Synthesis of Extracellular Proteinase by Pseudomonas fluorescens ...

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Synthesis of Extracellular Proteinase by Pseudomonas fluorescens. Under Conditions of Limiting Carbon, Nitrogen, and Phosphatet. R. C. McKELLAR* AND H.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1984, p. 1224-1227

Vol. 47, No. 6

0099-2240/84/061224-04$02.00/0 Copyright © 1984, American Society for Microbiology

Synthesis of Extracellular Proteinase by Pseudomonas fluorescens Under Conditions of Limiting Carbon, Nitrogen, and Phosphatet R. C. McKELLAR* AND H. CHOLETTE

Food Research Institute, Agriculture Canada, Research Branch, Central Experimental Farm, Ottawa, Ontario, Canada KIA 0C6 Received 21 September 1983/Accepted 9 March 1984

The influence of carbon, nitrogen, and phosphate concentrations on growth and proteinase production by Pseudomonas fluorescens 32A was examined. In mineral salts medium containing dialyzed skim milk supernatant as an inducer, maximum growth was obtained at 1.0 and 2.5 mM orthophosphate at 20 and 5°C, respectively. At both temperatures, 5 mM orthophosphate was required for maximum proteinase production, whereas significant inhibition was found at 10 mM. Orthophosphate was the only phosphate compound able to support growth. With sodium pyruvate as the carbon source, maximum enzyme synthesis was at 100 mM carbon at both temperatures. At both 20 and 5°C maximum growth and enzyme production was found with 10 mM NH4Cl. A bioassay for available phosphate based on the growth of P. fluorescens 32A in phosphate-limited mineral salts medium showed that skim milk and skim milk supernatant contained 50 and 10 mM orthophosphate, respectively. Proteinase production in skim milk was 2.6- and 12-fold greater than that in optimal mineral salts medium at 20 and 5°C, respectively. These results suggest that proteinase production in milk does not occur as a result of nutrient limitation and may be regulated in part by milk phosphates.

milk obtained from the Central Experimental Farm in Ottawa, Ontario, Canada. Milk was centrifuged at 5,000 x g for 10 min, and the cream was removed by aspiration. The pH was adjusted to 4.5 with concentrated HCl, and after centrifugation at 10,000 x g for 10 min at 25°C, the supernatant was adjusted to pH 7.0 with 10 N NaOH. The neutralized supernatant was centrifuged at 10,000 x g for 15 min at 25°C, filter sterilized, and stored at 4°C. Inducer was dialyzed for 18 h at 5°C against several changes of demineralized water as specified. Growth experiments. All experiments were done in 50-ml screw-capped Erlenmeyer flasks in a final volume of 10 ml. Cells were grown in mineral salts medium, and after shaking in a water bath shaker (model G86; New Brunswick Scientific Co., Inc., Edison, N.J.) at 180 rpm for 18 h at 20°C, cells were washed in sterile 50 mM BES buffer (pH 7.0) and suspended in the test medium at a concentration of 20 Klett units. Growth was monitored with a Klett-Summerson colorimeter fitted with a red filter (X = 640 to 700). In some experiments, growth was monitored by diluting cells in 0.1% peptone and plating on plate count agar (Difco Laboratories, Detroit, Mich.). Incubation was at 20°C for 48 h. When maximum growth was attained (2 days at 20°C and 7 days at 5°C), 2-ml volumes were centrifuged at 5,000 x g for 10 min at 4°C and used as the source of proteinase. Proteinase assay. Proteinase activity was determined in 50ml Erlenmeyer flasks containing, in a final volume of 5 ml: 50 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; Sigma Chemical Co.), pH 7.5; 1 mM CaC12; 50 mg of hide powder azure (HPA; Sigma Chemical Co.); 0.05% NaN3; and an appropriate dilution of cell-free culture fluid. After incubation in a 35°C water bath shaker (model G86; New Brunswick Scientific Co.) unreacted HPA was removed by filtration with Whatman no. 1 paper, and the absorbance of the filtrate was taken at 595 nm. Activity was expressed as HPA units/100 Klett units where 1 HPA unit was the amount of enzyme required to produce an increase

Psychrotrophic bacteria produce extracellular heat-stable proteinases in refrigerated milk (1). Sufficient enzyme may be present in raw milk destined for ultrahigh-temperature treatment to persist in the finished product, leading to gelation and the development of bitterness (9, 10, 15, 16). Little information is available regarding the factors influencing the ability of psychrotrophs to produce proteinase. A number of workers have examined nutritional factors necessary for proteinase production by Pseudomonas fluorescens (2, 12, 14, 19); however, only a limited number of studies have addressed the problem of proteinase production at low temperatures in milk (7, 8, 11). Proteinase synthesis has been demonstrated in a mineral salts medium containing a smallmolecular-weight inducing fraction isolated from skim milk (11). This same study showed that extensive proteinase synthesis occurred at both 5 and 20°C. Control of proteinase production by psychrotrophs in milk would be desirable. With this in mind, the present study examines the effect of limiting nutrients in a mineral salts medium on proteinase production by P. fluorescens 32A. MATERIALS AND METHODS Strain. The isolation, characterization and maintenance of P. fluorescens 32A has been described previously (11). Media. All growth experiments were done in mineral salts medium which contained, in a final volume of 1 liter: MgSO4 7H20, 0.8 mM, and N,N-bis[2-hydroxyethyl]-2aminoethane sulfonic acid (BES; Sigma Chemical Co., St. Louis, Mo.), 50 mM. Sodium pyruvate, NH4Cl, and K2HPO4 were added at the indicated concentrations (expressed as millimolar carbon, nitrogen, or phosphate) as required. The medium was adjusted to pH 7.0 before autoclaving. Preparation of acid-soluble skim milk supernatant. Acidsoluble fraction for use as an inducer was prepared from raw Corresponding author. t Contribution no. 560 from the Food Research Institute.

*

1224

SYNTHESIS OF PSYCHROTROPH PROTEINASE

VOL. 47, 1984 TABLE 1. Effect of acid-soluble inducer on proteinase production in phosphate-limited mediuma

RESULTS

Proteinase

activity (HPA units/ 100 Klett

Growth (A Klett units)

Phosphate' Inducerb Induce Phosphate"

units)

40 179 41 210 233 214

-

+

+ dialyzed _

+ + +

+

+ dialyzed a Carbon (as sodium pyruvate), 300 mM; nitrogen (as mM. b Adjusted to 0.03 A2XO units/ml. 2 mM orthophosphate.

1225

0.22 10.70 0.93 0.44 52.62 14.70

NH4Cl), 20

of 1.0 absorbance unit per h under standard assay conditions. When necessary, cell counts were converted to Klett units using the relationship 2.74 x 109 CFU/ml = 100 Klett units. Determination of phosphate. (i) Bioassay method. Appropriate dilutions of test samples were added to 10 ml of mineral salts medium containing 300 mM carbon and 20 mM nitrogen. Washed suspensions of cells were added, and growth was determined after 2 days at 20°C. A calibration curve was prepared with 0 to 2 mM orthophosphate. (ii) Chemical method. The method of Eibl and Lands (5) was used with 0 to 100 ,umol of orthophosphate as the standard.

Growth and proteinase production in phosphate-limited medium were slight in the absence of added phosphate or inducer (Table 1). The addition of inducer (0.03 absorbance at 280 nm [A280] units/ml) resulted in elevated levels of proteinase; however, these were not apparent if phosphate was removed from the inducer by dialysis (Table 1). Increased growth and proteinase production were found with medium containing 2 mM phosphate and inducer. In the presence of phosphate and dialyzed inducer, proteinase synthesis was decreased, whereas growth was unaffected. The ability of a number of ring- and straight-chain polyphosphates to act as sources of phosphate for growth was examined. These included pyrophosphate, metaphosphate, tripolyphosphate, tetrapolyphosphate, phosphate glass (P4P6 and P13-P18), and sodium ammonium phosphate. Slight but significant growth was observed in the absence of added orthophosphate. None of the other phosphate compounds, adjusted to 2 mM phosphorus, was able to support growth (data not shown). Results of the chemical assay for orthophosphate indicated that only three of the phosphate compounds mentioned above contain appreciable quantities of free phosphate. Expressed as a percentage of the total phosphate, free phosphate comprised 18.6% of the metaphosphate and tetrapolyphosphate and 90.8% of the sodium ammonium phosphate preparations. It is interesting to note that whereas sodium ammonium phosphate reacted as free phosphate in the chemical assay, it was not metabolized.

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