p -Galactosidase from Bacillus stearothermophilus Departtnenrs ofBiology and Chi~mistty,Californirr Srrrte College>, Son Bernrrrditto. Crrl(fiforr~irr92407
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Accepted February 20, 1976
R. E., and D. M. PEDERSON. 1976. P-Galactosidase from Bocillirs stc~rrrorhcvGOODMAN, mopl~ilrrs.Can. J . Microbiol. 22: 817-825. Several strains of thermophilic aerobic spore-forming bacilli synthesize P-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The p-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 "C and the Arrhenius activation energy is about 24 kcallmol below 47 "C and 16 kcallmol above that temperature. At 55 "C the K , is 0. l l M for lactose and 9.8 x M for o-nitrophenyl-P-D-galactopyranoside.The enzyme is strongly product-inhibited by galactose (Ki = 2.5 x M). It is relatively stable at 5OoC,losing only half of its activity after 20 days at this temperature. At 60 "C more than 60%of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein. and in the presence of 4 mglml of bovine serum albumin the enzyme is only 18% inactivated in I0 min at 60 "C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000. GOODMAN, R. E., et D. M. PEDERSON.1976. P-Galactosidase from Brrcillus sterrrothermophilus. Can. J . Microbiol. 22: 8 17-825. Quelques souches de bacilles sporuIants thermophiles aerobies synthitisent une P-galactosidase (EC 3.2.1.23) de f a ~ o nconstitutive. Cette propriett constitutive n'est apparernrnent pas due a un represseur thermosensible. La 0-galactosidase d'une souche, etudiie dans des extraits acellulaires a un pH optimal entre 6.0 et 6.4 et une dependance de pH tres nette du c6te acide pour son optimum. Pour cette enzyme, la temperature optimale est de 65 "C et I'energie d'activation d'ArrhCnius est d'environ 24 kcallmol au-dessous de 47 "C et de 16 kcallmol M au-dessus de cette ternpbrature. A 55 "C le K, est egal a 0.11 M pour le lactose et a 9.8 x pour 1'0-nitrophenyl-p-D-galactopyranoside.Pour cette enzyme, I'inhibition par le produit est M). L'enzyrne est relativement stable a 50 "C tres forte dans le cas du galactose (Ki = 2.5 x car, apres 20 jours a cette temperature, elle perd seulernent la moitie de son activite. A 60 "C plus de 60% de I'activite disparait en 10 min. Une proteine protege I'enzyme contre ['inactivation thermique. En presence de 4 mg/ml d'albumine skrique bovine, I'inactivation est reduite a 18% apres 10 rnin B 60 "C. EvaIut par electrophortse en gel le poids moleculaire est &gali 215 000. [Traduit par le journal]
Introduction The biochemical basis of thermophily, though widely investigated, has not yet been satisfactorily explained by any unitary hypothesis. Several recent excellent reviews (7, 23) have examined many possible bases for thermophily. The most frequently advanced (and supported) hypothesis is that the proteins of thermophiles are more thermostable than those of mesophiles, allowing the bacteria to survive a t temperatures at which many mesophilic proteins are denatured. However, no obvious chemical differences between thermophilic and mesophilic proteins have been demonstrated which might account for their differences in thermostability (23). So, the question is still open and new perspectives are continually being brought forth (2, 10, 12, 15, 25). 'Received December 2, 1975.
We have detected P-D-galactoside galactohydrolase (EC 3.2.1.23; P-galactosidase) in five of eight strains of Bacillus stearothermophilus. The five strains apparently produce this enzyme constitutively. Although a-amylases (17-19, 21, 26-28) from thermophilic members of the genus Bacillus have been examined, we have not encountered reports of other glycosidases from such bacteria. An inducible P-galactosidase has, however, been reported from an extreme thermophile resembling Thermus aquaticus (24). The present report describes some characteristics of the galactosidase of B. stearothermophilus.
Materials and Methods Bacterial Straitls
Eight strains of aerobic, obligately thermophilic, spore-forming bacilli were isolated from the Arrowhead Hot Springs located in the San Bernardino Mountains. The temperatures of the aqueous environments from which samples were taken ranged from 55 t o 80 "C. Each
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CAN. J. MICROBIOL. V O L . 22, 1976
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of the eight strains has a minimum growth temperature between 38 and 41 "C, and a maxinium between 67 and 74 "C. These temperature characteristics allow us tentatively to identify all eight strains as Bacilllrs stearotlre,n~ophil~rs (5). For reference, the strains are numbered AT-1 (Arrowhead thermophile No. 1) through AT-8. Media atzd Grotvtll Conditions Bacteria were routinely cultured at 55-60 "C on TYMn medium containing (per litre) tryptone(Difco), l o g ; yeast extract, 5 g; and MnCI2, mol. For a solid medium, TYMn was supplemented with 3% agar. Liquid cultures were aerated in Erlenmeyer flasks on a reciprocating waterbath shaker at 150-180 strokes/min. The medium comprised no more than 20% of the flask volume. Culture turbidity was monitored with a KlettSumnierson photoelectric colorinieter with a green filter (No. 52). Appropriate corrections were made for nonlinearity of Klett response above a reading of 100. Preparotiotr of Crrrdc Extracts Log-phase cells of strain AT-7 grown in TYMn broth were spread on TYMn agar and incubated overnight at 55-60 "C. The cell crop was suspended in 'extract buffer' (0.05 M sodium phosphate, pH 6.6 containing 5 x M MgCI2), centrifuged at about 6000 x g, weighed, resuspended in three volumes of extract buffer, and broken by one or two passages through a French Pressure cell (American Instrument Co.). The yield was about 1 g (wet weight) of ceIls/100 cm2 of solid medium. The broken cell suspension was centrifuged at about 16 000 x g for 40 min at 25 "C and the pellet was discarded. Extracts were stored, unfrozen, at 0 "C. Enzyt~reAssnj~s 0-Galactosidase activity was determined by means of either a fixed-time or initial-rate assay procedure. All fixed-time assays were performed in duplicate. For the fixed-time assay in cell-free extracts, a I-ml reaction mixture was prepared containing an amount of extract chosen such that reaction velocity was first order with respect to enzyme concentration, 6 x M o-nitrophenyl-p-Dgalactopyranoside (ONPG; obtained from Nutritional Biochemicals Corp. or Sigma Chemical Co.), 0.1 M sodium phosphate (pH 6.4), and at least 1.5 mg/ml of gelatin (Difco) or bovine serum albumin (BSA; Sigma fraction V). The extra protein was added to stabilize the enzyme against thermal denaturation. All reagents except the substrate were placed in a test tube and prewarmed for 5 min at the reaction temperature, usually 55 "C, and the similarly preheated ONPG was then added. After incubation for either IS or 20 min, the reaction was stopped with 1.5 ml of 0.8 M N a 2 C 0 3 and the optical density was determined at 420 nm. Under these assay conditions, 10 nmol/ml of o-nitrophenol has an optical density of 0.047. Initial-rate assays were performed with a Cary model 14 recording spectrophotometer equipped with thermostable cell jackets. Samples (0.98 ml) containing all components listed above except the extract were prewarmed for 5 min in a water bath at the desired temperature and transferred to the cuvettes. The reaction was initiated by addition of 0.02 ml of the appropriate enzyme solution and the absorbance at 420 nm was recorded against a blank containing all components except the enzyme. Under these assay conditions (pH 6.4), 10 nmol/ml of o-nitrophenol has as optical density of 0.01 1.
One unit of enzyme activity is defined as that amount of 0-galactosidase which hydrolyzes 1 nmol of ONPG in 1 min. A fixed-time assay of 0-galactosidase with lactose (reagent grade; Matheson Coleman and Bell) as a substrate was similar t o that using ONPG except that t h e substrate concentration was 5 x M and t h e incubation time was either 25 or 30 min. The reaction was stopped by transfer of the tubes to a n ice bath. Glucose, liberated by the enzyme, was determined by the glucose oxidase - peroxidase assay (Tech. Bull. No. 510, Sigma Chemical Co.). To assay intracellular 0-galactosidase, 0.5 ml or less o f a culture was treated with several drops of toluene a n d mixed vigorously. The volume was then brought to 1.5 ml with 0.1 M sodium phosphate (pH 6.4-6.6). After addition of 0.5 ml of 1.2 x lo-' M ONPG, the mixture was incubated at 60°C. When we judged the mixture to be suitably yellow, the reaction was stopped with 1 ml of 0.8 M Na2C03 and the time was recorded. The samples were centrifuged when necessary, and the optical density was determined at 420 nm. Modifications of the assay procedures are described a s they pertain to individual experiments. Measuren~cntof Proteitr Protein concentration was determined by a modification of the method of Lowry el a/. (16). Crystalline BSA (Sigma) was used a s a standard. Partial Prrrificaliot~of 0-Galoctosidose The enzyme from strain AT-7 was partially purified by ammonium sulfate fractionation followed by diethylaminoethyl (DEAE) cellulose chromatography. A 270-rnl volume of crude extract (obtained from 64 g of packed cells) was brought to 25% saturation with respect t o (NH4),S04 by addition of the solid a t room temperature. The pH was maintained between 6 and 8 by addition of concentrated N H 4 0 H . After incubation for at least 1 h at 0 "C, the precipitate was removed by centrifugation a n d discarded. The supernatant was brought t o 50% saturation with respect to (NH4),S04 as above, and the precipitate was allowed to form at 0 "C. This precipitate was centrifuged, resuspended in about 30 ml of 0.01 it4 sodium phosphate buffer, pH 6.5, containing 0.02 M 2-mercaptoethanol (2-ME), a n d dialyzed against 3 x 800ml of the same buffer. This solution was applied to a column (2 c m x 24.5 cm) of DEAE-cellulose (Eastman Organic Chemicals) previously equilibrated with 0.05 M sodium phosphate buffer, pH 6.5, containing 0.02 M 2-ME. T h e column was washed with 100 ml of the same buffer a n d the bound proteins were then liberated by step elution with increasing concentrations of NaCl (0.5 to 2.5% in 0.5% increments). The !3-galactosidase was eluted with 1.5-2% NaCI. Fractions of 20 ml were collected and all containing significant 0-galactosidase were pooled. T h e resulting solution was placed in a dialysis bag and concentrated to 100 ml by pervaporation. A sevenfold purification with a 50% yield was obtained by these procedures. Thermal Inaclivation A tube containing 4.8 ml of buffer (0.1 M sodium phosphate, pH 6.4) was equilibrated at the desired temperature. Then 0.2 ml of the partially purified enzyme solution was added. At the appropriate times, duplicate 0.2-ml samples
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were transferred to chilled tubes containing 0.3 ml of the buffer supplemented with 4 mg of BSA. The enzyme activity of these samples was determined by the fixed-time assay procedure.
r
I
Molecular Weight Estiniation by Disc Electrophoresis Disc gel electrophoresis was performed according to Broome's (4) modification of the method of Davis (8). All experiments were carried out at room temperature with a tris(hydroxymethyl)aminomethane (7ris)glycine buffer at pH 8.9 and a current of 3-4 mA per tube. Gels of different concentration were prepared and proteins of known molecular weight used as standards. The proteins were located in the gel either by staining with amido black o r by activity stains. Data were analyzed according to the method of Hedrick and Smith (11).
Results Constitutive P-Galactosidase Synthesis Of the eight bacterial strains investigated, three did not possess d~scernible ONPGhydrolyzing activity, whereas five exhibited significant activity regardless of the presence or absence of 0.4% lactose or 1.2 x M isopropyl- P-D-thiogalactopyranoside (IPTG, Sigma) in the growth medium. These latter strains are designated AT-2, AT-3, AT-4, AT-7, and AT-8. Strains AT-3 and AT-7 appeared to possess greater P-galactosidase activity than the others and were therefore selected for further study. To examine the kinetics of P-galactosidase synthesis, we grew these bacteria on TYMn at 60 "C and followed the culture turbidity and P-galactosidase activity. Strain AT-7 produced significantly greater P-galactosidase at 60 "C than strain AT-3 for an equivalent culture turbidity (Fig. 1). A lactose operon has not been demonstrated Should such an operon for B. stearotherr~~oplzilus. exist, however, one possible explanation for constitutive synthesis of P-galactosidase by these bacteria growing at high temperatures could be that they produce a temperature-sensitive repressor. To explore this hypothesis, we grew strains AT-3 and AT-7 at 46 "C on TYMn, either unsupplemented, or supplemented with possible inducers: lo-' M lactose or 5 x l o p 4 M IPTG. Culture turbidity and P-galactosidase were followed for over 4 h. The differential plot of P-galactosidase activity against culture turbidity (Fig. 2) shows that the hypothesis of a temperature-sensitive repressor is not supported; for both strains the differential rates of synthesis of P-galactosidase at 46 "C are apparently independent of the presence or absence of lactose or
TURBIDITY (Klelt units)
FIG.1 . Differential plot of 8-galactosidase against culture turbidity for strains AT-3 and AT-7 grown a t 60 "C. A, AT-3 ; A, AT-7.
TURBIDITY (Klett units)
FIG.2. Differential plot of 8-galactosidase against culture turbidity for strains AT-3 and AT-7 grown a t 46°C. AT-3: lactose present, B; IPTG present, 0 ; n o inducer, A. AT-7: lactose present, ; IPTG present, 0; no inducer, A.
IPTG at the concentrations used. We cannot, however, rule out the possibility of a repressor so heat-labile that it is denatured at 46 "C. Interestingly, strain AT-3 exhibits greater P-galactosidase
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CAN. J. MICROBIOL. VOL. 22, 1976
activity than AT-7 at this low temperature, whereas the reverse is true at 60 "C (Fig. I). Also of interest is the apparent acceleration in the rate of P-galactosidase synthesis during growth at 46 "C by both strains (Fig. 2). At 60 "C only strain AT-7 displayed this phenomenon (Fig. 1). These increasing differential rates of P-galactosidase synthesis are apparently real and are not simply the result of chain formation or clumping of cells. Had such cellular aggregations occurred, they could have resulted in spuriously low culture turbidities. However, this explanation was not supported either by direct microscopic examination nor, at 46 "C, by the exponential increase in culture turbidity which occurred simultaneously with the accelerating differential rate of Pgalactosidase synthesis. Finally, strain AT-7, when grown in the presence of lactose, showed increasing P-galactosidase in the culture for a time, but a significant progressive decline in the amount of enzyme activity after the Klett reading reached about 100. We subsequently found that the AT-7 P-galactosidase is productinhibited by galactose. We believe that the apparent decline in P-galactosidase activity in this culture was a result of galactose liberated from lactose by the enzyme. Characterizatiot1 of AT-7 p-Galactosidase it? Cellfree Extracts To establish a fixed-time assay we followed the catalytic response of the AT-7 enzyme with two substrates, ONPG and lactose. The response with ONPG was linear for at least 20 min and that with lactose for over 50 min. We then routinely used reaction times for these two assays for which the catalytic response was linear. To determine the optimum storage temperature for this enzyme, we placed portions of the extract at -20 "C, 0 "C, room temperature (22-24 "C), and 50 "C. A few drops of toluene (previously determined to have no deleterious effect on the enzyme) were added to the tubes stored at room temperature and 50 "C to prevent bacterial growth. The stored samples were assayed with ONPG at intervals. After a variable lag period, the P-galactosidase activity of all stored samples decayed exponentially with halflives of about 250 days at room temperature, 75 days at 0 "C, 60 days at -20 "C, and 20 days at 50 "C. For examination of the effect of varying pH on
AT-7 P-galactosidase activity we prepared a set of 0.1 M sodium phosphate buffers and carefully recorded their p H at the temperature of assay with a Corning model 112 research p H meter. The assay procedure with lactose as substrate was as described in Materials and Methods but the fixed-time assay with ONPG differed in that the ONPG concentration was 3 x lop3 M and the incubation temperature was 60 "C. The effect of varying pH was substantially the same for both substrates (Fig. 3). The pH optimum lies between 6.0 and 6.4. Of particular interest is the extremely sharp decline in enzyme activity below pH 5.9. Enzyme activity was determined with ONPG at several temperatures by the fixed-time assay procedure. Maximum activity was observed a t 65 "C. An Arrhenius plot (Fig. 4) shows two distinct slopes, above and below 47 "C, giving activation energies of about 16 kcal/mol and 24 kcal/mol, respectively. We investigated the effect of a variety of substances on the catalytic activity of the enzyme a t M ONPG. None of the 60 "C with 3 x following substances had a significant effect: M CaC1,; M CoC1,; M FeSO,; 5 x l o p 3M MgC1,; 2 x M MnC1,; 1.5 x lo-, M ethylenediaminetetraacetate (EDTA); lo-, M 2-ME; lo-, M L-cysteine. The enzyme was 92% inhibited by lo-, M CuSO, and 30% inhibited by lo-, M ZnC1,. Extra protein, either gelatin or BSA, in the reaction mixture consistently resulted in greater enzyme activity than that found in the absence of added protein.
-
loo -
80-
',\ 60
g 2
0 \:,\\'
40
=
a
O
,,' 20
6.0
7.0
8.0
PH
FIG.3. Effect of pH on p-galactosidase activity. 0 , ONPG; 0, lactose.
82 1
GOODMAN AND PEDERSON
TABLE 1. Effect of various sugars on the catalytic activity of AT-7 8-galactosidase" 8-Galactosidase activity as % of control
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-
Lactose Glucose Galactose Fructose Mannose Mannitol Xylose Cellobiose Sucrose Raffinose
88 80 29 98 97 99 91 67 98 98
OAssayed with 3 x lo-' M ONPG a t 60 "C. sugars were tested a t a concentration of
10-1 M.
1
.
FIG.4. Effect of temperature on 0-galactosidase activity (Arrhenius plot). Velocity, expressed as nmol of ONPG hydrolyzed/min is plotted against the reciprocal of the absolute temperature.
Maximal activity was observed when the protein concentration in the reaction mixture was 1.5 mg/ml or greater. Landman (13) and Rohlfing and Crawford (22) have reported a P-galactosidase from Bacillus megaterium which is stimulated by glucose. We were interested, therefore, in seeing whether or not the AT-7 P-galactosidase was stimulated by this or other sugars. We found (Table l), on the contrary, that several sugars, notably galactose, cellobiose, glucose, and to a lesser extent lactose, inhibited the enzyme (when tested with the substrate ONPG). The effect of lactose is probably a case of competitive inhibition by another substrate, but that of glucose and galactose is probably competitive inhibition by the products of lactose hydrolysis. The pronounced inhibition of this enzyme by galactose led us to compare the affinity between this sugar and the enzyme with the affinities of the substrates ONPG and lactose. We measured differences in reaction velocity as the galactose concentration was varied in the presence of two concentrations of ONPG, and plotted the data according to the method of Dixon (9). A graph of the data from a typical experiment is depicted in Fig. 5. The intersection of the two lines above
GALACTOSE CONCENTRATION ( m ~ )
5. D i x ~ nplot o f the effect of varying galactose concentration on reaction velocity of AT-7 8-galactosidase. Velocity was recorded as nmol of ONPG hydrolyzed/ 10-3 M ONPG; 0, ,in at 55 OC, symbols: . , 3 6 x 10-3 M O N P G .
the x-axis indicates that the inhibition is of either the competitive or complex noncompetitive type rather than of the simple noncompetitive type (9). Although we cannot rule out complex noncompetitive inhibition, we hypothesize that galactose is a competitive inhibitor because this sugar is a product of the reaction. Under this assumption, the inhibitor constant (K,) and the Michaelis constant (K,) can be determined graphically from the Dixon plot. Average values for K, and K,,, obtained from several experiments are 2.5 x M and 9.8 x M, re-
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C A N . J. MICROBSOL. VOL. 22, 1976
spectively. The product galactose apparently binds more tightly to the enzyme than does the substrate ONPG. (In all discussions of K,,, we have assumed that the rate of product formation is slow relative to the rate of dissociation of the enzyme-substrate complex.) I n other experiments we determined the Km for ONPG at 60 "C by the method of Lineweaver and Burk (14). The value obtained from either the initial-rate or fixed-time assay is 6 x M. We determined the K,,, for lactose at 55 "C to be 0.1 1 M. Lactose, therefore, seems to bind more weakly to the enzyme than either ONPG or galactose. Thermal inactivation of the galactosidase was investigated at three different temperatnres (Fig. 6). Added protein had a significant protective effect. Such a stabilizing effect on a thermophilic a-amylase has been reported (20) and is most likely a result of nonspecific protein-protein interactions. Our thermal inactivation data would appear to indicate that appreciable loss of activity had occurred during the time period ~ ~ s eind our fixed-time assays. However, as we
TIME (rnin)
FIG.6. Thermal inactivation of AT-7 (3-galactosidase at three temperatures. Inactivation teniperatures are 55 "C (circles), 60 "C (squares), and 65 'C (triangles). In one set of experiments the enzyme was exposed to the inactivation temperature without added protein (open symbols); in the other set, 4 niglml of BSA was present (closed symbols).
stated previously, the catalytic response is linear over that time period and, in addition, the value of Km for ONPG at 60 "C is the same whether determined by either initial-rate or fixed-time assay. It is possible that the substrate has a significant stabilizing effect on the enzyme. Disc electrophoresis with gels of different concentration gives an approximate molecular weight of 21 5 000 for the partially purified AT-7 P-galactosidase (Fig. 7).
Discussion The thermophilic bacilli we have investigated synthesize P-galactosidase constitutively. This stands in marked contrast to the normally inducible synthesis of P-galactosidase by other members of the genus Bacillus (1, 13) and by the extreme thermophile investigated by Ulrich et al. (24). However, Anema (1) has isolated a constitutive mutant of B. subtilis. We have noted that strain AT-3 produces more of the enzyme than strain AT-7 if the two are grown a t low temperat~lre(46 "C) while the reverse is true if the bacteria are grown at high temperature (60 "C). This may indicate that some part of the protein synthetic machinery is somewhat temperature-sensitive in strain AT-3. In any case, the growth temperature has a greater influence on the rate of synthesis of P-galactosidase by AT-7 than on that by AT-3. The increasing differential rate of P-galactosidase synthesis observed for strains AT-3 (Fig. 2) and AT-7 (Figs. 1 and 2) is similar to the pattern ofsynthesis observed by Coleman (6) for a-amylase and other extracellular enzymes in B. subtilis. We have not overlooked the possibility that Pgalactosidase might be associated with sporulation in B. stearother1~7ophilus. The pH optimum of the AT-7 P-galactosidase lies between 6.0 and 6.4 for the substrates O N P G and lactose. This compares with optima of 6.5 reported for the enzyme isolated from B. subtilis (1) and 5 for the one from the extreme thermophile investigated by Ulrich et al. (24). The optimum of 7.3-8 for the B. n7egaterium enzyme reported by Landman (13) may not be valid for reasons pointed out by Anema (1). A strikingly sharp decline in enzyme activity was observed below pH 5.9 (Fig. 3). Such a pronounced pH dependence of enzyme activity is uncommon and to our knowledge has not been noted for other P-galactosidases. The steepness of the curve on the acid side suggests t o us a
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GOODMAN AND PEDERSON
FIG.7. Molecular weight of AT-7 P-galactosidase estimated from disc gel electrophoresis. The partially purified enzyme was used. Egg white lysozyme,BSA, and glucose oxidase were obtained from Sigma. The Escherichio coli K-12 P-galactosidase was prepared in our laboratory. Electrophoresis of the proteins was performed with gels of different concentrations: 5, 6, 8, and 10%. For each protein the logarithm of its mobility relative to that of bron~ophenolblue was plotted against gel concentration. The negative slopes from these graphs are plotted here against niolecular weight. highly cooperative pH-dependent phenomenon. A Hill plot of our data indicates that a minim~lm of six protons is released during the transition from an inactive to an active form. The teniperature optim~lm of 65 "C for the AT-7 P-galactosidase is close to optima reported for many other enzymes of B. steurotherniophilw (23). The distinct break observed in the Arrhenius plot (Fig. 4) is not uncomlnon for thermophilic enzymes. This discontin~lityis not sin~plya result of thermal inactivation of the enzyme, except possibly above 60 "C, because, as we have demonstrated, the catalytic response is linear at 55 "C during tlie inc~lbationtime and K,,, values determined at 60 "Care independent of the lilethod of assay. Because of the relatively high K,, for O N P G (abo~lt0.01 M) it was not practical to perform P-galactosidase assays ~lnder sat~lratingconditions of substrate. Accordingly, one might argue that tlie non-linearity could be accounted for by a decreasing stability of the enzyme-substrate complex as the temperature is increased. However, our data d o not support this hypothesis. Our investigation of the effect of various substances on the AT-7 P-galactosidase must be considered preliminary pending repetition of these experiments with a purified enzyme preparation. Nevertheless, it does appear that no divalent cation tested stimulates the enzyme nor
is it inhibited by EDTA. By comparison, the P-galactosidase of B. /1ieguterh/?i is stiln~llated by M n 2 + (12) but the B. slrbtilis enzyme is not affected by divalent cations (I). The enzyme from the thermopliile resembling T. aq~ratic~rs is stim~llatedby both M n 2 + and Fe2+ (24). Also, this latter enzyme is stimulated by cysteine and 2-ME whereas tlie AT-7 P-galactosidase is not (at least in c r ~ l d eextracts). The B. tnegateriirni P-galactosidase is stimulated by gl~lcose(13, 22) but tlie AT-7 enzyme is inhibited by this sugar as well as by galactose and cellobiose. This galactosidase is, therefore, product-inhibited and, as we have shown, galactose binds more strongly to the active site than either of the substrates, O N P G or lactose. Anema ( I ) has investigated the relative binding affinities of ONPG, lactose, and galactose for the P-galactosidase of B. subtilis. Because his assay conditions were similar to ours (pH 6.5 and 50 "C conipared to our p H 6.4 and 55 "C), his reported values for K,,, ( O N P G ) and K i (lactose and galactose) may be compared to those for the AT-7 P-galactosidase we report here (Table 2). In all three cases the constants for the B. subtilis enzyme are many times higher than the corresponding constants for the AT-7 P-galactosidase. Brock (3) has suggested that enzymes of thermophilic bacteria may be inherently less efficient than mesophilic enzymes. The very high
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4. BROOME,J. 1963. A rapid method of disk electrophoresis. Nature (Lond.), 199: 179-180. 5. B U C H A N A N R., E., and N. E. GIBBONS.(Eilitors). 1974. Bergey's manual of determinative bacteriology. K , or K r for K , or K , for 8th ed. The Williams and Wilkins Co., Baltimore. pp. AT-7 B . srrbtilis 539-540. P-galactosidase P-galactosidase 6. COLEMAN, G. 1967. Studies on the regulation of exLigand (MY (MIb tracellular enzyme formation by Bacillrrs srrbrilis. J. Gen. Microbiol. 49: 421-431. ONPG 9 . 8 lo-3 ~ (K,) 4 . 2 ~ (K,) 7. CRISAN,E. V. 1973. Current concepts of thermoLactose 0 . I 1 (K,) 0.71 ( K , ) philism and the thermophilic fungi. Mycologia, 65: Galactose 2 . 5 ~ (K,) 4 . 2 ~ (K,) 1171-1 199. 'Measured at pH 6.4 and 55 "C. 8. DAVIS, B. J. 1964. Disc electrophoresis. 11. Method bMeasured at pH 6.5 and 5 0 ° C ; data from (1). and application to human serum proteins. Ann. N.Y. Acad. Sci. 121: 40&427. K, of the AT-7 P-galactosidase with lactose 9. DIXON, M., and E. C. WEBB.1964. Enzymes. 2nd ed. seems to support this possibility, although the Academic Press, Inc., New York. pp. 328-330. H.-U., and H. Z U B E R .1974 Thermobinding efficiency of the B. subrilis enzyme 10. HABERSTICH, adaptation of enzymes in thermophilic and mesophilic (Table 2) is also very low. It seems, however, that cultures of Boc~illrrssrc~nrorhermoplrilrrs and Bacill~rs the latter is a thermophilic enzyme residing in a c~i1doirna.r.Arch. Mikrobiol. 98: 275-287. mesophilic organism (1). 11. HEDRICK, J. L., and A. J. SMITH.1968. Size and charge isomer separation and estimation of molecular An alternate explanation of the high K, weights by disk gel electrophoresis. Arch. Biochem. values observed with these enzymes is that 125: 155-164. neither ONPG nor lactose is a normal substrate; 12. JBiophys. U N G ,L., R. JOST, E. STOLL,and H. ZUBER.1974. other galactosides such as D-galactans or galacMetabolic differences in Bncillrrs srocrrorhc~,mopi~iIrrs tolipids seem likely candidates. Indeed, these grown at 55 "C and 37°C. Arch. Mikrobiol. 95: 125-138. enzymes may not be primarily P-galacto~idases 13. L ANDMAN 0. . E. 1957. Properties and induction of at all, but glycosidases with a specific.ty broader P-galactosidase in Bncillrrs tnegrctc.rirrtn. Biochem. than is common with such enzymes. A loose Biophys. Acta. 23: 558-569. substrate specificity might be a property of some 14. L I N E W E A V EH., R , and D. BURK.1934. The determination of enzyme dissociation constants. J . Am. Chem. thermo~hilicenzvmes. owinp: " to the constraints s o c . 56: 658-666. on conformation of the proteins imposed by high 15. LJUNGER, C. 1973. Further investigations on the natemperatures. ture of the heat resistance of thermophilic bacteria. Physiol. Plant. 28: 415-418. Acknowledgments 16. LOWRY,0 . H.. N. J. ROSEBROUGH, A. L . FARR,and R. J. R A N D A L L1951. . Protein measurement with the We express our appreciation to Mr. Donald Folin phenol reagent. J. Biol. Chem. 193: 265-275. Marks for technical assistance and to Miss 17. M A N N I N GG. , B., and L. L. CAMPBELL.1961. Deborah Sullivan, whose observation of Thermostable a-amylase of Bncill~tssrearorhermopkigalactosidase in our strains of B. srearorhermo111s.I. Crystallization and some general properties. J. Biol. Chem. 236: 2952-2957. philus provided the initial stimulus for this reK., A. IMANISHI, and T . ISEMURA. 1970. search. We are also grateful to the Campus 18. OGASAHARA, Studies on thermophilic a-amylase from Bncillrrs Crusade for Christ for its cooperation during stenrorherrnopi~ilrrs. I. Some general and physicoour visits to the Arrowhead Hot Springs. chemical properties of thermophilic a-amylase. J. This research was supported in part by a Biochem. 67: 65-75. K., A. IMANISHI, and T . ISEMURA. 1970. National Science Foundation Institutional 19. OGASAHARA, Studies on thermophilic a-amylase from Bnc~illrrs Grant, GU3221-2. ~renroilrc~rtnophilrrs. 11. Thermal stability of thermophilic a-amylase. J. Biochem. 67: 77-82. 1. ANEMA,P. J. 1964. Purification and some properties K., K. YUTANI,A. I M A N I S Hand I. T . 20. OGASAHARA, ISEMURA.1970. Studies on thermophilic a-amylase of P-galactosidase of BnciNrrs srrbrilis. Biochem. Biophys. Acta, 89: 495-502. 111. Effect of tempfrom Bnc~illrrssrc~nrotl~c~rtnopI~iIr~s. erature on the renaturation of denatured thermophilic 2. BABEL,W.. H . A. ROSENTHAL, and S. RAPAPORT. 1972. A unified hypothesis on the causes of the cardia-amylase. J. Biochem. 67: 83-89. nal temperatures of microorganisms; the temperature 21. PFUELLER. S. L., and W. H . ELLIOTT.1969. The extracellular alpha-amylase of Bncillrrs srclnrorhrrActa Biol. minimum of Bnc,illrrs stc~nr~orhertnopi~iI~rs. mophilrrs. J. Biol. Chem. 244: 48-54. Med. Ger. 28: 565-576. S . R., and I. P. CRAWFORD. 1966. Partial 22. KOHLFING, 3. BROCK, T . D. 1967. Life at high temperatures. Science (Wash.), 158: 1012-1019. purification and physical properties of Bucillrrs
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TABLE2. Comparison of K,, and K , for 0-galactosidase from strain AT-7 and B. subtilis
P-
GOODMAN AND PEDERSON
N. E.. and L. I-. CAMPBELL. 1963. Effect of 26. WELKER. carbon sources on formation of alpha-amylase by 1259. Brrcillrrs s r ~ ~ ~ r o r l ~ ~ ~ t ? J. ~ oBacteriol. p I ~ i l i ~ s86: . 68123. SINGLETON, R., and R. E . A M E L U N X E N 1973. . Pro686. teins from thermophilic microorganisms. Bacteriol. 27. Y U T A N IK. , 1973. Molecular weight of thermostable Rev. 37: 320-342. a-amylase from B. sreclrorhemophil~rs.J. Biochem. 24. ULRICH, J. T . , G . A. MCFETERS,and K . L. TEMPLE. 74: 58 1-586. 1972. Induction and chal-acterization of p-galactosi28. Y U T A N IK., , I. S A S A K Iand , K. OGASAHARA. 1973. dase in an extreme thermophile. J. Bacteriol. 110: Comparison of thermostable B. src~orotlirrt~~ophil~ts 69 1-698. grown at different temperatures. J. Biochem. 74: 25. WEERKAMP, A , , and R . D. MACELROY. 1972. Lactate 573-579. dehydrogenase from an extremely thermophilic B N L . ~ / / IArch. I S . Mikrobiol. 85: 113-122. tncgaterirtm P-galactosidase. J. Bacteriol. 92: 125%
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825
I
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Accepted February 20, 1976
R. E., and D. M. PEDERSON. 1976. P-Galactosidase from Bocillirs stc~rrrorhcvGOODMAN, mopl~ilrrs.Can. J . Microbiol. 22: 817-825. Several strains of thermophilic aerobic spore-forming bacilli synthesize P-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The p-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 "C and the Arrhenius activation energy is about 24 kcallmol below 47 "C and 16 kcallmol above that temperature. At 55 "C the K , is 0. l l M for lactose and 9.8 x M for o-nitrophenyl-P-D-galactopyranoside.The enzyme is strongly product-inhibited by galactose (Ki = 2.5 x M). It is relatively stable at 5OoC,losing only half of its activity after 20 days at this temperature. At 60 "C more than 60%of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein. and in the presence of 4 mglml of bovine serum albumin the enzyme is only 18% inactivated in I0 min at 60 "C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000. GOODMAN, R. E., et D. M. PEDERSON.1976. P-Galactosidase from Brrcillus sterrrothermophilus. Can. J . Microbiol. 22: 8 17-825. Quelques souches de bacilles sporuIants thermophiles aerobies synthitisent une P-galactosidase (EC 3.2.1.23) de f a ~ o nconstitutive. Cette propriett constitutive n'est apparernrnent pas due a un represseur thermosensible. La 0-galactosidase d'une souche, etudiie dans des extraits acellulaires a un pH optimal entre 6.0 et 6.4 et une dependance de pH tres nette du c6te acide pour son optimum. Pour cette enzyme, la temperature optimale est de 65 "C et I'energie d'activation d'ArrhCnius est d'environ 24 kcallmol au-dessous de 47 "C et de 16 kcallmol M au-dessus de cette ternpbrature. A 55 "C le K, est egal a 0.11 M pour le lactose et a 9.8 x pour 1'0-nitrophenyl-p-D-galactopyranoside.Pour cette enzyme, I'inhibition par le produit est M). L'enzyrne est relativement stable a 50 "C tres forte dans le cas du galactose (Ki = 2.5 x car, apres 20 jours a cette temperature, elle perd seulernent la moitie de son activite. A 60 "C plus de 60% de I'activite disparait en 10 min. Une proteine protege I'enzyme contre ['inactivation thermique. En presence de 4 mg/ml d'albumine skrique bovine, I'inactivation est reduite a 18% apres 10 rnin B 60 "C. EvaIut par electrophortse en gel le poids moleculaire est &gali 215 000. [Traduit par le journal]
Introduction The biochemical basis of thermophily, though widely investigated, has not yet been satisfactorily explained by any unitary hypothesis. Several recent excellent reviews (7, 23) have examined many possible bases for thermophily. The most frequently advanced (and supported) hypothesis is that the proteins of thermophiles are more thermostable than those of mesophiles, allowing the bacteria to survive a t temperatures at which many mesophilic proteins are denatured. However, no obvious chemical differences between thermophilic and mesophilic proteins have been demonstrated which might account for their differences in thermostability (23). So, the question is still open and new perspectives are continually being brought forth (2, 10, 12, 15, 25). 'Received December 2, 1975.
We have detected P-D-galactoside galactohydrolase (EC 3.2.1.23; P-galactosidase) in five of eight strains of Bacillus stearothermophilus. The five strains apparently produce this enzyme constitutively. Although a-amylases (17-19, 21, 26-28) from thermophilic members of the genus Bacillus have been examined, we have not encountered reports of other glycosidases from such bacteria. An inducible P-galactosidase has, however, been reported from an extreme thermophile resembling Thermus aquaticus (24). The present report describes some characteristics of the galactosidase of B. stearothermophilus.
Materials and Methods Bacterial Straitls
Eight strains of aerobic, obligately thermophilic, spore-forming bacilli were isolated from the Arrowhead Hot Springs located in the San Bernardino Mountains. The temperatures of the aqueous environments from which samples were taken ranged from 55 t o 80 "C. Each
818
CAN. J. MICROBIOL. V O L . 22, 1976
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of the eight strains has a minimum growth temperature between 38 and 41 "C, and a maxinium between 67 and 74 "C. These temperature characteristics allow us tentatively to identify all eight strains as Bacilllrs stearotlre,n~ophil~rs (5). For reference, the strains are numbered AT-1 (Arrowhead thermophile No. 1) through AT-8. Media atzd Grotvtll Conditions Bacteria were routinely cultured at 55-60 "C on TYMn medium containing (per litre) tryptone(Difco), l o g ; yeast extract, 5 g; and MnCI2, mol. For a solid medium, TYMn was supplemented with 3% agar. Liquid cultures were aerated in Erlenmeyer flasks on a reciprocating waterbath shaker at 150-180 strokes/min. The medium comprised no more than 20% of the flask volume. Culture turbidity was monitored with a KlettSumnierson photoelectric colorinieter with a green filter (No. 52). Appropriate corrections were made for nonlinearity of Klett response above a reading of 100. Preparotiotr of Crrrdc Extracts Log-phase cells of strain AT-7 grown in TYMn broth were spread on TYMn agar and incubated overnight at 55-60 "C. The cell crop was suspended in 'extract buffer' (0.05 M sodium phosphate, pH 6.6 containing 5 x M MgCI2), centrifuged at about 6000 x g, weighed, resuspended in three volumes of extract buffer, and broken by one or two passages through a French Pressure cell (American Instrument Co.). The yield was about 1 g (wet weight) of ceIls/100 cm2 of solid medium. The broken cell suspension was centrifuged at about 16 000 x g for 40 min at 25 "C and the pellet was discarded. Extracts were stored, unfrozen, at 0 "C. Enzyt~reAssnj~s 0-Galactosidase activity was determined by means of either a fixed-time or initial-rate assay procedure. All fixed-time assays were performed in duplicate. For the fixed-time assay in cell-free extracts, a I-ml reaction mixture was prepared containing an amount of extract chosen such that reaction velocity was first order with respect to enzyme concentration, 6 x M o-nitrophenyl-p-Dgalactopyranoside (ONPG; obtained from Nutritional Biochemicals Corp. or Sigma Chemical Co.), 0.1 M sodium phosphate (pH 6.4), and at least 1.5 mg/ml of gelatin (Difco) or bovine serum albumin (BSA; Sigma fraction V). The extra protein was added to stabilize the enzyme against thermal denaturation. All reagents except the substrate were placed in a test tube and prewarmed for 5 min at the reaction temperature, usually 55 "C, and the similarly preheated ONPG was then added. After incubation for either IS or 20 min, the reaction was stopped with 1.5 ml of 0.8 M N a 2 C 0 3 and the optical density was determined at 420 nm. Under these assay conditions, 10 nmol/ml of o-nitrophenol has an optical density of 0.047. Initial-rate assays were performed with a Cary model 14 recording spectrophotometer equipped with thermostable cell jackets. Samples (0.98 ml) containing all components listed above except the extract were prewarmed for 5 min in a water bath at the desired temperature and transferred to the cuvettes. The reaction was initiated by addition of 0.02 ml of the appropriate enzyme solution and the absorbance at 420 nm was recorded against a blank containing all components except the enzyme. Under these assay conditions (pH 6.4), 10 nmol/ml of o-nitrophenol has as optical density of 0.01 1.
One unit of enzyme activity is defined as that amount of 0-galactosidase which hydrolyzes 1 nmol of ONPG in 1 min. A fixed-time assay of 0-galactosidase with lactose (reagent grade; Matheson Coleman and Bell) as a substrate was similar t o that using ONPG except that t h e substrate concentration was 5 x M and t h e incubation time was either 25 or 30 min. The reaction was stopped by transfer of the tubes to a n ice bath. Glucose, liberated by the enzyme, was determined by the glucose oxidase - peroxidase assay (Tech. Bull. No. 510, Sigma Chemical Co.). To assay intracellular 0-galactosidase, 0.5 ml or less o f a culture was treated with several drops of toluene a n d mixed vigorously. The volume was then brought to 1.5 ml with 0.1 M sodium phosphate (pH 6.4-6.6). After addition of 0.5 ml of 1.2 x lo-' M ONPG, the mixture was incubated at 60°C. When we judged the mixture to be suitably yellow, the reaction was stopped with 1 ml of 0.8 M Na2C03 and the time was recorded. The samples were centrifuged when necessary, and the optical density was determined at 420 nm. Modifications of the assay procedures are described a s they pertain to individual experiments. Measuren~cntof Proteitr Protein concentration was determined by a modification of the method of Lowry el a/. (16). Crystalline BSA (Sigma) was used a s a standard. Partial Prrrificaliot~of 0-Galoctosidose The enzyme from strain AT-7 was partially purified by ammonium sulfate fractionation followed by diethylaminoethyl (DEAE) cellulose chromatography. A 270-rnl volume of crude extract (obtained from 64 g of packed cells) was brought to 25% saturation with respect t o (NH4),S04 by addition of the solid a t room temperature. The pH was maintained between 6 and 8 by addition of concentrated N H 4 0 H . After incubation for at least 1 h at 0 "C, the precipitate was removed by centrifugation a n d discarded. The supernatant was brought t o 50% saturation with respect to (NH4),S04 as above, and the precipitate was allowed to form at 0 "C. This precipitate was centrifuged, resuspended in about 30 ml of 0.01 it4 sodium phosphate buffer, pH 6.5, containing 0.02 M 2-mercaptoethanol (2-ME), a n d dialyzed against 3 x 800ml of the same buffer. This solution was applied to a column (2 c m x 24.5 cm) of DEAE-cellulose (Eastman Organic Chemicals) previously equilibrated with 0.05 M sodium phosphate buffer, pH 6.5, containing 0.02 M 2-ME. T h e column was washed with 100 ml of the same buffer a n d the bound proteins were then liberated by step elution with increasing concentrations of NaCl (0.5 to 2.5% in 0.5% increments). The !3-galactosidase was eluted with 1.5-2% NaCI. Fractions of 20 ml were collected and all containing significant 0-galactosidase were pooled. T h e resulting solution was placed in a dialysis bag and concentrated to 100 ml by pervaporation. A sevenfold purification with a 50% yield was obtained by these procedures. Thermal Inaclivation A tube containing 4.8 ml of buffer (0.1 M sodium phosphate, pH 6.4) was equilibrated at the desired temperature. Then 0.2 ml of the partially purified enzyme solution was added. At the appropriate times, duplicate 0.2-ml samples
819
GOODMAN A1VD PEDERSON
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were transferred to chilled tubes containing 0.3 ml of the buffer supplemented with 4 mg of BSA. The enzyme activity of these samples was determined by the fixed-time assay procedure.
r
I
Molecular Weight Estiniation by Disc Electrophoresis Disc gel electrophoresis was performed according to Broome's (4) modification of the method of Davis (8). All experiments were carried out at room temperature with a tris(hydroxymethyl)aminomethane (7ris)glycine buffer at pH 8.9 and a current of 3-4 mA per tube. Gels of different concentration were prepared and proteins of known molecular weight used as standards. The proteins were located in the gel either by staining with amido black o r by activity stains. Data were analyzed according to the method of Hedrick and Smith (11).
Results Constitutive P-Galactosidase Synthesis Of the eight bacterial strains investigated, three did not possess d~scernible ONPGhydrolyzing activity, whereas five exhibited significant activity regardless of the presence or absence of 0.4% lactose or 1.2 x M isopropyl- P-D-thiogalactopyranoside (IPTG, Sigma) in the growth medium. These latter strains are designated AT-2, AT-3, AT-4, AT-7, and AT-8. Strains AT-3 and AT-7 appeared to possess greater P-galactosidase activity than the others and were therefore selected for further study. To examine the kinetics of P-galactosidase synthesis, we grew these bacteria on TYMn at 60 "C and followed the culture turbidity and P-galactosidase activity. Strain AT-7 produced significantly greater P-galactosidase at 60 "C than strain AT-3 for an equivalent culture turbidity (Fig. 1). A lactose operon has not been demonstrated Should such an operon for B. stearotherr~~oplzilus. exist, however, one possible explanation for constitutive synthesis of P-galactosidase by these bacteria growing at high temperatures could be that they produce a temperature-sensitive repressor. To explore this hypothesis, we grew strains AT-3 and AT-7 at 46 "C on TYMn, either unsupplemented, or supplemented with possible inducers: lo-' M lactose or 5 x l o p 4 M IPTG. Culture turbidity and P-galactosidase were followed for over 4 h. The differential plot of P-galactosidase activity against culture turbidity (Fig. 2) shows that the hypothesis of a temperature-sensitive repressor is not supported; for both strains the differential rates of synthesis of P-galactosidase at 46 "C are apparently independent of the presence or absence of lactose or
TURBIDITY (Klelt units)
FIG.1 . Differential plot of 8-galactosidase against culture turbidity for strains AT-3 and AT-7 grown a t 60 "C. A, AT-3 ; A, AT-7.
TURBIDITY (Klett units)
FIG.2. Differential plot of 8-galactosidase against culture turbidity for strains AT-3 and AT-7 grown a t 46°C. AT-3: lactose present, B; IPTG present, 0 ; n o inducer, A. AT-7: lactose present, ; IPTG present, 0; no inducer, A.
IPTG at the concentrations used. We cannot, however, rule out the possibility of a repressor so heat-labile that it is denatured at 46 "C. Interestingly, strain AT-3 exhibits greater P-galactosidase
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CAN. J. MICROBIOL. VOL. 22, 1976
activity than AT-7 at this low temperature, whereas the reverse is true at 60 "C (Fig. I). Also of interest is the apparent acceleration in the rate of P-galactosidase synthesis during growth at 46 "C by both strains (Fig. 2). At 60 "C only strain AT-7 displayed this phenomenon (Fig. 1). These increasing differential rates of P-galactosidase synthesis are apparently real and are not simply the result of chain formation or clumping of cells. Had such cellular aggregations occurred, they could have resulted in spuriously low culture turbidities. However, this explanation was not supported either by direct microscopic examination nor, at 46 "C, by the exponential increase in culture turbidity which occurred simultaneously with the accelerating differential rate of Pgalactosidase synthesis. Finally, strain AT-7, when grown in the presence of lactose, showed increasing P-galactosidase in the culture for a time, but a significant progressive decline in the amount of enzyme activity after the Klett reading reached about 100. We subsequently found that the AT-7 P-galactosidase is productinhibited by galactose. We believe that the apparent decline in P-galactosidase activity in this culture was a result of galactose liberated from lactose by the enzyme. Characterizatiot1 of AT-7 p-Galactosidase it? Cellfree Extracts To establish a fixed-time assay we followed the catalytic response of the AT-7 enzyme with two substrates, ONPG and lactose. The response with ONPG was linear for at least 20 min and that with lactose for over 50 min. We then routinely used reaction times for these two assays for which the catalytic response was linear. To determine the optimum storage temperature for this enzyme, we placed portions of the extract at -20 "C, 0 "C, room temperature (22-24 "C), and 50 "C. A few drops of toluene (previously determined to have no deleterious effect on the enzyme) were added to the tubes stored at room temperature and 50 "C to prevent bacterial growth. The stored samples were assayed with ONPG at intervals. After a variable lag period, the P-galactosidase activity of all stored samples decayed exponentially with halflives of about 250 days at room temperature, 75 days at 0 "C, 60 days at -20 "C, and 20 days at 50 "C. For examination of the effect of varying pH on
AT-7 P-galactosidase activity we prepared a set of 0.1 M sodium phosphate buffers and carefully recorded their p H at the temperature of assay with a Corning model 112 research p H meter. The assay procedure with lactose as substrate was as described in Materials and Methods but the fixed-time assay with ONPG differed in that the ONPG concentration was 3 x lop3 M and the incubation temperature was 60 "C. The effect of varying pH was substantially the same for both substrates (Fig. 3). The pH optimum lies between 6.0 and 6.4. Of particular interest is the extremely sharp decline in enzyme activity below pH 5.9. Enzyme activity was determined with ONPG at several temperatures by the fixed-time assay procedure. Maximum activity was observed a t 65 "C. An Arrhenius plot (Fig. 4) shows two distinct slopes, above and below 47 "C, giving activation energies of about 16 kcal/mol and 24 kcal/mol, respectively. We investigated the effect of a variety of substances on the catalytic activity of the enzyme a t M ONPG. None of the 60 "C with 3 x following substances had a significant effect: M CaC1,; M CoC1,; M FeSO,; 5 x l o p 3M MgC1,; 2 x M MnC1,; 1.5 x lo-, M ethylenediaminetetraacetate (EDTA); lo-, M 2-ME; lo-, M L-cysteine. The enzyme was 92% inhibited by lo-, M CuSO, and 30% inhibited by lo-, M ZnC1,. Extra protein, either gelatin or BSA, in the reaction mixture consistently resulted in greater enzyme activity than that found in the absence of added protein.
-
loo -
80-
',\ 60
g 2
0 \:,\\'
40
=
a
O
,,' 20
6.0
7.0
8.0
PH
FIG.3. Effect of pH on p-galactosidase activity. 0 , ONPG; 0, lactose.
82 1
GOODMAN AND PEDERSON
TABLE 1. Effect of various sugars on the catalytic activity of AT-7 8-galactosidase" 8-Galactosidase activity as % of control
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-
Lactose Glucose Galactose Fructose Mannose Mannitol Xylose Cellobiose Sucrose Raffinose
88 80 29 98 97 99 91 67 98 98
OAssayed with 3 x lo-' M ONPG a t 60 "C. sugars were tested a t a concentration of
10-1 M.
1
.
FIG.4. Effect of temperature on 0-galactosidase activity (Arrhenius plot). Velocity, expressed as nmol of ONPG hydrolyzed/min is plotted against the reciprocal of the absolute temperature.
Maximal activity was observed when the protein concentration in the reaction mixture was 1.5 mg/ml or greater. Landman (13) and Rohlfing and Crawford (22) have reported a P-galactosidase from Bacillus megaterium which is stimulated by glucose. We were interested, therefore, in seeing whether or not the AT-7 P-galactosidase was stimulated by this or other sugars. We found (Table l), on the contrary, that several sugars, notably galactose, cellobiose, glucose, and to a lesser extent lactose, inhibited the enzyme (when tested with the substrate ONPG). The effect of lactose is probably a case of competitive inhibition by another substrate, but that of glucose and galactose is probably competitive inhibition by the products of lactose hydrolysis. The pronounced inhibition of this enzyme by galactose led us to compare the affinity between this sugar and the enzyme with the affinities of the substrates ONPG and lactose. We measured differences in reaction velocity as the galactose concentration was varied in the presence of two concentrations of ONPG, and plotted the data according to the method of Dixon (9). A graph of the data from a typical experiment is depicted in Fig. 5. The intersection of the two lines above
GALACTOSE CONCENTRATION ( m ~ )
5. D i x ~ nplot o f the effect of varying galactose concentration on reaction velocity of AT-7 8-galactosidase. Velocity was recorded as nmol of ONPG hydrolyzed/ 10-3 M ONPG; 0, ,in at 55 OC, symbols: . , 3 6 x 10-3 M O N P G .
the x-axis indicates that the inhibition is of either the competitive or complex noncompetitive type rather than of the simple noncompetitive type (9). Although we cannot rule out complex noncompetitive inhibition, we hypothesize that galactose is a competitive inhibitor because this sugar is a product of the reaction. Under this assumption, the inhibitor constant (K,) and the Michaelis constant (K,) can be determined graphically from the Dixon plot. Average values for K, and K,,, obtained from several experiments are 2.5 x M and 9.8 x M, re-
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C A N . J. MICROBSOL. VOL. 22, 1976
spectively. The product galactose apparently binds more tightly to the enzyme than does the substrate ONPG. (In all discussions of K,,, we have assumed that the rate of product formation is slow relative to the rate of dissociation of the enzyme-substrate complex.) I n other experiments we determined the Km for ONPG at 60 "C by the method of Lineweaver and Burk (14). The value obtained from either the initial-rate or fixed-time assay is 6 x M. We determined the K,,, for lactose at 55 "C to be 0.1 1 M. Lactose, therefore, seems to bind more weakly to the enzyme than either ONPG or galactose. Thermal inactivation of the galactosidase was investigated at three different temperatnres (Fig. 6). Added protein had a significant protective effect. Such a stabilizing effect on a thermophilic a-amylase has been reported (20) and is most likely a result of nonspecific protein-protein interactions. Our thermal inactivation data would appear to indicate that appreciable loss of activity had occurred during the time period ~ ~ s eind our fixed-time assays. However, as we
TIME (rnin)
FIG.6. Thermal inactivation of AT-7 (3-galactosidase at three temperatures. Inactivation teniperatures are 55 "C (circles), 60 "C (squares), and 65 'C (triangles). In one set of experiments the enzyme was exposed to the inactivation temperature without added protein (open symbols); in the other set, 4 niglml of BSA was present (closed symbols).
stated previously, the catalytic response is linear over that time period and, in addition, the value of Km for ONPG at 60 "C is the same whether determined by either initial-rate or fixed-time assay. It is possible that the substrate has a significant stabilizing effect on the enzyme. Disc electrophoresis with gels of different concentration gives an approximate molecular weight of 21 5 000 for the partially purified AT-7 P-galactosidase (Fig. 7).
Discussion The thermophilic bacilli we have investigated synthesize P-galactosidase constitutively. This stands in marked contrast to the normally inducible synthesis of P-galactosidase by other members of the genus Bacillus (1, 13) and by the extreme thermophile investigated by Ulrich et al. (24). However, Anema (1) has isolated a constitutive mutant of B. subtilis. We have noted that strain AT-3 produces more of the enzyme than strain AT-7 if the two are grown a t low temperat~lre(46 "C) while the reverse is true if the bacteria are grown at high temperature (60 "C). This may indicate that some part of the protein synthetic machinery is somewhat temperature-sensitive in strain AT-3. In any case, the growth temperature has a greater influence on the rate of synthesis of P-galactosidase by AT-7 than on that by AT-3. The increasing differential rate of P-galactosidase synthesis observed for strains AT-3 (Fig. 2) and AT-7 (Figs. 1 and 2) is similar to the pattern ofsynthesis observed by Coleman (6) for a-amylase and other extracellular enzymes in B. subtilis. We have not overlooked the possibility that Pgalactosidase might be associated with sporulation in B. stearother1~7ophilus. The pH optimum of the AT-7 P-galactosidase lies between 6.0 and 6.4 for the substrates O N P G and lactose. This compares with optima of 6.5 reported for the enzyme isolated from B. subtilis (1) and 5 for the one from the extreme thermophile investigated by Ulrich et al. (24). The optimum of 7.3-8 for the B. n7egaterium enzyme reported by Landman (13) may not be valid for reasons pointed out by Anema (1). A strikingly sharp decline in enzyme activity was observed below pH 5.9 (Fig. 3). Such a pronounced pH dependence of enzyme activity is uncommon and to our knowledge has not been noted for other P-galactosidases. The steepness of the curve on the acid side suggests t o us a
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GOODMAN AND PEDERSON
FIG.7. Molecular weight of AT-7 P-galactosidase estimated from disc gel electrophoresis. The partially purified enzyme was used. Egg white lysozyme,BSA, and glucose oxidase were obtained from Sigma. The Escherichio coli K-12 P-galactosidase was prepared in our laboratory. Electrophoresis of the proteins was performed with gels of different concentrations: 5, 6, 8, and 10%. For each protein the logarithm of its mobility relative to that of bron~ophenolblue was plotted against gel concentration. The negative slopes from these graphs are plotted here against niolecular weight. highly cooperative pH-dependent phenomenon. A Hill plot of our data indicates that a minim~lm of six protons is released during the transition from an inactive to an active form. The teniperature optim~lm of 65 "C for the AT-7 P-galactosidase is close to optima reported for many other enzymes of B. steurotherniophilw (23). The distinct break observed in the Arrhenius plot (Fig. 4) is not uncomlnon for thermophilic enzymes. This discontin~lityis not sin~plya result of thermal inactivation of the enzyme, except possibly above 60 "C, because, as we have demonstrated, the catalytic response is linear at 55 "C during tlie inc~lbationtime and K,,, values determined at 60 "Care independent of the lilethod of assay. Because of the relatively high K,, for O N P G (abo~lt0.01 M) it was not practical to perform P-galactosidase assays ~lnder sat~lratingconditions of substrate. Accordingly, one might argue that tlie non-linearity could be accounted for by a decreasing stability of the enzyme-substrate complex as the temperature is increased. However, our data d o not support this hypothesis. Our investigation of the effect of various substances on the AT-7 P-galactosidase must be considered preliminary pending repetition of these experiments with a purified enzyme preparation. Nevertheless, it does appear that no divalent cation tested stimulates the enzyme nor
is it inhibited by EDTA. By comparison, the P-galactosidase of B. /1ieguterh/?i is stiln~llated by M n 2 + (12) but the B. slrbtilis enzyme is not affected by divalent cations (I). The enzyme from the thermopliile resembling T. aq~ratic~rs is stim~llatedby both M n 2 + and Fe2+ (24). Also, this latter enzyme is stimulated by cysteine and 2-ME whereas tlie AT-7 P-galactosidase is not (at least in c r ~ l d eextracts). The B. tnegateriirni P-galactosidase is stimulated by gl~lcose(13, 22) but tlie AT-7 enzyme is inhibited by this sugar as well as by galactose and cellobiose. This galactosidase is, therefore, product-inhibited and, as we have shown, galactose binds more strongly to the active site than either of the substrates, O N P G or lactose. Anema ( I ) has investigated the relative binding affinities of ONPG, lactose, and galactose for the P-galactosidase of B. subtilis. Because his assay conditions were similar to ours (pH 6.5 and 50 "C conipared to our p H 6.4 and 55 "C), his reported values for K,,, ( O N P G ) and K i (lactose and galactose) may be compared to those for the AT-7 P-galactosidase we report here (Table 2). In all three cases the constants for the B. subtilis enzyme are many times higher than the corresponding constants for the AT-7 P-galactosidase. Brock (3) has suggested that enzymes of thermophilic bacteria may be inherently less efficient than mesophilic enzymes. The very high
824
C A N . J. MICROBIOL. V O L . 22, 1976
4. BROOME,J. 1963. A rapid method of disk electrophoresis. Nature (Lond.), 199: 179-180. 5. B U C H A N A N R., E., and N. E. GIBBONS.(Eilitors). 1974. Bergey's manual of determinative bacteriology. K , or K r for K , or K , for 8th ed. The Williams and Wilkins Co., Baltimore. pp. AT-7 B . srrbtilis 539-540. P-galactosidase P-galactosidase 6. COLEMAN, G. 1967. Studies on the regulation of exLigand (MY (MIb tracellular enzyme formation by Bacillrrs srrbrilis. J. Gen. Microbiol. 49: 421-431. ONPG 9 . 8 lo-3 ~ (K,) 4 . 2 ~ (K,) 7. CRISAN,E. V. 1973. Current concepts of thermoLactose 0 . I 1 (K,) 0.71 ( K , ) philism and the thermophilic fungi. Mycologia, 65: Galactose 2 . 5 ~ (K,) 4 . 2 ~ (K,) 1171-1 199. 'Measured at pH 6.4 and 55 "C. 8. DAVIS, B. J. 1964. Disc electrophoresis. 11. Method bMeasured at pH 6.5 and 5 0 ° C ; data from (1). and application to human serum proteins. Ann. N.Y. Acad. Sci. 121: 40&427. K, of the AT-7 P-galactosidase with lactose 9. DIXON, M., and E. C. WEBB.1964. Enzymes. 2nd ed. seems to support this possibility, although the Academic Press, Inc., New York. pp. 328-330. H.-U., and H. Z U B E R .1974 Thermobinding efficiency of the B. subrilis enzyme 10. HABERSTICH, adaptation of enzymes in thermophilic and mesophilic (Table 2) is also very low. It seems, however, that cultures of Boc~illrrssrc~nrorhermoplrilrrs and Bacill~rs the latter is a thermophilic enzyme residing in a c~i1doirna.r.Arch. Mikrobiol. 98: 275-287. mesophilic organism (1). 11. HEDRICK, J. L., and A. J. SMITH.1968. Size and charge isomer separation and estimation of molecular An alternate explanation of the high K, weights by disk gel electrophoresis. Arch. Biochem. values observed with these enzymes is that 125: 155-164. neither ONPG nor lactose is a normal substrate; 12. JBiophys. U N G ,L., R. JOST, E. STOLL,and H. ZUBER.1974. other galactosides such as D-galactans or galacMetabolic differences in Bncillrrs srocrrorhc~,mopi~iIrrs tolipids seem likely candidates. Indeed, these grown at 55 "C and 37°C. Arch. Mikrobiol. 95: 125-138. enzymes may not be primarily P-galacto~idases 13. L ANDMAN 0. . E. 1957. Properties and induction of at all, but glycosidases with a specific.ty broader P-galactosidase in Bncillrrs tnegrctc.rirrtn. Biochem. than is common with such enzymes. A loose Biophys. Acta. 23: 558-569. substrate specificity might be a property of some 14. L I N E W E A V EH., R , and D. BURK.1934. The determination of enzyme dissociation constants. J . Am. Chem. thermo~hilicenzvmes. owinp: " to the constraints s o c . 56: 658-666. on conformation of the proteins imposed by high 15. LJUNGER, C. 1973. Further investigations on the natemperatures. ture of the heat resistance of thermophilic bacteria. Physiol. Plant. 28: 415-418. Acknowledgments 16. LOWRY,0 . H.. N. J. ROSEBROUGH, A. L . FARR,and R. J. R A N D A L L1951. . Protein measurement with the We express our appreciation to Mr. Donald Folin phenol reagent. J. Biol. Chem. 193: 265-275. Marks for technical assistance and to Miss 17. M A N N I N GG. , B., and L. L. CAMPBELL.1961. Deborah Sullivan, whose observation of Thermostable a-amylase of Bncill~tssrearorhermopkigalactosidase in our strains of B. srearorhermo111s.I. Crystallization and some general properties. J. Biol. Chem. 236: 2952-2957. philus provided the initial stimulus for this reK., A. IMANISHI, and T . ISEMURA. 1970. search. We are also grateful to the Campus 18. OGASAHARA, Studies on thermophilic a-amylase from Bncillrrs Crusade for Christ for its cooperation during stenrorherrnopi~ilrrs. I. Some general and physicoour visits to the Arrowhead Hot Springs. chemical properties of thermophilic a-amylase. J. This research was supported in part by a Biochem. 67: 65-75. K., A. IMANISHI, and T . ISEMURA. 1970. National Science Foundation Institutional 19. OGASAHARA, Studies on thermophilic a-amylase from Bnc~illrrs Grant, GU3221-2. ~renroilrc~rtnophilrrs. 11. Thermal stability of thermophilic a-amylase. J. Biochem. 67: 77-82. 1. ANEMA,P. J. 1964. Purification and some properties K., K. YUTANI,A. I M A N I S Hand I. T . 20. OGASAHARA, ISEMURA.1970. Studies on thermophilic a-amylase of P-galactosidase of BnciNrrs srrbrilis. Biochem. Biophys. Acta, 89: 495-502. 111. Effect of tempfrom Bnc~illrrssrc~nrotl~c~rtnopI~iIr~s. erature on the renaturation of denatured thermophilic 2. BABEL,W.. H . A. ROSENTHAL, and S. RAPAPORT. 1972. A unified hypothesis on the causes of the cardia-amylase. J. Biochem. 67: 83-89. nal temperatures of microorganisms; the temperature 21. PFUELLER. S. L., and W. H . ELLIOTT.1969. The extracellular alpha-amylase of Bncillrrs srclnrorhrrActa Biol. minimum of Bnc,illrrs stc~nr~orhertnopi~iI~rs. mophilrrs. J. Biol. Chem. 244: 48-54. Med. Ger. 28: 565-576. S . R., and I. P. CRAWFORD. 1966. Partial 22. KOHLFING, 3. BROCK, T . D. 1967. Life at high temperatures. Science (Wash.), 158: 1012-1019. purification and physical properties of Bucillrrs
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TABLE2. Comparison of K,, and K , for 0-galactosidase from strain AT-7 and B. subtilis
P-
GOODMAN AND PEDERSON
N. E.. and L. I-. CAMPBELL. 1963. Effect of 26. WELKER. carbon sources on formation of alpha-amylase by 1259. Brrcillrrs s r ~ ~ ~ r o r l ~ ~ ~ t ? J. ~ oBacteriol. p I ~ i l i ~ s86: . 68123. SINGLETON, R., and R. E . A M E L U N X E N 1973. . Pro686. teins from thermophilic microorganisms. Bacteriol. 27. Y U T A N IK. , 1973. Molecular weight of thermostable Rev. 37: 320-342. a-amylase from B. sreclrorhemophil~rs.J. Biochem. 24. ULRICH, J. T . , G . A. MCFETERS,and K . L. TEMPLE. 74: 58 1-586. 1972. Induction and chal-acterization of p-galactosi28. Y U T A N IK., , I. S A S A K Iand , K. OGASAHARA. 1973. dase in an extreme thermophile. J. Bacteriol. 110: Comparison of thermostable B. src~orotlirrt~~ophil~ts 69 1-698. grown at different temperatures. J. Biochem. 74: 25. WEERKAMP, A , , and R . D. MACELROY. 1972. Lactate 573-579. dehydrogenase from an extremely thermophilic B N L . ~ / / IArch. I S . Mikrobiol. 85: 113-122. tncgaterirtm P-galactosidase. J. Bacteriol. 92: 125%
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825
I
