JOURNAL OF BACTERIOLOGY, Dec. 1969, p. 1355-1363Copyright 0 1969 American Society for Microbiology

Vol. 100, No. 3Printed in U.S.A .

Purification and Characterization of ,3-Glucosidaseof Alcaligenes faecalis1

Y. W. HAN AND V. R. SRINIVASAN

Department of Microbiology, Louisiana State University, Baton Rouge, Louisiana 70803

Received for publication 19 September 1969

A cellobiose-utilizing bacterium isolated from sugar cane bagasse and identifiedas a strain of Alcaligenes faecalis (ATCC 21400) produced an inducible #3-glucoside-splitting enzyme. The enzyme was purified by a series of streptomycin and am-monium sulfate fractionations and by Sephadex and diethylaminoethyl columnchromatography. The final preparation was purified 130-fold, with a recovery ofabout 10% of the initial enzyme activity. The enzyme had a widepH range, with opti-mal activity atpH 6.0 to 7.0. The enzyme was stable in solution atpH 6.5 to 7.8 whenkept at 30 C for 2 hr, but it was destroyed by temperatures above 55 C. At 58 and 60C, the time required to inactivate 90% of the initial activity was 16 and 6.5 min, re-spectively. An activation energy of 9,500 cal/mole and a Km of 1.25 X 10-4 M were

obtained by using p-nitrophenyl ,B-glucoside as a substrate. The Ki value and hy-drolysis of cellobiose by the enzyme indicated a high affinity of the enzyme for thecellobiose. The enzyme had its specificity on,3-glucosidic linkage and the rate ofhydrolysis of glucosides depended upon the nature of the aglycon moiety. The inac-tivation studies showed the presence of sulfhydryl groups in the enzyme. The ac-

tivity of the enzyme was easily destroyed by the Cu++ and Hg++ ions. The Michaelis-Menten relationship and the rate of heat inactivation indicated the presence of onetype of noninteracting active site in the bacterial 3-glucosidase. Molecular weightof the enzyme was estimated by gel filtration (Sephadex G-200) and sucrose densitygradient, and a value of 120,000 to 160,000 was obtained.

,B-Glucosidases are known to be widely dis-tributed among plants, fungi, and yeasts. How-ever, only a few reports on bacterial #-glucosi-dases were found in the literature, and no attempthas been made so far to isolate and characterizeany bacterial ,3-glucosidase. f3-Glucosidase (,8-D-glucoside glucohydrolase, EC 3.2.1.21), generallycategorized as an enzyme which hydrolyzesf3-(1-4) glucosidic linkage, has a wide variety ofenzymatic properties, depending upon the originand conditions under which the organism wasgrown. Among the yeasts, for example, Candidatropicalis NCYC 4 hydrolyzes salicin but notarbutin, whereas several strains of Saccharomycescerevisiae are active on arbutin but act onlyweakly on salicin (5). Three strains of S. lactis,strains Y-123, Y-14, and Y-1057A, constitutivelyproduce high, medium, and low levels of f,-glu-cosidase and have different catalytic and immuno-logic properties (19-21). The properties of yeast,B-glucosidase were intensively studied by Duerk-sen et al. (5-8, 11, 18-20). Jermyn investigated,

1 Part of a dissertation submitted by Y. W. Han in partial ful-fillment of the requirements for the Ph.D. degree.

in detail, the properties of a fungal ,B-glucosidaseisolated from Stachybotrys atra (12-14). Schaeflerisolated spontaneous mutants of Escherichia coliK-12 capable of fermenting aryl-glucoside andstudied the induction pattern of the enzyme andthe genetics of the mutant system (25-26). The,B-glucosidases from other sources such as sweetalmonds (10), sheep rumen liquor (2), pig intes-tine (4), and a root rotting fungus (23) have alsobeen reported.We report here a procedure for purification of

an inducible bacterial ,B-glucosidase, and itsphysical chemical and enzymatic properties. Theenzyme was obtained from an organism isolatedfrom sugar cane bagasse and identified as a strainof Alcaligenes faecalis (9). Some of the typicalcharacteristics of bacterial ,B-glucosidase werecompared with that from other sources.

MATERIALS AND METHODSOrganisms, media, and growth. A j3-glucosidase-

producing bacterium was isolated from sugar canebagasse and was identified as a strain of A. faecalis.The organism was maintained and grown in a basalmedium containing: NaCl, 3.0 g; (NH4)2SO4, 1.0 g;

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KH2PO4, 0.5 g; K2HPO4, 0.5 g; MgSO4, 0.1 g; CaC12,0.1 g; and 30 g of lactose in 1 liter of distilled water.When large quantities of bacteria were required, theorganism was grown in a draft-tube fermentor. Aftersuccessive propagation, 30-gal (189-liter) quantities ofcells were harvested at an early stationary phase. Cellswere separated by centrifugation in a Sharples centri-fuge, and the resultant paste was stored at -15 C.The enzyme activity in the cells frozen in this mannerwas stable for nearly 6 months.

Purification of 8-glucosidase. The cell paste wasthawed in sufficient 0.1 M sodium phosphate buffer atneutral pH to obtain a 10% cell suspension. Thesuspended cells were ruptured with a Gaulin Labora-tory homogenizer (Manton-Gaulin ManufacturingCo., Inc., Everett, Mass.) at 6,000 psi. The rupturedcell suspensions were centrifuged at 10,000 X g for15 min. The precipitated cell debris was resuspendedin the same buffer and treated again with the homog-enizer to release the residual enzyme in unrupturedcells. These procedures were repeated several timesuntil the amount ofenzyme released in the supernatantfluid was considerably reduced. All the supernatantfluids thus obtained were combined and used as astock solution of crude B-glucosidase. The crudeenzyme was stored at -15 C until further use. Unlessotherwise indicated, all steps of the purification werecarried out in the cold. Nucleic acids were first pre-cipitated out with 1.5% streptomycin sulfate (Eli Lilly& Co., Indianapolis, Ind.) at neutral pH, and theprecipitate was removed by centrifugation at 20,000 Xg for 15 min. Powdered ammonium sulfate was thenadded slowly to the supernatant fluid with constantstirring. Most of the 8-glucosidase activity was pre-cipitated between 40 and 60% saturation. Afterammonium sulfate fractionation, the precipitatedprotein was resuspended in 0.02 M sodium phosphatebuffer, pH 7.0 (.0 mg of protein/ml), and 2 ml of thesuspension was filtered through high-porosity gels(Sephadex G-200) to desalt the preparations andobtain further purification. Columns (5 X 60 cm)were prepared from thin slurries of deaerated gelwhich had been washed and swelled for 3 days ac-cording to the manufacturer's recommendation. Frac-tions (5 ml) were collected, and the active fractionswere combined and subjected to ion-exchange chroma-tography. Diethylaminoethyl (DEAE) cellulose (CarlSchleicher & Schuell Co., Keene, N.H.) was washedwith 1 N NaOH, 1 N HCI, and 0.02 M sodium phos-phate buffer, pH 7.4, until the effluent showed nomore yellow coloration. Large volumes of bufferwere percolated slowly through the packed column(2.5 X 50 cm) to equilibrate the cellulose to bufferconditions. After the application of the enzyme,elution was achieved by a linear gradient of 0.5% ofNaCl.Enzyme and protein assay. ,B-Glucosidase activity

was estimated by measuring spectrophotometricallythe release of p-nitrophenol from p-nitrophenyl-,f-D-glucoside (PNPG). A 0.5-ml amount of a suitabledilution of the enzyme preparation was added to 2.5ml of a preincubated (10 min, 40 C) reaction mixturethat contained 2.0 ml of 0.1 M sodium phosphatebuffer, pH 6.5, and 0.5 ml of 5 X 10-3 M PNPG.

After a suitable reaction time, the enzyme activity wasstopped by adding 2 ml of a 1 M solution of sodiumcarbonate. The yellow color that developed duringthe hydrolysis of the substrate was read at 400 nm ina Beckman DB spectrophotometer. A unit of enzymewas defined as that amount of enzyme necessary tohydrolyze 1 ,umole of PNPG per min. Protein wasdetermined by the colorimetric procedure of Lowryet al. (17) with the Folin-Ciocalteau reagent, withcrystalline serum albumin as the standard.

Activation energy. The effect of temperature on theinitial velocity of PNPG hydrolysis was examinedover the range of 20 to 45 C with 5 degree increments,and was plotted in the conventional Arrheniusmanner (log K versus 1/T, wherein K is the initialrate of hydrolysis and T is the absolute temperature).Activation energy (Ea) for the enzyme was calculatedfrom the equation 2.3 log K1/K2 = Ea/R (1/T2 -1/T1).

Enzyme-inhibitor dissociation constant. The affini-ties of several j8-glucosides and related compounds tothe enzyme were determined by the inhibition of theenzymatic hydrolysis of PNPG by a fixed concentra-tion of the test compound over a wide range ofsubstrate concentrations. Initial velocities of substratehydrolysis were plotted against the various sub-strate concentrations in Lineweaver-Burk fashionto obtain apparent Km values for the inhibiting com-pounds. Enzyme-inhibitor dissociation constants, oraffinity constants (Ki), were then calculated from theequation:

1 Km 1+W 1 1

v Vmax \tKi, (S) Vmax

or Ki = (1) KmKp- Km

where (I) is the concentration of inhibitor and Kp isthe apparent Km.

Heat treatment. A thin-walled test tube was placedin a water bath at specified temperatures. After 10min, 5 ml of enzyme solution in 0.02 M sodium phos-phate buffer, pH 7.0, was added; 0.5-ml samples werewithdrawn at predetermined time intervals and addedto test tubes packed in ice. Enzyme samples wereassayed immediately to prevent possible renaturation.The rate of the thermal inactivation of the jO-glucosi-dase was determined graphically for each temperature.

Molecular weight determination. The molecularweight of ,-glucosidase was determined by gel filtra-tion and by sucrose density gradient centrifugation.Gel filtration was done on a column of cross-linkeddextran (Sephadex G-200) at pH 7.4. Cytochrome c(Nutritional Biochemicals Corp., Cleveland, Ohio)and catalase (beef-liver, Mann Research Laboratories,Inc., New York, N.Y.) were used as reference markers.For purposes of calculation, the cytochrome c wasassumed to have a molecular weight of 13,000, andthe catalase a molecular weight of 250,000. Themolecular weight of ,3-glucosidase was determinedaccording to the method of Andrews (1). Sucrosedensity gradient centrifugation was performed ac-cording to Martin and Ames (22) with the use of

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4.6-ml gradients. The linear gradients were preparedwith 5 and 20% sucrose solution in 0.05 M phosphatebuffer, pH 7.0, and were centrifuged in an SW-39rotor at 35,000 rev/min for 2 hr at 2 C with a Spincomodel L ultracentrifuge. Cytochrome c and catalasewere used as reference markers. After centrif-ugation, the bottom of each lusteroid tube waspunctured, and eight-drop fractions were collected.Cytochrome c was located by determining the ab-sorbancy at 415 nm, and the location of catalase wasdetermined by measuring the disappearance of ab-sorbancy at 240 nm after adding 0.3% hydrogenperoxide. The distance each protein migrated wasestimated by the location of the different enzymes,determined by the analysis of the fractions. Assumingthat the distance of migration of each enzyme isdirectly proportional to its S value, the molecularweight of the enzyme was calculated from the distanceof migration as follows:

MIIM2 = (SI/52)312 = (d1/d2)3'2

where M1 is molecular weight of a reference marker,M2 is molecular weight of a sample, S1 is sedimentationcoefficient of a reference marker, 52 is sedimentationcoefficient of a sample, di is distance migrated by areference marker, and d2 is distance migrated by a

sample.

RESULTS

The isolated f3-glucosidase-producing bac-terium was a gram-negative, short rod and wasmotile with peritrichous flagella. Gelatin was notliquefied, and litmus milk turned alkaline withoutpeptonization. Acid and gas were not producedfrom any of the carbohydrates tested, andacetylmethylcarbinol was not produced. Thesecharacteristics were identical to the Bergey'sManual description of genus Alcaligenes. Of thesix species described in the genus, only threespecies are motile, and two of the motile speciesliquefy gelatin. Therefore, the isolate is mostlikely A. faecalis. The organism produced differentlevels of the enzyme depending upon the substrateon which it was grown (Table 1). Cellobiose,lactose, and f3-methyl-D-glucoside induced almostequal amounts of 3-glucosidase, whereas only alow level of enzyme activity was noticed in thepresence of either glucose or melibiose (27, 28).Apparently the organism was paraconstitutive for,B-glucosidase, and the level of the enzyme couldbe increased by induction.

Table 2 is a resume of the procedure followed inthe purification of the ,B-glucosidase. The enzymewas purified 130-fold, with a recovery of about10% of the initial enzyme activity. The activefraction was eluted from a DEAE column withapproximately 4% NaCl. Partially purifiedenzyme could be stored at 4 C for weeks withminimal loss of activity. The activity of purifiedenzyme was completely destroyed by lyophiliza-

TABLE 1. Level of 8-glucosidase in organisms grownon different carbon sourcesa

Specific activitySubstrate (units/g of

protein)

Cellobiose........................ 11.18Lactose ........................... 10.82,3-Methyl-glucoside................ 10.01Glucose .......................... 1.92Melibiose ......................... 1.28Sodium acetate ................... 0.000Galactose ........................ 0.000Glycerol.......................... 0.000

a The organism was grown for 2 days on basalmedium containing each carbohydrate (0.1%) asthe sole carbon source.

tion. The activity was partially or completelyprotected by the incorporation of such com-pounds as inositol, albumin, glycerol, and car-boxymethyl cellulose. Long-chain hydrocarbonhad no protective effect (Table 3). Most of thestudies on the characteristics of the enzyme weremade with enzyme which had been purified about50- to 80-fold.The effect of pH on the hydrolysis of substrate

(PNPG) was studied with the use of sodiumacetate and sodium phosphate buffers. The en-zyme had a widepH range for its activity, with anoptimal pH range of 6.0 to 7.0. The enzyme wasstable in solution at pH 6.5 to 7.8 when kept at30 C for 2 hr. Rapid inactivation of the enzymeoccurred at pH values above 8.0 and below 6.0.Preincubation for 2 hr at pH 6.0 and 8.0 resultedin 60 and 40% loss of activity, respectively (Fig.1).

Heat-inactivation studies showed that thebacterial ,B-glucosidase was destroyed easily bytemperatures above 55 C. At 58 and 60 C, thetime required to inactivate 90% of the initialactivity was 16 and 6.5 min., respectively (Fig. 2).The effect of temperature on the initial veloc-

ities of substrate hydrolysis was studied over therange of 20 to 45 C at 5 degree increments. Figure3 shows a conventional Arrhenius plot of the data.A deviation from linearity was observed above40 C. An activation energy of 9,500 cal per molewas calculated from the slope of the linear portionof the line.

Hydrolysis of PNPG by the bacterial ,3-glu-cosidase followed the zero-order kinetics over theportion of the curve through 50% hydrolysis. Theinitial velocity of the reactions was directly pro-portional to the enzyme concentration. WithPNPG as a substrate, a typical Michaelis-Mentenrelationship was obtained between the substrateconcentration and the initial velocity of the

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TABLE 2. Purification offl-glucosidaseTreatmenta Total vol Total enzyme Total protein Specific activity Enzyme yield

ml units mg unitsIg S

Crude extract .................. 1,000 530 5,000 10.6 100Supernatant fluid of strepto-mycin (1.5%) treatment ...... 950 450 4,000 11.2 85

Precipitate of ammonium sul-fate (40-60%) treatment ...... 150 292 720 40.2 55

Sephadex (G-200) .............. 50 143 80 180 27DEAE (0-5% NaCI)............ 35 112 21 530 21DEAE (2-5% NaCI) ............ 10 100 3.5 1,450 9.5

a Each treatment is fully described in the text.

TABLE 3. Protective effect of various compoundson the activity of,B-glucosidase during

lyophilization

Enzyme activity

Additiona' afte ProtectionCototlyophili-45Conrla zation

units/ml units/ml %None ................ 6.89 0 0Kerosene (10%)b ...... 5.02 0 0Inositol (1%) ......... 6.89 6.89 100Inositol (0.1%)... 6.89 3.18 5Albumin (1%).6.62 2.65 40CM-cellulose (1%).... 4.14 0.74 17Glycerol (10%) ........ 6.62 2.75 40

a Each compound of designated concentrationwas dissolved in an enzyme solution (0.25 mg ofprotein/ml) and divided into two portions. Oneportion of each treatment was kept cold and usedas a control, and the other portion was lyophi-lized. After lyophilization, the enzyme was recon-stituted with distilled water to make the originalvolume, and the activity was measured understandard conditions.

b Kerosene and glycerol did not stay in solidphase at 0 C. Thus, it was necessary to freeze thesesamples frequently during the lyophilization.After the completion of the lyophilization, thekerosene and glycerol remained in the tubes.

reaction. The Lineweaver-Burk plot yielded astraight line, from which a Km value of 1.25 X10-4 M was obtained with PNPG as a substrate(Fig. 4). The affinity of various glucosides for theenzyme was measured in a series of tests in whichthe inhibition of PNPG hydrolysis by a fixed con-centration of inhibitor was tested at variousPNPG concentrations. Ki values of 3 X 10-,1 X 10- and 5 X 10-2 M were calculated forglucose, cellobiose, and f3-methyl-D-glucoside,respectively.To study the substrate specificity of the enzyme,

. 1.0EO O.$0It O.$@aO 0.7

)" 0.6I-

> 0.5

0.449

0.3

a 0.2

N o0.zhi

STABILITY

I 7 0

pH VALUE

0%,oo °

toro-0

*O-

20 aI130

30

toN

zhi

FIG. 1. Effect ofpH on the activity and stability ofbacterial ,B-glucosidase. The stability of the enzyme wasexamined by preincubating the enzyme solution, ad-justedto variouspH levels, for 2 hr at 30 C. The residualactivity in the sample was measured under standard con-ditions. The enzyme activity was measured as describedin the text with phosphate buffer of different pH levelsin the reaction mixture.

1.0 (S C)

E \ \0

(58' C)

0

Izo.. \(60-C)

_0I

zhi

10 es

TIME (min)

FIG. 2. Heat inactivalion of ,-glucosidase.

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1359,-GLUCOSIDASE OF ALCALIGENES FAECALIS

1.0

W-0

0

4.0

0.1U)

z

ca-i4

I-

0

3.0 3.1 3.2 3.3 3.4 35

IO3T

FIG. 3. Arrhenius plot ofPNPG hydrolysis.

ME-GLUCOSI DE(Ix io-2".)

INHIBITOR

-10 0 10 20 30 40

MS

FIG. 4. Competitive inhibition of PNPG hydrolysisby several (-glucosides. The reaction mixture (3 ml)contained the indicated concentration of inhibitor and

PNPG in 2.8 ml of 0.01 M phosphate buffer, pH 6.5,and 0.2 ml of enzyme. After 10 min, the reaction was

stopped and the activity was measured.

various glucosides and galactosides were sub-jected to enzymatic hydrolysis and the liberatedglucose was measured. As Table 4 shows, 13-

glucosidase hydrolyzed various ,B-glucosides.The rate of hydrolysis depended on the nature ofaglycon moiety and the type of linkage. Thespecificity of the enzyme was not restricted to thefl-1,4 linkage, for the enzyme hydrolyzed 3-1 ,2and ,B-1 , 3 linkages. The enzyme, however, had itshighest activity toward fl-1,4 linkage, and (3-1,6linkage was difficult to hydrolyze. Of the com-

pounds having,B-1,4 linkage, the rate of hydrol-ysis increased as the degree of polymerization

TABLE 4. Hydrolysis of various glucosides by the(3-glucosidase

Substrate Type of linkage liberatea

mg/miCellobiose........ Glucoside (,6-1,4) 0.48Cellotrioseb....... Glucoside ((3-1,4) 0.30Cellotetraoseb..... Glucoside ((3-1,4) 0.24Laminaribiose.... Glucoside (fl-1,3) 0.31Sophorose........ Glucoside ((3-1 ,2) 0.20Gentiobiose ...... Glucoside (,9-1,6) 0Lactose........... Galactoside (fl-1,4) 0.04Sucrose........... Glucoside (ft-1,4) 0.01Maltose .......... Glucoside (a-1,4) 0Melibiose. Galactoside (,(-1,6) 0Methyl-,O-D-glu-

coside .......... 0Salicin ((3-salicylalcohol gluco-side) ........... 0.02

a The reaction mixture contained 2.0 ml ofphosphate buffer, pH 6.5, 0.5 ml of 0.1 M substrate,and 0.5 ml of enzyme solution. After incubatingfor 20 min at 40 C, 1.0 ml of the reaction mixturewas withdrawn and heated for 1 min in boilingwater to inactivate the (-glucosidase activity, andthe amount of glucose liberated was determinedby the Glucostat method according to the manu-facturer's instructions (Worthington BiochemicalCorp., Freehold, N.J.).

b Chemicals obtained from E. T. Reese of theU.S. Army Natick Laboratory, Natick, Mass.

decreased. None of the compounds with an aconfiguration was hydrolyzed, except for a slighthydrolysis of sucrose. This may be due to theimpurity of the chemicals. One interesting ob-servation was the hydrolysis of ,B-galactoside(lactose) by the purified #-glucosidase, eventhough the rate of hydrolysis was only about 10%of the corresponding f3-glucoside, cellobiose. Veryweak hydrolysis of o-nitrophenyl-f3-D-galactosideby the purified f,-glucosidase was also observed.The bacterial f3-glucosidase was inhibited by

reagents reacting with sulfhydryl groups, such asp-chloromercuribenzoate and other mercurials.Mercurials at a level of 10-5 M completely in-hibited the enzyme activity. The reducing agentsdithiothreitol and mercaptoethanol were noteffective. However, the chelating agents eth-ylenediaminetetraacetate and o-phenanthrolineshowed a mild inhibitory activity. At the concen-trations studied, the only cations which had aneffect on the enzyme activity were mercuric,cupric, and ferric ions (Tables 5 and 6).The results of the molecular weight determina-

tion of j3-glucosidase are presented in Fig. 5 and 6.With gel ifitration, the activity peaks of catalase,

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TABLE 5. Effect of enzyme inhibitors onthe ,3-glucosidasea

Inhibitor Concn Inhibition

ju %Noneo. 0p-Chlormercuribenzoate... 1.7 X 10- 100EDTA................... 1.7 X 10-2 14EDTA................... 1.7X 10-3 01,10-Phenanthroline ...... 1.7 X 10-2 431, 10-Phenanthroline ...... 1.7 X 10-3 9Sodium diethyldithiocar-bamate................. 1.7 X 10-2 27

Sodium diethyldithiocar-bamate................. 1.7 X 10-3 0

Sodium azide............. 1.7 X 10-2 02-Mercaptoethanol ........ 1.7 X 10-2 0Dithiothreitol ............ 1.7 X 10-2 0

a The reaction mixture contained 2.0 ml of 0.1M phosphate buffer, pH 6.5, 0.5 ml of inhibitor,0.2 ml of enzyme solution, and 0.3 ml of PNPGat 5 X 10-' M. The PNPG was added after a 20-min preincubation of the other components at35 C.

TABLE 6. Effect of various cations on the activityoff3-glucosidase

Cationa Concn Inhibition

X %K .............. 1 X 10-3 0Co ............. 1 X 10-3 0Ni++........... 1 X 10-3 0Ca++.......... 1 X 10-3 0Mg++.......... 1 X 10-3 0Hg++.......... 1 X 104 100Cu ............. 1 X 10-4 100Fe++ .......... 1 X 10-3 100Fe++........... 1 X 10-4 20Fe+........... 1 X 10-4 0Zn............ 1 X 10-4 0

a All of the cations were added as chloridesexcept Cu++, Fe+++, and Zn++, which were addedas sulfates.

,B-glucosidase, and cytochrome c were found infractions 22, 24, and 39, respectively (Fig. 5).When elution volumes (fraction tube X 5 ml) ofeach enzyme were plotted against molecularweight on a log scale in Andrew's fashion (1), amolecular weight of 160,000 was obtained for(3-glucosidase from the graph. The molecularweight of ,B-glucosidase was also determined bysucrose density gradient centrifugation as shownin Fig. 6. The distances of migration of cyto-chrome c, ,B-glucosidase, and catalase were 5, 22,and 35, respectively, when determined by the

number of tubes in which activity peaks of eachenzyme was found. When catalase and cyto-chrome c were used independently as a referenceprotein, the molecular weight of f3-glucosidasewas calculated as 124,000 and 120,000, respec-

tively.

0.7

X 0.6 CATALASE

0

4.0iL 0.4 / --GLUCOSIDASEz

0 160E 0~~~~~~YOHRM

0.2 -

0.I

0 10 20 30 40 50 60

FRACTION NO.

FIG. 5. Gel filtration. A mixture of enzymes was

filtered through a Sephadex column (2.5 X 50 cm,

G-200) with the use of0.02Mphosphate buffer, pH 7.4,with an elution rate of 12 ml/hr. Fractions (5 ml) were

collected and analyzed.

0.6

'E OA0 P-GLUCOSIDASE

i CYTOCHROME C

IL

0 Ez ~~~~~~~~~~~~~~Zi*0.3 CATALASE

05

0.Zc

20 30 40FRACTION NO.

so 60

S:mpl*A-"Volum35 22 5 0

DISTANCE MIGRATED(NO. OF TUBES)

FIG. 6. Sucrose density gradient centrifugatior. Amixture ofenzymes was centrifuged on a sucrose densitygradient of 5 to 20% for 20 hr at 35,000 rev/min. Thelocations of enzymes were detected as described in thetext.

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DISCUSSIONThe characteristics of the isolated organisms

were very similar to those of A. faecalis as de-scribed in Bergey's Manual with the one exceptionthat the isolate could be induced to synthezise 13-

glucosidase with various 13-glucosides and lactose.The American Type Culture Collection culture(ATCC 8750) of A. faecalis could not be inducedto synthesize ,B-glucosidase with these com-pounds. According to Bergey's Manual, the genusAlcaligenes generally does not utilize carbohy-drates. A. faecalis is described as not producingany detectable amount of acid or gas from car-bohydrates. This was true for the isolate when itwas grown on peptone broth medium supple-mented with various carbohydrates. However,when the isolate was grown on synthetic basalmedium supplemented with lactose, a slightamount of acid was produced. The difference isconsidered to be due to the buffering action ofpeptone in the complex medium. The isolate, aswell as A. faecalis ATCC 8750, grew on lactose asa sole carbon source, even though the cell yieldwas low [1 g (wet weight) per liter of medium].Therefore, carbohydrate utilization is not a goodcriterion for the characterization of genus Alcali-genes.

Since, in the presence of certain substrates, theorganism produces 13-glucosidase to a greater ex-tent than its constitutive level, it is apparent thatthe production of 13-glucosidase in this organismis partially inducible. Induction of enzyme syn-

thesis occurred not only with the 13-glucosides, butalso with lactose, which is a 13-galactoside. Sincelactose induces 13-glucosidase synthesis and theorganism grows on lactose as a sole carbonsource, the possibility of hydrolysis of lactose bythe enzyme was investigated. Purified ,B-glucosi-dase did in fact hydrolyze lactose, which is 13-1,4galactosyl-glucose, at about 10% of the velocityof the hydrolysis of the corresponding ,B-glucoside,cellobiose. Veibel (29) also mentioned the hy-drolytic effect on 13-galactosides of many f3-glu-cosidase preparations. Even though the ,B-glu-cosidase catalyzes a low rate of hydrolysis oflactose, the main agent of hydrolysis of lactose inin this organism is not 13-glucosidase but 1B-galactosidase. A large quantity of 1-galactosidasewas found in the crude cell extract of the orga-nism grown on lactose medium. Therefore, thelactose induces both the synthesis of 13-glucosidaseand 13-galactosidase in this organism. 13-Methyl-glucoside was a good inducer for the synthesis of13-glucosidase. However, purified enzyme did nothydrolyze ,B-methyl-glucoside to any detectable

extent. If it were indeed the case that #-methyl-glucoside acted as a gratuitous inducer, it isdifficult to explain the fact that the organismgrows on ,3-methyl-glucoside as a sole carbonsource.The specificity of f3-glucosidase varies con-

siderably with the species of the organism pro-ducing the enzyme and the conditions underwhich the organism is grown. (3-Glucosidasesfrom many sources (3, 5, 7, 13,25) are reported tohave only aryl-j3-glucosidase activity and very lowor no cellobiase activity. The bacterial 13-glu-cosidase hydrolyzed both cellobiose and aryl-,3-glucoside (PNPG). The specificity of,-glucosi-dase with regard to the configuration aboutcarbon atom 1 of the glucosyl residue is con-sidered to be absolute. Bacterial ,B-glucosidasehydrolyzed only the (-configuration. Epimeriza-tion of the 4 carbon atom transforms the #-glucosides into 13-galactosides, which are reportedto be hydrolyzable by almond emulsin and/3-glucosidase preparations from almost everysource, even if the velocities are only about 10%of the velocity of hydrolysis of the corresponding,3-glucosides (29). Bacterial ,3-glucosidase alsohydrolyzed ,3-galactoside, o-nitrophenyl-13-D-ga-lactoside and lactose at about 10% of the velocityof hydrolysis of the PNPG and cellobiose. Theenzyme hydrolyzed various ,3-glucosides; highestactivity was toward the 3-1,4 linkage, but it alsohydrolyzed ,B-1, 2 and 1B-1,3 linkages. Of the13-1,4 glucosides, the rate of hydrolysis, measuredby liberation of glucose, increased as the degree ofpolymerization decreased. Thus, according toReese's criteria (24), the enzyme clearly belongs tothe j3-glucosidase group. If it were ,3-glucanase,which also liberates glucose from (3-glucan, therate of hydrolysis would increase as the degree ofpolymerization increased. Among the aryl-,B-glucosides, salicin was only slightly hydrolyzedwhereas PNPG was hydrolyzed very easily. Thedifference is probably due to the strong electro-negativity of the nitro group in PNPG.

It is difficult to compare directly the dataobtained in different laboratories, even on thesame enzyme, because of differences in exper-imental methods and conditions. Nevertheless,the characteristics of 13-glucosidase from A.faecalis are similar to those from other sources.Michaelis constant (Kin), energy of activation(Ea), heat resistance, pH optimum, and affinityconstant (Ki) for several glucosides are similar.However, the molecular weight of bacterial,8-glucosidase is one-half to one-third that ofyeast j8-glucosidase. Fleming and Duerksen (8)postulated that the yeast 13-glucosidase is com-posed of three or four structural subunits and

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HAN AND SRINIVASAN

calculated the molecular weight of the polymerand monomer as 325,000 and 110,000, respec-tively. Thus, it appears that the bacterial f3-glu-cosidase has a size similar to that of one subunitof yeast f3-glucosidase. /l-Glucosidase fromStachybotrys atra is reported to have at least twotypes of binding sites (13). The bacterial f3-glucosidase displays a typical Michaelis-Mentenrelationship, gives a slope of about 1 in a plot ofthe Hill equation, and shows a first-order rate ofheat inactivation (Fig. 2). All of these resultsindicate that only one type of noninteractingactive site is present in the bacterial /3-glucosidasemolecule. The activity of the purified enzyme wascompletely destroyed by lyophilization. Theactivity was completely or partially protected bythe addition of such compounds as inositol,glycerol, or albumin before lyophilization. Thus,it is speculated that the enzyme has exposedhydroxyl or sulfhydryl groups, which easily inter-act with each other to alter the native conforma-tion during the dehydration process. The existenceof sulfhydryl groups in this enzyme was shown byinhibition tests in which p-chloromercuribenzoateand other mercurials were used (Tables 5 and 6).The protecting compounds, thus, are believed tobind to the exposed reacting moiety of the enzymeand prevent the interaction of enzyme molecules.The inactivation of ,3-glucosidase by the addi-

tion of salts of heavy metals, especially of mer-cury, silver, and copper, is well known (18, 29).The inactivation might be due to nonspecific saltformation with the enzyme, since the activity isrestored if the heavy metal is precipitated byhydrogen sulfide. The activity of bacterial ,B-glucosidase was also affected by these chemicals.The inhibitory effect of various metals on enzymeactivity is controversial. Mandels and Reese (18)presented a table which shows wide discrepancyamong the various workers as to which metals areinhibitory. Well-known inhibitors, such as silverand copper, are listed among the inactive group,as well as in the active group. These differencesmay be due to variations in experimental con-ditions such as pH, ionic concentration of buffer,concentration of enzyme, and the presence ofimpurities in enzyme solution.A sulfhydryl inhibitor, p-chloromercuriben-

zoate, was an extremely effective inhibitor for theactivity of bacterial ,B-glucosidase, suggesting theexistence of sulfhydryl groups in the enzymemolecule. Cellulases have been reported (18) tolack sulfur-containing amino acids, even thoughsome of the cellulase was inactivated by p-chloromercuribenzoate at relatively high concen-trations (10-' to 10-2 M). Mandels and Reese (18a)found a strong inhibitory effect of zinc and di-

sodium ethylene bisdithiocarbamate (10-4 M) oncellulases from Trichoderma viride and Myro-thecium verrucaria, but they had no effect on anyf-glucosidase tested. Bacterial ,3-glucosidase alsowas unaffected by such compounds as reducingagents (dithiothreitol and mercaptoethanol),azide, and dithiocarbamate. Likewise, such cat-ions as Zn++, Mg++, Ca++, Co++, and Ni i, arenot effective inhibitors for the ,B-glucosidase.

,B-Glucosidase activity on PNPG was com-petitively inhibited by such compounds as cel-lobiose, glucose, and ,B-methylglucoside. Cel-lobiose showed the highest affinity (Ki) to theactive site of the enzyme and also liberated themaximal amount of glucose when the enzymeacted on the same molarity of various glucosides.Since cellobiose is the product of cellulase actionon cellulose, it is believed to be a good inhibitorfor most cellulases (18). Cellobiose, however,showed very low affinity to many other f3-glu-cosidases (5, 7, 14, 27).

ACKNOWLEDGMENTS

We gratefully acknowledge the expert technical assistance of J.Raymond and M. Bumm. We also thank Elwyn T. Reese for thestimulating discussions.

This investigation was supported by Public Health Servicegrant UI-00919-01 from the National Center for Urban andIndustrial Health, and by grants from the Graduate ResearchCouncil of the Louisiana State University and the American andFlorida Sugarcane Leagues.

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