Biochemistry Department, Memorial University ofNewfoundland, St. John's, .... buffer containing 0.3 M KCI) was used to elutebound ... Weber and Osborn (26).
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1983, p. 6-12
Vol. 46, No. 1
0099-2240/83/070006-07$02.00/0 Copyright C 1983, American Society for Microbiology
Heat-Stable Proteases from Psychrotrophic Pseudomonads: Comparison of Immunological Properties D. M. JACKMAN, F. M BARTLETT, AND T. R. PATEL* Biochemistry Department, Memorial University of Newfoundland, St. John's, Newfoundland, Canada AIB 3X9
Received 15 November 1982/Accepted 11 April 1983
A heat-stable extracellular protease from Pseudomonas fluorescens was purified by chromatography on a DEAE-cellulose column and gel filtration on a Sephadex G100 column. The homogeneous enzyme preparation was used to prepare antiserum in rabbits. The rabbit antiserum was used to study the antigenic relatedness of proteases from 19 psychrotrophic pseudomonads isolated from raw milk. The inhibition of the proteases by the antiserum and the gel precipitin reactions revealed similar antigenic determinants in proteases from different isolates. Rabbit antiserum to the purified protease gave precipitin bands with antigens (proteases) from 10 different isolates. However, the same antiserum did not inhibit the protease activity in cell extracts of isolates T10, T13, and T24. By determining serological cross-reactions, proteases from psychrotrophic pseudomonads were shown to be different from one another. MATERIALS AND METHODS Organisms. Raw milk from a local dairy was plated on plate count agar containing 1 to 2% skim milk powder. The plates were incubated at 7°C for 8 to 10 days, and colonies showing proteolysis, as indicated by clearing of casein around the colonies, were selected and purified by streaking on fresh plate count agar plates. Of the original 28 isolates, only 19 exhibited considerable proteolysis; these isolates were chosen for further study. Microbiological and biochemical identification was performed as previously described (19). All isolates were found to be gram negative, rod shaped, polarly flagellated, and oxidase and catalase positive. All hydrolyzed gelatin, casein, and Tween 60 but not starch. All grew at 0 to 35°C, but failed to grow at >40°C. The isolates were tentatively identified as
The introduction of modern technology to the dairy industry has altered the procedures used in handling raw milk. Because of these changes, raw milk is stored at refrigerated temperatures (ca. 4°C) for a prolonged time before processing. During the time at the farm, in transport, and at the dairy plant, psychrotrophs proliferate under these conditions. The psychrotrophs produce heat-stable extracellular proteases and lipases (1, 4-6, 9, 14-16, 18, 23). The heat-stable proteases are able to withstand the conditions employed in the ultrahigh-temperature processing of milk. Thus, such psychrotrophs influence the quality of milk and milk products. Alichanidis and Andrews (2) have purified an extracellular heat-stable protease from Pseudomonasfluorescens AR-11, and Barach et al. (4, 5) have purified a metalloprotease from Pseudomonas sp. strain MC60 which has a half-life at 149°C of 7.4 s. Recently, Gebre-Egziabher et al. (10) have reported several pseudomonads that produce heat-resistant proteases, all of which retain some activity after being heated at 121°C for 10 min. In a similar study, Richardson and TeWhaiti (21) have examined four types of heatstable extracellular proteases synthesized by Pseudomonas spp. To take a closer look at heat-stable proteases, we isolated 19 psychrotrophic pseudomonads which produce extracellular heat-stable proteases. In this report, we discuss immunological properties of the proteases from these pseudomonads.
strains of P. fluorescens. They produced proteases that differed in heat stability, pH optimum, and sensitivity to inhibitors. Enzyme preparation. The isolates were grown in 500-ml Erlenmeyer flasks containing 125 ml of sterile Trypticase soy broth plus 1 to 2% skim milk powder. The flasks were incubated at 25°C for 4 to 5 days on a shaker (Psychrotherm; New Brunswick Scientific Co., New Brunswick, N.J.). Cells were removed by centrifugation at 10,000 rpm for 15 min in a centrifuge (Ivan Sorvall, Inc., Norwalk, Conn.). The clear supematant was lyophilized, and the dry residue obtained was dissolved in a minimum quantity of 20 mM Trishydrochloride buffer, pH 7.2 (hereafter called Tris buffer). The concentrated samples thus obtained were extensively dialyzed against Tris buffer. The dialyzed extract was the source of the enzyme. Protease assay. The protease activity in enzyme preparations was estimated by a modification of the method of Hull (11). The substrate, soluble casein (1 to 6
VOL. 46, 1983
ANTIGENIC PROPERTIES OF HEAT-STABLE PROTEASES
2% solution; BDH Chemicals Ltd., Poole, England), was extensively dialyzed against Tris buffer to remove any free amino acids that may have been contaminating the casein. Similarly, it was found necessary to extensively dialyze enzyme samples for optimum enzyme activity. The reaction mixture contained 1.5 ml of Tris-hydrochloride (100 mM, pH 7.5), 0.2 to 0.4 mg of enzyme protein, and 0.5 ml of the substrate (1% soluble casein) (total volume, 2 ml). The reaction mixture was incubated at 25°C for 10 to 30 min in a temperature-regulated water bath. The reaction was terminated by adding 1.0 ml of 5% trichloroacetic acid. The precipitated proteins were removed by centrifugation, and the trichloroacetic acid-soluble free tyrosine and tryptophan in the clear supernatant were determined by absorbance measurements at 280 nm. Appropriate enzyme and substrate controls were always included. One enzyme unit was defined as the amount of extract that produced 1 ,umol of tyrosine equivalent per min per ml under the assay conditions. Protein in cell extracts was determined by the method of Lowry et al. (18). Purification of proteases. All procedures were performed at 0 to 4°C. Crude extract (1,300 ml) was dialyzed against Tris buffer and applied to a DEAEcellulose column (5 by 31 cm) that had been equilibrated with Tris buffer. Bound protein was released with a linear gradient consisting of 590 ml of Tris buffer and 590 ml of Tris buffer containing 0.3 M KCI. At pH 7.2, the protease was eluted at 0.2 to 0.3 M KCI. Fractions 322 through 348, which contained maximum protease activity, were pooled (Table 1, step 2). The pooled fractions were dialyzed against Tris buffer and chromatographed on a DEAE-Sephacel column (2.6 by 23 cm) that had been equilibrated with Tris buffer. A linear gradient (300 ml of Tris buffer and 300 ml of Tris buffer containing 0.3 M KCI) was used to elute bound protein. Fractions 126 through 139, which were the most active, were pooled and dialyzed against Tris buffer (Table 1, step 3). We added ammonium sulfate to the pooled fractions to obtain 80% saturation. Precipitated protein was collected by centrifugation, and the pellet was redissolved in a minimum quantity of Tris buffer and dialyzed against Tris buffer. The ammonium sulfate-concentrated fraction (Table 1, step 4) was chromatographed on a Sephadex G100 column (2 by 51 cm) (see Table 5, step 5) that had been equilibrated with Tris buffer containing 3 mM sodium azide. Fractions (ca. 2 ml) were collected. Protease was eluted in a single protein peak which coincided with a single activity peak (see Fig. 1). The purified protein had a specific activity of 2.11 enzyme units per
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mg of protein and was 106-fold more active than the crude preparation. Molecular weight determination. The molecular weight of the protease was determined by gel filtration on a Sephadex G150 column as described by Andrews (3). Polyacrylamide gel electrophoresis. The purified enzyme was examined by disc gel electrophoresis as described by Davis (8). Electrophoresis was performed with 7.5% polyacrylamide gels. Protein on the gels was stained with Coomassie brilliant blue (0.25% [wt/vol]; stain dissolved in methanol-acetic acid). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed in 10%o gels by the procedures of Weber and Osborn (26). Antisera. Antiserum to the purified protease was prepared in randomly bred New Zealand white rabbits. We administered 100- ,g injections of purified enzyme in Freund adjuvant (Difco Laboratories, Detroit, Mich.) subcutaneously at 2-week intervals for 8 weeks. Antibody titers were examined by Ouchterlony double-diffusion tests beginning 1 week after the final
injection.
The immunoglobulin G (IgG) fractions from the rabbit sera were obtained by the sodium sulfate precipitation method of Kekwick (13) and the DEAE-cellulose chromatography method of Levy and Sober (17). Immunodiffusion. The antigenic relatedness of the proteases from several isolates was examined by Ouchterlony double-diffusion testing as described by Stollar and Levine (24). The concentration of the IgG fraction of rabbit antiserum was adjusted to 10 mg/ml. The concentrations of antigens (crude enzyme) that gave the sharpest precipitin bands were determined in preliminary tests. To allow the precipitin bands to develop, the Ouchterlony plates were incubated in a humid atmosphere at 5 to 8°C for 24 h. Pictures of the precipitin bands were taken at the end of the incubation period. Immunoelectrophoresis. Immunoelectrophoresis of the samples of purified and crude enzyme was carried out by the method of Scheidegger (22) with Trisbarbital buffer (pH 8.6). Precipitin bands were developed with the IgG fraction of the antiserum to the purified isolate T25 enzyme. Inhibition studies. To evaluate inhibition of the proteases in cell extracts, various concentrations of the antiserum or the IgG fraction were incubated with cell extracts in reaction mixtures under standard assay conditions. Equivalent amounts of antiserum or IgG from an unimmunized rabbit were preincubated in reaction mixtures as controls. The percent inhibition
TABLE 1. Purification of P. fluorescens T25 protease Purification Protein Recovery Vol Sp Enzyme (%) (fold) actb Step (ml) unitsa (mg) 100 1 0.02 192 8,580 1,300 1. Obtain crude extract 72 7.5 138 0.15 928 290 2. DEAE-cellulose chromatography 45 11 86 0.22 391 120 3. DEAE-Sephacel chromatography 36 95.5 1.91 69 4 36 4. Ammonium sulfate (0-80%) concentration 12 106 23 2.11 11 15 5. Sephadex G-100 chromatography a One enzyme unit is the amount of extract that released 1 ,umol of tyrosine equivalent per min per ml at 25°C. b Expressed as enzyme units per milligram of protein.
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