Biosci. Biotechnol. Biochem., 70 (2), 535–537, 2006
Note
Partial Purification and Some Properties of a Phospholipase C from Pseudomonas sp. Strain KS3.2 Daisuke S UGIMORIy and Masatoshi N AKAMURA Department of Chemistry and Biology Engineering, Fukui National College of Technology, Geshi, Sabae, Fukui 916-8507, Japan Received September 20, 2005; Accepted October 23, 2005
An extracellular phospholipase C was partially purified from Pseudomonas sp. strain KS3.2. The enzyme was composed of an approximately 18-kDa peptide. Maximal enzyme activity was found at pH 7.2 and 50 C. The enzyme retained activity between pH 8 and 9, and 50% activity at about 52 C for 30 min. The enzyme sample showed the highest activity on phosphatidylcholine and low activity toward other phospholipids. Key words:
phospholipase C; metalloenzyme; hydrolase; Pseudomonas sp.
serine
Phospholipase C (PLC) [EC 3.1.4.3] have been found in a variety of microorganisms.1) With regard to gramnegative bacteria, there have been many reports of Pseudomonas PLC enzymes,1–4) but the catalytic functions and the molecular properties have not been fully elucidated. Furthermore, there is currently great interest in the industrial application of PLC to food processing and the degumming process, whereas no industrial PLC has been developed. We have embarked on research attempting to elucidate the catalytic functions and the properties of PLC and to develop an industrial PLC enzyme. A screening study yielded Pseudomonas sp. strain KS3.2 producing a novel extracellular PLC. This paper describes the partial purification and some properties of the PLC from Pseudomonas sp. strain KS3.2. A PLC-producing Pseudomonas sp. strain KS3.2 was found in our stock cultures of 256 triglyceride-assimilating microorganisms. Strain KS3.2 was incubated in a 500-ml baffle-walled shaking flask containing 100 ml of lecithin nutrient broth (1.0% extract beef, 1.0% Bacto Peptone, 1.0% soybean lecithin, 0.5% NaCl, pH 7.2) at 28 C for 48 h on a shaker at 166 rpm. The enzyme was purified from the 440-ml culture supernatant by ammonium sulfate fractionation, DEAE-Sepharose, and Phenyl-Sepharose column chromatography. The standard assay mixture, containing 10 ml of enzyme solution, 275 ml of 50 mM Tris–HCl buffer y
(pH 7.2), 9 ml of 9.5% (v/v) Triton X-100, and 6 ml of 10% (w/v) L- -phosphatidylcholine (PC), was incubated at 50 C for 10 min. The reaction was stopped by boiling for 5 min. Samples of 10 ml were subsequently added to 190 ml of 50 mM glycine–NaOH buffer (pH 9.6) containing 1 mM MgCl2 , 0.1 mM ZnCl2 , and 0.2 units of alkaline phosphatase from calf intestine. Incubation was at 37 C for 20 min. Samples of 0.1 ml was mixed with 0.9 ml of Biomol Green reagent (Biomol Research Laboratories, PA, USA) and subsequently incubated at room temperature for 20 min. The rates of inorganic phosphate release from enzyme reaction mixtures were determined. One unit (U) of activity was defined as the amount of enzyme that hydrolyzed 1 mmol of substrate per min. Protein concentrations were determined by the use of bicinchoninic acid protein assay reagent (Pierce Biotechnology, IL, USA) and bovine serum albumin as the standard. PLC was partially purified 107-fold to a specific activity of 186 U mg1 protein at pH 7.2 and 50 C with
Fig. 1. SDS–PAGE of the Partially Purified PLC Enzyme from Pseudomonas sp. KS3.2. Lane 1, standard protein maker; lane 2, the partially purified PLC (amount of protein loaded, 50 ng). Proteins in the gel were visualized by silver staining.
To whom correspondence should be addressed. Present address: Faculty of Symbiotic Systems Science, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan; Fax: +81-24-548-8206; E-mail:
[email protected] Abbreviations: PLC, phospholipase C; PC, L- -phosphatidylcholine; PE, phosphatidylethanolamine; U, unit
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D. SUGIMORI and M. NAKAMURA
A
C
B
D
Fig. 2. Effects of pH and Temperature on Activity (A, C) and Stability (B, D) of the PLC for PC Hydrolysis. A, The enzyme activity was assayed at 37 C in each buffer (46 mM). The buffers used were CH3 COONa–CH3 COOH (pH 4.15 to 6.0), bisTris (pH 6.0 to 7.2), Tris–HCl (pH 7.2 to 8.8), and glycine–NaOH (pH 8.8 to 11.0). B, The enzyme in each buffer (45 mM) was incubated at 5 C for 3 h. The residual activity was assayed at 50 C in 46 mM Tris–HCl (pH 7.2). The buffers used were CH3 COONa–CH3 COOH (pH 4.1 to 5.8), bis-Tris (pH 5.8 to 7.8), Tris–HCl (pH 7.8 to 9.7), and glycine–NaOH (pH 9.7 to 12.0). C, The enzyme activity was assayed at each temperature in 46 mM Tris–HCl (pH 7.2). D, The enzyme in 46 mM Tris–HCl (pH 7.2) containing 0.01% Triton X-100 and 0.186 mM ammonium sulfate was incubated at each temperature for 30 min and then immediately transferred to ice. The residual activity was assayed at 50 C in 46 mM Tris–HCl (pH 7.2).
36% recovery. The enzyme sample produced two polypeptide bands of approximately 17 and 18 kDa on SDS–polyacrylamide gel electrophoresis (Fig. 1). The molecular mass obviously differed from that of Pseudomonas PLCs.2–6) In contrast, it was similar to that of S. hachijoensis.7) The highest activity was observed at pH 7.2 and 50 C (Fig. 2A, C). The enzyme retained activity between pH 8 to 9 at 5 C for 3 h and 50% activity at about 52 C
for 30 min (Fig. 2B, D). The features of its pH and thermal stabilities were distinct from those of P. schuylkilliensis PLC; its thermal stability was similar to that of P. fluorescens strain A-3-1 PLC.8) KS3.2 PLC showed the highest activity on PC and low activity toward other phospholipids; the relative activities for PC, phosphatidic acid, phosphatidylethanolamine (PE), sphingomyelin, phosphatidylserine, and phosphatidylinositol were 100, 9.04, 0.65, 0.328, 0.302,
A Phospholipase C from Pseudomonas sp. KS3.2
and 0.221% respectively. This substrate specificity is most likely due to recognition of the glycerol side chains and the polar head group of the phospholipid substrate. KS3.2 PLC was thus specific for PC, and did not act on PE, whereas other Pseudomonad PLCs act on PE.2,3,5,6,8–10) The substrate-specific feature of KS3.2 PLC clearly differed from that of other Pseudomonad PLCs. Examination of the effects of metal ions (1 mM) and several chemicals (2 mM) on PLC activity showed that the enzyme was inhibited by ZnCl2 (relative activity, 10.8%), FeCl3 (10.3%), CoCl2 (10.3%), MnCl2 (7.83%), HgCl2 (7.62%), FeCl2 (5.85%), CuSO4 (4.15%), 2-mercaptoethanol (59.6%), SDS (9.65%), EDTA (8.41%), dithiothreitol (6.17%), and phenylmethylsulfonyl fluoride (5.52%). In contrast, CaCl2 (93.5%), MgCl2 (89.9%), and iodoacetamide (109%) had no marked effect on the enzyme activity. Stimulation of enzyme activity by divalent cations such as Zn2þ and Mg2 has been reported for other Pseudomonad PLCs.3,5,6,8) On the other hand, no activation of the KS3.2 PLC enzyme by divalent cations has been observed. Phenylmethylsulfonyl fluoride and EDTA strongly inhibited KS3.2 PLC activity, showing that the PLC enzyme belongs to a group of metallophospholipases C and a class of serine hydrolases. In addition, dithiothreitol and 2-mercaptoethanol inhibited KS3.2 PLC, suggesting that sulfhydryl groups are involved in the catalytic activity. Finally, we conclude that several features of KS3.2 PLC are different from those of known PLCs. To discover the relationship between these differences and the catalytic functions, further studies are in progress to purify the PLC and to clone its gene.
ChemteX Corporation for their generous financial assistance. Part of this study was supported by a grant from the Fukushima University Research Promotion Fund (no. 17-A-10-07).
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Acknowledgments 10)
This paper owes much to the thoughtful and helpful comments of Dr. Xiaoli Liu and Ms. Misa Shiihara of Nagase ChemteX Corporation. We wish to thank Nagase
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Titball, R. W., Bacterial phospholipase C. Microbiol. Rev., 57, 347–366 (1993). Crevel, I. U. S., Carne, A., and Katan, M., Purification and properties of zinc-metallophospholipase C from Pseudomonas fluorescens. Eur. J. Biochem., 224, 845– 852 (1994). Ivanov, A., Titball, R. W., and Kostadinova, S., Charactarisation of a phospholipase C produced by Pseudomonas fluorescens. Microbiologica, 19, 113–121 (1996). Preuss, I., Kaiser, I., and Gehring, U., Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C. Eur. J. Biochem., 268, 5081–5091 (2001). Sonoki, S., and Ikezawa, H., Studies on phospholipase C from Pseudomonas aureofaciens. Biochim. Biophys. Acta, 403, 412–424 (1975). Arai, M., Matsunaga, K., and Murao, S., Purification and some properties of phospholipase C of Pseudomonas schuylkilliensis. Nippon No¯geikagaku Kaishi (in Japanese), 48, 409–415 (1974). Okawa, Y., and Yamaguchi, T., Studies on phospholipases from Streptomyces. J. Biochem., 78, 537–545 (1975). Doi, O., and Nojima, S., Phospholipase C from Pseudomonas fluorescens. Biochim. Biophys. Acta, 248, 234– 244 (1971). Lysenko, O., Phospholipase C of Pseudomonas chlororaphis and its toxicity for insects. Folia Microbiol., 18, 125–132 (1973). Taguchi, R., and Ikezawa, H., Hydrolytic action of phospholipases on bacterial membranes. J. Biochem., 82, 1225–1230 (1977).
