JOURNAL OF BACTERIOLOGY, Mar. 1975, p. 933-941 Copyright 0 1975 American Society for Microbiology
Vol. 121, No. 3 Printed in U.S.A.
Purification and Properties of a Serine Protease from Pseudomonas maltophilia ROBERT S. BOETHLING Department of Bacteriology, University of California, Los Angeles, California 90024 Received for publication 18 December 1974
The extracellular protease of Pseudomonas maltophilia was partially purified by ammonium sulfate precipitation and chromatography on Sephadex G-75 and Bio-rex 70. Gel electrophoresis revealed minor impurities. The enzyme exhibited the following properties: (i) molecular weight, 35,000; (ii) A I%O = 10.8; (iii) isoelectric point, 9.3; (iv) pH optimum, 10.0; (v) S2o.11 = 3.47. The enzyme was rapidly inactivated by ethylenediaminetetracetate, but activity could be partially restored with divalent cations. Of those tested, Ca,2+ Sr2+, Ba2+, Co2+, Cu2+, Mg2+, and Zn2+ were all effective. Both phenylmethylsulfonylfluoride and diisopropylfluorophosphate were powerful inhibitors of protease activity, but L-1-tosylamide-2-phenylethylchloromethyl ketone, iodoacetic acid, and iodoacetamide were without effect. The enzyme hydrolyzed the esters N-acetyl-Ltyrosine ethyl ester and a-N-benzoyl-L-arginine ethyl ester (BAEE) with Km values of 10.4 and 3.4 mM, respectively. The hydrolysis of BAEE was also inhibited by phenylarsonic acids. The kinetics of inhibition by m-nitrophenylarsonate were of the mixed type, and the K, was 1.8 mM. The data followed a theoretical curve for a 1:1 enzyme-inhibitor complex with a dissociation constant of 1.8 mM. Inhibition by m-nitrophenylarsonate was pH dependent and followed a theoretical curve for the titration of a protonated group with a pKa of 7.0. Extracellular proteases of microbial origin are thought to be instrumental in the degradation of complex protein substrates to amino acids and peptides in nature. For the most part, these enzymes are small molecules that can be isolated with comparative ease and in good yield. As a result, many of these enzymes have been crystallized and extensively characterized with respect to physicochemical properties and substrate specificity. The best studied as a group are the microbial serine proteases, and among these the best known are the subtilisins. The subtilisins are alkaline proteases of broad specificity produced by different strains of Bacillus subtilis. The most striking feature of these enzymes is the structure of the active site, which is similar to that found in the pancreatic serine proteases, a group of enzymes of independent evolutionary origin (21). There are few reports of extracellular proteases from gram-negative bacteria. Several of these appear to be metalloproteases that resemble bovine carboxypeptidase in their requirement for Zn2+ (11, 12, 13, 16, 17). The enzymes from the psychrophile Escherichia freundii (16) and from Serratia sp. (12) resemble the subtilisins in that all have alkaline pH optima with casein as substrate. In other respects, however, they are not similar. Extracellular enzymes
from Pseudomonas aeruginosa have been studied by Morihara et al. (14, 15). This organism apparently produces at least two distinct proteases, one on alkaline protease (14) and the other an elastase (15) which also has caseinolytic activity. Both are inhibited by ethylenediaminetetracetate (EDTA) but not by diisopropylfluorophosphate (DFP), and are otherwise dissimilar to the subtilisins. In a recent publication, Kato et al. (7) reported the purification of four different enzymes from a marine psychrophile, Pseudomonas sp. no. 548. One of these was an alkaline protease that was most active on casein at pH 10. The enzyme was inhibited by both DFP and chelating agents. In this communication, the partial purification and properties of an extracellular protease produced by a gram-negative isolate are described. The enzyme is a serine protease that in a number of ways appears to be closely related to the subtilisins. This work is the first step in an investigation of the process of enzyme secretion in bacteria. MATERIALS AND METHODS Bacteria and growth conditions. The organism was isolated from enrichment culture containing denatured hemoglobin as the sole carbon source by J. 9:33
934
BOETHLING
Lascelles. The inoculum was material derived from sewage. The organism was identified as a strain of Pseudomonas maltophilia (6, 22) and was maintained on slopes of 1.5% nutrient agar. Cells were grown in a salt-succinate-yeast extract medium which contained the following: 0.2% (wt/vol) yeast extract, 0.2% (NH4)2SO4, 50 mM disodium succinate, 8.8 mM NaH2PO4, 25 mM K2HPO4, 1 mM CaCl2, and 1 mM MgCl2. The CaC12 and MgCl2 were added together as a concentrated sterile solution after autoclaving. The pH was 7.2 and required no adjustment. P. maltophilia is a strict aerobe. The bacteria were grown to stationary phase (about 12 h) at 30 C in 15-liter volumes of medium aerated with a sparger. Inoculation was made with 300 ml of a 12-h culture grown at 30 C with aeration. Growth was followed turbidimetrically at 540 nm; an absorbance of 1.0 at 540 nm was equivalent to 0.21 mg of cell protein/ml. Enzyme assays. Proteolytic activity in crude culture filtrates was assayed spectrophotometrically by the Azocoll procedure. Sample volumes contained 0.1 ml of enzyme solution and distilled water to a total of 2.0 ml. The reaction was initiated by the addition of 1.0 ml of a suspension of Azocoll at 10 mg/ml in 0.1 M tris(hydroxymethyl)-aminomethane (Tris) buffer at pH 7.4, and the tubes were placed in a reciprocal shaker bath at 37 C for 15 min. The reaction was terminated by filtration of the suspension through cotton-plugged Pasteur pipets, and the absorbance was measured at 520 nm. During the purification of the enzyme and subsequent experiments proteolytic activity was determined by modifications of the method of Kunitz (8). In the purification, each assay contained 10 gl of enzyme solution, 0.1 ml of 0.15 M CaCl2, and 1.0 ml of casein (Hammarsten) at 10 mg/ml in 0.1 M Tris buffer, pH 7.4, in a final volume of 3.0 ml. Enzyme, CaCl2 and water to make 2.0 ml were preincubated for 5 min at 37 C, and the reaction was initiated by addition of the substrate. After incubation for 15 min, the assay was terminated by plunging the tubes into ice and immediately adding 2.0 ml of 10% trichloroacetic acid. After at least 1 h, the precipitated casein was removed by filtration with Whatman GF/C disks and the absorbance was measured at 280 nm. The enzyme preparation obtained after the final purification step will hereafter be referred to as purified enzyme. For the determination of enzyme activity as a function of pH, assays contained 0.1 ml of 0.15 M CaCl2, 1.0 ml of casein at 10 mg/ml in 50 mM NaCl adjusted to pH 9.0, and 1.6 ml of buffer in a final volume of 3.0 ml. Buffers were of varying pH but the conductance was held constant at 3,000 + 300 umhos as measured by a Yellow Springs Instrument model 31 Conductivity Bridge (Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio). The assay tubes were preincubated for 5 min, and the reaction was allowed to proceed for 15 min as before. The reaction was initiated by the addition of 10 ml of purified enzyme at 0.5 mg/ml and was terminated with 2.0 ml of 15% trichloroacetic acid. In other experiments in which purified enzyme was used, the casein assay procedure was similar to that
J. BACTERIOL.
described for the purification, except that the substrate was made up in 50 mM glycine buffer at pH 10.0, and assays were initiated by addition of enzyme
rather than substrate. One unit of protease activity is defined as the amount sufficient to produce an increase in absorbance at 280 nm of 1.0 as measured by the casein assay. In all of the above procedures, (i) blank values were obtained by assay without enzyme and were substracted from experimental values, (ii) the absorbance of the blank never exceeded 0.1, and (iii) the assay was linear to an absorbance of about 0.7. For the determination of esterase activity the rates of hydrolysis of N-acetyl-L-tyrosine ethyl ester (ATEE) and a-N-benzoyl-L-arginine ethyl ester (BAEE) were measured with a Radiometer model TTT lc pH-stat equipped with a thermostatted reaction vessel (Radiometer, Copenhagen, Denmark). Reaction volumes were 5 ml, and titrations were performed in the absence of buffers at 37 C with 0.02 N NaOH as titrating agent. All assays contained 0.1 M KCl and 5 mM CaCl2. Standardization of the titrating agent was performed with KH(103)2 as primary standard. The assay was linear over a range of 20 to 200 Ag of enzyme per assay with BAEE, and 2 to 20 jg with ATEE as substrate. Activity is expressed as milliequivalents of OH- released/minute per milligram of protein. Protein assays. Protein was determined by the method of Lowry et al. (10) with crystalline bovine serum albumin as standard. Polyacrylamide gel electrophoresis. Electrophoresis was conducted in native gels of 7.5% acrylamide according to the method of Laemmli (9). A Hoefer model SE-500 slab gel apparatus was used (Hoefer Scientific Instruments, San Francisco, Calif.). Gels of 1.5-mm thickness were run at pH 8.9 for 3 h with no stacking gel, at a constant current of 20 mA. The gels were stained with 0.1% Coomassie brilliant blue in 50% methanol-10% trichloroacetic acid for 2 h at 37 C, and destained in 10% methanol-10% acetic acid. Isoelectric focusing. Electrofocusing was conducted in an LKB column of 110-ml capacity according to the recommendations of the manufacturer (LKB Produkter AB, Bromma, Sweden). A pH gradient of 3.5 to 10 was established by the use of 1% (vol/vol) carrier ampholytes (LKB) covering this range. The column contained as the anticonvectant medium a stepwise gradient of 0 to 50% (wt/vol) sucrose, to which was added CaCl2 to a final concentration of 5 mM. Purified enzyme (11.7 mg in 0.5 ml) was added to one of the middle fractions during preparation of the sucrose gradient. Enzyme was focused at 4 C for 4 h at a constant voltage of 300 V, followed by 36 h at 400 V. Fractions of 2 ml were collected. Ultraviolet spectra. The ultraviolet spectrum of the purified enzyme was obtained with a Cary model 14R spectrophotometer (Applied Physics Corp., Monrovia, Calif.). Molecular weight determination. The molecular weight of the purified enzyme was estimated by the method of Andrews (1). A column of Sephadex G-100 (1 by 100 cm) was equilibrated with 10 mM Tris-5
VOL. 121, 1975
P. MALTOPHILIA SERINE PROTEASE
935
mM CaCl2, pH 7.4, and run at 10.8 ml/h. Calibration standards included ribonuclease A, myoglobin, achymotrypsinogen, ovalbumin, and bovine serum albumin. Each standard was applied as a solution of 4 mg in 0.5 ml of buffer. All procedures were carried out at 4 C. Sedimentation analysis. Sedimentation velocity experiments were performed at 20 C in a Beckman model E analytical ultracentrifuge with an AN-D rotor and Schlieren optics. Ultracentrifugation was carried out at 44,770 rpm at a protein concentration of 4 mg/ml, and at 52,640 rpm at 1, 2, and 3 mg/ml. The purified protease was first dialyzed exhaustively against 10 mM Tris-5 mM CaCl2, pH 7.4; the buffer made no measurable contribution to viscosity or density in the centrifugation. Reagents. Sodium cacodylate, DFP, phenylmethylsulfonylfluoride (PMSF), L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK), iodoacetic acid, TIME (lHZ) iodoacetamide, ATEE, BAEE, bovine pancreatic ribonuclease A, ovalbumin, and bovine serum alFIG. 1. Growth and production of extracellular bumin were obtained from Sigma. Iodoacetic acid protease by P. maltophilia. The organism was grown Phenylarsonic use. before recrystallized was twice in batch culture, and protease activity was deteracid, o-nitrophenylarsonic acid, m-nitrophenyl- mined by the Azocoll procedure. Samples of clear arsonic acid, p-arsanilic acid, and primary standard supernatant fluid were obtained at intervals by cenKH(10,)2 were gifts from A. N. Glazer. Casein (Ham- trifugation at 12,000 x g for 10 min. Symbols: cell marsten) was obtained from Nutritional Biochemi- density, 0; protease activity, 0. cals, Azocoll from Calbiochem, ethylenediamine from Eastman, and methylamine from Matheson, Coleman, and Bell. Bovine pancreatic a-chymotrypsino- dialysate was concentrated by pressure ultrafilgen and sperm whale myoglobin were purchased from Mann. Bio-rex 70 was a product of Bio-Rad. Sephadex tration with an Amicon UM-2 filter.the previous The concentrated material from G-25, G-75, and G-100 were products of Pharmacia. G-75 of Sephadex to a column was applied step of grade. were reagent All other chemicals
RESULTS Purification. Extracellular protease activity was measured during the growth of the organism in batch culture (Fig. 1). In the complex medium in which the cells were grown, protease activity was not detected until early stationary phase. When protease activity had reached a maximum, the culture was immediately chilled in an ice bath, and the cells- were removed by centrifugation at 10,000 x g for 45 min. The cells were discarded and the supernatant fluid was retained. Unless indicated otherwise, all operations during the purification of the enzyme were carried out at 4 C. Solid ammonium sulfate (561 g/liter of supernatant fluid) was then added with stirring, and the solution was allowed to sit undisturbed overnight. The resulting precipitate was collected by centrifugation at 10,000 x g for 30 min. The precipitate was dissolved in approximately 100 ml of 10 mM Tris-5 mM CaCl2, pH 7.4, and dialyzed against 4 liters of the same buffer for 48 h with at least three changes of buffer. A small insoluble residue was removed by centrifugation. The
(3 by 170 cm) equilibrated with Tris-CaCl2 buffer as before. The column was operated at 32.4 ml/h, and 5-ml fractions were collected. Two components were eluted after the void volume; both contained activity but the second was much smaller and was more heterogeneous when examined by polyacrylamide gel electrophoresis. Fractions from the first component with activity were pooled and concentrated by pressure ultrafiltration, and desalted at room temperature with a column of Sephadex G-25 (2 by 20 cm) equilibrated with 10 mM sodium cacodylate-5 mM CaCl2, pH 6.0. The Sephadex-derived material was fractionated by chromatography on a column (2 by 20 cm) of Bio-rex 70, equilibrated with cacodylateCaCl2 buffer as before. The column was operated at 32.4 ml/h, and 3-ml fractions were collected. When the column was washed with the same buffer, a small amount of protein without protease activity emerged and was discarded. A linear gradient of 0 to 0.2 M NaCl in a total of 600 ml was then applied, and the enzyme emerged as a single component of activity at about 0.14 M NaCl. The peak fractions were pooled and concentrated by pres-
936
BOETHLING
sure ultrafiltration, and desalted with Sephadex G-25 as before. The enzyme could be stored at -70 C for at least 6 months with no loss of activity. A typical purification is summarized in Table 1. Criteria of homogeneity. Molecular sieve chromatography was performed in connection with the determination of' molecular weight by gel filtration and did not reveal the presence of contaminating protein. Sedimentation analysis also revealed a single protein component. Polyacrylamide gel electrophoresis did, however, reveal minor impurities immediately below the major band in the stained gel (Fig. 2). It is possible that the contaminating material is a product of autodigestion since the intensity of staining increased with time of storage at 4 C of the purified enzyme and with the number of times that the preparation was thawed and refrozen. A nearly identical gel pattern has been observed for the autodigestion products of subtilisin Carlsberg (23). Ultraviolet spectra. The ultraviolet spectrum of the purified protease is shown in Fig. 3. The vaiue of A "'0 calculated from the figure is 10.8. The ratio of the absorbance at 280 nm to that at 260 nm is 2.05, indicating the absence of a significant quantity of nucleic acid. Sedimentation analysis. Sedimentation velocity runs were performed at protein concentrations of 1, 2, 3, and 4 mg/ml, and the values for s20,, obtained were plotted versus protein concentration. A value for s201, of 3.47 was calculated by extrapolation to zero protein concentration. A single symmetrical peak was observed in the Schlieren patterns at all protein concentrations. Molecular weight. Gel f'iltration analysis gave a value of 35,000 for the molecular weight of the purified protease (Fig. 4). Isoelectric point. The apparent isoelectric point of the enzyme was 9.3. Failure to include CaCl2 during the electrotocusing procedure resulted in a total loss of activity and of TABLE 1. Purification of protease Step
Step
1. Ammonium
Total units
Protein Sp act (mg) (U/mg)
17,080
448
38.1
100
13,200 6,270
313 84
42.2 74.7
77 37
sulfatea 2. Sephadex G-75 3. Bio-rex 70
Yield (%)
a The protein concentration and caseinolytic activity in the crude culture fluid were not measured because of the high background absorbance at 280 nm.
J. BACTERIOL.
.M ..0
+
MARKER DYE
FIG. 2. Polyacrylamide gel analysis of purified pro-
tease. The direction of migration is from top to
bottom.
reproducibility in the pattern of absorbance at 280 nm of the eluted material; this effect was prevented by the addition of 5 mM CaCl2. Effect of pH and ionic strength on protease activity. Maximum protease activity was observed at pH 10.0 with casein as substrate (Fig. 5). Despite the high pH optimum the enzyme demonstrated a broad range of activity; at pH values of 6 and 12 the enzyme retained nearly 50% of its peak activity. Since the ionic strength was held constant, the actual concentrations of buffer species necessarily varied with pH. The conductance of the casein assay mixture was approximately 4,500 ,mhos; concentrations of NaCl as high as 0.25 M, which corresponded to a conductance of 20,000 gmhos, were shown to have no effect on protease activity. Temperature stability. The effect of temperature on the stability of the purified protease was determined with purified enzyme at 0.5 mg/ml in 10 mM Tris-5 mM CaCl2, pH 7.4.
937
P. MALTOPHILIA SERINE PROTEASE
VOL. 121, 1975
enzyme can withstand temperatures as high as 60 C for 15 min with no loss of activity. Effect of EDTA and reactivation by metal ions. In the presence of EDTA the enzyme is rapidly inactivated (Fig. 6). Although it is possible to recover 100% of the initial activity if the assay is carried out immediately in the presence of Ca2+, the extent to which Ca2+stimulated reactivation is possible also declines and reaches a minimum of approximately 17% at 60 min. The ability of other divalent cations to reactivate EDTA-treated enzyme is demonstrated in Table 2. All of the tested cations except Hg2+ produce significant reactivation; in fact, Ca2 , Sr2 , Co2+, and Ba2+ are essentially
0.4
0.0
0.4
0.3
0.2
0.1
240
230
240
320
300
WAVELENOTH (n"-
0.5
FIG. 3. Ultraviolet spectrum of purified protease. The spectrum was obtained with purified enzyme at 0.50 mg/ml in 10 mM cacodylate-5 mM CaCl2 buffer, pH 6.0.
E4 064
z
0.3
(IK2)
I'll0
4
02 D-
0.6
m.ohyleinm
*yngdimin.
\
x
RNas. A
0-
myoglobin 0.1
0.5
oi
-
0.4
0.3
chymotrypsinog*n
*
I
I
I
I
7
a
9
10
11
12
FIG. 5. Effect of pH on protease activity. The carried out at constant ionic strength as described. The ionic strength corresponded to a NaCI concentration of approximately 50 mM. Symbols: ethylenediamine, 0; Tris, 0; methylamine, 0. assays were
PROTEASE
0.3
voalbumin 0.2
SSA O .4
0.1
z
I.
0.3
z
20
4s0 MOLECULAR WEIGHT
s0
ao
10.0
10-4 by gel filmolecular weight 4. Estimation of FIG. tration with Sephadex G-100. K. is a measure of the elution volume and is equal to ve - v0/vt - v0, where Ve is the elution volume, v, is the bed volume, and v. is the void volume as determined by blue dextran. The estimated molecular weight is 35,000.
rt
x
Samples of the enzyme solution were incubated at various temperatures for 15 min and were assayed by the casein procedure at pH 10.0. The
7 10
20
40
50
00
0
120
100
T1ZE 6in )
FIG. 6. Effect of EDTA on protease activity, and reactivation by Ca2+. Purified enzyme at 0.5 mg/ml was incubated with 10 mM EDTA in 10 mM Tris buffer, pH 7.4. At intervals samples of 10 ,l (5 ,g of enzyme) were removed and assayed with or without 5 mM CaCI2 by the casein procedure, at pH 10.0. Symbols: with 5 mM CaCI2, 0; without CaCI2, 0.
938
BOETHLING
equivalent in this respect. These results suggest that metal ion is required only for stability and does not participate in catalysis. An alternative explanation is that the addition of metal ions other than Ca2+ to the assay mix results in the formation of a ternary complex of enzyme, metal ion, and EDTA at the enzyme surface, permitting the release of Ca2+ from EDTA. Effect of protease inhibitors. The effects of inhibitors of protease activity are summarized in Table 3. Both PMSF and DFP are powerful inhibitors of protease activity, but PMSF inhibits more rapidly, as complete inhibition is
J. BACTERIOL.
observed within 30 min at only a 4 times molar excess of inhibitor over enzyme. The chymotrypsin inhibitor TPCK has no effect at 20 times molar excess. The sulfhydryl protease inhibitors iodoacetic acid and iodoacetamide are also without effect, under conditions that would be expected to reveal any inhibitory activity. Esterase activity. The initial rates of hydrolysis of the ester substrates ATEE and BAEE were measured as a function of substrate concentration in a pH-stat, at pH 8.0. The values for the kinetic parameters Km and Vmax were calculated from the Lineweaver-Burk plots. These are, for ATEE, 10.4 mM and 16.7 x 10-2
TABLE 2. Restoration of protease activity by metal ionSa
Reactivation (%)b
Ion
Ca2 ............................ Sr2 ...................... ......
100
106 99 96 91 84 79 33 4 8
.. Ba2.
Co2 . ...................... Cu2+ ............................. Mg2 .. ......................... Zn2+ .......................... Mn2 .......................... Hg2.. .................... . None ..........................
a Purified enzyme at 0.5 mg/ml was incubated for 5 min at 4 C with 10 mM EDTA in 10 mM Tris buffer, pH 7.4. Ten-microliter samples (5 ug of enzyme) were then assayed in the presence of 5 mM metal ions at pH 10.0 as described. b The values given are the average of two determinations, normalized to the value for Ca2+ (i.e., Ca2+ = 100%).
mEq of OH- released/min per mg of protein, and for BAEE, 3.4 mM and 0.37 x 10- 2 mEq/min per mg. It is evident that ATEE is a
much better substrate for the purified protease than BAEE. The kinetic data for ATEE must be interpreted with caution, however, since the assay volumes contained 10% (vol/vol) p-dioxane. No inhibitory effects from peroxides were observed, but p-dioxane is known to be a competitive inhibitor of subtilisin. Effect of phenylarsonic acids on esterase activity. The pentavalent organic arsenicals phenylarsonic acid and its derivatives are potent inhibitors of serine esterases (3). For this reason, a study of the effect of various phenylarsonates on esterase activity was undertaken with BAEE as substrate. The kinetics of hydrolysis of BAEE in the presence and absence of 2 mM phenylarsonates are depicted in Fig. 7. Phenylarsonic acid appeared to be the most powerful inhibitor, but only m-nitrophenylarsonate attained an apparent zero order rate of
TABLE 3. Effect of inhibitors on protease activitya Relative sp actb
Inhibitor
20
x
molar excess of inhibitor
30 min
DFP ......................... PMSF ........................ TPCK ........................ lodoacetic acid Iodoacetamide None ........................
60 min
4
x
molar excess of inhibitor
30 min
60 min
0.10
0.03
0.62
Vol. 121, No. 3 Printed in U.S.A.
Purification and Properties of a Serine Protease from Pseudomonas maltophilia ROBERT S. BOETHLING Department of Bacteriology, University of California, Los Angeles, California 90024 Received for publication 18 December 1974
The extracellular protease of Pseudomonas maltophilia was partially purified by ammonium sulfate precipitation and chromatography on Sephadex G-75 and Bio-rex 70. Gel electrophoresis revealed minor impurities. The enzyme exhibited the following properties: (i) molecular weight, 35,000; (ii) A I%O = 10.8; (iii) isoelectric point, 9.3; (iv) pH optimum, 10.0; (v) S2o.11 = 3.47. The enzyme was rapidly inactivated by ethylenediaminetetracetate, but activity could be partially restored with divalent cations. Of those tested, Ca,2+ Sr2+, Ba2+, Co2+, Cu2+, Mg2+, and Zn2+ were all effective. Both phenylmethylsulfonylfluoride and diisopropylfluorophosphate were powerful inhibitors of protease activity, but L-1-tosylamide-2-phenylethylchloromethyl ketone, iodoacetic acid, and iodoacetamide were without effect. The enzyme hydrolyzed the esters N-acetyl-Ltyrosine ethyl ester and a-N-benzoyl-L-arginine ethyl ester (BAEE) with Km values of 10.4 and 3.4 mM, respectively. The hydrolysis of BAEE was also inhibited by phenylarsonic acids. The kinetics of inhibition by m-nitrophenylarsonate were of the mixed type, and the K, was 1.8 mM. The data followed a theoretical curve for a 1:1 enzyme-inhibitor complex with a dissociation constant of 1.8 mM. Inhibition by m-nitrophenylarsonate was pH dependent and followed a theoretical curve for the titration of a protonated group with a pKa of 7.0. Extracellular proteases of microbial origin are thought to be instrumental in the degradation of complex protein substrates to amino acids and peptides in nature. For the most part, these enzymes are small molecules that can be isolated with comparative ease and in good yield. As a result, many of these enzymes have been crystallized and extensively characterized with respect to physicochemical properties and substrate specificity. The best studied as a group are the microbial serine proteases, and among these the best known are the subtilisins. The subtilisins are alkaline proteases of broad specificity produced by different strains of Bacillus subtilis. The most striking feature of these enzymes is the structure of the active site, which is similar to that found in the pancreatic serine proteases, a group of enzymes of independent evolutionary origin (21). There are few reports of extracellular proteases from gram-negative bacteria. Several of these appear to be metalloproteases that resemble bovine carboxypeptidase in their requirement for Zn2+ (11, 12, 13, 16, 17). The enzymes from the psychrophile Escherichia freundii (16) and from Serratia sp. (12) resemble the subtilisins in that all have alkaline pH optima with casein as substrate. In other respects, however, they are not similar. Extracellular enzymes
from Pseudomonas aeruginosa have been studied by Morihara et al. (14, 15). This organism apparently produces at least two distinct proteases, one on alkaline protease (14) and the other an elastase (15) which also has caseinolytic activity. Both are inhibited by ethylenediaminetetracetate (EDTA) but not by diisopropylfluorophosphate (DFP), and are otherwise dissimilar to the subtilisins. In a recent publication, Kato et al. (7) reported the purification of four different enzymes from a marine psychrophile, Pseudomonas sp. no. 548. One of these was an alkaline protease that was most active on casein at pH 10. The enzyme was inhibited by both DFP and chelating agents. In this communication, the partial purification and properties of an extracellular protease produced by a gram-negative isolate are described. The enzyme is a serine protease that in a number of ways appears to be closely related to the subtilisins. This work is the first step in an investigation of the process of enzyme secretion in bacteria. MATERIALS AND METHODS Bacteria and growth conditions. The organism was isolated from enrichment culture containing denatured hemoglobin as the sole carbon source by J. 9:33
934
BOETHLING
Lascelles. The inoculum was material derived from sewage. The organism was identified as a strain of Pseudomonas maltophilia (6, 22) and was maintained on slopes of 1.5% nutrient agar. Cells were grown in a salt-succinate-yeast extract medium which contained the following: 0.2% (wt/vol) yeast extract, 0.2% (NH4)2SO4, 50 mM disodium succinate, 8.8 mM NaH2PO4, 25 mM K2HPO4, 1 mM CaCl2, and 1 mM MgCl2. The CaC12 and MgCl2 were added together as a concentrated sterile solution after autoclaving. The pH was 7.2 and required no adjustment. P. maltophilia is a strict aerobe. The bacteria were grown to stationary phase (about 12 h) at 30 C in 15-liter volumes of medium aerated with a sparger. Inoculation was made with 300 ml of a 12-h culture grown at 30 C with aeration. Growth was followed turbidimetrically at 540 nm; an absorbance of 1.0 at 540 nm was equivalent to 0.21 mg of cell protein/ml. Enzyme assays. Proteolytic activity in crude culture filtrates was assayed spectrophotometrically by the Azocoll procedure. Sample volumes contained 0.1 ml of enzyme solution and distilled water to a total of 2.0 ml. The reaction was initiated by the addition of 1.0 ml of a suspension of Azocoll at 10 mg/ml in 0.1 M tris(hydroxymethyl)-aminomethane (Tris) buffer at pH 7.4, and the tubes were placed in a reciprocal shaker bath at 37 C for 15 min. The reaction was terminated by filtration of the suspension through cotton-plugged Pasteur pipets, and the absorbance was measured at 520 nm. During the purification of the enzyme and subsequent experiments proteolytic activity was determined by modifications of the method of Kunitz (8). In the purification, each assay contained 10 gl of enzyme solution, 0.1 ml of 0.15 M CaCl2, and 1.0 ml of casein (Hammarsten) at 10 mg/ml in 0.1 M Tris buffer, pH 7.4, in a final volume of 3.0 ml. Enzyme, CaCl2 and water to make 2.0 ml were preincubated for 5 min at 37 C, and the reaction was initiated by addition of the substrate. After incubation for 15 min, the assay was terminated by plunging the tubes into ice and immediately adding 2.0 ml of 10% trichloroacetic acid. After at least 1 h, the precipitated casein was removed by filtration with Whatman GF/C disks and the absorbance was measured at 280 nm. The enzyme preparation obtained after the final purification step will hereafter be referred to as purified enzyme. For the determination of enzyme activity as a function of pH, assays contained 0.1 ml of 0.15 M CaCl2, 1.0 ml of casein at 10 mg/ml in 50 mM NaCl adjusted to pH 9.0, and 1.6 ml of buffer in a final volume of 3.0 ml. Buffers were of varying pH but the conductance was held constant at 3,000 + 300 umhos as measured by a Yellow Springs Instrument model 31 Conductivity Bridge (Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio). The assay tubes were preincubated for 5 min, and the reaction was allowed to proceed for 15 min as before. The reaction was initiated by the addition of 10 ml of purified enzyme at 0.5 mg/ml and was terminated with 2.0 ml of 15% trichloroacetic acid. In other experiments in which purified enzyme was used, the casein assay procedure was similar to that
J. BACTERIOL.
described for the purification, except that the substrate was made up in 50 mM glycine buffer at pH 10.0, and assays were initiated by addition of enzyme
rather than substrate. One unit of protease activity is defined as the amount sufficient to produce an increase in absorbance at 280 nm of 1.0 as measured by the casein assay. In all of the above procedures, (i) blank values were obtained by assay without enzyme and were substracted from experimental values, (ii) the absorbance of the blank never exceeded 0.1, and (iii) the assay was linear to an absorbance of about 0.7. For the determination of esterase activity the rates of hydrolysis of N-acetyl-L-tyrosine ethyl ester (ATEE) and a-N-benzoyl-L-arginine ethyl ester (BAEE) were measured with a Radiometer model TTT lc pH-stat equipped with a thermostatted reaction vessel (Radiometer, Copenhagen, Denmark). Reaction volumes were 5 ml, and titrations were performed in the absence of buffers at 37 C with 0.02 N NaOH as titrating agent. All assays contained 0.1 M KCl and 5 mM CaCl2. Standardization of the titrating agent was performed with KH(103)2 as primary standard. The assay was linear over a range of 20 to 200 Ag of enzyme per assay with BAEE, and 2 to 20 jg with ATEE as substrate. Activity is expressed as milliequivalents of OH- released/minute per milligram of protein. Protein assays. Protein was determined by the method of Lowry et al. (10) with crystalline bovine serum albumin as standard. Polyacrylamide gel electrophoresis. Electrophoresis was conducted in native gels of 7.5% acrylamide according to the method of Laemmli (9). A Hoefer model SE-500 slab gel apparatus was used (Hoefer Scientific Instruments, San Francisco, Calif.). Gels of 1.5-mm thickness were run at pH 8.9 for 3 h with no stacking gel, at a constant current of 20 mA. The gels were stained with 0.1% Coomassie brilliant blue in 50% methanol-10% trichloroacetic acid for 2 h at 37 C, and destained in 10% methanol-10% acetic acid. Isoelectric focusing. Electrofocusing was conducted in an LKB column of 110-ml capacity according to the recommendations of the manufacturer (LKB Produkter AB, Bromma, Sweden). A pH gradient of 3.5 to 10 was established by the use of 1% (vol/vol) carrier ampholytes (LKB) covering this range. The column contained as the anticonvectant medium a stepwise gradient of 0 to 50% (wt/vol) sucrose, to which was added CaCl2 to a final concentration of 5 mM. Purified enzyme (11.7 mg in 0.5 ml) was added to one of the middle fractions during preparation of the sucrose gradient. Enzyme was focused at 4 C for 4 h at a constant voltage of 300 V, followed by 36 h at 400 V. Fractions of 2 ml were collected. Ultraviolet spectra. The ultraviolet spectrum of the purified enzyme was obtained with a Cary model 14R spectrophotometer (Applied Physics Corp., Monrovia, Calif.). Molecular weight determination. The molecular weight of the purified enzyme was estimated by the method of Andrews (1). A column of Sephadex G-100 (1 by 100 cm) was equilibrated with 10 mM Tris-5
VOL. 121, 1975
P. MALTOPHILIA SERINE PROTEASE
935
mM CaCl2, pH 7.4, and run at 10.8 ml/h. Calibration standards included ribonuclease A, myoglobin, achymotrypsinogen, ovalbumin, and bovine serum albumin. Each standard was applied as a solution of 4 mg in 0.5 ml of buffer. All procedures were carried out at 4 C. Sedimentation analysis. Sedimentation velocity experiments were performed at 20 C in a Beckman model E analytical ultracentrifuge with an AN-D rotor and Schlieren optics. Ultracentrifugation was carried out at 44,770 rpm at a protein concentration of 4 mg/ml, and at 52,640 rpm at 1, 2, and 3 mg/ml. The purified protease was first dialyzed exhaustively against 10 mM Tris-5 mM CaCl2, pH 7.4; the buffer made no measurable contribution to viscosity or density in the centrifugation. Reagents. Sodium cacodylate, DFP, phenylmethylsulfonylfluoride (PMSF), L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK), iodoacetic acid, TIME (lHZ) iodoacetamide, ATEE, BAEE, bovine pancreatic ribonuclease A, ovalbumin, and bovine serum alFIG. 1. Growth and production of extracellular bumin were obtained from Sigma. Iodoacetic acid protease by P. maltophilia. The organism was grown Phenylarsonic use. before recrystallized was twice in batch culture, and protease activity was deteracid, o-nitrophenylarsonic acid, m-nitrophenyl- mined by the Azocoll procedure. Samples of clear arsonic acid, p-arsanilic acid, and primary standard supernatant fluid were obtained at intervals by cenKH(10,)2 were gifts from A. N. Glazer. Casein (Ham- trifugation at 12,000 x g for 10 min. Symbols: cell marsten) was obtained from Nutritional Biochemi- density, 0; protease activity, 0. cals, Azocoll from Calbiochem, ethylenediamine from Eastman, and methylamine from Matheson, Coleman, and Bell. Bovine pancreatic a-chymotrypsino- dialysate was concentrated by pressure ultrafilgen and sperm whale myoglobin were purchased from Mann. Bio-rex 70 was a product of Bio-Rad. Sephadex tration with an Amicon UM-2 filter.the previous The concentrated material from G-25, G-75, and G-100 were products of Pharmacia. G-75 of Sephadex to a column was applied step of grade. were reagent All other chemicals
RESULTS Purification. Extracellular protease activity was measured during the growth of the organism in batch culture (Fig. 1). In the complex medium in which the cells were grown, protease activity was not detected until early stationary phase. When protease activity had reached a maximum, the culture was immediately chilled in an ice bath, and the cells- were removed by centrifugation at 10,000 x g for 45 min. The cells were discarded and the supernatant fluid was retained. Unless indicated otherwise, all operations during the purification of the enzyme were carried out at 4 C. Solid ammonium sulfate (561 g/liter of supernatant fluid) was then added with stirring, and the solution was allowed to sit undisturbed overnight. The resulting precipitate was collected by centrifugation at 10,000 x g for 30 min. The precipitate was dissolved in approximately 100 ml of 10 mM Tris-5 mM CaCl2, pH 7.4, and dialyzed against 4 liters of the same buffer for 48 h with at least three changes of buffer. A small insoluble residue was removed by centrifugation. The
(3 by 170 cm) equilibrated with Tris-CaCl2 buffer as before. The column was operated at 32.4 ml/h, and 5-ml fractions were collected. Two components were eluted after the void volume; both contained activity but the second was much smaller and was more heterogeneous when examined by polyacrylamide gel electrophoresis. Fractions from the first component with activity were pooled and concentrated by pressure ultrafiltration, and desalted at room temperature with a column of Sephadex G-25 (2 by 20 cm) equilibrated with 10 mM sodium cacodylate-5 mM CaCl2, pH 6.0. The Sephadex-derived material was fractionated by chromatography on a column (2 by 20 cm) of Bio-rex 70, equilibrated with cacodylateCaCl2 buffer as before. The column was operated at 32.4 ml/h, and 3-ml fractions were collected. When the column was washed with the same buffer, a small amount of protein without protease activity emerged and was discarded. A linear gradient of 0 to 0.2 M NaCl in a total of 600 ml was then applied, and the enzyme emerged as a single component of activity at about 0.14 M NaCl. The peak fractions were pooled and concentrated by pres-
936
BOETHLING
sure ultrafiltration, and desalted with Sephadex G-25 as before. The enzyme could be stored at -70 C for at least 6 months with no loss of activity. A typical purification is summarized in Table 1. Criteria of homogeneity. Molecular sieve chromatography was performed in connection with the determination of' molecular weight by gel filtration and did not reveal the presence of contaminating protein. Sedimentation analysis also revealed a single protein component. Polyacrylamide gel electrophoresis did, however, reveal minor impurities immediately below the major band in the stained gel (Fig. 2). It is possible that the contaminating material is a product of autodigestion since the intensity of staining increased with time of storage at 4 C of the purified enzyme and with the number of times that the preparation was thawed and refrozen. A nearly identical gel pattern has been observed for the autodigestion products of subtilisin Carlsberg (23). Ultraviolet spectra. The ultraviolet spectrum of the purified protease is shown in Fig. 3. The vaiue of A "'0 calculated from the figure is 10.8. The ratio of the absorbance at 280 nm to that at 260 nm is 2.05, indicating the absence of a significant quantity of nucleic acid. Sedimentation analysis. Sedimentation velocity runs were performed at protein concentrations of 1, 2, 3, and 4 mg/ml, and the values for s20,, obtained were plotted versus protein concentration. A value for s201, of 3.47 was calculated by extrapolation to zero protein concentration. A single symmetrical peak was observed in the Schlieren patterns at all protein concentrations. Molecular weight. Gel f'iltration analysis gave a value of 35,000 for the molecular weight of the purified protease (Fig. 4). Isoelectric point. The apparent isoelectric point of the enzyme was 9.3. Failure to include CaCl2 during the electrotocusing procedure resulted in a total loss of activity and of TABLE 1. Purification of protease Step
Step
1. Ammonium
Total units
Protein Sp act (mg) (U/mg)
17,080
448
38.1
100
13,200 6,270
313 84
42.2 74.7
77 37
sulfatea 2. Sephadex G-75 3. Bio-rex 70
Yield (%)
a The protein concentration and caseinolytic activity in the crude culture fluid were not measured because of the high background absorbance at 280 nm.
J. BACTERIOL.
.M ..0
+
MARKER DYE
FIG. 2. Polyacrylamide gel analysis of purified pro-
tease. The direction of migration is from top to
bottom.
reproducibility in the pattern of absorbance at 280 nm of the eluted material; this effect was prevented by the addition of 5 mM CaCl2. Effect of pH and ionic strength on protease activity. Maximum protease activity was observed at pH 10.0 with casein as substrate (Fig. 5). Despite the high pH optimum the enzyme demonstrated a broad range of activity; at pH values of 6 and 12 the enzyme retained nearly 50% of its peak activity. Since the ionic strength was held constant, the actual concentrations of buffer species necessarily varied with pH. The conductance of the casein assay mixture was approximately 4,500 ,mhos; concentrations of NaCl as high as 0.25 M, which corresponded to a conductance of 20,000 gmhos, were shown to have no effect on protease activity. Temperature stability. The effect of temperature on the stability of the purified protease was determined with purified enzyme at 0.5 mg/ml in 10 mM Tris-5 mM CaCl2, pH 7.4.
937
P. MALTOPHILIA SERINE PROTEASE
VOL. 121, 1975
enzyme can withstand temperatures as high as 60 C for 15 min with no loss of activity. Effect of EDTA and reactivation by metal ions. In the presence of EDTA the enzyme is rapidly inactivated (Fig. 6). Although it is possible to recover 100% of the initial activity if the assay is carried out immediately in the presence of Ca2+, the extent to which Ca2+stimulated reactivation is possible also declines and reaches a minimum of approximately 17% at 60 min. The ability of other divalent cations to reactivate EDTA-treated enzyme is demonstrated in Table 2. All of the tested cations except Hg2+ produce significant reactivation; in fact, Ca2 , Sr2 , Co2+, and Ba2+ are essentially
0.4
0.0
0.4
0.3
0.2
0.1
240
230
240
320
300
WAVELENOTH (n"-
0.5
FIG. 3. Ultraviolet spectrum of purified protease. The spectrum was obtained with purified enzyme at 0.50 mg/ml in 10 mM cacodylate-5 mM CaCl2 buffer, pH 6.0.
E4 064
z
0.3
(IK2)
I'll0
4
02 D-
0.6
m.ohyleinm
*yngdimin.
\
x
RNas. A
0-
myoglobin 0.1
0.5
oi
-
0.4
0.3
chymotrypsinog*n
*
I
I
I
I
7
a
9
10
11
12
FIG. 5. Effect of pH on protease activity. The carried out at constant ionic strength as described. The ionic strength corresponded to a NaCI concentration of approximately 50 mM. Symbols: ethylenediamine, 0; Tris, 0; methylamine, 0. assays were
PROTEASE
0.3
voalbumin 0.2
SSA O .4
0.1
z
I.
0.3
z
20
4s0 MOLECULAR WEIGHT
s0
ao
10.0
10-4 by gel filmolecular weight 4. Estimation of FIG. tration with Sephadex G-100. K. is a measure of the elution volume and is equal to ve - v0/vt - v0, where Ve is the elution volume, v, is the bed volume, and v. is the void volume as determined by blue dextran. The estimated molecular weight is 35,000.
rt
x
Samples of the enzyme solution were incubated at various temperatures for 15 min and were assayed by the casein procedure at pH 10.0. The
7 10
20
40
50
00
0
120
100
T1ZE 6in )
FIG. 6. Effect of EDTA on protease activity, and reactivation by Ca2+. Purified enzyme at 0.5 mg/ml was incubated with 10 mM EDTA in 10 mM Tris buffer, pH 7.4. At intervals samples of 10 ,l (5 ,g of enzyme) were removed and assayed with or without 5 mM CaCI2 by the casein procedure, at pH 10.0. Symbols: with 5 mM CaCI2, 0; without CaCI2, 0.
938
BOETHLING
equivalent in this respect. These results suggest that metal ion is required only for stability and does not participate in catalysis. An alternative explanation is that the addition of metal ions other than Ca2+ to the assay mix results in the formation of a ternary complex of enzyme, metal ion, and EDTA at the enzyme surface, permitting the release of Ca2+ from EDTA. Effect of protease inhibitors. The effects of inhibitors of protease activity are summarized in Table 3. Both PMSF and DFP are powerful inhibitors of protease activity, but PMSF inhibits more rapidly, as complete inhibition is
J. BACTERIOL.
observed within 30 min at only a 4 times molar excess of inhibitor over enzyme. The chymotrypsin inhibitor TPCK has no effect at 20 times molar excess. The sulfhydryl protease inhibitors iodoacetic acid and iodoacetamide are also without effect, under conditions that would be expected to reveal any inhibitory activity. Esterase activity. The initial rates of hydrolysis of the ester substrates ATEE and BAEE were measured as a function of substrate concentration in a pH-stat, at pH 8.0. The values for the kinetic parameters Km and Vmax were calculated from the Lineweaver-Burk plots. These are, for ATEE, 10.4 mM and 16.7 x 10-2
TABLE 2. Restoration of protease activity by metal ionSa
Reactivation (%)b
Ion
Ca2 ............................ Sr2 ...................... ......
100
106 99 96 91 84 79 33 4 8
.. Ba2.
Co2 . ...................... Cu2+ ............................. Mg2 .. ......................... Zn2+ .......................... Mn2 .......................... Hg2.. .................... . None ..........................
a Purified enzyme at 0.5 mg/ml was incubated for 5 min at 4 C with 10 mM EDTA in 10 mM Tris buffer, pH 7.4. Ten-microliter samples (5 ug of enzyme) were then assayed in the presence of 5 mM metal ions at pH 10.0 as described. b The values given are the average of two determinations, normalized to the value for Ca2+ (i.e., Ca2+ = 100%).
mEq of OH- released/min per mg of protein, and for BAEE, 3.4 mM and 0.37 x 10- 2 mEq/min per mg. It is evident that ATEE is a
much better substrate for the purified protease than BAEE. The kinetic data for ATEE must be interpreted with caution, however, since the assay volumes contained 10% (vol/vol) p-dioxane. No inhibitory effects from peroxides were observed, but p-dioxane is known to be a competitive inhibitor of subtilisin. Effect of phenylarsonic acids on esterase activity. The pentavalent organic arsenicals phenylarsonic acid and its derivatives are potent inhibitors of serine esterases (3). For this reason, a study of the effect of various phenylarsonates on esterase activity was undertaken with BAEE as substrate. The kinetics of hydrolysis of BAEE in the presence and absence of 2 mM phenylarsonates are depicted in Fig. 7. Phenylarsonic acid appeared to be the most powerful inhibitor, but only m-nitrophenylarsonate attained an apparent zero order rate of
TABLE 3. Effect of inhibitors on protease activitya Relative sp actb
Inhibitor
20
x
molar excess of inhibitor
30 min
DFP ......................... PMSF ........................ TPCK ........................ lodoacetic acid Iodoacetamide None ........................
60 min
4
x
molar excess of inhibitor
30 min
60 min
0.10
0.03
0.62
