Mar 4, 1983 - the bound protein was eluted with 0.1 M Tris-hydro- ... the method of Weber and Osborn (35) and by gel ..... Juan, S. M., and J. J. Cazzulo. 1976.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1983, p. 333-337
Vol. 46, No. 2
0099-2240/83/080333-05$02.00/0 Copyright C 1983, American Society for Microbiology
Heat-Stable Protease from Pseudomonas fluorescens T16: Purification by Affinity Column Chromatography and Characterization T. R. PATEL,* D. M. JACKMAN, AND F. M. BARTLETT Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada AIB 3X9
Received 4 March 1983/Accepted 19 May 1983
A heat-stable extracellular protease from Pseudomonas fluorescens T16, a psychrotroph, was purified by affinity column chromatography on a carbobenzoxy-D-phenylalanine-triethylene tetramine-Sepharose-4B column. The purified 2,000. In an analytical enzyme is a monomer with a molecular weight of 38,905 ultracentrifuge, the Schlieren profile revealed a single symmetrical peak. The sedimentation coefficient was estimated to be 3.93S. Alpha-casein was the preferred substrate, with a Km of 0.05 mM. Heating crude enzyme and purified enzyme in buffer at 50, 90, and 120°C resulted in a rapid initial loss of more than 50% of the initial activity followed by a gradual inactivation which exhibited firstorder kinetics. The activation energy for the hydrolysis of casein was calculated to be 3.2 kcal/mol (13.4 kJ/mol). ±
The current practice of refrigerated storage of milk for prolonged periods is selective for psychrotrophs, which become the predominant microflora. Many psychrotrophs excrete extracellular proteinases and lipases which are heat stable. The bacteria themselves are eliminated by pasteurization, but these heat-stable lipases (9, 12, 16, 20) and proteases (1, 5-8, 13, 24, 27-29, 31, 32, 34; A. C. Malik and A. M. Swanson, J. Dairy Sci. 57:591) survive heat treatments. Consequently, they cause spoilage of milk and many dairy products (10, 11, 21, 36, 37). Reports describing purification of heat-stable proteases from psychrotrophic pseudomonads of milk origin are limited. Only three heat-stable proteases from Pseudomonas fluorescens B52 (28, 30), P. fluorescens AR-11 (3), and Pseudomonas sp. strain MC60 (5, 6) have been examined. These proteases differ in many of their biochemical and physicochemical properties. We report in this communication the purification and some properties of a heat-stable protease from P. fluorescens T16, a psychrotroph isolated from raw milk. MATERIALS AND METHODS Cultures. A gram-negative rod isolated from raw
milk was identified as P. fluorescens T16. Microbiological and biochemical tests performed for identifica-
tion purposes for T16 and the other 18 isolates have been published elsewhere (26). All of the isolates were gram-negative rods which were polarly flagellated and oxidase and catalase positive and which utilized trehalose and beta-alanine. All of the isolates grew between 0 and 35°C but not at 41°C. 333
Enzyme preparation. P. fluorescens T16 was grown in Trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.) containing 1 to 2% skim milk powder incubated at 25°C for 96 to 120 h on a shaker (Psychrotherm; New Brunswick Scientific Co., New Brunswick, N.J.). For maximum enzyme production, the culture (0.1 to 0.2%, vol/vol) was inoculated into several 500-ml Erlenmeyer flasks, each containing 125 ml of sterile medium. Cells were removed by centrifugation of the culture at 12,000 x g for 15 min in a centrifuge (Ivan Sorvall, Inc., Norwalk, Conn.). The clear supernatant solution was lyophilized, and the dry residue was dissolved in a minimum of 20 mM Trishydrochloride buffer (pH 7.2). This concentrated extract was extensively dialyzed in the same buffer and formed the source of the enzyme. Protease assay. The protease activity was determined by a modified method of Hull (14). The substrate, soluble casein, and enzyme samples were extensively dialyzed before use. The reaction mixture contained the following (in a total volume of 2 ml): 1.5 ml of Tris-hydrochloride buffer (100 mM, pH 7.5); 0.2 to 0.4 mg of enzyme protein; and 0.5 ml of substrate (1% soluble casein solution). The reaction mixture was incubated at 25°C for 10 to 30 min in a temperatureregulated water bath. The reaction was stopped by adding 1.0 ml of 5% trichloroacetic acid solution. The precipitated proteins were removed by centrifugation, and the trichloroacetic acid-soluble free aromatic amino acids in the clear supernatant solution were determined by optical density measurements at 280 nm. Tubes containing either substrate and no enzyme or enzyme but no substrate were included as controls. One enzyme unit is the amount of extract that releases 1 ,Ljmol of tyrosine per min per ml under the experimental conditions. Specific activity is enzyme units per milligram of protein. Protein in cell extracts was determined by the
334
PATEL, JACKMAN, AND BARTLETT
method of Lowry et al. (23), using bovine serum albumin as a standard. Enzyme purification. Crude extract (500 ml) was lyophilized, and the dry residue obtained was dissolved in 50 ml of 25 mM sodium acetate buffer (pH 5.8) containing 0.1 M NaCI and 0.01 M CaCl2. The concentrated extract was applied to an affinity column of carbobenzoxy-D-phenylalanine-triethylene tetramine-Sepharose-4B (Pierce Chemical Co., Rockford, Ill). The column (0.8 by 25 cm) containing about 10 ml of the affinity material was previously washed with about 200 ml of the same buffer. The unbound protein was eluted with about 400 ml of the acetate buffer, and the bound protein was eluted with 0.1 M Tris-hydrochloride (pH 8.0) containing 0.5 M NaCl and 0.01 M CaC12. The protein profile and the protease distribution in the fractions are shown in Fig. 1. Fractions 44 through 54, containing maximum protease activity, were pooled and concentrated to 3 ml by ultrafiltration in an ultrafiltration cell with a UM-10 membrane (Amicon Corp., Oakville, Ontario, Canada). This concentrated protein solution was used as a source of enzyme. Molecular weight determination. The relative molecular weight of the enzyme was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by the method of Weber and Osborn (35) and by gel chromatography on a Sephadex G150 column (4). Heat stability. Crude extract (1 ml) was sealed in glass ampoules which were immersed in an oil bath at 50, 90, and 120°C for times varying from 0 to 10 min. After cooling in ice water, the samples were centrifuged to remove any denatured insoluble proteins. The residual protease activity was determined by standard assay procedures. The rate constants (k) for the inactivation of protease were calculated from the slope for
Fraction Number FIG. 1. Affinity chromatography of partially purified protease from P. fluorescens T16 on a column of carbobenzoxy-D-phenylalanine-triethylene tetramineSepharose-4B. Protein in the eluate was determined by absorbance at 280 nm. Protease activity in the fractions was determined as described in the text. The arrow indicates the change of the eluting buffer to 0.1 M Tris-hydrochloride buffer (pH 8.0) containing 0.5 M NaCl and 0.01 M CaCI2.
APPL. ENVIRON. MICROBIOL.
first-order plots, obtained by plotting log(percent remaining activity) against time of heating (28). Chromatofocusing. The method of Lampson and Tytell (19) was used to determine the isoelectric point (pI) of the protease. Ultracentrifugation. The sedimentation velocity of the purified enzyme was determined from the Schlieren patterns obtained when a solution (1.5 mg of protein per ml) of enzyme was centrifuged at 20°C and 60,000 rpm in a Spinco model E ultracentrifuge (34). Antisera. Antisera to the purified protease were prepared in random-bred New Zealand white rabbits. A total of four 100-,ug injections of the purified enzyme in 1 ml of complete Freund adjuvant (Difco Laboratories, Detroit, Mich.) were given intraperitoneally at 2-week intervals. The immunoglobulin G from the rabbit sera was prepared by sodium sulfate precipitation (16) followed by chromatography on a DEAEcellulose column (22). Immunodiffusion and immunoelectrophoresis. The Ouchterlony double-diffusion tests were performed as described previously by Stollar and Levine (33). The concentration of the immunoglobulin G fraction of rabbit antiprotease was adjusted to 5 mg/ml. All reactions were evaluated after a 24-h incubation under a humid atmosphere at 4°C. Immunoelectrophoresis was carried out by the method of Scheidegger (31), using Tris-barbital buffer (pH 8.6). Precipitin bands were developed with the immunoglobulin G fraction of the antiserum to the purified enzyme.
RESULTS Protease purification. The affinity chromatography of the crude, partially purified extract gave two protein peaks. The first major peak contained most of the nonabsorbed material, whereas the second minor peak released by the eluting buffer contained most of the protease activity. A summary of the purification procedures is shown in Table 1. The purified protease repeatedly showed a decrease in the specific activity during the ultrafiltration step. A 137-fold purification was achieved, with a final yield of 22%. Polyacrylamide gel electrophoresis with 7.5% gels at pH 9.5 revealed a single band that stained for protein (Fig. 2). Immunoelectrophoresis of the crude, impure enzyme and of the purified enzyme gave a single precipitin band with the antiserum to the purified enzyme. The Schlieren pattern observed when the purified enzyme was subjected to analysis in an ultracentrifuge showed a single symmetrical peak with a sedimentation coefficient of 3.9S. These observations indicated that the protease obtained from the affinity column was a homogeneous protein. Molecular weight and subunit structure. The relative molecular weight of the purified protease was determined by disc gel electrophoretic analysis of the reduced enzyme in the presence of 0.1% sodium dodecyl sulfate and 0.1% 2mercaptoethanol. A single band was obtained.
PSEUDOMONAS FLUORESCENS HEAT-STABLE PROTEASE
VOL. 46, 1983
335
TABLE 1. Purification of protease from P. fluorescens T16 Total Vol
Step
(ml)
Crude extract Concentrated and
550 50
protein
(mg) 189 163
Enzyme
Enzyme
69 63
0.025 0.385
~~~Degree of
punfication Enzyme Recovery Enzyme nisspat(fold)Reors
100 91
1 15
lyophilized 51 27 4.50 180 6.2 35 Affinity column 22 3.43 137 3 4.8 15 Concentrated and ultrafiltered a One unit is the amount of enzyme required to produce 1 1tmol of tyrosine equivalent per min under standard assay conditions. b Units per milligram of protein.
The molecular weight of the subunit was calculated to be 37,800 1,500. Thus, the purified protease seems to be a monomer with a single subunit. Properties of the protease. The pH optimum for protease activity was 7.3 to 7.5. Consistently higher activity was observed with Tris-hydrochloride buffer than with potassium phosphate buffer. At a final concentration of 2 mM, the metal ions Zn2+, Co2+, Cu2+, and Hg2+ were highly inhibitory, whereas Ca2 Mg2+, and Mn2+ had little effect on the protease activity. The effect of some of the inhibitors on the protease activity is shown in Table 2. Metal-chelating agents caused considerable inhibition of the protease. Under similar conditions, soybean trypsin inhibitor and phenylmethylsulfonyl fluoride gave no inhibi±
,
tion. Inhibition with p-chloromercuribenzoate indicated the presence of cysteine in the active site. An inactive form, an apoenzyme, was obtained by dialyzing the protease against 0.1 M EDTA in Tris-hydrochloride buffer. Almost 100% of the protease activity was regained by the addition of divalent cations (Ca2+, Mn2+) to the apoenzyme in the reaction mixture. Some of the substrates hydrolyzed by the protease included alpha-, beta-, and gammacasein, ovalbumin, bovine serum albumin, and hemoglobin (Table 3). The protease exhibited a preference for milk proteins, caseins, as substrates. The activity observed in the presence of alpha-casein and soluble casein was maximum. Only partial activity was observed in the presence of other substrates. The apparent Km value for alpha-casein calculated from the double reciprocal plots was 5 x 10-5 M. The protease was found to have optimum activity between 40 and 45°C when assayed by standard procedures. Above 50°C the protease activity decreased rapidly. No tests were performed to examine the effect of calcium ions on the heat stability of the protease. Heat stability of the protease. When partially purified enzyme was subjected to heat denaturation at 50, 90, and 120°C, the reduction in the protease activity followed first-order kinetics. TABLE 2. Effect of inhibitor on protease activity Micromoles per assay
Inhibitiona
p-Chloromercuribenzoate
2 2 4 10 2 2 10
58 100 75 75 92 0 0
o-Phenanthroline
8-Hydroxyquinoline 2,2'-Bipyridyl EDTA FIG. 2. Polyacrylamide gel electrophoresis of purified protease. The enzyme (50 ,ug of protein) was subjected to gel electrophoresis for 4 h at 3 mA in Trisglycine buffer (pH 9.5). The arrow indicates the position of the tracking dye (bromophenol blue).
t
Inhibitor
PMSFb Soybean trypsin inhibitor a Percent inhibition is based without the inhibitors. b
Phenylmethylsulfonyl
on a
fluoride.
control assay
336
PATEL, JACKMAN, AND BARTLETT
TABLE 3. Substrate specificity of the protease Substrate Sp acta Soluble casein ............... 0.71 Bovine serum albumin ........... .... 0.0 Ovalbumin ............... 0.03 0.57 Alpha-casein ............... Beta-casein ............... 0.30 Gamma-casein ............... 0.13 0.0 Hemoglobin ............... a Specific activity is expressed as units per milligram of protein. A partially purified enzyme preparation was used in the above tests.
First-order rate constants (k) of 0.085, 0.026, and 0.106 min-' were obtained at 50, 90, and 120°C, respectively, by linear regression analysis. Corresponding values for the half-life of the enzyme during heat inactivation were estimated to be 7.1, 30.8, and 7.6 min, respectively. The activation energy for the hydrolysis of soluble casein was calculated from an Arrhenius plot (Fig. 3), using the slope of the plot obtained by linear regression analysis. The activation energy for the hydrolysis of the soluble casein was 3.2 kcal/mol (13.4 kJ/mol). Immunological studies. The serological crossreactions of the crude protein and the purified protein were examined by qualitative precipitin bands with the homologous antigen. Strong precipitin bands were detected in Ouchterlony double-diffusion tests. The antisera to the purified enzyme also inhibited the protease activity. The percent inhibition increased with increasing concentration of the antisera in the reaction mixture. Maximum (100%) inhibition was observed with 13 mg of antisera in the reaction mixture.
DISCUSSION The molecular weight of the T16 enzyme was estimated to be 38,905 2,000. In this respect it is similar to the enzymes from P. fluorescens R12 (15) and P.fluorescens AR-11 (3) but different from proteases from other pseudomonads (1, 24, 27). Proteases from different Pseudomonas strains (13) exhibit various degrees of heat resistance. Heating at 121°C for 2 min destroys less than 40% of the initial activity of most of these proteases. At 1210C, proteases from strains 23 and 16 are inactivated in 10 and 8 min, respectively (13). In the present case, the protease retained 8% activity after being heated at 1200C for 10 min. Different conditions used by various workers makes the comparison of heat stabilities of proteases difficult. However, in the majority of the cases a rapid initial loss of activity has been attributed to autolytic degradation during heating and cooling (27). The addition of milk to ±
APPL. ENVIRON. MICROBIOL.
0.8 0.6
0.41+ 0 -J
0.2
a6 3.4 1lTx 10 K FIG. 3. Effect of temperature on rate of hydrolysis of soluble casein by the protease. The standard protease assays were performed with ammonium sulfateconcentrated extracts. V, Enzyme units per milliliter; T, absolute temperature. 3
3.2
the protease solution before heating prevents this initial loss of activity, perhaps because the milk proteins prevent the autodigestion of the enzyme. Preferential hydrolysis of beta- (17, 25) and kappa-casein (2, 20, 29) by protease have been reported. The T16 protease exhibited maximum activity in the presence of alpha-casein, although beta- and gamma-casein were attacked to a lesser degree. The majority of proteases from psychrotrophs are metalloproteases requiring either Ca2" or Zn2+ for optimum activity (5, 6, 27). The protease from P. fluorescens T16 showed optimum activity in the presence of either Ca2+ or Mn2+ and thus differs in this respect from other proteases. The T16 protease was inhibited by p-chloromercuribenzoate, whereas the enzymes from strains B52 (27) and R-12 (15) are not. The T16 enzyme appears to be well adapted for growth at low temperatures. This is reflected in the low activation energy (3.2 kcal/mol) required for the hydrolysis of casein. Under similar conditions the activation energy for hydrolysis of casein by a mesophilic peptidase, trypsin, is 12 kcallmol (50.21 kJ/mol), a two- or threefold difference. Activation energies for other purified heat-stable proteases have not been reported. The antisera to the purified T16 enzyme inhibited the protease activity as well as giving strong precipitin bands when analyzed by the Ouchterlony double-diffusion test. The crude enzyme gave only a single precipitin band in immunoelectrophoretic analysis, indicating the presence of a single protease in the cell extract. Work is under progress to determine the antigenic relatedness between heat-stable proteases of bacterial origin.
VOL. 46, 1983
PSEUDOMONAS FLUORESCENS HEAT-STABLE PROTEASE
ACKNOWLEDGMENT This research was supported by the National Science and Engineering Research Council of Canada operating grant no. A-1934. LITERATURE CITED 1. Adams, D. M., J. T. Barach, and M. L. Speck. 1975. Heat resistant protease produced in raw milk by psychrotrophic bacteria of dairy origin. J. Dairy Sci. 58:828-834. 2. Adams, D. M., J. T. Barach, and M. L. Speck. 1976. Effect of psychrophilic bacteria from raw milk on milk proteins and stability of milk proteins to ultra-high temperature treatment. J. Dairy Sci. 59:823-827. 3. Alichanidis, E., and A. T. Andrews. 1977. Some properties of the extracellular protease produced by the psychrotrophic bacterium Pseudomonas fluorescens strain Ar-11. Biochim. Biophys. Acta 485:424-433. 4. Andrews, P. 1964. Determination of molecular weight of proteins. Biochem. J. 91:222-233. 5. Barach, J. T., and D. M. Adams. 1977. Thermostability at ultrahigh temperature of thermolysin and a protease from a psychrotrophic Pseudomonas. Biochim. Biophys. Acta 485:417-423. 6. Barach, J. T., D. M. Adams, and M. L. Speck. 1976. Stabilization of a psychrotrophic Pseudomonas protease by calcium against thermal inactivation in milk at ultrahigh temperature. Appl. Environ. Microbiol. 31:875-879. 7. Barach, J. T., D. M. Adams, and M. L. Speck. 1978. Mechanism of low temperature inactivation of a heatresistant bacterial protease in milk. J. Dairy Sci. 61:523528. 8. Bengtsson, K., L. Gardhage, and B. Isaksson. 1973. Gelation in UHT treated milk, whey and casein solution. The effect of heat resistant protease. Milchwissenschaft 28:495-499. 9. Chrisope, G. L., and T. R. Marshall. 1976. Combined action of lipase and microbial phospholipase-C on a model fat globule emulsion and raw milk. J. Dairy Sci. 59:20242030. 10. Cousin, M. A., and E. H. Marth. 1977. Changes in milk proteins caused by psychrotrophic bacteria. Milchwissenschaft 32:337-341. 11. DeBeukelar, N. J., M. A. Cousin, R. L. Bradley, Jr., and E. H. Marth. 1977. Modification of milk proteins by psychrotrophic bacteria. J. Dairy Sci. 60:857-861. 12. Downey, W. K. 1980. Review of progress of dairy science: flavour impairment from pre- and post-manufacture lipolysis in milk and dairy products. J. Dairy Res. 47:237-252. 13. Gebre-Egziabher, A., E. S. Humbert, and G. Blankenagel. 1980. Heat-stable protease from psychrotrophs in milk. J. Food Prot. 43:197-200. 14. Hull, M. E. 1974. Studies on milk proteins. II. Colorimetric determination of the partial hydrolysis of the proteins in milk. J. Dairy Sci. 30:881-884. 15. Juan, S. M., and J. J. Cazzulo. 1976. The extracellular
protease from Pseudomonas fluorescens. Experientia 32:1120-1122. 16. Kekwkk, R. A. 1940. The serum protein in multiple myelomas. Biochem. J. 34:1248-1257. 17. Kishontl, E. 1975. Influence of heat resistant lipases and proteases in psychrotrophic bacteria on product quality. Ann. Bull. Int. Dairy Fed. 36:121-124.
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18. Knaut, T., and G. Bruderer. 1965. Elecktrophoretische Veranderungen das Kaseins beim Washstum von Pseudomonas-Arten der Milch. Milschwissenschaft 20:315-317. 19. Lampson, G. P., and A. A. Tytell. 1965. A simple method for determining isoelectric points. Anal. Biochem. 11:374-377. 20. Law, B. A., A. T. Andrews, and M. E. Sharpe. 1977. Gelation of ultrahigh temperature-sterilized milk by protease from a strain of Pseudomonas fluorescens isolated from raw milk. J. Dairy Res. 44:145-148. 21. Law, B. A., M. E. Sharpe, and H. R. Chapman. 1976. The effect of lipolytic gram-negative psychrotrophs in stored milk on the development of rancidity in Cheddar cheese. J. Dairy Res. 43:459-468. 22. Levy, H. B., and H. A. Sober. 1980. A simple chromatographic method for preparation of gammaglobulin. Proc. Soc. Exp. Biol. Med. 103:250-259. 23. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 24. Mayerhofer, H. J., R. T. Marshall, C. H. White, and M. Lu. 1973. Characterization of a heat-stable protease of Pseudomonas fluorescens P26. Appl. Microbiol. 25:4448. 25. Moreno, V., and F. V. Kosikowshi. 1973. Degradation of beta-casein by micrococcal cell-face preparation. J. Dairy Sci. 56:33-38. 26. Patel, T. R., F. M. Bartlett, and J. Hamid. 1983. Extracellular heat-resistant proteases of psychrotrophic pseudomonads. J. Food Prot. 46:90-94. 27. Purschell, M., and M. Posplsil. 1968. Proteolytischer Abbau de Milcheiweisstoffe durch Bakterien. 1. Mitt. Dei Wirkung von aeroben sporenbildnern auf fie Eiweisstoffe in der Milch. Nahrung 12:775-781. 28. Richardson, B. C. 1981. The purification of a heat-stable protease from Pseudomonas fluorescens B52. N.Z. J. Dairy Sci. Technol. 16:195-207. 29. Richardson, B. C., and D. F. Newstead. 1979. Effect of heat-stable protease on the storage life of UHT milk. N.Z. J. Dairy Sci. Technol. 14:273-279. 30. Rkhardson, B. C., and I. E. TeWhait. 1978. Partial characterization of heat-stable extracellular proteases for some psychrotrophic bacteria from raw milk. N.Z. J. Dairy Sci. Technol. 13:172-176. 31. Scheidegger, J. J. 1955. Une micromethode de l'immunoelectrophoresis. Int. Arch. Allergy Appl. Immunol. 7:103110. 32. Speck, M. L., and D. M. Adams. 1976. Heat resistant proteolytic enzymes from bacterial sources. J. Dairy Sci. 59:786-789. 33. Stollar, D., and L. Levine. 1963. Two dimensional immunodiffusion. Methods Enzymol. 6:845-854. 34. Vmvada, P. C., and C. H. White. 1979. Quality of commercial buttermilk. J. Dairy Sci. 62:802-806. 35. Weber, K., and M. Osborn. 1969. The reliability of molecular determination by dodecyl sulfate polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412. 36. White, C. H., and R. T. Marshall. 1973. Reduction of shelf-life of dairy products by a heat-stable protease from
Pseudomonasfluorescens P26. J. Dairy Sci. 56:849-853. 37. Yanaglya, T., M. Mikami, and H. Miura. 1973. Changes of milk protein by psychrotrophic organisms. J. Agric. Chem. Soc. Jpn. 47:259-266.
