APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1976, p. 138-144Copyright © 1976 American Society for Microbiology

Vol. 32, No. 1Printed in U.S.A.

Isolation and Characterization of an Enzyme with EsteraseActivity from Micropolyspora faeni

ELIZABETH N. BANNERMAN* AND J. NICOLETInstitute of Veterinary Bacteriology, University ofBerne, CH-3012 Berne, Switzerland

Received for publication 3 February 1976

The isolation and the characterization ofone ofthe enzymes ofMicropolysporafaeni that hydrolyzes the substrate N-benzoyl-DL-phenylalanine-beta-naphthylester and that seems to be of medical importance are described. This enzyme(enzyme 1) was isolated with an 86-fold purification by using the following sevensteps: ammonium sulfate precipitation, gel filtration through Sephadex G-150,heat treatment, chromatography on diethylaminoethyl-cellulose, rechromatog-raphy on diethylaminoethyl-Sephadex, gel filtration through Sephadex G-200,and affinity chromatography. Enzyme 1 has a molecular weight of approxi-mately 500,000 and maximum activity at pH 7.8 to 8.0 and at 20°C. The enzymeis stable between pH 7.5 and 10.5 and at temperatures up to 60°C. Its activity isnot inhibited by ethylenediaminetetraacetic acid. It is, however, sensitive todiisopropyl phosphofluoride and phenylmethyl sulfonyl fluoride. These proper-ties and the ability to hydrolyze the esters of phenylalanine, tyrosine, andtryptophan without endopeptidasic activity and no marked proteolytic activitysuggest that the enzyme is an esterase.

Although several reports (32, 33, 38), with acomprehensive review from Singleton andAmelunxen (33), have been made on the enzy-matic activities of thermophilic bacteria, onlyvery few of these reports are on enzymes fromthermophilic actinomycetes (8, 24).

Micropolyspora faeni is a facultative thermo-philic actinomycete (growth temperature rangebetween 37 and 60°C), which is actually classi-fied in the family Nocardiaceae (7). This gram-positive branched bacterium with short chainsof 5 to 10 spores is a saprophyte known to beinvolved, under well-defined conditions, in themolding of hay. This phenomenon raised con-siderable medical interest when it was recog-nized that inhalation of dust from moldy haymay cause an allergic respiratory disease calledfarmer's lung, an extrinsic allergic alveolitis inhumans and animals.The immunological response to the inhala-

tion of moldy hay shows the formation of pre-cipitins principally against metabolic productsof the molding agent M. faeni (9, 10, 36).Walbaum et al. (36) and Biguet et al. (2)

reported that a large number of these metabolicantigens were enzymes, which they identifiedas aminopeptidases, beta-galactosidases, beta-glucuronidases, carboxypeptidases, chymotryp-sin-like enzymes, esterases, lipases, trypsin-like enzymes, catalases, malic dehydrogenase,

and peroxidase. The most interesting group ofthese enzymes proved to be those that hydro-lyze the substrate N-benzoyl-DL-phenylalanine-beta-naphthyl ester. This substrate is usuallyused in the estimation of chymotrypsin activityand, hence, these enzymes were called chymo-trypsin-like enzymes. These enzymes werefound by the same authors (2, 36) to be highlyimmunogenic. In a serological survey theyfound that 30 out of 33 sera reacted with at leastone and up to a maximum of four fractionspresent in their M. faeni extract. Later, Nicoletet al. (27), testing sera from cattle affected withfarmer's lung, obtained similar results. Theseenzymes have also been shown to be releasedextracellularly on hay during molding andmay thus be inhaled as free active enzymeswith the moldy hay dust (26).Some microbial enzymes are known to be of

medical importance. Those ofBacillus subtilis,for example, may cause allergic reactions indetergent factory workers exposed to them (11,12, 20, 28, 39). Other microbial enzymes mayalso cause the emphysema syndrome (14).Thus, the present study was undertaken to

isolate good immunogenic enzymes ofM. faeni,known in the medical literature as chymotryp-sin-like enzymes, with the aim of acquiring abetter understanding of the nature of allergensand their role in the pathogenesis of farmer's

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ENZYME WITH ESTERASE ACTIVITY FROM M. FAENI 139

lung. In this paper, only the isolation, purifica-tion, and general properties of one of theseenzymes are presented.

MATERIALS AND METHODSPreparation of extract. M. faeni strain 9535 (ori-

gin, London School of Hygiene and Tropical Medi-cine), kindly supplied by S. Walbaum (Lille), wasused.

Nineteen batches of culture were grown. For eachbatch, 15 to 30 petri dishes (diameter, 18 cm) wereused. The organism was inoculated on V8 agar,using the method of Walbaum and Biguet (35),which was, however, slightly adapted by coveringthe agar surface aseptically with cellophane sheetsbefore inoculation.

After incubation at 40°C for 6 days, the cultureswere harvested and extracted with 10 times theirweight in a volume of 1 M glycine, pH 7.5, by themethod of Macula and Cowles (19). After centrifuga-tion at 14,000 rpm for 20 min, the supernatant of theglycine extract was then dialyzed against runningtap water for 48 h. From a total wet weight of 989 g,10,000 ml of dialyzed extract was obtained. The gly-cine extraction was used to enhance the yield ofmetabolic enzymes, since this proved to be superiorto the method of Walbaum and Biguet (35).

Protein assay. Estimation of protein content inthe extract and enzyme samples was based on theHartree (16) modification of the Lowry method. Bo-vine serum albumin (Fluka, Buchs, Switzerland)was used to plot a standard curve.Enzyme assays. Enzymatic activity was assayed

with N-benzoyl-DL-phenylalanine-beta-naphthyl es-ter (Sigma Chemical Co., St. Louis, Mo.) as sub-strate by a modification of the method of Ravin et al.(29).To 0.5 ml of a test solution appropriately diluted

in 0.05 M veronal buffer, pH 7.8, 2.5 ml of thesubstrate (0.1 mg/ml) in the same buffer was added,and the reaction mixture was incubated at 25°C for 1h. After incubation, 0.5 ml of a freshly preparedsolution of Fast Blue B salt (Merck, Darmstadt,Germany) (4 mg/ml) was added. This was followed 3min later by the addition of 80% trichloroacetic acid.The resulting reddish pigment was extracted byshaking the reaction mixture with 5 ml of ethylacetate. After centrifugation, the organic layer wasmeasured at 550 nm with a Hitachi UV/Vis spectro-photometer. From a calibration curve of pure /3-naphthol, the color density was converted to milli-grams of f3-naphthol. The unit of enzymatic activitywas defined as micromoles of f-naphthol releasedper minute at 25°C.

Esterase activity was assayed with beta-naphthylacetate (Serva, Heidelberg, Germany) as substrate,using the method of Seligman and Nachlas (31). Theunit of esterase activity was defined as micromolesof beta-naphthol released at 25°C in 1 min.The hydrolytic activity of the purified enzyme on

synthetic peptides and esters was determined byincubating 1 ml of peptide or ester solution [0.1 mg/ml of 0.05 M tris(hydroxymethyl)aminomethane

(Tris)-hydrochloride buffer, pH 8.01 with 0.1 ml ofthe enzyme solution at 25°C for 1 h. After incuba-tion, a suitable portion of the assay mixture waschromatographed on Merck cellulose thin-layerchromatography plates in the solvent system n-bu-tanol-acetic acid-water (4:1:5, by volume) and de-veloped with ninhydrin as well as Pauly reagent.Bovine f8-chymotrypsin was used as the model.

Electrophoresis. Analytical disk electrophoresiswas carried out by the method of Bloemendal et al.(3, 4), using a 5.6% gel. A current of 5 mA/tube wasused, and the electrophoresis was carried out for 40min. Then 20-,u1 amounts of the test solution weresubjected to electrophoresis.

After electrophoresis, the gels were stained forenzymatic activity as well as for protein. The enzy-matic activity staining was carried out by themethod of Uriel (34), using N-acetyl-DL-phenylala-nine-beta-naphthyl ester (Schwarz/Mann, Div. ofBecton, Dickinson & Co., Orangeburg, N. Y.) as sub-strate. Protein staining was carried out with Coom-assie brilliant blue R 250 (Serva, Heidelberg, Ger-many), using the method of Chrambach et al. (6).Enzyme purification. Step 1. Precipitation with

ammonium sulfate. To 1,000 ml of the dialyzed M.faeni glycine extract, 518 g of ammonium sulfatewas added (to give a 75% ammonium sulfate concen-tration) with stirring and left overnight at 4°C. Theprecipitate was collected by centrifugation at 10,000rpm for 20 min. The sediment was dissolved in 1/10of its original volume of 0.025 M Tris-hydrochloridebuffer, pH 7.2, and then dialyzed against the samebuffer for 48 h.

Step 2. Fractionation on Sephadex G-150. A col-umn (2.6 by 95 cm) of Sephadex G-150 was equili-brated in 0.025 M Tris-hydrochloride buffer, pH 7.2,containing 0.1 M KCl. A total of 50 ml of the di-alyzed ammonium sulfate precipitate solution wasintroduced into the column and washed with theequilibrating buffer. Both test solution and bufferwere introduced from below, by means of a flowadapter. A mariotte flask was used to maintain anoperating pressure of 25 cm of water. The ultravioletabsorption was recorded at 280 nm on a Hitachi UV/Vis spectrophotometer, and the protein profile wasrecorded with a W + W recorder (series 1100). Atotal of 22 runs were carried out, and 6- to 8-mlfractions were collected at a flow rate of 30 ml/h.

Step 3. Heat treatment. The pools of the first G-150 peak containing the enzyme to be studied weredivided into 545-ml portions, heated at 56°C for 60min in a water bath, and then cooled in an ice bath.The cooled solutions were then centrifuged at 14,000rpm for 20 min. The supernatant was retained anddialyzed against 0.05 M Tris-hydrochloride buffer,pH 7.2, for 48 h.

Step 4. Chromatography on DEAE-cellulose. Acolumn (5 by 20 cm) of diethylaminoethyl (DEAE)-cellulose-SS (Serva, Heidelberg) was equilibratedwith 0.05 M Tris-hydrochloride buffer, pH 7.2. Atotal of 545 ml of the dialyzed test solution wasplaced on it and washed with the buffer until theultraviolet absorption at 280 nm was near zero. Theactive substance was then eluted with a linear NaCl

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140 BANNERMAN AND NICOLET

gradient (500 ml of 0.05 M Tris-hydrochloride, pH7.2, plus 0.7 M NaCl added to 500 ml of 0.1 M NaClin the same buffer). Ten-milliliter fractions werecollected at a rate of 40 ml/h.

Step 5. Rechromatography on DEAE-Sephadex.A column (5 by 20 cm) of DEAE-Sephadex A-50(Pharmacia, Uppsala) was equilibrated in 0.05 MTris-hydrochloride buffer, pH 7.2. The test solution(eluate from step 4) was dialyzed against 0.05 MTris-hydrochloride, pH 7.2, and then placed on thecolumn and washed with the equilibrating bufferuntil the absorption at 280 nm was near zero. Theactive fraction was eluted with a linear NaCl gra-dient (0.8 M NaCI in 500 ml of buffer added to 0.1 MNaCl in 500 ml of buffer), and 9-ml fractions werecollected. The rate of flow was 45 ml/h.

Step 6. Gel filtration on Sephadex G-200. Therequired active fractions from step 5 were pooled andconcentrated to 30 ml and dialyzed for 48 h against0.05 M Tris-hydrochloride containing 0.1 M KCl.The dialyzed solution was placed on a Sephadex G-200 column (2.6 by 95 cm) that had been equilibratedin 0.05 M Tris-hydrochloride buffer, pH 7.2, contain-ing 0.1 M KCl. This was then fractionated. Bothenzyme and buffer solutions were introduced frombelow by means of a flow adapter. The rate of flowwas 24 ml/h, and 4-ml fractions were collected.

Step 7. Affinity chromatography. Equilibrationand chromatography on a column (1.5 by 8 cm) ofagarose-epsilon-amino-caproyl-D-tryptophan methylester (Miles Yeda Ltd., Israel) was carried out perinstructions (Miles Yeda Ltd.). Fractions (3 ml)were collected, and the rate of flow was 12 ml/h.

RESULTSGlycine whole extract. The glycine extract

of M. faeni cultures revealed, after polyacryl-amide gel disc electrophoresis, at least six frac-tions that reacted with the substrate N-acetyl-DL-phenylalanine-f-naphthyl ester. The bandswere numbered 1 to 6, and the first slow-mov-ing band (cathodic) numbered 1 was arbitrarilytermed enzyme 1 (Fig. lb). The glycine extractalso proved to be rich in other protein fractionsas well (Fig. la), since about 25 bands could becounted.

Purification of enzyme 1. The quantitativeresults of the steps followed during the purifica-tion of the enzyme are shown in Table 1. Theeluate from the affinity chromatography (step7) showed only one band in disc electrophoresis.Step 1. Precipitation with ammonium sul-

fate. A 75% ammonium sulfate precipitationgave the maximum yield in enzymatic activityin the sediment and almost negligible yield inthe supernatant. After the sediment had beenresuspended, it was subjected to polyacryl-amide gel electrophoresis, which subsequentlyrevealed an increase in the number of bandsthat reacted with the substrate N-acetyl-DL-phenylalanine-,3-naphthyl ester. At least one

I2

3

+

a bFIG. 1. Polyacrylamide gel pattern after electro-

phoresis of glycine extract of M. faeni. (a) Proteinstaining with Coomassie blue R 250 (6); (b) enzy-matic activity staining using N-acetyl-DL-phenylala-nine-,fnaphthyl ester as substrate and Fast Blue Bsalt as stain (34).

other fraction appeared that was not previouslydetected in the glycine extract. Bands 1 and 6also reacted with the acetyl esterase substrate,3-naphthyl acetate.Step 2. Gel filtration on Sephadex G-150.

Initial separation of the enzyme 1 fraction wasachieved at this step. Three peaks were ob-tained. Peak I (from 28 to 41) and II (from 42 to100) fractions showed enzymatic activity withboth N-benzoyl-DL-phenylalanine-,3-naphthylester and beta-naphthyl acetate, whereas peakIII (from 106 to 140) did not. The polyacryl-amide gel patterns showed that the enzyme 1fraction was in the first peak and that thisfraction was eluted together with other frac-tions that also hydrolyze the substrate N-ben-zoyl-DL-alanine-,f-naphthyl ester. Fractions 28to 41 from all 22 runs, which contained amongother enzymes the required enzyme 1 fraction,

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ENZYME WITH ESTERASE ACTIVITY FROM M. FAENI

TABLE 1. Purification of enzyme 1

Yield in Sp actVol Total protein U/ml Total units (U/mg ofStep no. Fraction (ml) (mg) (x 103) (x 10'3) activity protein)%) (X 103)

1 75% ammonium sulfate precipi- 1,100 10,450 15.0 16,500 1.58tate

2 Sephadex G-150 pool I 2,180 3,488 1.7 3,706 100 1.063 Heating at 60°C 2,180 3,161 1.83 3,989.4 107.1 1.264 DEAE-cellulose 1,200 192 3.03 3,636.0 97.6 18.945 DEAE 290 38.5 4.8 1,392.0 37.4 36.166 Sephadex G-200 120 12.0 5.04 604.8 16.3 50.407 Agarose-epsilon-amino-caproyl- 80 2.0 3.4 272.0 7.2 136.0

D-tryptophan methyl ester

were combined and subjected to temperaturetreatment (next step).

Step 3. Heat treatment. The protein contentof the peak I pool, after heating at 60°C for 1 h,decreased from 1.6 to 1.45 mg/ml, whereas theunits of activity increased slightly from 1.7 to1.83/ml (Table 1). The disc electrophoretic pat-tern showed a slight decrease in intensity of theprotein bands, with the complete disappearanceof about four bands.Step 4. Chromatography on DEAE-cellu-

lose-SS. Fractions 91 to 120 were found to beactive when assayed with the substrate N-ben-zoyl-DL-phenylalanine-f8-naphthyl ester. Thedisc electrophoretic pattern showed that most ofthe protein, as well as the required enzymaticfraction, was eluted at the same time althoughthere was a decrease in protein content with aconcomitant increase in the units of chymotryp-sin activity per milliliter of eluate (Table 1).Step a. Rechromatography on DEAE-Seph-

adex A-50. Two active peaks (from 93 to 101 andfrom 102 to 140) were obtained. Fractions 105 to136 contained the required enzyme 1. In thedisc electrophoretic patterns, only four proteinbands could be seen, and the enzymatic stain-ing of gel revealed only the enzyme 1 band.Step 6. Gel filtration on Sephadex G-200.

Further purification from the accompanyingproteins was achieved by gel filtration throughSephadex G-200. Only one other protein bandwas eluted together with enzyme 1. The re-quired enzyme was found in fractions 18 to 32.Step 7. Affinity chromatography. Purifica-

tion was achieved by chromatography onagarose-epsilon-amino-caproyl - D- tryptophanmethyl ester. Enzyme 1 was found in fractions 6to 15, and the contaminating protein was foundin fractions 18 to 30.The purification procedure is summarized in

Table 1. The purified enzyme (enzyme 1) gave afinal yield of 7.2%. From the 989 g, net weight,of M. faeni culture, 418 mg of lyophilized en-zyme containing 1.7 mg of total protein was

obtained. An 86-fold increase in specific activityover the ammonium sulfate precipitate fractionwas obtained.

Properties of enzyme 1. (i) Proteolyticproperties. Enzyme 1 slightly hydrolyzes he-moglobin and, to a much lesser extent, casein.Whereas 10 g.g of chymotrypsin released 4mmol of tyrosine from hemoglobin, 1 mg ofenzyme released the same amount of tyrosine.In the case of casein, 1 mg of enzyme hydro-lyzed the substrate to the same extent as did 2Ag of chymotrypsin. An increase in-the amountofenzyme used did not produce any appreciableincrease in the hydrolysis of casein.

(ii) Other enzymatic properties. Enzyme 1hydrolyzes both N-acetyl- and N-benzoyl-DL-phenylalanine-beta-naphthyl ester. It also hy-drolyzes the acetyl esterase substrate beta-naphthyl acetate. A 1-mg amount of enzymeproduced 145 x 10:1 U of esterase activity fromnaphthyl acetate.

(iii) Hydrolysis of synthetic peptides andesters revealed on thin-layer chromatogra-phy. Enzyme 1 was found to hydrolyze the es-ters of phenylalanine, tyrosine, and tryptophanbut not the analogous amides. Bovine alpha-chymotrypsin, which was used as a model, wasfound to hydrolyze the substrate d,l-aspara-gine(NH.,) I-valine5-D-histidine"-hypertensin IIas well as the esters and amides of tyrosine andphenylalanine but not those of tryptophan. Theenzyme also did not hydrolyze the trypsin sub-strates N-benzoyl-arginine amide and its analo-gous ester nor the collagenase substrate 4-phenylazobenzyloxy-carbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine. It also does not hy-drolyze d,l-asparagine'-valine-D-histidine'-hy-pertensin II.

(iv) Effect of pH. The optimum pH for en-zyme 1 was determined with the N-benzoyl-DL-phenylalanine-f3-naphthyl ester. Activity wasassayed under standard conditions (see above)in 0.05 M Tris-maleate buffer, pH 4.0 to 8.0, andin 0.05 M Tris-hydrochloride buffer, pH 7.0 to

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142 BANNERMAN AND NICOLET

10.0. The optimal pH for the hydrolysis of thesubstrate was found at pH 7.8 and 8.0. Enzyme1 was stable, with 100% activity, between pH7.0 and 10.5 at 25°C.

(v) Effect of temperature. For the effect oftemperature on the stability of enzyme 1, 1-mlportions of the enzyme solution were incubatedat temperatures ranging from 20 to 80°C for 60min. After incubation, the enzyme solutionswere immediately cooled in an ice bath andthen centrifuged at 10,000 rpm for 10 min. Thesupernatants were assayed. The maximal enzy-matic activity ofenzyme 1 was observed from 20up to 60°C. At 70°C, about 61% of the activitywas still present, and at 80°C about 100% waspresent.

(vi) Effect of known inhibitors. Ethylenedi-aminetetraacetic acid had negligible effect onthe enzymatic activity. Only 1.8% of the totalactivity was inhibited by a 10-' M concentra-tion of ethylenediaminetetraacetic acid. Diiso-propyl phosphofluoride totally inhibited the en-zyme at a 10-4 M concentration. Phenylmethylsulfonyl fluoride totally inhibited the enzymeat a 10-3 M concentration. At a 10-4 M concen-tration, 87% of the enzyme was inhibited.

(vii) Estimation of molecular weight. BySephadex G-200 gel filtration, using blue dex-tran 2000 and bovine /8-chymotrypsinogen (mo-lecular weight, 25,000), bovine serum albumin(molecular weight, 65,000 to 70,000), humangamma globulin (molecular weight, 150,000 to170,000), apoferritin (molecular weight, 460,000to 490,000), thyroglobulin (molecular weight,660,000 to 680,000), and ferritin (molecularweight, 700,000 to 800,000) as marker proteinsand the method of Andrews (1), the molecularweight was estimated to be approximately720,000.To determine the subunit composition of en-

zyme 1, the purified enzyme solution washeated at 37°C for 2 h in the presence of 1%sodium dodecyl sulfate and 1% ,3-mercaptoetha-nol and subjected to electrophoresis in acrylam-ide gels containing 0.1% sodium dodecyl sulfate(37). The migration distance relative to the mi-gration distance of the above marker proteinswas recorded. Four bands, one of which showedstrong activity on staining for enzymatic activ-ity, were observed. The molecular weight of thestrongly reactive band was estimated to be500,000. The molecular weights of the threeinactive bands were estimated to be approxi-mately 200,000, 20,000, and 18,000.

DISCUSSIONA few reports have been made on the pres-

ence of esterases in fungi (5) and in bacteria(17, 25, 30). Although the relationship of these

esterases to our enzyme 1 from M. faeni is notknown, some of the properties of the esterasesfrom bacilli are, however, similar. Recently,Higerd and Spizizen (17) isolated two esterasesfrom B. subtilis. These esterases, like enzyme1, showed very low proteolytic activity. Like-wise, other enzymes from B. subtilis (15, 22)and a cytoplasmic endopeptidase from B. mega-terium (23) have been reported to be highlyactive on ester substrates but to have low activ-ity on protein substrates.Another property of enzyme 1 that is compa-

rable with that of one of the esterases isolatedby Higerd and Spizizen (17) is its thermostabil-ity. Esterase A of B. subtilis, like enzyme 1,retained all of its original activity after 10 minat 60°C. Enzyme 1, however, shows a higherthermal stability than does the esterase fromB. subtilis in retaining 100% of its activity after1 h at 60°C. Even after 1 h at 80°C, 10% of theactivity was still retained.

The metal chelating reagent ethylenedia-minetetraacetic did not inhibit enzyme 1, whichindicates that the enzyme is not a metalloen-zyme. It is, however, inhibited by the organo-phosphorus reagent diisopropyl phosphofluor-ide and by phenylmethyl sulfonyl fluoride, sug-gesting that a serine residue is essential for theactivity. The sensitivity towards these inhibi-tors and the ability to hydrolyze various esters,especially benzoyl or acetyl phenylalanine-L3-naphthyl esters and the ethyl ester of benzoylor acetyl tyrosine, ally enzyme 1 to the "chymo-trypsin-like" enzymes of Walbaum et al. (36)and especially to the Chy 2 fraction. Like theseenzymes, enzyme 1 is found in culture filtrates(36; E. S. Bannerman, Ph.D. thesis, Univ. ofBerne, Berne, Switzerland, 1975) and thusseems to be extracellular. However, the en-zyme is found in higher concentrations whenthe cells are disrupted with glycine. Since es-terases are known to be mostly intracellular,this seems to suggest that the enzyme may bemembrane bound and can be released duringgrowth and, as such, is not a true extracellularenzyme.

Since enzyme 1 did not show any peptidaseactivity as did chymotrypsin, it must be re-garded as an esterase. The enzyme probablybelongs to the same group as the B-esterases ofthe carboxylic esters hydrolases (18). It is inter-esting to note that to this group belong severalimportant endopeptidases, such as chymotryp-sin, trypsin, elastase, subtilisin, and thrombin,all of which show esterase activity toward cer-tain substrates and which have a serine residueat the active site.A property unique to enzyme 1 is its high

molecular weight. Higerd and Spizizen (17) re-

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ENZYME WITH ESTERASE ACTIVITY FROM M. FAENI 143

ported that the molecular weight of esterase Afrom B. subtilis was estimated at 160,000 by gelfiltration but was subsequently found to be31,000 after electrophoresis in sodium dodecylsulfate gel. Matsubara and Feder (21) reportedthat the molecular weight of most microbialproteases with esterolytic activity lies in therange of 20,000 to 25,000, with very few excep-

tions, the highest recorded being 52,000 for a

protease of Aspergillus oryzae. The molecularweight ofenzyme 1 was estimated to be approx-

imately 720,000 by gel filtration and 500,000after sodium dodecyl sulfate-acrylamide gelelectrophoresis. Like acetylcholinesterase (mo-lecular weight, about 250,000), enzyme 1 was

found to have unidentical subunit composition(13). Although the carboxylic ester hydrolasesdo possess subunits, the molecular weights are

in the range of 162,000 to 168,000 (18).The pH optimum for enzyme 1 falls within

the range reported for most carboxyl esterases(pH 7.5 to 9.0) (18). Enzyme 1 has optimal activ-ity at pH 7.8 and 8.0. It is stable between pH 7.5to 10.5 and up to 60°C.

This enzyme has also been found to be highlyimmunogenic for rabbits and guinea pigs afterexperimental aerogen exposition with M. faeni(M. Wanner, DMV thesis, Univ. of Berne,Berne, Switzerland, 1975). It seems also thatpatients affected with farmer's lung constantlyharbor precipitins against this enzyme (36),also suggesting a high immunogenicity afternatural exposition. On the basis of the presentcharacterization of this enzyme, it seems thatthe molecular weight is one of the determinantfactors for the stimulation of the immune re-

sponse. The exact role played by enzyme I inthe pathogenesis of the extrinsic allergic alveo-litis due to inhalation of M. faeni and its prod-ucts is, however, still not clear. With the purifi-cation of this seemingly important esterase ofM. faeni, we seem to have a tool for furtherexperimental investigations that could throwmore light on the complex problems ofthe inha-lation of biological substances.

ACKNOWLEDGMENTSWe thank Herbert Zuber (ETH Zurich) for his help and

advice and also Margrit Krawinkler for her technical assist-ance.

This investigation was supported by grant no. 3.0140.73of the Swiss National Foundation for Scientific Research.

LITERATURE CITED

1. Andrews, P. 1965. The gel-filtration behaviour of pro-teins related to their molecular weights over a widerange. Biochem. J. 96:595-606.

2. Biguet, J., S. Walbaum, and A. Vernes. 1974. Physico-chemical aspects and antigenic structure of Micropo-lyspora faeni, p. 169. In R. de Haller and F. Suter(ed.), Aspergillosis and farmer's lung in man and

animal. Hans Huber Publishers, Berne, Stuttgart,Vienna.

3. Bloemendal, H. 1963. Zone-elektroforese: de toe-pass-ing van enkele recente snelle scheidingsmethoden.Chem. Weekbl. 59.281-287.

4. Bloemendal, H., J. F. Jongkind, and J. H. Wisse. 1962.Polyacrylamide: an nieuw stabilisierend mediumvoor zoneelektroforese. Chem. Weekbl. 58:501-504.

5. Byrde, R. J. W., and A. H. Fielding. 1955. Studies onacetylesterase ofSclerotina laxa. Biochem. J. 61:337-341.

6. Chrambach, A., R. A. Reisfeld, M. Wyckoff, and J.Zaccari. 1967. A procedure for rapid and sensitivestaining of protein fractionated by polyacrylamide gelelectrophoresis. Anal. Biochem. 20:150-154.

7. Cross, T., and M. Goodfellow. 1973. Taxonomy andclassification of Actinomycetes, p. 57. In G. Sykes andF. A. Skinner (ed.), Actinomycetales: characteristicsand practical importance. Academic Press Inc., Lon-don.

8. Desai, A. J., and S. A. Dhala. 1969. Purification andproperties of proteolytic enzymes from thermophilicactinomycetes. J. Bacteriol. 100:149-155.

9. Edwards, J. H. 1971. The production of farmer's lungantigens. Med. Lab. Technol. 28:172-174.

10. Fletcher, S. M., C. J. M. Rondle, and I. G. Murray.1970. The extracellular antigens of Micropolysporafaeni: their significance in farmer's lung disease. J.Hyg. 68:401-409.

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