Purification, Biochemical Characterization, and Cloningof Phospholipase D from Streptomyces racemochromogenesStrain 10-3

Yozo Nakazawa • Yoshimasa Sagane • Teppei Kikuchi •

Masataka Uchino • Takeshi Nagai • Hiroaki Sato •

Kazuki Toeda • Katsumi Takano

Published online: 17 November 2010

� Springer Science+Business Media, LLC 2010

Abstract We previously isolated Streptomyces racemo-

chromogenes strain 10-3, which produces a phospholipase

D (PLD) with high transphosphatidylation activity. Here,

we purified and cloned the PLD (PLD103) from the strain.

PLD103 exerted the highest hydrolytic activity at a slightly

alkaline pH, which is in contrast to the majority of known

Streptomyces PLDs that have a slightly acidic optimum pH.

PLD103 shares only 71–76% amino acid sequence identity

with other Streptomyces PLDs that have a slightly acidic

optimum pH; thus, the diversity in the primary structure

might explain the discrepancy observed in the optimum

pH. The purified PLD displayed high transphosphatidyla-

tion activity in the presence of glycerol, L-serine, and

2-aminoethanol hydrochloride with a conversion rate of

82–97% in a simple one-phase system, which was

comparable to the rate of other Streptomyces PLDs in a

complicated biphasic system.

Keywords Transphosphatidylation � Phospholipase D �Streptomyces racemochromogenes � Phospholipid

modification

Abbreviations

PLD Phospholipase D

pI Isoelectric point

PL Phospholipid

PC Phosphatidylcholine

PG Phosphatidylglycerol

PE Phosphatidylethanolamine

PA Phosphatidic acid

IEF Isoelectric focusing

MOPS 3-(N-Morpholino) propanesulfonic acid

TLC Thin-layer chromatography

2-ME 2-Mercaptoethanol

ORF Open reading frame

Rbs Ribosome binding site

1 Introduction

Phospholipids (PLs) are ubiquitous molecules that are

widely distributed among the plant and animal kingdoms.

In industrial fields, PLs can be abundantly derived from

lecithin, the mixture of PLs with other minor components

that arise as by-products from an oil-production process

from animal fats or vegetable oils. The PL content in lec-

ithin is predominantly phosphatidylcholine (PC) and small

amounts of phosphatidylserine (PS), phosphatidylglycerol

(PG), phosphatidylethanolamine (PE), and phosphatidyl-

inositol. The lecithin-derived PLs have been included in

food products, cosmetics, and pharmaceuticals because of

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10930-010-9292-y) contains supplementarymaterial, which is available to authorized users.

Y. Nakazawa (&) � T. Nagai � H. Sato � K. Toeda

Department of Food and Cosmetic Science,

Faculty of Bioindustry, Tokyo University of Agriculture,

196 Yasaka, Abashiri, Hokkaido 099-2493, Japan

e-mail: y3nakaza@bioindustry.nodai.ac.jp

Y. Sagane

Sars International Centre for Marine Molecular Biology,

Thormøhlensgate 55, 5008 Bergen, Norway

T. Kikuchi � M. Uchino � K. Takano

Department of Applied Biology and Chemistry, Faculty of

Applied Bioscience, Tokyo University of Agriculture, 1-1-1

Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

123

Protein J (2010) 29:598–608

DOI 10.1007/s10930-010-9292-y

their interfacial activity, emulsification properties, and

liposome-formation ability [7, 13, 22, 26]. In addition to

these common properties of PLs, PG can improve the

emulsification capacity of lecithin over wide ranges of pH

and salt conditions [20]. Moreover, PS, which can be

obtained from animal brain, has been proposed as a food

supplement to decrease and/or prevent senile dementia

[15, 34]. However, the yields of such PLs from lecithin and

other natural resources are limited. In particular, the use of

animal brain has been avoided because of the occurrence of

bovine spongiform encephalopathy. Even though chemi-

cally synthesized PLs are available, they are too expensive

to use in daily food products. Phospholipase D (PLD, EC

3.1.4.4) is an enzyme that catalyzes the hydrolysis of the

phosphodiester bond of PLs to generate phosphatidic acid

(PA) and its corresponding alcohol moiety. In addition to

its hydrolytic activity, PLD also catalyzes the intercon-

version of the polar head groups of PLs by a reaction called

transphosphatidylation [38]. The transphosphatidylation

reaction synthesizes various phosphatidyl derivatives of

hydroxyl compounds, such as alcohols, phenols, sugars,

nucleotides, vitamins, and others [6, 18, 19, 23, 31, 35].

Therefore, this reaction is an economical means of PL

modification. The reactions performed by PLD can occur

with different degrees of selectivity between hydrolysis

and transphosphatidylation, depending on the enzymatic

source [16, 17]. In particular, PLD from Streptomyces

predominantly displays high selectivity for the transphos-

phatidylation reaction [24, 37] and, accordingly, PLDs

from this bacterium are already used in industrial fields.

The transphosphatidylation by PLD is often carried out

in a biphasic system composed of an aqueous solution of

PLD enzyme and an organic solution of the PL substrate. In

the aqueous reaction mixture, PLD is exposed not only to

substrate but also to product PLs and can competitively

react to de novo synthesized PLs as it hydrolyzes the

products. This might result in low production of the

phosphatidyl derivatives [5]. However, the biphasic sys-

tems require enormous quantities of organic solvents and

considerable energy to make an emulsion with strong

agitations. Additionally, the organic solvents in this system

often include toxic compounds, which are undesirable for

industrial food, cosmetic, and medical fields. To identify

effective reaction conditions that do not use organic sol-

vents, we recently screened six Streptomyces strains,

derived from soil samples that produce PLD with high

transphosphatidylation capacity even in the one-phase

system. In this system, the substrate PC is dispersed in

aqueous buffer with a small amount of detergent [24, 25].

The culture supernatants of the strains displayed high PG-,

PS-, and PE-producing transphosphatidylation from the

substrate PC with conversion rates ranging from 74 to 97%,

which are comparable to those of Streptomyces PLDs

assayed in biphasic systems. Furthermore, they showed low

hydrolysis activities against PG and PS. Therefore, the

PLDs produced by these strains are expected to be avail-

able for PL modification through their high transphospha-

tidylation capacity in the aqueous system. In this study, we

purified, characterized and cloned the PLD enzyme from

S. racemochromogenes strain 10-3.

2 Materials and Methods

2.1 Materials

Egg yolk lecithin (PC-98N) was kindly provided by Kewpie

Fine Chemicals (Tokyo, Japan). The PL composition of the

lecithin was the following: 98.9% PC, 0.1% lysophospha-

tidylcholine, and others, according to the manufacturer’s

specification assessment. Other PLs were obtained from

Avanti Polar Lipids (Alabaster, AL, USA).

2.2 Bacterial Strains and Cultural Conditions

Streptomyces racemochromogenes strain 10-3 was isolated

from soil samples collected in Japan as previously reported

[24, 25]. Strains were cultured in GYP medium, which was

composed of 5 g/L glucose, 5 g/L Bacto yeast extract (BD

Difco, Franklin Lakes, NJ, USA), 5 g/L Polypepton (Nihon

Pharmaceutical, Tokyo, Japan), 2 g/L K2HPO4, and 0.5 g/L

MgSO4�7H2O, with shaking at 28 �C for 2 days.

2.3 PLD Assay

Phospholipase D activity was assayed using PC as a sub-

strate and measuring the formation of choline with choline

oxidase and peroxidase [12]. A one milliliter reaction

mixture that was composed of 1 mg/mL PC-98N, 4 mM

sodium deoxycholate, 20 mM Tris–HCl buffer (pH 7.5),

10 mM CaCl2, and enzyme was incubated at 37 �C for

10 min. The reaction was immediately terminated by

adding 0.2 mL of solution (1 M Tris–HCl, pH 8.0,

100 mM EDTA) and incubating in a boiling water bath for

20 min. After chilling at room temperature, the reaction

mixture was mixed with 0.2 mL of the choline assay

solution, containing 0.1 M Tris–HCl buffer (pH 8.0), 1.25

units of choline oxidase (Sigma–Aldrich, St. Louis, MO,

USA), 1 unit of peroxidase (Wako Pure Chemicals),

1.6 lmol of phenol, and 1 lmol of 4-aminoantipyrine, and

then incubated at 37 �C for 60 min to allow for color

development. After adding 2 mL of 10 g/L Triton X-100,

the absorbance of the reaction mixture was measured at

500 nm. A calibration curve was obtained by adding a

standard solution of choline chloride to the assay mixture

instead of the enzyme solution. One unit (U) was defined as

Purification, Biochemical Characterization, and Cloning of Phospholipase D 599

123

the amount of enzyme that liberated 1 lmol of choline per

min. To determine the optimum pH, PLD activity was

assayed using reaction mixtures made with 20 mM buffer

at different pHs (pH 4, 5, and 6 = sodium acetate-acetic

acid; pH 7, 7.5, and 8 = Tris–HCl; pH 9 and 10 = boric

acid-NaOH). To determine the optimum temperature, PLD

assay experiments were carried out at several temperatures

for 10 min each.

2.4 Protein Assay

Protein concentration was determined using the Bio-Rad

(Hercules, CA, USA) Protein Assay based on the method

of Bradford [2], using bovine serum albumin as a standard.

2.5 Enzyme Purification

The cultured broth (1 L) of strain 10-3 was centrifuged,

and the supernatant was brought to 65% saturation with

(NH4)2SO4. After standing overnight at 4 �C, the sus-

pension was centrifuged at 10,000 rpm for 30 min at 4 �C.

The precipitate was dissolved and dialyzed to 20 mM

sodium acetate buffer (pH 5.5) at 4 �C. The dialyzed

solution was centrifuged to remove insoluble materials.

The supernatant was applied to a CM Sepharose Fast Flow

(GE Healthcare Bio-Sciences, Little Chalfont, Bucking-

ham, England) column (2.0 mm I.D. 9 80 mm) equili-

brated with 20 mM sodium acetate buffer (pH 5.5). After

washing the column with the equilibration buffer, the

bound proteins were eluted using a stepwise gradient with

0.1 M NaCl and 0.25 M NaCl in 20 mM sodium acetate

buffer (pH 5.5). The active fractions were pooled, dia-

lyzed against distilled water at 4 �C, and lyophilized. The

lyophilized material was dissolved in 2 mL of 20 mM

MOPS-NaOH buffer (pH 7.0) and loaded onto a TOYO-

PEARL DEAE-650M (Tosoh, Tokyo, Japan) column

(1.6 mm I.D. 9 50 mm) equilibrated with 20 mM MOPS-

NaOH buffer (pH 7.0). After washing the column with the

equilibration buffer, the bound proteins were eluted with

0.5 M NaCl in 20 mM MOPS-NaOH buffer (pH 7.0). The

PLD activity fractions were pooled, desalted by dialysis in

cold water, and lyophilized.

2.6 Electrophoresis Analyses

SDS–PAGE was performed using the method of Laemmli

[21] on a 12.5% polyacrylamide gel using a discontinuous

buffer system. The molecular mass under denaturing con-

ditions was determined using an LMW-SDS Marker Kit

(GE Healthcare Bio-Science). Isoelectric focusing (IEF)

electrophoresis was carried out in a Multiphor II apparatus

(GE Healthcare Bio-sciences) at 10 �C using an Ampho-

line PAG plate (pH 3.5–9.5, GE Healthcare Bio-sciences).

The isoelectric point (pI) of purified PLD was determined

using a Broad pI Kit (GE Healthcare Bio-sciences). The

proteins were detected by Coomassie blue staining.

2.7 Determination of the pH and Thermal Stability

Profiles

To determine the pH stability of the PLD, 100 lL of PLD

solution (5 U/mL) was added to 100 lL of 0.2 M buffer at

different pHs (pH 2 and 3 = glycine–HCl; pH 4, 5, 6, 7,

7.5, 8, 9 and 10 = same buffer used in pH optimum

determination; pH 11 and 12 = Na2HPO4-NaOH). Test

tubes containing the above solutions were incubated at

25 �C for 4 h. Ten microliters of the solution were then

used to determine the residual activity (Tris–HCl, pH 7.5).

To determine the thermal stability of the PLD, 100 lL

of PLD solution (2.5 U/mL) was incubated at different

temperatures (10, 20, 30, 37, 40, 50, 60, 70, and 80 �C) for

10 min, and then cooled on ice immediately. Ten micro-

liters of the solution were then used to determine the

residual activity (Tris–HCl, pH 7.5).

2.8 Amino Acid Sequencing

The NH2-terminal amino acid sequence of the purified PLD

protein was determined by the direct protein sequencing

method described by Hirano and Watanabe [11] using a

protein sequencer (model PPSQ-23A; Shimadzu, Kyoto,

Japan) equipped with online HPLC (model LC-10A;

Shimadzu).

2.9 PLD Cloning

Chromosomal DNA was prepared from S. racemochrom-

ogenes strain 10-3 according to the method described

previously [25]. Based on the NH2-terminal amino acid

sequence (NH2-ASPTPHL) of the purified PLD103, two

forward primers (N1 and N2; Supplementary Table 1) were

designed. Furthermore, a reverse primer (S300 Rv; Sup-

plementary Table 1) designed in a previous report [25] was

also employed for PCR. PCR amplification was performed

in a 50 lL reaction mixture, containing genomic DNA

(20 ng), primers (0.2 lM each), dNTP mixture (0.2 mM

each) and 1.25 U PrimeSTAR DNA Polymerase (Takara

Bio, Ohtsu, Japan), with the following cycle parameters:

denaturing for 10 s at 98 �C, primer annealing for 5 s at

55 �C, and primer extension for 1 min 30 s at 72 �C, for 30

cycles. The products were purified and then treated with T4

polynucleotide kinase. The 50 terminally phosphorylated

DNA was subcloned into the HincII-digested site of

pUC118. Plasmid DNA was subjected to cycle sequencing,

and then the sequences were analyzed. To amplify the

upstream and downstream regions of the pld gene, inverse

600 Y. Nakazawa et al.

123

PCR was performed using the circular DNA made from the

SacI or BamHI-digested DNA (50 ng) followed by self-

ligation using a T4 DNA ligase overnight at 16 �C. The

primers for the inverse PCR and nested PCR were designed

based on the nucleotide sequences of the amplified DNA

fragments (Supplementary Table 1).

The amplified PCR products were subcloned into the

pUC118 vector and subjected to cycle sequencing using the

ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit

(Applied Biosystems), universal vector primers, and gene-

specific primers (Supplementary Table 1). The nucleotide

sequence of pld gene reported in this article has been sub-

mitted to the DDBJ/EMBL/Genbank under accession

number AB573232.

2.10 In Silico Analysis

Potential ribosome binding site (rbs) and rho-independent

transcription terminator signals were predicted using

GENETYX software (ver. 9; Genetyx, Tokyo, Japan).

Alignments of the nucleotide sequences of the pld genes

and the amino acid sequences of PLDs were generated

using CLUSTAL X [36]. A 3D model of the PLD103 was

predicted by utilizing structure of Streptomyces sp. strain

PMF PLD (PMF-PLD; PDB ID: 1V0W) as template, using

the homology-modeling server SWISS-MODEL [9] at

http://swissmodel.expasy.org/. Template structure was

downloaded from the Protein Data Bank. The image was

generated using the UCSF Chimera version 1.4.1 (available

at http://www.cgl.ucsf.edu/chimera/) [28] to display ribbon

diagrams of 3D structure of the PLDs. The model exhibited

good geometry having 95.8% of favored regions according

to Ramachandran plots generated using MolProbity

(available at http://molprobility.biochem.duke.edu) [4].

2.11 Transphosphatidylation Analysis

Six milliliters of reaction mixture composed of 1 mg/mL

PC-98N, 4 mM sodium deoxycholate, 20 mM MOPS-

NaOH buffer (pH 7.5), 10 mM CaCl2, 0.12 U PLD, and

200 mg/mL glycerol, L-serine, or 2-aminoethanol hydro-

chloride were incubated at 37 �C. One milliliter of reaction

mixture was transferred at intervals of 30–300 min into the

test tube containing 0.2 mL of 0.1 M NaOH to terminate

the enzyme reaction. The PLs in the reaction mixture were

extracted using the method of Bligh and Dyer [1]. The

chloroform phase was separated and evaporated, and the

resultant lipid residue was dissolved with 100 lL of

chloroform. After centrifugation, 10 lL of the clear chlo-

roform phase was applied onto a silica gel 60 thin-layer

chromatography (TLC) plate (Merck, Darmstdt, Germany).

The plate was developed with chloroform/methanol/acetic

acid (40:15:6, v/v). Spots on the plate were visualized by

iodine vapor, and the intensities of the visualized spots

were measured with the Gel-Pro Analyzer software (ver-

sion 3.1; Media Cybernetics, Silver Spring, MD, USA).

The transphosphatidylation conversion rate (%) was

defined as: [PX] 9 100/[PX] ? [PC] ? [PA], and the

selectivity (%) was defined as: [PX] 9 100/[PX] ? [PA],

where the PX in the formula represents the transphospha-

tidylation product.

3 Results

3.1 Purification and Characterization of PLD Produced

by S. racemochromogenes 10-3

Phospholipase D secreted by S. racemochromogenes 10-3

(PLD103) was purified from the culture supernatant

by anion- and cation-exchange chromatographic steps

(Table 1). The purity of the enzyme was confirmed by both

of SDS–PAGE and IEF. The active fractions eluted from the

final chromatography ran as a single band at 55 kDa on

SDS–PAGE in the presence of 2-mercaptoethanol (Fig. 1a,

lane 4) and at pI 8.0 on IEF (Fig. 1b, lane 4). Starting with

1 L of the culture medium, 300 lg of the purified PLD103,

with a specific activity of 374 U/mg, was obtained. The

NH2-terminal amino acid sequence of PLD103 was deter-

mined to be NH2-ASPTPHLDSVEQTLRQVSPG. This

sequence displayed high homology with other streptomy-

cete PLD sequences (60–85% identity).

The PC-hydrolytic activity of PLD103 is stable in the

pH 4–9 range; over 70% of the original activity remained

Table 1 Summary of the results of the purification of PLD from S. racemochromogenes 10-3

Purification step Protein (mg) Activity (U) Specific activity (U/mg) Purification (-fold) Yield (%)

Culture supernatant 17.1 ± 0.7 365.9 ± 10.5 21.5 ± 0.6 1 100

(NH4)2SO4 precipitation 2.0 ± 0.1 290.5 ± 22.0 142.3 ± 3.3 6.7 ± 0.3 79.9 ± 8.5

CM Sepharose FF 0.5 ± 0.1 200.6 ± 11.1 344.7 ± 7.1 16.1 ± 0.6 55.0 ± 4.2

TOYOPEARL DEAE 650M 0.3 ± 0.02 118.3 ± 5.1 374.3 ± 12.0 17.4 ± 0.5 32.5 ± 2.3

Values are represented as mean ± SE of three independent preparations in 1,000 mL of culture

Purification, Biochemical Characterization, and Cloning of Phospholipase D 601

123

after a 4-h exposure to this pH range (Fig. 2a). The opti-

mum reaction temperature of PLD103 was 50 �C, and the

enzyme remained active with over 80% of the original

activity after 10-min exposures to a 10–60 �C temperature

range (Fig. 2b). Therefore, PLD103 exhibited similarities

in pH stability, optimum temperature, thermal stability of

the hydrolytic activity, and molecular mass to previously

reported enzymes from other Streptomyces strains

(Table 2). On the other hand, PLD103 exhibited significant

differences in optimum pH for the PC-hydrolytic activity to

those observed in other Streptomyces PLDs. While the

majority of the Streptomyces PLD enzymes show the

highest PC-hydrolysis activity at pH 5–6, our enzyme was

the most active at pH 7.5. In acidic conditions (pH 5–6),

PLD103 displayed only 10–50% of the activity compared

to its activity at the optimum pH. Therefore PLD103 dis-

played the highest activity at the slightly alkaline condi-

tion, which is in contrast to the majority of Streptomyces

PLDs that exert the highest activity at slightly acidic

conditions.

3.2 Nucleotide Sequence of the PLD103 Gene

To clarify the primary structure of PLD103 and compare it

to other Streptomyces PLDs, we isolated the PLD103 gene

from the genomic DNA of strain 10-3. We previously

cloned a 274-bp partial fragment of the pld103 gene [25].

In this study, to isolate the entire ORF of pld103, we

designed degenerate primers based on the NH2-terminal

amino acid sequence of the purified enzyme. PCR using

degenerate primers (Supplementary Table 1) yields a

1,390-bp product. To amplify the downstream and

upstream regions of the PCR product, inverse PCR was

performed. Finally, we determined the nucleotide sequence

of a 2,770-bp genomic DNA fragment. In the sequence,

there is a potential rbs (AAGGAA), but the general start

codon, ATG, could not be found near the binding site.

Instead, a TTG codon was located 5-bp downstream from

the rbs and could be a good candidate for the start codon.

The rho-independent transcription terminator signal [29],

consisting of a 36-bp stem-loop sequence, was also found

37-bp downstream of the pld103 gene. Therefore, the

determined sequence contained the entire putative 1,587-bp

ORF that encodes 528 amino acid residues (Fig. 3). The

NH2-terminal amino acid sequence of the purified PLD103

exactly matched with the nucleotide-deduced amino acid

sequence starting at Ala-27. The SignalP 3.0 sever program

[8] predicted that the signal cleavage site is between Ala-26

and Ala-27, which coincides with the NH2-terminal amino

acid sequence of the purified PLD103. Computer-assisted

motif analysis using the protein family database, InterPro,

predicted that the enzyme contains a set of dual PLD active

Fig. 1 Electrophoretic analysis of the purified Streptomyces racemo-chromogenes 10-3 strain phospholipase D (PLD103). a SDS–PAGE

banding profiles of the PLD103 preparation at each purification step.

The molecular mass of the purified enzyme was estimated to be

55 kDa based on the electrophoretic mobility of the purified PLD103

(lane 4) and molecular mass standard proteins (lane M). Lane 1culture supernatant, lane 2 ammonium sulfate precipitation, lane 3

elutant from CM Sepharose and lane 4 elutant from TOYOPEARL

DEAE. b Isoelectric focusing (IEF) banding profile of the purified

PLD103. The purified enzyme eluted from TOYOPEARL DEAE

corresponds to lane 4 in panel a. The isoelectric point (pI) of PLD103

was estimated to be 8.0 based on the electrophoretic mobility of the

pI standard proteins (lane M) concomitantly applied to the IEF

602 Y. Nakazawa et al.

123

site HKD motifs at the residue segments His-192–Asp-199

and His-462–Asp-469.

A comparison of the sequences of Streptomyces PLDs,

whose enzymatic properties have been published (Table 2),

using CLUSTAL X multiple-sequence alignment analysis

indicated that PLD103 shares relatively low sequence

homology (71–76% identical) with other Streptomyces

PLDs (Fig. 4a). Hence, the difference in the optimum pH

for hydrolytic activity observed among enzymes might

result from the diversity of their primary structures. To find

out the difference in 3D structures of the PLD103 and the

other Streptomyces PLDs, the computer modeled 3D

structures of the enzymes were generated (Fig. 4b). The

comparison of the models indicated that the diversity in the

primary structure of the PLD103 generates the disruption

of one of 18 a helices.

3.3 PG-, PS-, and PE-Producing

Transphosphatidylation by PLD103

The PG-, PS-, and PE-producing transphosphatidylation

activities of PLD103 were examined in an aqueous one-

phase reaction mixture in which 1 mg/mL egg yolk lecithin

(98.9% PC) was used as a substrate and was dispersed with

a small amount of detergent (4 mM deoxycholate). In the

PG-producing reaction, 97.1% of the substrate PC was

converted into PG by using glycerol as the acceptor. The

selectivity for transphosphatidylation instead of hydrolysis

was significantly high; 97.7% of the product resulted from

transphosphatidylation; only 2.3% was the hydrolytic

product PA (Fig. 5a). In the PS-producing transphosphati-

dylation reaction, 82.5% of the substrate PC was converted

into PS. Of the reaction products, 88.9% resulted from the

Fig. 2 Effect of pH (a) and temperature (b) on the activity and

stability of PLD103. The maximal enzyme activities observed were

set as 100% relative activity. a To determine the optimum pH

(indicated with closed circles), enzyme activity was measured under

the standard assay conditions with various pH values. To determine

the pH stability of PLD103 (indicated with open circles), the enzyme

was incubated in buffers with various pHs at 25 �C for 4 h prior to the

standard assay. b To determine the optimum temperature (indicated

with closed circles), enzyme activity was measured under standard

assay conditions at varying temperatures. To determine the heat

stability of PLD103 (indicated with open circles), the enzyme was

incubated at different temperatures for 10 min prior to the standard

assay. Both experiments were repeated in triplicate, and the errorbars represent the standard error of the means

Table 2 Enzymatic properties of streptomycete PLD enzymes

Specific activity

(U/mg)

Molecular

mass (kDa)

pI Optimum pH Optimum

temperature (�C)

pH stability Thermal

stability (�C)

References

PLD103 392 55 8.0 7.5 50 4.0–9.0 \60 This work

PMF-PLD 42 54 9.1 5.0 60 4.0–9.0 ND [3]

TH2-PLD 46 55 6.5 5.0 55 4.0–9.0 \60 [10]

Sci PLD 468 54 ND 6.0 50 ND ND [27]

San PLD 1,437 64 6.5 5.5 60 4.0–8.0 \50 [30]

PLD103, Streptomyces racemochromogenes strain 10-3 PLD; PMF-PLD, Streptomyces sp. strain PMF PLD; TH2-PLD, Streptomyces septatusstrain TH-2 PLD; Sci PLD, Streptovelticillium cinnamoneum PLD; San PLD, Streptomyces antibioticus strain S-170

Purification, Biochemical Characterization, and Cloning of Phospholipase D 603

123

transphosphatidylation reaction, and 10.3% resulted from

the hydrolytic reaction. Thus, the apparent transphospha-

tidylation efficiency for PS production was inferior to that

for PG production. Moreover, PS production reached a

plateau after 120 min, indicating that increased production

of PS could not be expected even with the additional

reaction time (Fig. 5b). Recently, Iwasaki et al. [14]

attempted efficient PS production in various aqueous

Fig. 3 Genomic nucleotide sequence of the S. racemochromogenes10-3 pld gene open reading frame (ORF) and the deduced amino acid

sequence of PLD103. The potential ribosome binding site (rbs) is

underlined upstream of the ORF. A putative initiation TTG codon is

boxed. Arrows below the nucleotide sequence show the region of

dyad symmetry for the rho-independent transcription terminator

signal, which consists of a stem-loop sequence. A double underlinebelow the deduced amino acid sequence indicates the determined

NH2-terminal amino acid sequence based on the direct sequencing of

purified PLD103. The dotted lines below the deduced amino acid

sequence indicate the HKD motifs in the PLD enzyme. The putative

signal cleavage site predicted is indicated with an arrowhead

604 Y. Nakazawa et al.

123

a

b

Purification, Biochemical Characterization, and Cloning of Phospholipase D 605

123

reaction systems using S. antibioticus PLD produced by

recombinant E. coli. In an aqueous mixture in which the

soybean lecithin was simply dispersed in aqueous buffer,

PLD generated only 20% PS. However, they improved the

production of PS and generated 80% PS, using an ‘‘aque-

ous suspension system’’ in which calcium sulfate in a fine

powder form and lecithin were suspended. In this study,

PLD103 produced an equivalent yield even in a simple

aqueous system without any additives, and the efficiency

slightly outperformed the S. antibioticus PLD in the

‘‘aqueous suspension system’’. In the PE-producing trans-

phosphatidylation reaction, 98.3% of the reaction product

resulted from the transphosphatidylation reaction, gener-

ating only 1.5% hydrolytic product. Although the conver-

sion rate from PC to PE reached 88.1%, a considerable

amount of unconverted PC (10.4%) remained after a 300-

min reaction. However, as PE production did not reach a

Fig. 5 Time course for the

phospholipid (PL) products in

the phosphatidylglycerol (PG)-,

phosphatidylserine (PS)-, and

phosphatidylethanolamine

(PE)-producing

transphosphatidylation reaction

(a–c, respectively). Left panels,

the PL standard (STD) and

reaction mixture were

developed on a thin-layer

chromatography (TLC) plate as

described in Sect. 2. Rightpanels, the PL contents (%),

derived from the spot intensities

in TLC and are indicated by a

closed circle for PC, a closed

square for

transphosphatidylation

products, and an open triangle

for PA. The selectivity (%),

which was calculated as

described in Sect. 2, is indicated

by an x-mark. All experiments

were repeated in triplicate, and

the error bars represent the

standard error of the means

Fig. 4 Alignment of the amino acid sequences and computer

modeled 3D structures of the Streptomyces PLDs. a The multiple-

sequence alignment was generated by CLUSTAL X using the amino

acid sequences of PLD103 and the PLDs whose enzymatic properties

were characterized previously, as shown in Table 2. The PLD

residues that match the PLD103 sequence are boxed. Shaded residues

represent the a9 helix domain of each PLD. The amino acid sequence

identities between PLD103 and each enzyme are indicated as

percentages in the bottom column. The accession numbers used in

this analysis are as follows: Streptomyces racemochromogenes strain

10-3 PLD (PLD103), AB573232; Streptovelticillium cinnamoneumPLD (Sci PLD), AB007132; Streptomyces septatus strain TH-2 PLD

(TH2-PLD), AB058783; Streptomyces antibioticus strain S-170 (San

PLD), D16444; Streptomyces sp. strain PMF PLD (PMF-PLD),

P84147. b The models of PLD103 and PMF-PLD 3D structures.

Catalytic His and Lys residues are indicated with black and gray in

the dotted circle, respectively. NH2– and COOH–termini are

indicated with N and C, respectively. The a9 helices indicated with

solid arrow in the PMF-PLD model are observed in all PLDs, but is

disrupted only in the PLD103 (dotted arrow)

b

606 Y. Nakazawa et al.

123

plateau and the hydrolytic product was scarcely observed

after 300 min, further PE production would be achieved

after additional reaction time (Fig. 5c).

The transphosphatidylation conversion rates of the com-

mercially available Streptomyces sp. PLD-P (Asahi-Kasei,

Tokyo, Japan) and S. chromofuscus PLD (Asahi-Kasei) in

the same aqueous one-phase system were 54.1 and 10.7% for

PG-, 80.4 and 0.6% for PS-, and 75.4 and 4.5% for the PE-

producing reactions after a 300 min incubation, respectively

(data not shown). Therefore, the transphosphatidylation

conversion rates of PLD103 were much higher than already

known Streptomyces PLDs in the aqueous one-phase system.

4 Discussion

In this study, we identified diversity among the amino acid

sequences of PLD103 and other Streptomyces PLDs, which

might affect on the optimum pH of the enzyme. Notably,

the diversity found in the a9 helix domain generates the

significant alternation of the secondary structure in the

computer modeled 3D structure. The a9 helix, which is

disrupted in the PLD103 model, is located on opposite side

of the active pocket. Therefore, this domain would not

directly contact to the substrate molecule, but may be

responsible for the accessibility to the substrate due to

structural change of the enzyme in a pH dependent manner.

Very recently, Simkhada et al. [32, 33] isolated alkaline

PLDs, exerting highest activity at pH 8, from Streptomyces

species. Although the primary structures of these PLDs are

not available at present, comparison of the amino acid

sequences of our PLD103 and these enzymes would be

worthwhile to clarify the relation of the primary structure

and pH optimum of the enzyme.

PLD103 transfers lecithin-derived PC into PG, PS, and

PE with high efficiency and selectivity even in the one-

phase aqueous system at a rate that is comparable to the

efficiency of other Streptomyces PLDs, which were

assayed in biphasic systems. Because of its ubiquitous

distribution, the in vivo functions of plant and animal PLD

have been well investigated. Furthermore, the dual reac-

tions performed by PLD have been a longstanding and

well-known property of the enzyme, regardless of the

source. Despite this, elucidating the detailed reaction

mechanism of transphosphatidylation and hydrolysis by

PLD had been hampered partially due to low productivity

of the enzyme. Further investigation of PLD103, including

overexpression in proper bacterium, may not only con-

tribute to its use in industrial fields because of its unique

property of transphosphatidylation activity in an aqueous

system, but may also reveal the transphosphatidylation

mechanism of the PLD enzyme.

Acknowledgments This work was partially supported by Grant-in-

aid for Scientific Research from the Kieikai Foundation (Tokyo,

Japan), which is greatly appreciated. We thank Mr. K. Hirabayashi

and Mr. S. Sakurai for their technical assistances. We also thank

Professor T. Nagashima (Tokyo University of Agriculture, Japan) for

his excellent contributions to this work.

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