio of 2: 75,wt/wt).

.. ...::

A B ::..:.

' fX:

:: .:*:: ;; ........... ....

... ... :: . : .. ... i :.

.:

.. .. ..

_._:

FIG. 5. Comparison of molecular sieving effect (A)and sodium dodecyl sulfate (B) in polyacrylamide gelelectrophoresis on undecyl acetate esterase. Proteinbands were stained with Coomassie brilliant blue.

1.01.8

.6

.

4

.2

1 2 4 6 8 10

Molecular Weight x 104

FIG. 6. Plot of standard proteins and undecyl ace-

tate esterase chromatographed on Sephadex G150.

1.9

1.8

1.7 ~~~~~~~~~~~~~~~Est.ras.1= 5.12

1.66 7 8 9 10 11 12

R0ibowclese A

Chymotrypsinog*n A

~|ndcyl ocott*e *teras

lose

2 a 0 1 a IllI20

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1098

7BS A

6

50~~~~vabumi'n4

1 \t Undecyl acetote esterose

() 3

Chymotrypsinogen A

°2

Ribon ucleose A

0 .2 .4 .6 .8 1.0MOBILITY

FIG. 7. Plot of standard proteins and undecyl ace-tate esterase in SDS-polyacrylamide gel electrophore-sis. Proteins were reduced with 2-mercaptoethanol.

against percent of gel concentration. A calibra-tion curve was plotted with negative slopes ofthe standard proteins versus molecular weight(Fig. 8). Two esterase bands were detected, asdescribed above, with negative slope values of5.12 and 5.13, respectively, which correspondedto a molecular weight reading of 34,600 on thecalibration curve.Sedimentation equilibrium was also used to

estimate the molecular weight. By plotting theloge absorbance at 280 nm against the square ofthe distance (r) from the axis of rotation to thepoint where the absorbance was recorded, astraight line was formed (Fig. 9), and the slopewas used for molecular weight calculations.Undecyl acetate esterase had a molecularweight of 34,000, as estimated by this methodwhen 0.74 was used as the value for the partialspecific volume (v) of the protein. Since v of theesterase was not determined and v = 0.70 to0.75 for most proteins (34), the range for theestimated molecular weight may be from 29,000to 35,000. The density of the solution wasdetermined to be 1.004 g/ml at 20 C.

Table 2 presents a summary of the differentprocedures used to determine the molecularweight of undecyl acetate esterase. The molecu-lar weight was approximately 34,500 for each ofthe two fractions exhibiting a single band (Aand C), as well as for the fraction containingboth bands (B) (Fig. 3).

10

8

6

4

4

2 3 4 5Moleculor Weight x 104

6 7

FIG. 8. Plot of standard proteins and undecyl ace-

tate esterase in polyacrylamide gel electrophoresisusing molecular sieving effect. The negative slope ofeach protein was obtained by a plot of the log of theelectrophoretic mobility versus percent of gel concen-

tration.

-1.8 F

c- -2.0CZ4

co

O -2

It'I'

i -24 F-

48 49 50 51 52

r2 (cm2)

FIG. 9. LogeC versus r2 plot used for the analysis ofsedimentation equilibrium data. C = absorbance at280 nm; r = distance from the axis of rotation to thepoint where the absorbance was recorded.

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vOvllbumin

Udecyl acetate esterose

hymotrypsinogen A

a a

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VOL. 118,1974 UNDECYL ACETATE ESTERASE FROM P. CEPACIA. I. 887

TABLE 2. Molecular weight determination on undecyl 5.0acetate esterase I

Procedure Mol wt

Gel filtration ............................. 34,500

Polyacrylamide gel electrophoresisSDS .................................. 34,800Molecular sieve .............. .......... 34,600

Sedimentation equilibrium ....... ........ 34,000

pH optimum. Undecyl acetate esterase activ-ity was determined at various pH values from5.5 to 8.75 using phosphate buffer. Optimumactivity was at pH 7.5 with a rather broadplateau from 7.0 to 8.5 (Fig. 10). A sharp drop inenzyme activity occurred below pH 6.5. Theeffect of pH higher than 8.75 was not deter-mined owing to hydrolysis of undecyl acetate athigh pH.

Kinetic studies. Effect of increased substrateconcentration on esterase activity was carriedout using undecyl acetate emulsified in TritonX-155. A plot of velocity versus substrate con-centration gave a typical Michaelis-Mentencurve (Fig. 11). The Lineweaver-Burk plotshown in Fig. 11 gave a straight line, and theapparent Km value calculated from the plot was2.3 x 10- 2 M.

4

E 3

0

E

4.0

_ 3.08

U

0.

,2.0

E

z

1.0

5 6 7 8 9pH

FIG. 10. Effect of pH on undecyl acetate esteraseactivity. Unit = micromoles of undecyl acetate hydro-lyzed per minute. Enzyme activities were determinedby the radioactive assay using undecyl [2- "C]acetateat a final concentration of 0.2M as substrate. Proteinconcentration was 0.1 mg/1.5 ml.

0 50 100 150 200 250 300 350

[UDA](mM)FIG. 11. Velocity-concentration plot for undecyl acetate esterase. Velocities of enzyme reactions were

determined by the radioactive assay using undecyl [2-'4C]acetate as substrate. The insert shows aLineweaver-Burk plot of the same data.

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SHUM AND MARKOVETZ

Temperature stability. The effect of temper-ature on esterase activity towards undecyl ace-tate was checked by incubating the enzyme at adesignated temperature for varying time pe-riods. Figure 12 shows that undecyl acetateesterase activity markedly decreased uponpreincubation at increased temperature.Reversal of esterase. Whether undecyl ace-

tate esterase could synthesize undecyl acetatefrom 1-undecanol and acetate was tested withnonradioactive 1-undecanol and sodium [1-"C ]acetate. No radioactive undecyl acetate wasdetected by thin-layer radiochromatographywhen a hexane extract of the reaction mixturewas chromatographed alone or with added non-radioactive undecyl acetate as carrier.

DISCUSSIONCiting only a few of numerous reports, multi-

ple forms of esterases have been found inanimals (2, 24, 25, 27, 31, 44), plants (11, 32),fungi (17, 30, 39), and bacteria (3, 5, 10, 18, 22,26, 29). Throughout the course of the purifica-tion reported here, two esterase bands werealways present. The separation of these twoesterase bands was only achieved in the finalelectrophoretic step that was monitored byspot-checking fractions by analytical polyacryl-amide gel electrophoresis. These two esterasesexhibited similar specific activities towardundecyl acetate. The molecular weight for eachesterase was approximately the same, and mo-lecular sieve polyacrylamide gel electrophoresisdemonstrated that these two esterases had thesame molecular weight but different charge,

1.2Cy - 3 0 C300

1.0

\.8

.6

.2 50060C70C

0 10 20 30 40 50 60

Time (min.)FIG. 12. Effect of temperature on the stability of

undecyl acetate esterase. Enzyme activity was deter-mined by the manometric method using 0.25 Mundecyl acetate as substrate.

i.e., isoenzymes. Also, both of these esteraseswere induced together (36).The purity of the esterase was indicated by

polyacrylamide gel electrophoresis (Fig. 5) offraction B (Table 1). Faint "contaminant"bands appeared in the gel of native enzyme butnot in the SDS gel. Absence of any upwardcurvature in the plot of sedimentation equilib-rium data (Fig. 9) was indicative of homogenei-ty.A definitive interpretation of the observed

kinetics of undecyl acetate hydrolysis (Fig. 11)cannot be made at the present time. Owing tothe poor solubility of the ester in aqueousmedia, the substrate was supplied as an emul-sion and so the limitations of using enzymekinetics established for water-soluble systemsfor water-insoluble substrates must apply.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grant

5RO1-GM19809 from the National Institute of General Medi-cal Sciences. A. J. M. is a recipient of a Public Health ServiceDevelopment Award-Research Career Program (5KO4GM14681) from the National Institute of General MedicalSciences.

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