Biotechnology Letters 22: 1587–1590, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
1587
Isolation of phospholipase D producing microorganisms with high transphosphatidylation activity Tairo Hagishita∗∗ , Masanobu Nishikawa & Tadashi Hatanaka∗ Research Institute for Biological Sciences, Okayama (RIBS), 7549-1, Kayo-cho, Jyobo-gun, Okayama 716-1241, Japan ∗ Author for correspondence (Fax: +81 866 56 9454; E-mail:
[email protected]) ∗∗ Present address: Tokyo Metropolitan Institute of Gerontology, Sakae-machi, Itabashi-ku, Tokyo 173-0015, Japan Received 31 July 2000; Accepted 9 August 2000
Key words: Actinomycetes, phospholipase D, phospholipid, transphosphatidylation
Abstract The transphosphatidylation and hydrolytic activities of phospholipase D in culture supernatants of soil isolates were evaluated by a specific spectrophotometric method for quantitative determination using an artifical substrate, phosphatidyl-p-nitrophenol. Phospholipase D from strain TH-2 showed the highest specific activity and ratio of transphosphatidylation activity to hydrolytic activity among those from the eight soil isolates and commercial Actinomycetes phospholipase D.
Introduction Phospholipase D (PLD) hydrolyzes phospholipids to form phosphatidic acid and the relevant head groups. This enzyme is also unique because of its capacity for transphosphatidylation, i.e., it catalyzes the transfer of phosphatidyl groups to various acceptors. There have been numerous studies on phospholipid modification by the transphosphatidylation using PLD from Actinomycetes (D’Arrigo & Servi 1997). PLD with high transphosphatidylation activity is expected to be a powerful tool for the synthesis of various phospholipids employed in many biological and industrial processes. The selectivity of PLD-catalyzed transphosphatidylation depends on the production of phosphatidic acid, which is a side-reaction during the hydrolysis of phospholipids. The reaction selectivity is an important factor in industrial applications. Therefore, it is important to assess the ratio of transphosphatidylation activity to hydrolytic activity of PLD. Previous methods used in measuring of PLD activity have provided only a qualitative assessment of PLD activity. To gain a better understanding of the reaction characteristics of PLD, we recently developed a spe-
cific spectrophotometric method for the quantitative determination of PLD-catalyzed transphosphatidylation activity (Hagishita et al. 1999). The method measures the amount of p-nitrophenol liberated by the transphosphatidylation reaction of phosphatidyl-pnitrophenol (PpNP) and ethanol in an aqueous-organic emulsion system. Combined with the assay for PLDcatalyzed hydrolytic activity with PpNP (D’Arrigo et al. 1995), this method for the quantitative determination of transphosphatidylation activity is useful for the evaluation of the transphosphatidylation and hydrolytic activities of PLD. The purpose of this study was to assess the transphosphatidylation activity of PLD in culture supernatants of microorganisms. In this study, a preliminary screening was performed by transphosphatidylation with natural phospholipid, followed by evaluation of the two activities of PLD using PpNP. We identified strain TH-2 as the microorganism that produced PLD and had a high transphosphatidylation activity.
1588 Table 1. Phospholipase
D
activities in culture supernatant of soil isolates.
Strains
Growth
Culture temperature ( ◦ C)
Protein concentration (mg ml−1 )
Transphosphatidylation activity (unit mg−1 )
Hydrolytic activity (unit mg−1 )
Trans/Hydro ratio
TH1 TH-2 K2 K3 K4 K5 K6 K7
++ + ++ +++ +++ ++ +++ +++
28 37 28 28 28 28 28 28
0.4 0.04 0.3 0.11 0.10 0.15 0.37 0.34
8.8 90 22.7 10.9 18 23.3 8.1 7.1
1.48 7.0 2.37 1.18 1.7 2.53 1.32 1.03
5.9 12.9 9.6 9.2 10.6 9.2 6.1 6.9
The degree of growth is indicated as +, ++ and + + + for various degrees, progressing from slow to rapid growth.
Materials and methods Materials PpNP was prepared from soybean phosphatidic acid and p-nitrophenol according to the procedure described by D’Arrigo et al. (1995). Dipalmitoylphosphatidylcholine was obtained from Nippon Fine Chemical (Osaka, Japan). All other chemicals were of reagent grade and purchased from commercial sources. Isolation of PLD producing microorganisms The isolation medium employed consisted of the following: 2 g lecithin, 10 g glycerol, 2 g KNO3 , 1 g K2 HPO4 , 0.5 g NaCl, 0.5 g MgSO4 · 7H2 O and 10 mg FeSO4 · 7H2 O in 1 l tap water, pH 7.0. Soil samples were suspended in tap water and plated on the isolation medium. After 5–10 days incubation at 28 ◦ C or 37 ◦ C, the colonies formed were inoculated on a plate containing the isolation medium and incubated at 28 ◦ C or 37 ◦ C for 3–5 days. For screening, the following production medium was used: 5 g glucose, 5 g yeast extract, 5 g Polypepton, 2 g K2 HPO4 and 0.5 g MgSO4 · 7H2 O in 1 l distilled water. Strains that grew on the plate were inoculated in 3 ml of the production medium and incubated at 28 ◦ C for 3 days with shaking. Strain TH-2 was cultured at 37 ◦ C. The culture was centrifuged to remove cells, and the supernatant was used for the detection of PLD activity. For the screening of PLD producing microorganisms, PLD activity was determined based on transphosphatidylation activity. Transphosphatidylation activity was assayed by detecting the production of dipalmitoylphosphatidyl-2-phenoxyethanol on
TLC plates (silica gel plates, Merck, 60 F254 ). The reaction mixture was composed of 200 µl benzene containing 1 mg dipalmitoylphosphatidylcholine and 8.5 µl of 2-phenoxyethanol, 20 µl sodium acetate buffer (2 mM, pH 5.5) and 180 µl culture supernatant. The reaction was carried out with shaking at 37 ◦ C for 30 min. The organic layer (5 µl) of the reaction mixture was spotted on a TLC plate and developed in chloroform/methanol/water (30:10:1 by vol) solution. Phospholipids on TLC plates were detected by UV irradiation or spraying the plates with 5% (w/v) sodium phosphomolybdate in ethanol, heating at 250 ◦ C. PLD assays PLD-catalyzed transphosphatidylation activity was determined by measuring the production of pnitrophenol from PpNP and ethanol according to the method described by Hagishita et al. (1999). The reaction mixture consisted of 200 µl benzene containing 4 µmol PpNP, and 150 µl 20 mM sodium acetate buffer (pH 5.5) containing 200 µmol ethanol and 1 mg bovine serum albumin in a 1.5 ml sample tube. The mixture was sonicated for 15 min and incubated at 37 ◦ C for 5 min. The supernatant (50 µl) from the culture was added to the reaction mixture, and the mixture was incubated at 37 ◦ C for 10 min. The reaction was terminated by the addition of 100 µl 1 M HCl and then followed by the addition of 150 µl 1 M NaOH. The phospholipids were then extracted with chloroform/methanol (3:1, v/v) solution. After centrifugation at 4 ◦ C for 10 min, 20 µl of the aqueous phase was taken and to this, 0.1 M Tris/HCl buffer (pH 8.0) was added to make 200 µl. This solution was then dispensed in a 96-well microtiter plate. The amount of
1589 liberated p-nitrophenol was estimated based from the absorbance at 405 nm using a microplate reader (Biolumin 960, Amersham Pharmacia Biotech, USA). The assay for PLD-catalyzed hydrolysis was performed using 4 mM PpNP as a substrate by a modification of the method described by D’Arrigo et al. (1995). The reaction was performed at 37 ◦ C with 4 mM PpNP in 0.1 M sodium acetate buffer (pH 5.5). The assay was carried out at 360 nm (molecular extinction coefficient of p-nitrophenol , ε = 6800). One unit of enzyme activity was defined as the amount of the enzyme that catalyzed the release of 1 µmol p-nitrophenol in 1 min under the above conditions. The amount of protein in the culture supernatant was determined according to the method of Bradford, using dye reagent from Bio-Rad (USA).
Results and discussion Isolation of PLD producing-microorganisms About 6000 strains were isolated from soils and those were screened for their ability to produce PLD. Twenty strains produced PLD and transphosphatidylation activity was obtained. Of these, the following eight strains, TH-1, TH-2, K2, K3, K4, K5, K6 and K7 were used in the experiments because of their high transphosphatidylation activity and good growth. The eight strains belonged to the class Actinomycetes (data not shown). The activity of PLD from these eight strains was examined from the transphosphatidylation of phosphatidylcholine using various substrates, such as 2-propanol, 3-propanol, t-butanol, p-methoxyphenol, L-serine and D-serine. There was no difference in the catalytic ability of PLD from the eight strains, and the phosphatidyl group was similarly transferred to 2-propanol, p-methoxyphenol, L-serine and D-serine. Evaluation of the ratio of transphosphatidylation activity to hydrolytic activities of PLD The transphosphatidylation and hydrolytic activities of PLD in the supernatant of the eight strains were examined using PpNP as the substrate. The transphosphatidylation and hydrolytic activities of PLD were detected in the range of 7.1–90.0 U mg−1 and 1.03– 7.00 U mg−1 , respectively (Table 1). Strain TH-2 showed the highest specific activity among the eight strains because of the lowest protein concentration of its supernatant. This property of TH-2 strain made it
Fig. 1. Relationship between transphosphatidylation activity and hydrolytic activity in culture supernatants of PLD-producing microorganisms.
a suitable microorganism for concentration and purification of the PLD enzyme. The ratio of transphosphatidylation activity to hydrolytic activity was calculated for each PLD (Table 1). The ratios were in the range of 5.9–12.9. Under the same conditions, when the transphosphatidylation and hydrolytic activities of PLD from Streptomyces sp. (Sigma type VII) were measured using PpNP as the substrate, the specific activity for transphosphatidylation (68 U mg−1 ) was only two fold higher than hydrolytic activity (35 U mg−1 ) (Hagishita et al. 1999). PLDs from the eight strains showed higher ratios of transphosphatidylation activity to hydrolytic activity than PLD from Streptomyces sp. (Sigma type VII). The transphosphatidylation activities in the culture supernatants of the eight strains were plotted against the hydrolytic activity. There was good correlation between the two activities of PLD (Figure 1). Shimbo et al. (1989) screened PLD-producing microorganinsms based on hydrolytic activity, and obtained 29 strains. However, no correlation between transphosphatidylation and hydrolytic activities was observed (Shimbo et al. 1989). This is contrary to our result showing good correlation of the two activities. One of the reasons for these contradicting results may be the difference in the screening methods used. The Streptomyces sp. that produced Sigma-type-VII PLD
1590 had been screened based on hydrolytic activity similar to that used as Shimbo et al. (1989) (Tsuchiya et al. 1992). It seemed that PLDs from microorganisms screened based on hydrolytic activity possessed various ratios of the two activities. However, in our case, Actinomycetes strains that were screened based on transphosphatidylation activity showed similar ratios of the two activities. Another possible reason may be the difference in the assay methods for PLD activities. Shimbo et al. (1989) used TLC analysis for determining transphosphatidylation activity. This method, however, is unsuitable for rate assay; thus, transphosphatidylation activity may not have been accurately measured by them. Four sequences encoding PLD genes from Streptomyces species have already been cloned. The deduced primary structures are very similar to each other (Iwasaki et al. 1994, Ogino et al. 1999). One of these is assumed to be identical with Sigma-type-VII PLD. The catalytic site of the PLD is proposed to be two separated copies of an invariant charged motif, HxKxxxxD (Hammond et al. 1995). This HKD motif is also found in Actinomycetes PLDs (Iwasaki et al. 1994, Ogino et al. 1999). It is interesting to determine whether the ratios of transphosphatidylation activity to hydrolytic activity are dependent on or independent of the differences in the primary structure of Actinomycetes PLDs. Further work is needed in elucidating the ratio of the two activities of Actinomycetes PLDs with highly homologous amino acids sequences. The comparison of the transphosphatidylation and hydrolytic activities of PLD is important for extending the applications of PLD. An extracellular PLD from strain TH-2 showed the highest specific activity and the highest ratio of the transphosphatidylation activity to the hydrolytic activity in this experiment. The PLD from strain TH-2 may be industrially useful.
Acknowledgements We thank Dr M. Takami, Technosoft Co., Ltd. for helpful advice during this study. We are also grateful to Dr Y. Katsuragi, Food Products Research Laboratory of Kao Corporation, for providing us with soybean phosphatidic acid. We thank Dr M. Iwabuchi, Research Institute for Biological Sciences, Okayama, for his critical reading of this manuscript.
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