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Purification and characterization of organic solvent-stable protease from organic solvent-tolerant Pseudomonas aeruginosa PST-01
J O U R N A L OP B I O S C I E N C E A N D BIOENGINEERING Vol. 87, No. 1, 61-68. 1999
Purification and Characterization of Organic Solvent-Stable Protease from Organic Solvent-Tolerant Pseudomonas aeruginosa PST-01 HIROYASU OGINO, * FUMITAKE WATANABE, MITSUHARU YAMADA, SATOSHI NAKAGAWA, TOSHIYA HIROSE, ATSUSHI NOGUCHI, MASAHIRO YASUDA, AND HARUO ISHIKAWA Department of Chemical Engineering, Osaka Prefecture University, I-I Gakuen-cho, Sakai, Osaka 599-8.531, Japan Received 30 July 1998/Accepted
12 October 1998
An organic solvent-stable protease (PST-01 protease) in a culture broth of organic solvent-tolerant Pseudomonas aeruginosa PST-01 was purified by successive hydrophobic interaction chromatography using ButylToyopearl gels. The purified enzyme was homogeneous as determined by SDS-polyacrylamide gel electrophoresis. PST-01 protease had a molecular mass of 38 kDa. The optimum temperature and pH for casein hydrolysis were 55°C and 8.5, respectively. PST-01 protease was stable at pH 8-12 and below 50°C and was determined to be a metalloprotease which was inhibited by EDTA, l,lO-phenanthroline, and phosphoramidon. PST-01 protease inhibited by EDTA was reactivated completely by the addition of zinc or cobalt ions. The stability of PST-01 protease in solutions containing water-soluble organic solvents or alcohols was higher than that in the absence of organic solvent. Furthermore, in general, PST-01 protease was more stable than commercially available proteases, namely, subtilisin Carlsberg, thermolysin, and a-chymotrypsin, in the presence of watersoluble organic solvents or alcohols. [Keywords: organic solvent-stable enzyme, metalloprotease, Pseudomonas aeruginosa, organic solventtolerant microorganism] Proteases are among the most valuable catalysts used in the food, pharmaceutical and detergent industries because they hydrolyze peptide bonds in aqueous environments and synthesize peptide bonds in microaqueous environments. There is also current interest in the application of proteases to the production of certain oligopeptides without producing by-products. Microorganisms are the most important source for enzyme production. Proteases are produced by a variety of microorganisms. To obtain valuable new proteases, new strains have been screened from nature. Proteases which are stable under high temperature or alkaline conditions are often found among thermophilic or alkalophilic microorganisms. Although many researchers and engineers are interested in the synthesis of peptides using protease, there have been no reports concerning the screening of microorganisms which produce organic solvent-stable proteases. So far several well-known proteases such as thermolysin, papain and chymotrypsin have been used as biocatalysts of peptide synthesis in the presence of organic solvents. However, most of these proteases were not sufficiently stable in the presence of organic solvents. We have screened organic solvent-tolerant microorganisms which produce organic solvent-stable enzymes (1, 2). Pseudomonas aeruginosa PST-01 isolated from soil, grew in the presence of a high concentration of several organic solvents and secreted an organic solvent-stable proteolytic enzyme (1). It is expected that both the solvent-tolerant microorganism and the organic solventstable enzyme produced by this strain could be used as catalysts for reactions in the presence of organic solvents. In this paper, we report the purification and characterization, including the organic solvent-stability, of the P. aeruginosa PST-01 protease.
MATERIALS AND METHODS Organism P. aeruginosa PST-01, an organic solvent-tolerant microorganism that produced a proteolytic enzyme, was isolated from soil as described in a previous paper (1). Hydrolysis of casein Protease activity was routinely determined by the casein hydrolysis method, as described previously (1). In brief, a reaction mixture of 5 ml of 50mM Borax-HCl buffer (pH 8.5) containing 0.6% (w/v) Hammarsten casein (E. Merck Darmstadt, Germany) and 0.1 ml of an enzyme solution was incubated at 30°C for 10min. The reaction was stopped by the addition of 1 ml of TCA solution consisting of 5.44% (w/v) trichloroacetic acid, 6% (w/v) acetic acid and 5.46% (w/v) sodium acetate. The mixture was further incubated at 4°C for 30 min and then filtered using no. 5C filter paper (Toy0 Roshi Kaisha Ltd., Tokyo). The concentration of digested casein in the filtrate was determined by measuring absorbance at 280 nm using tyrosine as a standard. One unit of proteolytic activity was defined as the amount of enzyme which produces the casein digest equivalent of 1 pmol of tyrosine in the filtrate per min at 30°C. Hydrolysis of elastin-orcein Determination of the elastase activity using elastin as a substrate was carried out by the method described by Morihara (3). Twenty mg of elastin-orcein (Sigma Chemical Company, St. Louis, USA) was suspended in 2.9ml of 30 mM TrisHCl buffer (pH 8.5) containing 2 mM CaC&. After preincubation for 5 min, a PST-01 protease solution was added to the solution and the reaction mixture was shaken at 160 strokes per min at 30°C. After 2 or 4 h, the reaction was terminated by the addition of 2 ml of 700 mM sodium phosphate buffer (pH 6.0), and then the reaction mixture was filtered using no. 5C filter paper. The absorbance of the filtrate was detected at 590nm. The complete hydrolysis of 20 mg elastin-orcein results in an in-
* Corresponding author. 61
62
OGINO ET AL.
crease in absorbance by 0.27. One unit of the hydrolytic activity of elastin-orcein was defined as the amount of the enzyme that produces an increase in absorbance of 0.0135 per hour under the conditions of the assay. Hydrolysis of elastin-like synthetic peptides The elastin-like synthetic peptides, namely, N-succinyl-AlaAla-Ala-p-nitroanilide, N-succinyl-Ala-Pro-Ala-p-nitroanilide, N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide, and glutaryl-Ala-Ala-Pro-Leu-p-nitroanilide, all of which were purchased from Peptide Institute, Inc. (Osaka), were used. The reaction mixture containing 1.0 mg/ml elastin-like synthetic peptide, 50 mM Tris-HCl (PH 8.0), 10 mM CaC12, and 1 .O U/ml (measured using casein as a substrate) PST-01 protease (50 mg/ml) or 30 mg/ml pancreatic elastase (EC 3.4.21.36, Type I, Sigma Chemical Co.) was incubated at 30°C. The increase in absorbance due to liberation of p-nitroaniline was measured at 410nm. One unit of the activity was defined as the amount of enzyme that produces 1 pmol of hydrolysate of the elastin-like synthetic peptide per minute. Hydrolysis of FA-Gly-Leu-amide One milliliter of 1 mM N-(3-[2-furyl]acryloyl)-Gly-Leu amide (FA-GlyLeu-amide, Sigma Chemical Co.) in 200mM Tris-HCl (pH 8.0) and 0.02 ml of 1.0 mg/ml PST-01 protease, pancreatic elastase, or thermolysin (EC 3.4.24.27, Protease type X, Sigma Chemical Co.) were mixed together and incubated at 25°C. The decrease in absorbance was measured at 345 nm. One unit of the activity was defined as the amount of enzyme that produces 1 /*mol of hydrolysate of FA-Gly-Leu-amide per minute. Determination of the protein concentration The protein concentrations in the course of enzyme purification were determined by measuring the absorbance at 280 nm using bovine serum albumin (BSA, Sigma Chemical Co.) as a standard. The absorbance of 1 mg/ml BSA was 0.622 at 280nm. In other procedures, the protein concentrations were determined by the method of Lowry et al. (4) using BSA as a standard. Determination of the concentration of ammonium sulfate The concentration of ammonium sulfate in the course of enzyme purification was determined by measuring the refractive index using an Abbe refractometer. Culture conditions and preparation of crude enzyme solution P. aeruginosa PST-01 was aerobically grown for 15 h at 30°C in a medium containing 1.0% (w/v) Polypepton (Nihon Pharmaceutical Co. Ltd., Tokyo), 1.0% (w/v) yeast extract (Dried Yeast Extract-S, Nihon Pharmaceutical Co. Ltd.), 0.2% (w/v) NaCl, and 0.8% (w/v) MgS04.7H20, which was adjusted to pH 8.5 with 1 M NaOH. The culture was used to inoculate 2 I of the medium in a 3-l jar fermentor. The size of the inoculum was 1% (v/v). Incubation was performed at 30°C with agitation at 250 rpm and an aeration rate of 3 vvm. After 40 h of cultivation, cells were removed by centrifugation at 10,000 x g for 10 min at 4°C to obtain the culture supernatant. Purification of PST-01 protease Purification of PST-01 protease in the culture supernatant was performed at 4°C. Solid ammonium Ammonium sulfate fractionation sulfate was added to the culture supernatant to 60% saturation. The resulting precipitate was collected by filtration with Hyflo Super-Cel as a filter aid. The collected precipitate was dissolved in 10mM Borax-HCI buffer (pH 8.5) containing 1 M ammonium sulfate. For the Butyl-Toyopearl column chromatography
J. BIOSCI. BIOENG.,
preparation of PST-01 protease, two different sizes of TSKgel Butyl-Toyopearl gels (Tosoh, Tokyo) were used; TSKgel Butyl-Toyopearl 650C (50 to 150 ,um) and 650M (40 to 90 pm). The enzyme solution containing 1 M ammonium sulfate was loaded onto a column (2.5 cm in diameter and 16.5 cm in height) of the TSKgel ButylToyopearl gel which was equilibrated with 10 mM BoraxHCl buffer (pH 8.5) containing 1 M ammonium sulfate. After the column was washed with 500ml of the same buffer used for the equilibration, proteins bound to the hydrophobic gel were eluted by reducing the concentration of ammonium sulfate in the 10mM Borax-HCl buffer (pH 8.5) from 1 to 0 M, with a linear gradient using 500 ml of the 10 mM Borax-HCl buffer (PH 8.5) containing 1 M ammonium sulfate and 500 ml of the 10 mM Borax-HCl buffer (pH 8.5). Fractions of approximately 1Oml were collected. Each of the fractions exhibiting protease activity was collected and pooled after addition of ammonium sulfate to a final concentration of 1 M. Determination of purity and molecular mass of purified PST-01 protease using SDS-polyacrylamide gel electrophoresis The purified PST-01 protease was dissolved in an SDS treatment solution consisting of 1% (w/v) SDS, 1% (w/v) 2-mercaptoethanol, 10% (w/v) glycerol and 31.25 mM Tris-HCl (pH 6.8), and was heated in a water bath at 100°C for 3 min. The sample was run in a stacking gel containing 4.35% (w/v) polyacrylamide and 0.3% (w/v) N,N-methylene-bis (acrylamide) and a separating gel containing 14.8% (w/v) polyacrylamide and 0.2% (w/v) N,N’-methylene-bis (acrylamide), under the conditions developed by Laemmli (5). The molecular mass standards used were rabbit muscle phosphorylase b (Mm = 94 kDa), bovine serum albumin (67 kDa), egg white ovalbumin (43 kDa), bovine erythrocyte carbonic-anhydrase (30 kDa), soybean trypsin-inhibitor (20.1 kDa), and bovine a-lactalbumin (14.4 kDa) purchased from Pharmacia (Uppsala, Sweden). Proteins were stained by the silver staining method using 2D-Silver Stain.11 “DAIICHI” (Daiichi Pure Chemicals Co. Ltd., Tokyo). Effect of protease inhibitors The caseinolytic activity in an enzyme solution (5.OU/ml) prepared with 10 mM Borax-HCl buffer (pH 8.5) was measured using a solution containing 0.6% (w/v) Hammarsten casein and 1, 5 or 50 mM of protease inhibitor. Although the conditions for the hydrolysis reaction of casein were the same as those for the assay of protease activity described above, the concentration of the digested casein in the filtrate was determined by the method of Lowry et al. (4), to minimize the effect of inhibitors because some inhibitors have been shown to absorb at 280 nm. The protease inhibitors used were diisopropyl fluorophosphate (DFP), phenylmethylsulfonyl fluoride (PMSF), p-chloromercuribenzoic acid (PCMB), iodoacetic acid (MIA), 2-mercaptoethanol (2-ME), L-cysteine (Cys), 1,2-epoxy-3(p-nitrophenoxy)propane ( E P N P ) , etylenediaminetetraacetic acid (EDTA), 1, lo-phenanthroline (o-Phe), and phosphoramidon (PR). Reactivation of PST-01 protease inhibited by EDTA A solution containing 5.0U/ml purified PST-01 protease, 10mM Borax-HCl buffer (pH 8.5) and 5 mM EDTA was stored at 4°C for 24 h. After addition of metal chloride, the enzyme solution was kept at room temperature for 10 min, and then the caseinolytic activity was measured. A 10mM Organic solvent-stabibty of enzyme
ORGANIC
VOL . 87, 1999 50
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FIG. 1. Column chromatography on Butyl-Toyopearl 65OC. Protein was collected by ammonium sulfate precipitation of culture supernatant and was applied to a Butyl-Toyopearl 650C column. It was eluted by reducing the concentration of ammonium sulfate from 1 to OM in 10mM Borax-HCI (pH 8.5). In each fraction, IOml of eluate was collected. Proteolytic activity (0) was determined by measuring the hydrolysis activity against casein. Lines: -, concentration of protein; ----, concentration of ammonium sulfate.
Borax-HCl buffer (pH 8.5) containing 5 U/ml of protease, namely, purified PST-01 protease (0.14 mg/ml), subtilisin Carlsberg (EC 3.4.21.62, protease Type VIII, Sigma Chemical Co., 0.67 mg/ml), thermolysin (EC 3.4.24.27, protease type X, Sigma Chemical Co., 0.20 mg/ml), or a-chymotrypsin (EC 3.4.21.1, type II, Sigma Chemical Co., 0.040 mg/ml), was filtered with a cellulose acetate membrane filter (pore size: 0.2 pm). One milliliter of an organic solvent was added to 3 ml of the filtrate in a test tube (16.5 mm in diameter) with a screw cap and was incubated at 30°C with shaking at 160 strokes per min. The time courses of the remaining proteolytic activity were determined by the casein hydrolysis method. RESULTS Purikation of protease To 1850ml of the culture supernatant of which the proteolytic activity was 11.1 U/ml, ammonium sulfate was added to 60% saturation. The suspension was filtrated using the Hyflo SuperCel and the collected precipitate was dissolved in 10mM Borax-HCI buffer (pH 8.5) containing 1 M ammonium sulfate. This enzyme solution containing 1 M ammonium sulfate was loaded onto a column packed with the TSKgel Butyl-Toyopearl 650C previously equilibrated with 10 mM Borax-HCl buffer (pH 8.5) containing 1 M ammonium sulfate. The enzyme with the proteolytic activity was completely adsorbed on the Butyl-Toyopearl 650C gel. After washing the Butyl-Toyopearl 650C column with 10 mM Borax-HCl buffer (pH 8.5) containing 1 M ammonium sulfate, the enzyme was eluted with TABLE 1.
FIG. 2. SDS-PAGE of purified PST-01 protease. The boiled PST-01 protease in the presence of 1% (w/v) SDS and 1% (w/v) 2mercaptoethanol was applied to polyacrylamide gel containing 14.8% (w/v) polyacrylamide, 0.2% (w/v) N,hP-methylene-bis(acrylamide). The molecular mass standards used were rabbit muscle phosphorylase b (Mm=94 kDa), bovine serum albumin (67 kDa), egg white ovalbumin (43 kDa), bovine erythrocyte carbonic-anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and bovine a-lactalbumin (14.4 kDa). Proteins were stained by the silver staining method.
a linear gradient from 1 M to 0 M ammonium sulfate in the buffer. The enzyme was eluted at about 0.6 M ammonium sulfate, as shown in Fig. 1. Ammonium sulfate was added to the effluent to a final concentration of 1 M before loading onto the TSKgel Butyl-Toyopearl 650M column previously equilibrated with 10 mM Borax-HCI buffer (pH 8.5) containing 1 M ammonium sulfate. The fractions containing protease, which were eluted by reducing the ammonium sulfate concentration from 1 to 0 M, were pooled and again loaded onto the ButylToyopearl 650M column. For the second ButylToyopearl 650M column run, the protein and protease activity elution curves coincided. Figure 2 shows the silver stained SDS-polyacrylamide gel of the effluent of the second run of the Butyl-Toyopearl 650M column. As shown in the figure, a single protein band was obtained. The molecular mass of the protein, PST-01 protease, was determined to be 38 kDa from the semi-logarithmic plot of molecular mass vs. mobilities for the standard proteins. The protease was purified 10Zfold with an overall
Purification table for protease in culture supernatant of P. aenrginosu
PST-01
Total volume Protein concentration Total activity Specific activity Purification Yield (fold) (ml) @w/ml) w (U/w9 (%) Culture supernatant 1850 32.0 20.5 x 103 0.347 1 100 Ammonium sulfate precipitation 800 6.56 22.4 x lo3 4.27 12.3 109 Butyl-Toyopearl65OC chromatography 550 0.880 11.3 x 103 23.4 67.5 55.2 First Butyl-Toyopearl65OM chromatography 450 0.665 9.43 x 103 31.5 90.8 46.0 Second Butyl-Toyopearl650M chromatography 113 1.31 5.24 x 103 35.3 102 25.6 Proteolytic activity was determined by assaying hydrolysis of casein at pH 8.5 and 30°C. One unit of the protease activity was defined as the amount of the enzyme which produces digested casein equivalent to 1 ,umol of tyrosine per min. Protein concentration was determined by measuring absorbance at 280 nm using bovine serum albumin as a standard. Purification step
64
OGINO ET AL.
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PH FIG. 3. Effect of pH on proteolytic activity at 30°C. Activities of the PST-01 protease at various pHs were measured at 30°C using casein as a substrate. The following buffer systems were used: 50 mM sodium acetate @H 3.9-5.7); 50 mM sodium phosphate (pH 5.8-7.6); 50 mM glycine-NaOH @H 8X5-10.5); 50 mM disodium hydrogenphosphate-NaOH (PH 11.0-l 1.7); 50 mM KCI-NaOH @H 12.8). Activities at various pHs relative to that at pH 8.6 are shown.
yield of 25.6%. The results of the purification are summarized in Table 1. Effect of pH on activity of PST-01 protease The effect of pH on the activity of PST-01 protease was investigated. The relative activity values at various pH values are shown in Fig. 3, in which the maximal PST-01 protease activity is taken as 1 .O. As can be seen in the figure, the optimum pH is 8.5. The pH-stability pH-stability of PST-01 protease of the enzyme was studied by assaying the residual activity of the enzyme after incubation at 4 or 30°C for 10min in buffers of various pH values. The relative remaining activity values at various pH values are shown
FIG. 5. Effect of temperature on the activity of purified PST-01 protease. Activity was determined by assaying the proteolytic activity using casein as a substrate at pH 8.5. Activities at various temperatures relative to that at 55°C are shown.
in Fig. 4, in which the remaining activity of PST-01 protease in 50 mM Borax-HCl (pH 8.5) after lo-min incubation at 30°C was taken as 1.0. The remaining activity of the enzyme in buffers of various pH values after incubation at 4°C for 10min were similar to those at 30°C (data not shown). PST-01 protease was stable in the pH range 8-12. Effect of temperature on PST-01 protease activity
The activity values of PST-01 protease at pH 8.5 were measured at various temperatures using casein as a substrate. The relative activities at various temperatures are shown in Fig. 5, taking the maximum activity to be 1.0. The maximum activity was observed at approximately 55°C. Heat stability of PST-01 protease
The heat stability of the enzyme was tested by assaying the residual activity after lo-min incubation at various temperatures at pH 8.5. As shown in Fig. 6, the enzyme was stable below 50°C and about 50% of the activity remained after
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PH FIG. 4. pH stability of the PST-01 protease at 30°C. Solutions containing the PST-01 protease were incubated at 30°C for 10 min in the following buffer systems: 50 mM sodium acetate (pH 3.5-6.0); 50mM sodium phosphate (pH 6.0-7.5); 50mM Tris-HCl (pH7.08.5); 50 mM Borax-HCl (pH 8.0-9.0); 50 mM Borax-NaOH (pH 9.511 .O); 50 mM disodium hydrogenphosphate-NaOH (pH 11.5-12.0). The remaining activity was measured using casein in 50 mM BoraxHCl (pH 8.5). The remaining activities of PST-01 protease after lomin incubation at 30°C in buffers with various pHs relative to that in 50 mM Borax-HCl (pH 8.5) are shown.
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Temperature [“Cl FIG. 6. Heat stability of the PST-01 protease. Purified PST-01 protease solution (pH 8.5) was incubated at various temperatures for 10 min. Remaining activity was measured at 3O’C. Remaining activities after incubation at various temperatures relative to the activity before incubation are shown.
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TABLE 2. Hydrolysis activities of the PST-01 protease and other two proteases against elastin orcein and synthetic peptides Substrate Elastin-orcein N-Succinyl-Ala-Ala-Alap-nitroanilid N-Succinyl-Ala-Pro-Alap-nitroanilide N-Succinyl-Ala-Ala-Pro-Leup-nitroanilide Glutaryl-Ala-Ala-Pro-Leup-nitroanilide FA-Gly-Leu-amide
Hydrolysis . . activity _.(U/ma) -. PST-01 Pancreatic Thermolysin protease elastase 44 28 n.t.
7.1
n.t.
16.8
n.t.
n.t.
n.t.
15.2 n.t.
n.t.
2.2
6.4
00 10 20 30 40 50
n.t., Not tested.
lo-mm incubation at 70°C. It is a well-known fact that for many proteases the addition of calcium ions increases heat stability. However, an increase in the heat stability of PST-01 protease was not observed on addition of calcium ions (data not shown). Hydrolysis activity against elastin-orcein and synthetic peptides Elastase activities determined using elastin-
orcein or elastin-like synthetic peptides are shown in Table 2. The specific activities of PST-01 protease and pancreatic elastase against elastin-orcein were 44 and 28 U/mg, respectively. Elastin-like synthetic peptides, such as iV-succinyl-Ala-Ala-Ala-p-nitroanilide, N-succinyl-Ala-Pro-Ala-p-nitroanilide, N-succinyl-Ala-Ala-ProLeu-p-nitroanilide, and glutaryl-Ala-Ala-Pro-Leu-p-nitroanilide, were not significantly hydrolyzed by PST-01 protease (less than 0.01 U/mg), while these were hydrolyzed by pancreatic elastase. The hydrolysis activities of PST-01 protease and thermolysin against FA-Gly-Leuamide were 2.2 and 6.4 U/mg, respectively. Effect of protease inhibitor The effect of various protease inhibitors on the activity was investigated. The activity values relative to those measured in the absence of the inhibitor are shown in Table 3. Serine protease inhibitors such as DFP and PMSF, and the carboxyl protease inhibitor EPNP had almost no effect on PST-01 protease. Although MIA, a sulfhydryl reagent used as a thiol protease inhibitor, had almost no effect, some other sulfhydryl reagents such as PCMB, 2-ME and Cys partially inhibited PST-01 protease activity. 5 mM EDTA also partially inhibited PST-01 protease activity, TABLE 3.
Effect of inhibitors on the activity of purified PST-01 protease
Inhibitor Diisopropyl fluorophosphate Phenylmethylsulfonyl fluoride p-Chloromercuribenzonic acid Iodoacetic acid 2-Mercaptoethanol L-Cysteine 1,2-Epoxy-3-@-nitrophenoxy)propane Etylenediaminetetraacetic acid 1, IO-Phenanthroline Phosphoramidon None
Concentration Relative activity 5mM 0.73 5mM 0.86 5mM 0.29 5mM 0.92 5mM 0.66 5mM 0.38 5 mM 0.80 5mM 0.49 50mM 0.12 5mM 0.01 1mM 0.10 1
The inhibitor was added to the substrate solution to the desired concentration. Activities in the presence of various inhibitors relative to that in the absence of inhibitor are shown.
Cont.
of ZnCI, in the enzyme solution [mM]
FIG. 7. Recovery of proteolytic activity of the PST-01 protease treated with 5 mM EDTA by addition of ZnC&. Solution containing purified PST-01 protease and 5 mM EDTA was incubated at 4°C for 24 h. After addition of various concentrations of ZnClz, the proteolytic activity was measured.
while o-Phe, PR and concentrated EDTA (50mM) strongly inhibited PST-01 protease activity. EDTA is a metal-chelating reagent and can be used as a metal protease inhibitor. PST-01 protease was most effectively inhibited by the metal-chelating reagents and phosphoramidon which specifically inhibits metalloproteases, therefore PST-01 protease can be classified as a metalloprotease. Reactivation of PST-01 protease inhibited with EDTA
The reactivation of PST-01 protease inhibited with 5 mM EDTA was studied by the addition of various concentrations of zinc chloride. In Fig. 7, the results are shown as a plot of the activity vs. the concentration of zinc chloride. The activity after incubation at 4°C for 24 h in the presence of 5 mM EDTA was about 18% of that measured before the addition of EDTA. The addition of zinc chloride to a final concentration of 5 mM reactivated the enzyme completely. The addition of zinc chloride to a final concentration of greater than 5 mM None WC4 aI E5
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CaCI,
z 5 a
MnCI, COCI, ZIlCI, 0
20
40
60
80
100
Activity [%] FIG. 8. Recovery of proteolytic activity of PST-01 protease treated with 5 mM EDTA by addition of 5 mM metal chloride. A solution containing purified PST-01 protease and 5 mM EDTA was incubated at 4°C for 24 h. After addition of 5 mM of metal chloride, the proteolytic activity was measured.
66
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apoenzyme of PST-01 protease, which lost the activity on addition of EDTA, was reactivated by the addition of zinc or cobalt ions. Effect of organic solvents on proteolytic activity The effect of various organic solvents on the stability of various proteases, that is, purified PST-01 protease, subtilisin Carlsberg, thermolysin, and cw-chymotrypsin, was studied. Mixtures of 3 ml of an enzyme solution (5 U/ml) and 1 ml of an organic solvent were incubated at 30°C with shaking and the remaining activity was measured at appropriate time intervals. Figure 9 shows the typical time courses of the remaining activity of various proteases in the presence of 1,5-pentanediol. Deactivation of all the proteases tested in the presence or absence of organic solvents obeyed first order kinetics. The half-lives of the activity of various proteases are summaTime [d] FIG. 9. Time courses of relative remaining activity of various proteases in the presence of 1,5pentanediol. The filtered purified PST-01 protease (0), subtilisin Carlsberg (A), thermolysin ( 0 ), and a-chymotrypsin (V) solutions (3 ml) were incubated at 30°C with shaking in the presence of 1,5-pentanediol. The relative remaining activities based on the activity before addition of organic solvent (5 U/ml) are shown.
inhibited the complete recovery of the activity. When 50mM zinc chloride was added (the final concentration of zinc chloride in the reaction mixture was 0.98 mM), the remaining activity was 33%. The effect of addition to 5 mM of other metal chlorides on reactivation of PST-01 protease is shown in Fig. 8. Magnesium ions, iron ions, and calcium ions were not effective for the reactivation of PST-01 protease. The addition of 5 mM cobalt ions had the same effect as that of zinc ions. The
rized in Table 4. The half-life of PST-01 protease in the
absence of organic solvent was about 9.7 d. The half-life of PST-01 protease in the presence of water-insoluble organic solvents such as toluene, benzene, n-heptane, pxylene, n-hexane, n-decane, and cyclohexane was similar to or shorter than that in the absence of organic solvent. However, the half-life of PST-01 protease in the presence of water-soluble organic solvents or alcohols such as ethylene glycol, 1 ,Cbutanediol, 1,5-pentanediol, ethanol, 1-hexanol, methanol, dimethyl sulfoxide, 2propanol, triethylene glycol, tert-butanol, 1-heptanol, N,N-dimethylformamide, 1-octanol, I-butanol, acetone, 1-decanol, and 1,6dioxane, was longer than that in the absence of organic solvent. Inactivation of the enzyme in the presence of ethylene glycol, l,Cbutanediol, 1,5-pentanediol, ethanol, and 1-hexanol was not observed at all
during the 15 day-experiment. When organic solvents of which the log P (6) values are between -0.23 and 3.1
TABLE 4. Effect of organic solvents on the stability of the purified PST-01 Organic solvent Ethyleneglycol 1,6Butanediol 1,5-Pentanediol Ethanol I-Hexanol Methanol Dimethyl sulfoxide (DMSO) 2-Propanol Triethyleneglycol tert-Butanol I-Heptanol N,N-Dimethylformamide (DMF) 1-Octanol 1-Butanol Acetone I-Decanol 1,CDioxane Toluene Benzene n-Heptane p-Xylene n-Hexane n-Decane Cyclohexane None
protease
Half-life (d) PST-01 protease >lOO > 100 >lOO >lOO >50 >50 >50 >50 >50 >50 >50 25.3 24.2 24.2 23.1 19.4 11.7 12.0 7.8 4.8 4.4 3.8 2.4 2.3 9.1
Subtilisin 14.3 25.0 >50 >50 13.7 26.2 6.4 47.6 39.7 41.6 8.6 39.8 n.t. 47.6 24.8 n.t. 29.3 5.7 n.t. n.t. n.t. n.t. n.t. n.t. 0.3
Thermolysin >50 4.4 1.7 3.0 18.2 4.6 2.6 1.2 5.1 0.8 13.1 0.9 n.t. 0.9 0.7 n.t. 0.8 22.5 n.t. n.t. n.t. n.t. n.t. nt. 10.8
a-Chymotrypsin 6.9 0.7 0.4 27.0 14.2 6.0 33.6 0.6 11.1 0.5 3.8 2.2 n.t. 0.3 0.6 n.t. 0.5 >lOO at. n.t. n.t. n.t. n.t. n.t. 13.2
The filtered solutions containing various proteases (3 ml), purified PST-01, subtilisin Carlsberg, thermolysin, and a-chymotrypsin, were incubated at pH 8.5 and 30°C with shaking at 160 strokes per min in the absence or presence of 1 ml of organic solvent for 15 d. The remaining activity was measured after 0, 1, 2, 3, 5, 7 , 10, and 15 d . The half-life was calculated from the exponential regression curve. n.t.. Not tested.
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were added to the filtered culture supernatant of P. aeruginosa PST-01 (the crude enzyme solution of PST-01 protease), the enzyme was inactivated a little more rapidly than when no organic solvents were added. When organic solvents of which the log P values are less than -0.23 or more than 3.1 were added, the inactivation rates of the enzyme were the same as those in the absence of organic solvent (1). However, in the case of the purified PST-01 protease, the relation between the stability against organic solvent and the log P value of the added organic solvent was not found. In the presence of watersoluble organic solvents or alcohols, the stability of PST-01 protease was much higher than in the absence of organic solvent. In general, the half-life of the PST-01 protease activity is longer than that of any other proteases tested in the presence of water-soluble organic solvents or alcohols. However, in the presence of toluene, the half-life of PST-01 protease is shorter than that of a-chymotrypsin or thermolysin. DISCUSSION
This paper describes the purification and characterization of the organic solvent-tolerant P. aeruginosa PST01 protease. The P. aeruginosa PST-01 protease was purified from the culture supernatant by ammonium sulfate precipitation and successive hydrophobic interaction chromatographies. In each of the purification steps, proteolytic activity appeared as a single peak. Some strains of P. aeruginosa produce three kinds of proteases (7, 8). The discrepancy in the number of proteases produced may be attributed to the difference in the strains of P. aeruginosa used, the cultivation medium, and/or the method of enzyme purification. The molecular mass of the purified PST-01 protease was determined to be 38 kDa by SDS-polyacrylamide gel electrophoresis. The molecular mass value that we determined for PST-01 protease was in agreement with that of the P. aeruginosa PA01 elastase determined by SDSpolyacrylamide gel electrophoresis (9). The optimum pH values of the caseinolytic activity of proteases from P. aeruginosa ranged from 8 to 9 (7, 8, 10, 11). The optimum pH, 8.5, of the caseinolytic activity of PST-01 protease was in agreement with the reported optimum pH range for other P. aeruginosa proteases. PST-01 protease was stable in alkaline conditions @H 8.0-12.0). PST-01 protease hydrolyzed casein rapidly at about 55°C and was stable in the temperature range below 50°C at pH 8.5. The protease (elastase) which is produced by P. aeruginosa is called pseudolysin (EC 3.4.24.26) (3, 12). PST-01 protease also hydrolyzes elastin. The specific activity of PST-01 protease for elastin-orcein (44U/mg) was very similar to that of pseudolysin (35 U/mg) (3). However, PST-01 protease did not significantly hydrolyzed elastin-like peptides, such as N-succinyl-Ala-AlaAla-p-nitroanilide, N-succinyl-Ala-Pro-Ala-p-nitroanilide, N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide, and glutarylAla-Ala-Pro-Leu-p-nitroanilide. PST-01 protease hydrolyzed FA-Gly-Leu-amide, and its specific activity was about one-third that of thermolysin. PST-01 protease was inhibited by metal-chelating reagents such as EDTA and o-Phe, and the peptide inhibitor PR which specifically inhibits metalloproteases. PST01 protease inhibited with EDTA was reactivated by the addition of zinc or cobalt ions. Therefore, PST-01 pro-
SOLVENT-STABLE
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tease can be classified as a metalloprotease which requires zinc or cobalt ions for a manifestation of the activity. Proteases are often used as catalysts for peptide syntheses. To shift the thermodynamic equilibrium in favor of the peptide synthesis, the reaction solution should contain organic solvent. However, enzymes are usually inactivated by the addition of organic solvents to the reaction solution. Crude PST-01 protease was stable in the presence of organic solvents (1). Purified PST-01 protease was also very stable in the presence of water-soluble organic solvents or alcohols such as ethylene glycol, l&butanediol, 1,5-pentanediol, ethanol, 1-hexanol, methanol, dimethyl sulfoxide, 2-propanol, triethylene glycol, tert-butanol, 1-heptanol, N,N-dimethylformamide, loctanol, 1-butanol, acetone, 1-decanol, and 1,Cdioxane. In general, PST-01 protease was more stable than the other proteases tested, namely, subtilisin Carlsberg, thermolysin, and a-chymotrypsin, in the presence of water-soluble organic solvents or alcohols. However, in a few cases, the half-life of the activity of PST-01 protease in the presence of organic solvent was shorter than those of the other proteases. Therefore, it is concluded that the organic solvent-stability of enzymes depends on the kind of enzyme and organic solvent. In this paper, to compare the stabilities of various proteases under the same conditions in the presence of organic solvent, the proteases were dissolved in 1OmM Borax-HCl buffer (pH 8.5), although this is not necessarily the best medium for each protease. The stability of enzyme in the presence of organic solvent can depend on the buffer solution or additives. It may be necessary to compare the stabilities of various enzymes measured under their optimal conditions for stability. We believe that PST-01 protease is a very useful biocatalyst for peptide syntheses in the presence of a water-soluble organic solvent because it is very stable. We are currently performing a variety of peptide synthetic reactions using PST-01 protease in the presence of water-soluble organic solvent. As a preliminary result, a dipeptide N-carbobenzoxy-Arg-Leu-NH2 was synthesized in a high yield (approximately 78%) from N-carbobenzoxy-Arg as an amine component and Leu-NH2 as a carboxy1 component in the presence of 50% (v/v) N,Ndimethylformamide. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Encouragement of Young Scientists (09750880) from the Japanese Ministry of Education, Science, Sports and Culture. REFERENCES 1. Ogino, H., Yasui, K., Shiotani, T., Ishihara, T., and Isbikawa, H.: Organic solvent-tolerant bacterium which secretes an organic solvent-stable proteolytic enzyme. Appl. Environ. Microbial., 61, 4258-4262 (1995). 2. Ogino, H., Miyamoto, K., and Ishikawa, H.: Organic-solventtolerant bacterium which secretes organic-solvent-stable lipolytic enzyme. Appl. Environ. Microbial., 60, 3884-3886 (1994). 3. Morihara, K.: Pseudolysin and other pathogen endopeptidases of thermolysin family. Methods Enzvmol.. 248. 242-253 (1995). 4. Lowry, 0. H., Rosebrougb, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275 (1951). .
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5. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature (London), 227, 680-685 (1970). 6. Laane, C., Boeren, S., Vos, K., and Veeger, C.: Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng., 30, 81-87 (1987). 7. Morihara, K.: Production of elastase and proteinase by Pseudomonas aeruginosa. J. Bacterial., 88, 745-757 (1964). 8. Wretlind, B. and Wadstrom, T.: Purification and properties of a protease with elastase activity from Pseudomonas aeruginosa. J. Gen. Microbial., 103, 319-327 (1977). 9. Goldberg, J. B. and Ohman, D. E.: Activation of an elastase
J. BIOSCI. BIOENG.. precursor by the 1asA gene product of Pseudomonas aeruginosa. J. Bacterial., 169, 4532-4539 (1987). 10. Morihara, K.: Pseudomonas aeruginosa proteinase. 1. Purification and general properties. Biochim. Biophys. Acta, 73, 113124 (1963). 11. Morihara, K., Tsuzuki, H., Oka, T., Inoue, H., and Ebata, M.: Pseudomonas aeruginosa elastase: isolation, crystallization, and preliminary characterization. J. Biol. Chem., 240, 32953304 (1965). 12. Rawliogs, N. D. and Barrett, A. J.: Evolutionary families of metallopeptidases. Methods Enzymol., 248, 183-228 (1995).
Purification and Characterization of Organic Solvent-Stable Protease from Organic Solvent-Tolerant Pseudomonas aeruginosa PST-01 HIROYASU OGINO, * FUMITAKE WATANABE, MITSUHARU YAMADA, SATOSHI NAKAGAWA, TOSHIYA HIROSE, ATSUSHI NOGUCHI, MASAHIRO YASUDA, AND HARUO ISHIKAWA Department of Chemical Engineering, Osaka Prefecture University, I-I Gakuen-cho, Sakai, Osaka 599-8.531, Japan Received 30 July 1998/Accepted
12 October 1998
An organic solvent-stable protease (PST-01 protease) in a culture broth of organic solvent-tolerant Pseudomonas aeruginosa PST-01 was purified by successive hydrophobic interaction chromatography using ButylToyopearl gels. The purified enzyme was homogeneous as determined by SDS-polyacrylamide gel electrophoresis. PST-01 protease had a molecular mass of 38 kDa. The optimum temperature and pH for casein hydrolysis were 55°C and 8.5, respectively. PST-01 protease was stable at pH 8-12 and below 50°C and was determined to be a metalloprotease which was inhibited by EDTA, l,lO-phenanthroline, and phosphoramidon. PST-01 protease inhibited by EDTA was reactivated completely by the addition of zinc or cobalt ions. The stability of PST-01 protease in solutions containing water-soluble organic solvents or alcohols was higher than that in the absence of organic solvent. Furthermore, in general, PST-01 protease was more stable than commercially available proteases, namely, subtilisin Carlsberg, thermolysin, and a-chymotrypsin, in the presence of watersoluble organic solvents or alcohols. [Keywords: organic solvent-stable enzyme, metalloprotease, Pseudomonas aeruginosa, organic solventtolerant microorganism] Proteases are among the most valuable catalysts used in the food, pharmaceutical and detergent industries because they hydrolyze peptide bonds in aqueous environments and synthesize peptide bonds in microaqueous environments. There is also current interest in the application of proteases to the production of certain oligopeptides without producing by-products. Microorganisms are the most important source for enzyme production. Proteases are produced by a variety of microorganisms. To obtain valuable new proteases, new strains have been screened from nature. Proteases which are stable under high temperature or alkaline conditions are often found among thermophilic or alkalophilic microorganisms. Although many researchers and engineers are interested in the synthesis of peptides using protease, there have been no reports concerning the screening of microorganisms which produce organic solvent-stable proteases. So far several well-known proteases such as thermolysin, papain and chymotrypsin have been used as biocatalysts of peptide synthesis in the presence of organic solvents. However, most of these proteases were not sufficiently stable in the presence of organic solvents. We have screened organic solvent-tolerant microorganisms which produce organic solvent-stable enzymes (1, 2). Pseudomonas aeruginosa PST-01 isolated from soil, grew in the presence of a high concentration of several organic solvents and secreted an organic solvent-stable proteolytic enzyme (1). It is expected that both the solvent-tolerant microorganism and the organic solventstable enzyme produced by this strain could be used as catalysts for reactions in the presence of organic solvents. In this paper, we report the purification and characterization, including the organic solvent-stability, of the P. aeruginosa PST-01 protease.
MATERIALS AND METHODS Organism P. aeruginosa PST-01, an organic solvent-tolerant microorganism that produced a proteolytic enzyme, was isolated from soil as described in a previous paper (1). Hydrolysis of casein Protease activity was routinely determined by the casein hydrolysis method, as described previously (1). In brief, a reaction mixture of 5 ml of 50mM Borax-HCl buffer (pH 8.5) containing 0.6% (w/v) Hammarsten casein (E. Merck Darmstadt, Germany) and 0.1 ml of an enzyme solution was incubated at 30°C for 10min. The reaction was stopped by the addition of 1 ml of TCA solution consisting of 5.44% (w/v) trichloroacetic acid, 6% (w/v) acetic acid and 5.46% (w/v) sodium acetate. The mixture was further incubated at 4°C for 30 min and then filtered using no. 5C filter paper (Toy0 Roshi Kaisha Ltd., Tokyo). The concentration of digested casein in the filtrate was determined by measuring absorbance at 280 nm using tyrosine as a standard. One unit of proteolytic activity was defined as the amount of enzyme which produces the casein digest equivalent of 1 pmol of tyrosine in the filtrate per min at 30°C. Hydrolysis of elastin-orcein Determination of the elastase activity using elastin as a substrate was carried out by the method described by Morihara (3). Twenty mg of elastin-orcein (Sigma Chemical Company, St. Louis, USA) was suspended in 2.9ml of 30 mM TrisHCl buffer (pH 8.5) containing 2 mM CaC&. After preincubation for 5 min, a PST-01 protease solution was added to the solution and the reaction mixture was shaken at 160 strokes per min at 30°C. After 2 or 4 h, the reaction was terminated by the addition of 2 ml of 700 mM sodium phosphate buffer (pH 6.0), and then the reaction mixture was filtered using no. 5C filter paper. The absorbance of the filtrate was detected at 590nm. The complete hydrolysis of 20 mg elastin-orcein results in an in-
* Corresponding author. 61
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crease in absorbance by 0.27. One unit of the hydrolytic activity of elastin-orcein was defined as the amount of the enzyme that produces an increase in absorbance of 0.0135 per hour under the conditions of the assay. Hydrolysis of elastin-like synthetic peptides The elastin-like synthetic peptides, namely, N-succinyl-AlaAla-Ala-p-nitroanilide, N-succinyl-Ala-Pro-Ala-p-nitroanilide, N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide, and glutaryl-Ala-Ala-Pro-Leu-p-nitroanilide, all of which were purchased from Peptide Institute, Inc. (Osaka), were used. The reaction mixture containing 1.0 mg/ml elastin-like synthetic peptide, 50 mM Tris-HCl (PH 8.0), 10 mM CaC12, and 1 .O U/ml (measured using casein as a substrate) PST-01 protease (50 mg/ml) or 30 mg/ml pancreatic elastase (EC 3.4.21.36, Type I, Sigma Chemical Co.) was incubated at 30°C. The increase in absorbance due to liberation of p-nitroaniline was measured at 410nm. One unit of the activity was defined as the amount of enzyme that produces 1 pmol of hydrolysate of the elastin-like synthetic peptide per minute. Hydrolysis of FA-Gly-Leu-amide One milliliter of 1 mM N-(3-[2-furyl]acryloyl)-Gly-Leu amide (FA-GlyLeu-amide, Sigma Chemical Co.) in 200mM Tris-HCl (pH 8.0) and 0.02 ml of 1.0 mg/ml PST-01 protease, pancreatic elastase, or thermolysin (EC 3.4.24.27, Protease type X, Sigma Chemical Co.) were mixed together and incubated at 25°C. The decrease in absorbance was measured at 345 nm. One unit of the activity was defined as the amount of enzyme that produces 1 /*mol of hydrolysate of FA-Gly-Leu-amide per minute. Determination of the protein concentration The protein concentrations in the course of enzyme purification were determined by measuring the absorbance at 280 nm using bovine serum albumin (BSA, Sigma Chemical Co.) as a standard. The absorbance of 1 mg/ml BSA was 0.622 at 280nm. In other procedures, the protein concentrations were determined by the method of Lowry et al. (4) using BSA as a standard. Determination of the concentration of ammonium sulfate The concentration of ammonium sulfate in the course of enzyme purification was determined by measuring the refractive index using an Abbe refractometer. Culture conditions and preparation of crude enzyme solution P. aeruginosa PST-01 was aerobically grown for 15 h at 30°C in a medium containing 1.0% (w/v) Polypepton (Nihon Pharmaceutical Co. Ltd., Tokyo), 1.0% (w/v) yeast extract (Dried Yeast Extract-S, Nihon Pharmaceutical Co. Ltd.), 0.2% (w/v) NaCl, and 0.8% (w/v) MgS04.7H20, which was adjusted to pH 8.5 with 1 M NaOH. The culture was used to inoculate 2 I of the medium in a 3-l jar fermentor. The size of the inoculum was 1% (v/v). Incubation was performed at 30°C with agitation at 250 rpm and an aeration rate of 3 vvm. After 40 h of cultivation, cells were removed by centrifugation at 10,000 x g for 10 min at 4°C to obtain the culture supernatant. Purification of PST-01 protease Purification of PST-01 protease in the culture supernatant was performed at 4°C. Solid ammonium Ammonium sulfate fractionation sulfate was added to the culture supernatant to 60% saturation. The resulting precipitate was collected by filtration with Hyflo Super-Cel as a filter aid. The collected precipitate was dissolved in 10mM Borax-HCI buffer (pH 8.5) containing 1 M ammonium sulfate. For the Butyl-Toyopearl column chromatography
J. BIOSCI. BIOENG.,
preparation of PST-01 protease, two different sizes of TSKgel Butyl-Toyopearl gels (Tosoh, Tokyo) were used; TSKgel Butyl-Toyopearl 650C (50 to 150 ,um) and 650M (40 to 90 pm). The enzyme solution containing 1 M ammonium sulfate was loaded onto a column (2.5 cm in diameter and 16.5 cm in height) of the TSKgel ButylToyopearl gel which was equilibrated with 10 mM BoraxHCl buffer (pH 8.5) containing 1 M ammonium sulfate. After the column was washed with 500ml of the same buffer used for the equilibration, proteins bound to the hydrophobic gel were eluted by reducing the concentration of ammonium sulfate in the 10mM Borax-HCl buffer (pH 8.5) from 1 to 0 M, with a linear gradient using 500 ml of the 10 mM Borax-HCl buffer (PH 8.5) containing 1 M ammonium sulfate and 500 ml of the 10 mM Borax-HCl buffer (pH 8.5). Fractions of approximately 1Oml were collected. Each of the fractions exhibiting protease activity was collected and pooled after addition of ammonium sulfate to a final concentration of 1 M. Determination of purity and molecular mass of purified PST-01 protease using SDS-polyacrylamide gel electrophoresis The purified PST-01 protease was dissolved in an SDS treatment solution consisting of 1% (w/v) SDS, 1% (w/v) 2-mercaptoethanol, 10% (w/v) glycerol and 31.25 mM Tris-HCl (pH 6.8), and was heated in a water bath at 100°C for 3 min. The sample was run in a stacking gel containing 4.35% (w/v) polyacrylamide and 0.3% (w/v) N,N-methylene-bis (acrylamide) and a separating gel containing 14.8% (w/v) polyacrylamide and 0.2% (w/v) N,N’-methylene-bis (acrylamide), under the conditions developed by Laemmli (5). The molecular mass standards used were rabbit muscle phosphorylase b (Mm = 94 kDa), bovine serum albumin (67 kDa), egg white ovalbumin (43 kDa), bovine erythrocyte carbonic-anhydrase (30 kDa), soybean trypsin-inhibitor (20.1 kDa), and bovine a-lactalbumin (14.4 kDa) purchased from Pharmacia (Uppsala, Sweden). Proteins were stained by the silver staining method using 2D-Silver Stain.11 “DAIICHI” (Daiichi Pure Chemicals Co. Ltd., Tokyo). Effect of protease inhibitors The caseinolytic activity in an enzyme solution (5.OU/ml) prepared with 10 mM Borax-HCl buffer (pH 8.5) was measured using a solution containing 0.6% (w/v) Hammarsten casein and 1, 5 or 50 mM of protease inhibitor. Although the conditions for the hydrolysis reaction of casein were the same as those for the assay of protease activity described above, the concentration of the digested casein in the filtrate was determined by the method of Lowry et al. (4), to minimize the effect of inhibitors because some inhibitors have been shown to absorb at 280 nm. The protease inhibitors used were diisopropyl fluorophosphate (DFP), phenylmethylsulfonyl fluoride (PMSF), p-chloromercuribenzoic acid (PCMB), iodoacetic acid (MIA), 2-mercaptoethanol (2-ME), L-cysteine (Cys), 1,2-epoxy-3(p-nitrophenoxy)propane ( E P N P ) , etylenediaminetetraacetic acid (EDTA), 1, lo-phenanthroline (o-Phe), and phosphoramidon (PR). Reactivation of PST-01 protease inhibited by EDTA A solution containing 5.0U/ml purified PST-01 protease, 10mM Borax-HCl buffer (pH 8.5) and 5 mM EDTA was stored at 4°C for 24 h. After addition of metal chloride, the enzyme solution was kept at room temperature for 10 min, and then the caseinolytic activity was measured. A 10mM Organic solvent-stabibty of enzyme
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FIG. 1. Column chromatography on Butyl-Toyopearl 65OC. Protein was collected by ammonium sulfate precipitation of culture supernatant and was applied to a Butyl-Toyopearl 650C column. It was eluted by reducing the concentration of ammonium sulfate from 1 to OM in 10mM Borax-HCI (pH 8.5). In each fraction, IOml of eluate was collected. Proteolytic activity (0) was determined by measuring the hydrolysis activity against casein. Lines: -, concentration of protein; ----, concentration of ammonium sulfate.
Borax-HCl buffer (pH 8.5) containing 5 U/ml of protease, namely, purified PST-01 protease (0.14 mg/ml), subtilisin Carlsberg (EC 3.4.21.62, protease Type VIII, Sigma Chemical Co., 0.67 mg/ml), thermolysin (EC 3.4.24.27, protease type X, Sigma Chemical Co., 0.20 mg/ml), or a-chymotrypsin (EC 3.4.21.1, type II, Sigma Chemical Co., 0.040 mg/ml), was filtered with a cellulose acetate membrane filter (pore size: 0.2 pm). One milliliter of an organic solvent was added to 3 ml of the filtrate in a test tube (16.5 mm in diameter) with a screw cap and was incubated at 30°C with shaking at 160 strokes per min. The time courses of the remaining proteolytic activity were determined by the casein hydrolysis method. RESULTS Purikation of protease To 1850ml of the culture supernatant of which the proteolytic activity was 11.1 U/ml, ammonium sulfate was added to 60% saturation. The suspension was filtrated using the Hyflo SuperCel and the collected precipitate was dissolved in 10mM Borax-HCI buffer (pH 8.5) containing 1 M ammonium sulfate. This enzyme solution containing 1 M ammonium sulfate was loaded onto a column packed with the TSKgel Butyl-Toyopearl 650C previously equilibrated with 10 mM Borax-HCl buffer (pH 8.5) containing 1 M ammonium sulfate. The enzyme with the proteolytic activity was completely adsorbed on the Butyl-Toyopearl 650C gel. After washing the Butyl-Toyopearl 650C column with 10 mM Borax-HCl buffer (pH 8.5) containing 1 M ammonium sulfate, the enzyme was eluted with TABLE 1.
FIG. 2. SDS-PAGE of purified PST-01 protease. The boiled PST-01 protease in the presence of 1% (w/v) SDS and 1% (w/v) 2mercaptoethanol was applied to polyacrylamide gel containing 14.8% (w/v) polyacrylamide, 0.2% (w/v) N,hP-methylene-bis(acrylamide). The molecular mass standards used were rabbit muscle phosphorylase b (Mm=94 kDa), bovine serum albumin (67 kDa), egg white ovalbumin (43 kDa), bovine erythrocyte carbonic-anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and bovine a-lactalbumin (14.4 kDa). Proteins were stained by the silver staining method.
a linear gradient from 1 M to 0 M ammonium sulfate in the buffer. The enzyme was eluted at about 0.6 M ammonium sulfate, as shown in Fig. 1. Ammonium sulfate was added to the effluent to a final concentration of 1 M before loading onto the TSKgel Butyl-Toyopearl 650M column previously equilibrated with 10 mM Borax-HCI buffer (pH 8.5) containing 1 M ammonium sulfate. The fractions containing protease, which were eluted by reducing the ammonium sulfate concentration from 1 to 0 M, were pooled and again loaded onto the ButylToyopearl 650M column. For the second ButylToyopearl 650M column run, the protein and protease activity elution curves coincided. Figure 2 shows the silver stained SDS-polyacrylamide gel of the effluent of the second run of the Butyl-Toyopearl 650M column. As shown in the figure, a single protein band was obtained. The molecular mass of the protein, PST-01 protease, was determined to be 38 kDa from the semi-logarithmic plot of molecular mass vs. mobilities for the standard proteins. The protease was purified 10Zfold with an overall
Purification table for protease in culture supernatant of P. aenrginosu
PST-01
Total volume Protein concentration Total activity Specific activity Purification Yield (fold) (ml) @w/ml) w (U/w9 (%) Culture supernatant 1850 32.0 20.5 x 103 0.347 1 100 Ammonium sulfate precipitation 800 6.56 22.4 x lo3 4.27 12.3 109 Butyl-Toyopearl65OC chromatography 550 0.880 11.3 x 103 23.4 67.5 55.2 First Butyl-Toyopearl65OM chromatography 450 0.665 9.43 x 103 31.5 90.8 46.0 Second Butyl-Toyopearl650M chromatography 113 1.31 5.24 x 103 35.3 102 25.6 Proteolytic activity was determined by assaying hydrolysis of casein at pH 8.5 and 30°C. One unit of the protease activity was defined as the amount of the enzyme which produces digested casein equivalent to 1 ,umol of tyrosine per min. Protein concentration was determined by measuring absorbance at 280 nm using bovine serum albumin as a standard. Purification step
64
OGINO ET AL.
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PH FIG. 3. Effect of pH on proteolytic activity at 30°C. Activities of the PST-01 protease at various pHs were measured at 30°C using casein as a substrate. The following buffer systems were used: 50 mM sodium acetate @H 3.9-5.7); 50 mM sodium phosphate (pH 5.8-7.6); 50 mM glycine-NaOH @H 8X5-10.5); 50 mM disodium hydrogenphosphate-NaOH (PH 11.0-l 1.7); 50 mM KCI-NaOH @H 12.8). Activities at various pHs relative to that at pH 8.6 are shown.
yield of 25.6%. The results of the purification are summarized in Table 1. Effect of pH on activity of PST-01 protease The effect of pH on the activity of PST-01 protease was investigated. The relative activity values at various pH values are shown in Fig. 3, in which the maximal PST-01 protease activity is taken as 1 .O. As can be seen in the figure, the optimum pH is 8.5. The pH-stability pH-stability of PST-01 protease of the enzyme was studied by assaying the residual activity of the enzyme after incubation at 4 or 30°C for 10min in buffers of various pH values. The relative remaining activity values at various pH values are shown
FIG. 5. Effect of temperature on the activity of purified PST-01 protease. Activity was determined by assaying the proteolytic activity using casein as a substrate at pH 8.5. Activities at various temperatures relative to that at 55°C are shown.
in Fig. 4, in which the remaining activity of PST-01 protease in 50 mM Borax-HCl (pH 8.5) after lo-min incubation at 30°C was taken as 1.0. The remaining activity of the enzyme in buffers of various pH values after incubation at 4°C for 10min were similar to those at 30°C (data not shown). PST-01 protease was stable in the pH range 8-12. Effect of temperature on PST-01 protease activity
The activity values of PST-01 protease at pH 8.5 were measured at various temperatures using casein as a substrate. The relative activities at various temperatures are shown in Fig. 5, taking the maximum activity to be 1.0. The maximum activity was observed at approximately 55°C. Heat stability of PST-01 protease
The heat stability of the enzyme was tested by assaying the residual activity after lo-min incubation at various temperatures at pH 8.5. As shown in Fig. 6, the enzyme was stable below 50°C and about 50% of the activity remained after
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PH FIG. 4. pH stability of the PST-01 protease at 30°C. Solutions containing the PST-01 protease were incubated at 30°C for 10 min in the following buffer systems: 50 mM sodium acetate (pH 3.5-6.0); 50mM sodium phosphate (pH 6.0-7.5); 50mM Tris-HCl (pH7.08.5); 50 mM Borax-HCl (pH 8.0-9.0); 50 mM Borax-NaOH (pH 9.511 .O); 50 mM disodium hydrogenphosphate-NaOH (pH 11.5-12.0). The remaining activity was measured using casein in 50 mM BoraxHCl (pH 8.5). The remaining activities of PST-01 protease after lomin incubation at 30°C in buffers with various pHs relative to that in 50 mM Borax-HCl (pH 8.5) are shown.
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Temperature [“Cl FIG. 6. Heat stability of the PST-01 protease. Purified PST-01 protease solution (pH 8.5) was incubated at various temperatures for 10 min. Remaining activity was measured at 3O’C. Remaining activities after incubation at various temperatures relative to the activity before incubation are shown.
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TABLE 2. Hydrolysis activities of the PST-01 protease and other two proteases against elastin orcein and synthetic peptides Substrate Elastin-orcein N-Succinyl-Ala-Ala-Alap-nitroanilid N-Succinyl-Ala-Pro-Alap-nitroanilide N-Succinyl-Ala-Ala-Pro-Leup-nitroanilide Glutaryl-Ala-Ala-Pro-Leup-nitroanilide FA-Gly-Leu-amide
Hydrolysis . . activity _.(U/ma) -. PST-01 Pancreatic Thermolysin protease elastase 44 28 n.t.
7.1
n.t.
16.8
n.t.
n.t.
n.t.
15.2 n.t.
n.t.
2.2
6.4
00 10 20 30 40 50
n.t., Not tested.
lo-mm incubation at 70°C. It is a well-known fact that for many proteases the addition of calcium ions increases heat stability. However, an increase in the heat stability of PST-01 protease was not observed on addition of calcium ions (data not shown). Hydrolysis activity against elastin-orcein and synthetic peptides Elastase activities determined using elastin-
orcein or elastin-like synthetic peptides are shown in Table 2. The specific activities of PST-01 protease and pancreatic elastase against elastin-orcein were 44 and 28 U/mg, respectively. Elastin-like synthetic peptides, such as iV-succinyl-Ala-Ala-Ala-p-nitroanilide, N-succinyl-Ala-Pro-Ala-p-nitroanilide, N-succinyl-Ala-Ala-ProLeu-p-nitroanilide, and glutaryl-Ala-Ala-Pro-Leu-p-nitroanilide, were not significantly hydrolyzed by PST-01 protease (less than 0.01 U/mg), while these were hydrolyzed by pancreatic elastase. The hydrolysis activities of PST-01 protease and thermolysin against FA-Gly-Leuamide were 2.2 and 6.4 U/mg, respectively. Effect of protease inhibitor The effect of various protease inhibitors on the activity was investigated. The activity values relative to those measured in the absence of the inhibitor are shown in Table 3. Serine protease inhibitors such as DFP and PMSF, and the carboxyl protease inhibitor EPNP had almost no effect on PST-01 protease. Although MIA, a sulfhydryl reagent used as a thiol protease inhibitor, had almost no effect, some other sulfhydryl reagents such as PCMB, 2-ME and Cys partially inhibited PST-01 protease activity. 5 mM EDTA also partially inhibited PST-01 protease activity, TABLE 3.
Effect of inhibitors on the activity of purified PST-01 protease
Inhibitor Diisopropyl fluorophosphate Phenylmethylsulfonyl fluoride p-Chloromercuribenzonic acid Iodoacetic acid 2-Mercaptoethanol L-Cysteine 1,2-Epoxy-3-@-nitrophenoxy)propane Etylenediaminetetraacetic acid 1, IO-Phenanthroline Phosphoramidon None
Concentration Relative activity 5mM 0.73 5mM 0.86 5mM 0.29 5mM 0.92 5mM 0.66 5mM 0.38 5 mM 0.80 5mM 0.49 50mM 0.12 5mM 0.01 1mM 0.10 1
The inhibitor was added to the substrate solution to the desired concentration. Activities in the presence of various inhibitors relative to that in the absence of inhibitor are shown.
Cont.
of ZnCI, in the enzyme solution [mM]
FIG. 7. Recovery of proteolytic activity of the PST-01 protease treated with 5 mM EDTA by addition of ZnC&. Solution containing purified PST-01 protease and 5 mM EDTA was incubated at 4°C for 24 h. After addition of various concentrations of ZnClz, the proteolytic activity was measured.
while o-Phe, PR and concentrated EDTA (50mM) strongly inhibited PST-01 protease activity. EDTA is a metal-chelating reagent and can be used as a metal protease inhibitor. PST-01 protease was most effectively inhibited by the metal-chelating reagents and phosphoramidon which specifically inhibits metalloproteases, therefore PST-01 protease can be classified as a metalloprotease. Reactivation of PST-01 protease inhibited with EDTA
The reactivation of PST-01 protease inhibited with 5 mM EDTA was studied by the addition of various concentrations of zinc chloride. In Fig. 7, the results are shown as a plot of the activity vs. the concentration of zinc chloride. The activity after incubation at 4°C for 24 h in the presence of 5 mM EDTA was about 18% of that measured before the addition of EDTA. The addition of zinc chloride to a final concentration of 5 mM reactivated the enzyme completely. The addition of zinc chloride to a final concentration of greater than 5 mM None WC4 aI E5
FeCI,
Eo
CaCI,
z 5 a
MnCI, COCI, ZIlCI, 0
20
40
60
80
100
Activity [%] FIG. 8. Recovery of proteolytic activity of PST-01 protease treated with 5 mM EDTA by addition of 5 mM metal chloride. A solution containing purified PST-01 protease and 5 mM EDTA was incubated at 4°C for 24 h. After addition of 5 mM of metal chloride, the proteolytic activity was measured.
66
J. BIOSCI. BIOENG.,
OGINO ET AL.
apoenzyme of PST-01 protease, which lost the activity on addition of EDTA, was reactivated by the addition of zinc or cobalt ions. Effect of organic solvents on proteolytic activity The effect of various organic solvents on the stability of various proteases, that is, purified PST-01 protease, subtilisin Carlsberg, thermolysin, and cw-chymotrypsin, was studied. Mixtures of 3 ml of an enzyme solution (5 U/ml) and 1 ml of an organic solvent were incubated at 30°C with shaking and the remaining activity was measured at appropriate time intervals. Figure 9 shows the typical time courses of the remaining activity of various proteases in the presence of 1,5-pentanediol. Deactivation of all the proteases tested in the presence or absence of organic solvents obeyed first order kinetics. The half-lives of the activity of various proteases are summaTime [d] FIG. 9. Time courses of relative remaining activity of various proteases in the presence of 1,5pentanediol. The filtered purified PST-01 protease (0), subtilisin Carlsberg (A), thermolysin ( 0 ), and a-chymotrypsin (V) solutions (3 ml) were incubated at 30°C with shaking in the presence of 1,5-pentanediol. The relative remaining activities based on the activity before addition of organic solvent (5 U/ml) are shown.
inhibited the complete recovery of the activity. When 50mM zinc chloride was added (the final concentration of zinc chloride in the reaction mixture was 0.98 mM), the remaining activity was 33%. The effect of addition to 5 mM of other metal chlorides on reactivation of PST-01 protease is shown in Fig. 8. Magnesium ions, iron ions, and calcium ions were not effective for the reactivation of PST-01 protease. The addition of 5 mM cobalt ions had the same effect as that of zinc ions. The
rized in Table 4. The half-life of PST-01 protease in the
absence of organic solvent was about 9.7 d. The half-life of PST-01 protease in the presence of water-insoluble organic solvents such as toluene, benzene, n-heptane, pxylene, n-hexane, n-decane, and cyclohexane was similar to or shorter than that in the absence of organic solvent. However, the half-life of PST-01 protease in the presence of water-soluble organic solvents or alcohols such as ethylene glycol, 1 ,Cbutanediol, 1,5-pentanediol, ethanol, 1-hexanol, methanol, dimethyl sulfoxide, 2propanol, triethylene glycol, tert-butanol, 1-heptanol, N,N-dimethylformamide, 1-octanol, I-butanol, acetone, 1-decanol, and 1,6dioxane, was longer than that in the absence of organic solvent. Inactivation of the enzyme in the presence of ethylene glycol, l,Cbutanediol, 1,5-pentanediol, ethanol, and 1-hexanol was not observed at all
during the 15 day-experiment. When organic solvents of which the log P (6) values are between -0.23 and 3.1
TABLE 4. Effect of organic solvents on the stability of the purified PST-01 Organic solvent Ethyleneglycol 1,6Butanediol 1,5-Pentanediol Ethanol I-Hexanol Methanol Dimethyl sulfoxide (DMSO) 2-Propanol Triethyleneglycol tert-Butanol I-Heptanol N,N-Dimethylformamide (DMF) 1-Octanol 1-Butanol Acetone I-Decanol 1,CDioxane Toluene Benzene n-Heptane p-Xylene n-Hexane n-Decane Cyclohexane None
protease
Half-life (d) PST-01 protease >lOO > 100 >lOO >lOO >50 >50 >50 >50 >50 >50 >50 25.3 24.2 24.2 23.1 19.4 11.7 12.0 7.8 4.8 4.4 3.8 2.4 2.3 9.1
Subtilisin 14.3 25.0 >50 >50 13.7 26.2 6.4 47.6 39.7 41.6 8.6 39.8 n.t. 47.6 24.8 n.t. 29.3 5.7 n.t. n.t. n.t. n.t. n.t. n.t. 0.3
Thermolysin >50 4.4 1.7 3.0 18.2 4.6 2.6 1.2 5.1 0.8 13.1 0.9 n.t. 0.9 0.7 n.t. 0.8 22.5 n.t. n.t. n.t. n.t. n.t. nt. 10.8
a-Chymotrypsin 6.9 0.7 0.4 27.0 14.2 6.0 33.6 0.6 11.1 0.5 3.8 2.2 n.t. 0.3 0.6 n.t. 0.5 >lOO at. n.t. n.t. n.t. n.t. n.t. 13.2
The filtered solutions containing various proteases (3 ml), purified PST-01, subtilisin Carlsberg, thermolysin, and a-chymotrypsin, were incubated at pH 8.5 and 30°C with shaking at 160 strokes per min in the absence or presence of 1 ml of organic solvent for 15 d. The remaining activity was measured after 0, 1, 2, 3, 5, 7 , 10, and 15 d . The half-life was calculated from the exponential regression curve. n.t.. Not tested.
ORGANIC
VOL . 87, 1999
were added to the filtered culture supernatant of P. aeruginosa PST-01 (the crude enzyme solution of PST-01 protease), the enzyme was inactivated a little more rapidly than when no organic solvents were added. When organic solvents of which the log P values are less than -0.23 or more than 3.1 were added, the inactivation rates of the enzyme were the same as those in the absence of organic solvent (1). However, in the case of the purified PST-01 protease, the relation between the stability against organic solvent and the log P value of the added organic solvent was not found. In the presence of watersoluble organic solvents or alcohols, the stability of PST-01 protease was much higher than in the absence of organic solvent. In general, the half-life of the PST-01 protease activity is longer than that of any other proteases tested in the presence of water-soluble organic solvents or alcohols. However, in the presence of toluene, the half-life of PST-01 protease is shorter than that of a-chymotrypsin or thermolysin. DISCUSSION
This paper describes the purification and characterization of the organic solvent-tolerant P. aeruginosa PST01 protease. The P. aeruginosa PST-01 protease was purified from the culture supernatant by ammonium sulfate precipitation and successive hydrophobic interaction chromatographies. In each of the purification steps, proteolytic activity appeared as a single peak. Some strains of P. aeruginosa produce three kinds of proteases (7, 8). The discrepancy in the number of proteases produced may be attributed to the difference in the strains of P. aeruginosa used, the cultivation medium, and/or the method of enzyme purification. The molecular mass of the purified PST-01 protease was determined to be 38 kDa by SDS-polyacrylamide gel electrophoresis. The molecular mass value that we determined for PST-01 protease was in agreement with that of the P. aeruginosa PA01 elastase determined by SDSpolyacrylamide gel electrophoresis (9). The optimum pH values of the caseinolytic activity of proteases from P. aeruginosa ranged from 8 to 9 (7, 8, 10, 11). The optimum pH, 8.5, of the caseinolytic activity of PST-01 protease was in agreement with the reported optimum pH range for other P. aeruginosa proteases. PST-01 protease was stable in alkaline conditions @H 8.0-12.0). PST-01 protease hydrolyzed casein rapidly at about 55°C and was stable in the temperature range below 50°C at pH 8.5. The protease (elastase) which is produced by P. aeruginosa is called pseudolysin (EC 3.4.24.26) (3, 12). PST-01 protease also hydrolyzes elastin. The specific activity of PST-01 protease for elastin-orcein (44U/mg) was very similar to that of pseudolysin (35 U/mg) (3). However, PST-01 protease did not significantly hydrolyzed elastin-like peptides, such as N-succinyl-Ala-AlaAla-p-nitroanilide, N-succinyl-Ala-Pro-Ala-p-nitroanilide, N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide, and glutarylAla-Ala-Pro-Leu-p-nitroanilide. PST-01 protease hydrolyzed FA-Gly-Leu-amide, and its specific activity was about one-third that of thermolysin. PST-01 protease was inhibited by metal-chelating reagents such as EDTA and o-Phe, and the peptide inhibitor PR which specifically inhibits metalloproteases. PST01 protease inhibited with EDTA was reactivated by the addition of zinc or cobalt ions. Therefore, PST-01 pro-
SOLVENT-STABLE
PROTEASE
67
tease can be classified as a metalloprotease which requires zinc or cobalt ions for a manifestation of the activity. Proteases are often used as catalysts for peptide syntheses. To shift the thermodynamic equilibrium in favor of the peptide synthesis, the reaction solution should contain organic solvent. However, enzymes are usually inactivated by the addition of organic solvents to the reaction solution. Crude PST-01 protease was stable in the presence of organic solvents (1). Purified PST-01 protease was also very stable in the presence of water-soluble organic solvents or alcohols such as ethylene glycol, l&butanediol, 1,5-pentanediol, ethanol, 1-hexanol, methanol, dimethyl sulfoxide, 2-propanol, triethylene glycol, tert-butanol, 1-heptanol, N,N-dimethylformamide, loctanol, 1-butanol, acetone, 1-decanol, and 1,Cdioxane. In general, PST-01 protease was more stable than the other proteases tested, namely, subtilisin Carlsberg, thermolysin, and a-chymotrypsin, in the presence of water-soluble organic solvents or alcohols. However, in a few cases, the half-life of the activity of PST-01 protease in the presence of organic solvent was shorter than those of the other proteases. Therefore, it is concluded that the organic solvent-stability of enzymes depends on the kind of enzyme and organic solvent. In this paper, to compare the stabilities of various proteases under the same conditions in the presence of organic solvent, the proteases were dissolved in 1OmM Borax-HCl buffer (pH 8.5), although this is not necessarily the best medium for each protease. The stability of enzyme in the presence of organic solvent can depend on the buffer solution or additives. It may be necessary to compare the stabilities of various enzymes measured under their optimal conditions for stability. We believe that PST-01 protease is a very useful biocatalyst for peptide syntheses in the presence of a water-soluble organic solvent because it is very stable. We are currently performing a variety of peptide synthetic reactions using PST-01 protease in the presence of water-soluble organic solvent. As a preliminary result, a dipeptide N-carbobenzoxy-Arg-Leu-NH2 was synthesized in a high yield (approximately 78%) from N-carbobenzoxy-Arg as an amine component and Leu-NH2 as a carboxy1 component in the presence of 50% (v/v) N,Ndimethylformamide. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Encouragement of Young Scientists (09750880) from the Japanese Ministry of Education, Science, Sports and Culture. REFERENCES 1. Ogino, H., Yasui, K., Shiotani, T., Ishihara, T., and Isbikawa, H.: Organic solvent-tolerant bacterium which secretes an organic solvent-stable proteolytic enzyme. Appl. Environ. Microbial., 61, 4258-4262 (1995). 2. Ogino, H., Miyamoto, K., and Ishikawa, H.: Organic-solventtolerant bacterium which secretes organic-solvent-stable lipolytic enzyme. Appl. Environ. Microbial., 60, 3884-3886 (1994). 3. Morihara, K.: Pseudolysin and other pathogen endopeptidases of thermolysin family. Methods Enzvmol.. 248. 242-253 (1995). 4. Lowry, 0. H., Rosebrougb, N. J., Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275 (1951). .
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5. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature (London), 227, 680-685 (1970). 6. Laane, C., Boeren, S., Vos, K., and Veeger, C.: Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng., 30, 81-87 (1987). 7. Morihara, K.: Production of elastase and proteinase by Pseudomonas aeruginosa. J. Bacterial., 88, 745-757 (1964). 8. Wretlind, B. and Wadstrom, T.: Purification and properties of a protease with elastase activity from Pseudomonas aeruginosa. J. Gen. Microbial., 103, 319-327 (1977). 9. Goldberg, J. B. and Ohman, D. E.: Activation of an elastase
J. BIOSCI. BIOENG.. precursor by the 1asA gene product of Pseudomonas aeruginosa. J. Bacterial., 169, 4532-4539 (1987). 10. Morihara, K.: Pseudomonas aeruginosa proteinase. 1. Purification and general properties. Biochim. Biophys. Acta, 73, 113124 (1963). 11. Morihara, K., Tsuzuki, H., Oka, T., Inoue, H., and Ebata, M.: Pseudomonas aeruginosa elastase: isolation, crystallization, and preliminary characterization. J. Biol. Chem., 240, 32953304 (1965). 12. Rawliogs, N. D. and Barrett, A. J.: Evolutionary families of metallopeptidases. Methods Enzymol., 248, 183-228 (1995).