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M. Saito, E. Bourque, J. Kanfer (1974)
Phosphatidohydrolase and base-exchange activity of commercial phospholipase D.Archives of biochemistry and biophysics, 164 2
O. Lowry, N. Rosebrough, A. Farr, R. Randall (1951)
Protein measurement with the Folin phenol reagent.The Journal of biological chemistry, 193 1
M. Hosokawa, T. Shimatani, T. Kanada, Y. Inoue, K. Takahashi (2000)
Conversion to docosahexaenoic acid-containing phosphatidylserine from squid skin lecithin by phospholipase D-mediated transphosphatidylation.Journal of agricultural and food chemistry, 48 10
P. Comfurius, R. Zwaal (1977)
The enzymatic synthesis of phosphatidylserine and purification by CM-cellulose column chromatography.Biochimica et biophysica acta, 488 1
S. Shuto, S. Imamura, K. Fukukawa, H. Sakakibara, J. Murase (1987)
A facile one-step synthesis of phosphatidylhomoserines by phospholipase D-catalyzed transphosphatidylation.Chemical & pharmaceutical bulletin, 35 1
L. Juneja, E. Taniguchi, S. Shimizu, T. Yamane (1992)
Increasing productivity by removing choline in conversion of phosphatidylcholine to phosphatidylserine by phospholipase DJournal of Fermentation and Bioengineering, 73
T. Uhm, Tao Li, J. Bao, Gukhoon Chung, D. Ryu (2005)
Analysis of phospholipase D gene from Streptoverticillium reticulum and the effect of biochemical properties of substrates on phospholipase D activityEnzyme and Microbial Technology, 37
C. Ponting, I. Kerr (1996)
A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: Identification of duplicated repeats and potential active site residuesProtein Science, 5
Hongying Yang, M. Roberts (2002)
Cloning, overexpression, and characterization of a bacterial Ca2+‐dependent phospholipase DProtein Science, 11
T. Hagishita, M. Nishikawa, T. Hatanaka (2000)
Isolation of phospholipase d producing microorganisms with high transphosphatidylation activityBiotechnology Letters, 22
L. Juneja, T. Kazuoka, N. Goto, T. Yamane, S. Shimizu (1989)
Conversion of phosphatidylcholine to phosphatidylserine by various phospholipases D in the presence of l- or d-serineBiochimica et Biophysica Acta, 1003
M. Bradford (1976)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical biochemistry, 72
A. Souček, C. Michalec, A. Soucková (1967)
Enzymic hydrolysis of sphingomyelins by a toxin of Corynebacterium ovis.Biochimica et biophysica acta, 144 1
C. Ogino, M. Kanemasu, Y. Hayashi, A. Kondo, N. Shimizu, S. Tokuyama, Y. Tahara, S. Kuroda, K. Tanizawa, H. Fukuda (2004)
Over-expression system for secretory phospholipase D by Streptomyces lividansApplied Microbiology and Biotechnology, 64
T. Hatanaka, T. Negishi, Megumi Kubota-Akizawa, T. Hagishita (2002)
Study on thermostability of phospholipase D from Streptomyces sp.Biochimica et biophysica acta, 1598 1-2
T. Hatanaka, T. Negishi, Megumi Kubota-Akizawa, T. Hagishita (2002)
Purification, characterization, cloning and sequencing of phospholipase D from Streptomyces septatus TH-2Enzyme and Microbial Technology, 31
E. Gottlin, A. Rudolph, Y. Zhao, H. Matthews, J. Dixon (1998)
Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate.Proceedings of the National Academy of Sciences of the United States of America, 95 16
C. Ponting (1996)
Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3‐kinases: Binding partners of SH3 domains?Protein Science, 5
S. Yang, S. Freer, A. Benson (1967)
Transphosphatidylation by phospholipase D.The Journal of biological chemistry, 242 3
I. Leiros, Francesco Secundo, Carlo Zambonelli, Stefano Servi, Edward Hough (2000)
The first crystal structure of a phospholipase D.Structure, 8 6
V. Shnigir, M. Kisel (2004)
Transformation of Phospholipids by Cabbage Phospholipase D in Mixed Micelles Containing 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonateApplied Biochemistry and Microbiology, 40
(2000)
The first crystal structure of a phospholipase
D. Hanahan, I. Chaikoff (1947)
The phosphorus-containing lipides of the carrot.The Journal of biological chemistry, 168 1
Annals of Microbiology, 58 (2) 227-231 (2008) Cloning and expression of phospholipase D gene pld from Streptomyces chromofuscus Bin LI, Fu-Ping LU*, Lin TIAN, Yu LI, Lianxiang DU Tianjin Key Lab of Industrial Microbiology, College of Bioengineering, Tianjin University of Science and Technology, 300222, Tianjin, P.R. China Received 28 August 2007 / Accepted 25 February 2008 Abstract - The phospholipase D (PLD) gene pld encoding the mature PLD enzyme from Streptomyces chromofuscus was cloned and sequenced. The recombinant protein has been expressed in Escherichia coli and purified. The yield of aim pro- tein was up to 27.5 mg/l culture medium. Because it was targeted with a 6 x his-tag, the recombinant protein could be much more easily purified by using Ni-NTA His•Bind Purification Kits. After concentration, the concentration of rPLD was 1.0 mg/ml buffer. With transphosphatidylation activity of rPLD, phosphatidylserine (PS) could be produced from phospha- tidylcholine (PC). The optimum organic phase was chloroform, optimum pH was 7.5, optimum temperature and time of transphosphatidylation reaction that catalysed by rPSS were 30 °C and 6 h, respectively. The highest transphosphatidy- lation rate of rPSS was up to 31% and the specific enzyme activity was up to 39 U/mg. Key words: phospholipase D (PLD), phosphatidylserine (PS), transphosphatidylation, Streptomyces chromofuscus,HPLC assay. PLD was firstly discovered in plants (Hanahan and INTRODUCTION Chaikoff, 1947) and later identified in microorgan- isms (Soucek et al., 1967) and mammals (Satio et The importance of large scale production of phos- al., 1974). PLD hydrolyzes phosphatidylcholine to phatidylserine is increasing because of their func- phosphatidic acid and choline by breaking its phos- tions in industrial processes, health and nutrition phodiester bond (Ponting and Kerr, 1996). PLD also applications (Lekh et al., 1992; Masashi et al., acts on other phosphatidylesters and catalyses a 2000). For example, in clinical trials conducted in transphosphatidylation reaction when alcohol is the United States and Europe, it indicated that present as a nucleophilic donor (Uhm, 2005). In phosphatidylserine (PS) supplemented in the diet particular, Actinomycetes (especially Streptomyces) plays important roles in the support of mental func- PLDs showed high transphosphatidylation activities tions in the aging brain. So the benefits of PS have than plant and mammalian PLDs (Tadashi et al., received a great deal of attention (Masashi et al., 2002). Although the biosynthesis has many advan- 2000). tages in phosphatidylserine production, there are The isolation of PS in pure and homogeneous also some limitations for industrial scale: the yield forms from natural sources and also PS synthesis by of PLD from Streptomyces was low and cost nearly chemical reactions are complicated, laborious and one week to culture (Tadashi et al., 2002, Ogino et expensive. Although PS has been prepared by a al., 2004). variety of methods, most of them have limitations To over-express the enzyme rapidly, we cloned at the industrial-scale (Lekh et al., 1992). Enzymatic conversion of phosphatidylcholine PLD gene from Streptomyces chromofuscus into (PC) to PS using phospholipase D (PLD, EC 3.1.4.4) Escherichia coli BL21 (DE3) with pET-22b (+) vec- from different resources, including from plants and tor. And the recombinant protein was targeted with microorganisms, has been examined by many a 6 x his-tag, it could be much more easily purified researchers (Yang et al., 1967; Comfurius and by using Ni-NTA His•Bind Purification Kits. With its Zwaal, 1987; Shuto et al., 1987; Masashi et al., transphosphatidylation activity, the rPLD could gen- 2000; Tairo et al., 2000; Shnigir and Kisel, 2004). erate PS by using PC and serine as a substrate. This research also investigated the optimum organic phase, pH, temperature and time of the transphos- * Corresponding author. E-mail: libin820213@hotmail.com B. LI et al. phatidylation reaction by the rPLD. It has found a and stored at -80 °C until needed. foundation for biosynthesis PS by rPLD from E. coli. Frozen pellets or fresh cells were thawed at room temperature and resuspended in 30 ml PBS (pH 7.0). The suspensions were sonicated for 2 x 4 MATERIALS AND METHODS s (total 5 min) on ice, and the supernatant was sep- arated from cell debris by centrifugation (3600 rpm Materials. L-Serine was purchased from Biodev for 20 min). The recombinant protein was purified (Shanghai, China). PS and PC (from soybean) were by Ni-NTA His•Bind Purification Kits. The super- purchased from Sigma Chemical Co. LA Taq with GC natant was slowly (1 ml/min) loaded onto an affini- buffer was purchased from TaKaRa (Japan). Ni-NTA ty column of the nickel gelose gel (10 ml). After His•Bind Purification Kits was purchased from loading, the column was washed with 50 ml of 50 Novagen. mmol/l PBS pH 7.4. rPLD was eluted from the resin with 50 mmol/l PBS pH 7.4 containing 50 mmol/l Strains and plasmid. The gene encoding PLD was and 400 mmol/l imidazole, respectively. isolated from Streptomyces chromofuscus AS Total proteins, supernatant or the soluble pro- 4.331. Escherichia coli JM109 was used for cloning tein fraction, and precipitate or the insoluble protein and E. coli BL21 (DE3) was used to express PLD fraction after sonication were subjected to SDS- protein. The vector pET-22b (+) was used for PAGE (12% resolving gel). Proteins were stained expression in E. coli BL21. with Coomassie Brilliant Blue. Protein concentration was estimated by the method of Bradford (1976). Culture medium. Streptomyces chromofuscus was grown on YME medium (4 g yeast extract/100 ml, Assay of PLD activity. Transphosphatidylation 10 g malt extract/100 ml, 4 g glucose/100 ml, pH reaction from PC and L-serine to PS was carried out 7.3~7.5). Escherichia coli K and BL21 (DE3) were in a biphasic system because water miscible serine grown on LB (Luria Bertani) medium. The recombi- and immiscible PC were used as substrate. In this nant E. coli was selected on LB medium containing study, the activity of the enzyme was determined as 50 Ìg/ml ampicillin. follows: 30 ml of 0.1 mol/l sodium acetate buffer pH 7.0 (containing 5 mmol/l CaCl , 0.15 mol of serine) Plasmid construction. Two oligonucleotide and 1 ml purified rPLD enzyme solution were added primers were designed based on the S. chromofus- with 15 ml acetonitrile containing 0.75 mmol (0.56 cus PLD DNA sequence recently reported (Yugo et g) phosphatidylcholine (Lekh et al., 1989; Masashi al., 1999; Yang and Roberts, 2002). 5’- et al., 2000). The mixture was stirred vigorously cgGGATCCggccgaccaggcccccgcctccct-3’ (containing (200 rpm) at 30 °C to obtain a homogeneous emul- a BamHIsite as indicated by the capitals) and 5’- sion. After 3 h reaction, the reaction mixture was cccAAGCTTctactcggggtcgtaggtgcgc-3’ (containing a stopped to demix dual-liquid phase and 1 ml organ- HindIII site as indicated by the capitals) were used ic phase was extracted. After volatilisation, the to amplify the 1530 bp PLD gene with LA Taq. The remainder was washed by chloroform twice and 30-cycle PCR products were digested by BamHI and resolved by 50 Ìl chloroform. Samples (15 Ìl) were HindIII?then ligated to BamHI-HindIII linearised analysed by HPLC operated at 206 nm. The eluting pET-22b (+) vector. The purified recombinant plas- solvent was acetonitrile/methane/85% phosphoric mid, pET-S, was transformed into E. coli JM109. The acid (100:10:0.8, v/v). The flow rate was 1 ml/min. recombinants were selected on the ampicillin resist- Each run took 30 min. ant plate. Sequencing, PCR and double restriction In particular, one unit of rPLD was defined as the enzyme digestion were carried out to confirm the amount of enzyme trasphosphatidylation 1 Ìmol of positive transformants. Then, the recombinant plas- pure PC per hour at optimum temperature and opti- mid was transformed into BL21 (DE3) competent mum pH in the biphasic reaction time and the spe- cells for pld gene expression. cific activity is defined as U/mg of protein. The optimum organic phase (dichloromethane, Expression and purification of recombinant chloroform, ethyl acetate and hexane), pH (ranging PLD cloned in pET-22b (+). A single colony of from 5 to 8.5), temperature (ranging from 20 to 40 BL21 (DE3) containing pET-S plasmid was grown in °C), and reaction time (ranging from 1 to 8 h) of 10 ml of LB medium containing ampicillin until OD transphosphatidylation reaction were tested at the reached 1.0. This culture was used to inoculate into conditions described above. 1 l fresh LB medium. Culture were grown with rapid shaking (200 rpm) at 37 °C to OD = 0.8. Overexpression of PLD was induced by the addi- RESULTS AND DISCUSSION tion of IPTG to a final concentration of 0.8 mmol/l. After induction, cultures were incubated at 25 °C Construction of recombinant plasmid pET-S overnight. Cells were harvested by centrifugation The amplified PLD gene (1530bp) was inserted into Ann. Microbiol., 58 (2) 227-231 (2008) 229 (Fig. 1). After concentration, the concentration of BamHI and HindIII restriction sites of the pET-22b rPLD was up to 1.0 mg/ml buffer. (+) vector to construct an expression vector pET-S, which harbours PLD gene under the control of T7 promoter. 12 3 4 5 Sequence analysis KDa The sequence of the pld gene cloned from S. chro- mofuscus AS 4.331 had 4 differences from the counterpart of S. chromofuscus from ATCC 35.0 (#23616) (Yang and Roberts, 2002). The identity is 69 198 99.22%. Gly was replaced by Glu, Gly by Ala, 206 285 Val by Ile, and Ser by Ala. 45.0 Several PLDs from Streptomyces have been sequenced and shown significant sequence similari- ty. In particular, the duplicated HxK(x) D(or HKD) motifs were identified (Yugo et al., 1999; Leiros et 66.2 al., 2000; Uhm et al., 2005). But PLD that isolated from S. chromofuscus, has very little sequence 116.2 homology with other Streptomyces PLD enzymes. The primary amino acid sequence of S. chromofus- cus PLD exhibits no HxK(x) D motif. However, 187 193 200 210 FIG. 1 - SDS-PAGE showing purification of PLD from pET HxK(x) D and HxK(x) D in the same region 3 7 expression. Lane 1: molecular masses for the of the protein as the HxK(x) D motifs in the other standard proteins, lane 2: protein content of the Streptomyces PLDs might be variations of that cat- crude cell extract, lane 3: protein content of wash from the purification column, lane 4: protein elu- alytic motif. And some experiments showed that the ted after incubation of column with 50 mM imida- mutant of the single Cys123 in S. chromofuscus to zole, lane 5: protein eluted after incubation of Ala or Ser generated well-folded protein with great- column with 400 mM imidazole. ly reduced activity (Yang and Roberts, 2002). Actually, the amino acidic sequence variation deduced for the pld gene of Streptomyces chromo- Standard curve for transphosphatidylserine fuscus AS 4.331 are not located in region that Phosphatidylserine, the product of transphos- should affect the enzymatic activity of the phospho- phatidylation reaction, was determined to calculate lipase. the transphosphatidylation rate of rPLD. When the standard PS solution was subjected to the present Expression and Purification of PLD transphosphatidylation-activity assay, a linear stan- The recombinant PLD protein had a calculated dard curve exhibiting a correlation coefficient of molecular size of 55~58 kDa similar with the report 0.9965 (R published previously (Tadashi et al., 2002). In con- ) was obtained (Fig. 2). trast, no band was detectable in the uninduced con- trol culture. With the method of Lowry et al. (1951), the total protein concentration of the crude lysed cell supernatant is 125 mg/l culture. And the expression of the S. chromofuscus PLD gene in BL21 (DE3) is about 20% (27.5 mg/l), based on the SDS-PAGE of the crude lysed cell supernatant. Because of short culture time (12 h) and high expression of the rPLD in E. coli, the rPLD could be fully restored, easily. As to the initial strain, highest yield of PLD is 30 mg/l culture medium (Hatanaka et al., 2002). But the production periods was very long, for 5 days. Its relative yield was no more than FIG. 2 - Phosphatidylserine concentration standard curve 3 mg/12 h in one litre medium. The relative yield by HPLC. was higher than the results reported previously (Yang and Roberts, 2002; Ogino et al., 2004). The recombinant protein was expressed with a Assay of PLD activity six-histidine tag at the C-terminus, allowing purifi- Four organic reagents were chose to investigate the cation by Nickel-chelate affinity chromatography. effect of organic phase to the transphosphatidyla- After 400 mM imidazole washed from the affinity tion reaction. And the polarity of the reagents men- column, the PLD protein was more than 90% pure tioned above increased as the same sequence. B. LI et al. Figure 3 showed that chloroform and ethyl acetate Transphosphatidylation rare was slightly were suitable for organic phase medium. It might increased by increasing the temperature from 20 to seem that the polarity of phospholipid compounds 30 °C. At the higher temperature, the rate of was similar to the polarity of the two reagents. So it transphosphatidylation decreased prominently. The could dissolve in chloroform and ethyl acetate. result proved that recombinant PLD has had well Chloroform was chose as the organic phase of the transphosphatidylation ability at 28~32 °C (Fig. 5). transphosphatidylation reaction for the reason that Through the transformation curve (Fig. 6), the phosphatidylcholine emulsified better in chloroform reaction time could be determined: after 6 h the than in ethyl acetate, so the PC could disperse reaction stabilised, and the transphosphatidylation homogeneously in chloroform. rate was up to 31%. It meant that 233 Ìmol PC was Figure 4 showed transformation curve for pH transformed to PS in 6 h. The enzyme activity was 5~8.5 Transphosphatidylation rare was slightly 39 U/mg proteins. increased by increasing the pH from 5 to 7.5. At the higher pH, the rate of transphosphatidylation decreased prominently. The result proved that recombinant PLD has had well transphosphatidyla- tion ability at pH 7~8. And the optimum growth pH of S. chromofuscus is pH 7.3~7.5. The alkaline con- dition was suitable for enzyme stability. Actually, at low pH, the PLD would occur to hydrolyzation more easily than transphosphatidylation (Gottlin, 1998). So the optimum pH of transphosphatidylation car- ried with rPLD is about 7.5. FIG. 5 - The effect of temperature to transphosphatidyla- tion reaction. FIG. 6 - The effect of reaction time to transformation rate. FIG. 3 - The effect of organic phase to transphosphatidyla- tion reaction. CONCLUSION In this research, the PLD gene encoding the mature PLD was cloned from the genomic DNA of S. chro- mofuscus and its gene sequence was analysed. There were four mutants appeared, Gly Glu, 198 206 285 Gly Ala, Val Ile, and Ser Ala. With the method described previously, the transphosphatidy- lation rate of PC to PS by rPLD could be calculated. And the optimum organic phase, pH, temperature and time of rPLD transphosphatidylation reaction are: chloroform, 7.5, 30 °C and 5~6 h, respective- ly. The highest transphosphatidylation rate is up to FIG. 4 - The effect of pH to transphosphatidylation reac- 31%. In other word, the IU of the rPLD is up to 39 tion. Ann. Microbiol., 58 (2) 227-231 (2008) 231 Ogino C., Kanemasu M., Hayashi Y., Kondo A. Shimizu N., U/mg. And because of short culture time of expres- Tokuyama S., Tahara Y., Kuroda S., Tanizawa K., sion host and high expression of the rPLD in E. coli, Fukuda H. (2004). Over-expression system for secre- the rPLD could be fully restored, easily. This tory phospholipase D by Streptomyces lividans. Appl. research has found a foundation for industrial-scale Microbiol. Biotechnol., 64: 823-828. biosynthesis PS by rPLD. Ponting C.P., Kerr I.D. (1996). 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Annals of Microbiology – Springer Journals
Published: Nov 21, 2009
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