Purification of phospholipase D by two-phase affinity extraction.

Abstract

An aqueous two-phase system of polyethylene glycol (PEG)-salt was used for purification of phospholipase D (PLD) from peanuts and carrots. Alginate, a known macroaffinity ligand for PLD, was incorporated in the PEG phase and resulted in 91 and 93% of the enzyme activity (from peanuts and carrots, respectively) getting partitioned in the PEG phase. The elution of the enzyme from alginate was facilitated by exploiting the fact that the latter can be reversibly precipitated in the presence of Ca2+. The enzyme was eluted from the polymer by using 0.5 M NaCl. Peanuts and carrots PLD could be purified 78- and 17-fold with 82 and 85% activity recovery, respectively. The purified enzyme from both sources gave a single band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.

Full text

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Purification of phospholipase D by two-phase affinity extraction
S. Teotia, M.N. Gupta*
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India
Received 15 August 2003; received in revised form 28 October 2003; accepted 28 October 2003
Abstract
An aqueous two-phase system of polyethylene glycol (PEG)-salt was used for purification of phospholipase D (PLD) from peanuts and
carrots. Alginate, a known macroaffinity ligand for PLD, was incorporated in the PEG phase and resulted in 91 and 93% of the enzyme
activity (from peanuts and carrots, respectively) getting partitioned in the PEG phase. The elution of the enzyme from alginate was facilitated
by exploiting the fact that the latter can be reversibly precipitated in the presence of Ca2+. The enzyme was eluted from the polymer by using
0.5 M NaCl. Peanuts and carrots PLD could be purified 78- and 17-fold with 82 and 85% activity recovery, respectively. The purified enzyme
from both sources gave a single band on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.
Keywords: Affinity extraction; Aqueous two-phase systems; Phospholipases; Enzymes; Alginate
1. Introduction
Two-phase affinity extractions have been widely used for
separation of enzymes/proteins [1-3]. Such an approach
combines the high selectivity of an affinity step with the well
known advantage of two-phase aqueous system that one
can directly deal with crude broths containing suspended
matter (e.g. cell debris). Kamihira et al. [4] had outlined a
useful strategy in which the affinity ligand incorporated in
the polyethylene glycol (PEG) phase of PEG-Reppal PES
200 (modified starch) was a smart macroaffinity ligand. A
smart macroaffinity ligand [5-7] in this context is an affinity
ligand linked to a reversibly soluble-insoluble polymer, the
solubility of which can be altered by an external stimulus
such as change inpH, temperature or presence of chemicals.
The approach of Kamihira et al. [4] allowed easy separation
of complex of affinity ligand and the target protein from
PEG phase. Thus, both PEG as well as affinity ligand could
be reused. More recently, we have shown that this strategy
could be utilized to purify wheat germ a-amylase [8], sweet
potato (3-amylase [8], xylanase [9] and pullulanase [9]. For
all starch degrading enzymes, alginate was used as such
as the smart macroaffinity ligand [10-12]. Alginate has
also shown to be a smart macroaffinity ligand for peanut
phospholipase D (PLD) [13]. PLD hydrolyses lecithin into
phosphatidic acid and choline. The enzyme has been impli-
cated in a number of cellular processes [14,15]. Recently,
Servi [16] has described a number of important synthetic
applications of PLD. Thus, a facile purification method for
PLD should further the study of its enzymology and ap-
plications. The present work describes such a method for
purification of phospholipase D from peanuts and carrots.
2. Materials and methods
2.1. Materials
Sodium alginate (catalog no. A-2158, composed primar-
ily of mannuronic acid residues) and standard molecular
weight markers were purchased from Sigma, St. Louis, MO,
USA. Soybean lecithin (phosphatidyl choline) was obtained
from BDH, E. Merck, Mumbai, India. Polyethylene glycol
(PEG 6000) was from E. Merck, Mumbai, India. Peanuts
and carrots were purchased from the local market. All other
chemicals were of analytical grade.
2.2. Methods
2.2.1. Preparation of phospholipase D crude extracts
The crude extracts of the enzyme from peanuts and carrots
were prepared as described earlier [13]. Dry peanut seeds

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(200 g) were washed with a commercial detergent containing
approximately 4% (w/v) SDS, rinsed thoroughly with water
and soaked overnight in water at 26 °C in an orbital shaker.
The covers (testae) were removed and the dehusked seeds
were homogenized for 1 min at 4 °C in a chilled Waring
blender with 600 ml of a solution containing 0.25 M sucrose,
50 mM Tris, 2 mM EDTA and 3 mM 2-mercaptoethanol
(pH 7.0).
Carrot roots (200 g) were washed, chopped and then ho-
mogenized in distilled water in a pre-chilled Waring blender
at 4°C for 3 min.
The actual purification work was carried out with a non-
clarified crude extract. For optimization work, the clarified
extract after centrifugation was used.
2.2.2. Preparation of alginate solution
Sodium alginate solution (2%) was prepared by dissolving
200 mg of sodium alginate in 10 ml of distilled water, pH
adjusted to 7.0 [13].
2.2.3. Preparation of PEG-salt two-phase system
To 10 ml of graduated centrifuge tubes, the desired phase
components in the order of PEG solution (2.5 ml from the
stock solution of 22% (w/v) polyethylene glycol 6000 in
10 mM phosphate buffer, pH 7.0) and salt solution (2.5 ml
stock solution of 10% K2HPO4 (w/v), and 12% (w/v), NaCl
in 10 mM phosphate buffer, pH 7.0) were added. After vor-
texing for a minute, two distinct phases were formed within
5 min at 25 °C.
2.2.4. Enzyme assay
Phospholipase D activity was assayed by a titrimetric
method using soybean lecithin as substrate. One unit is de-
fined as the amount of enzyme which liberates one micro-
mole of acid from soybean lecithin per minute at 25 °C at
assay pH [17]. The activity measurements for various sys-
tems were carried out by withdrawing aliquots in the range
of 100-500 fxl.
2.2.5. Glycoprotein assay (phenol sulphuric acid test)
Twenty-five microliters of 80% phenol solution in distilled
water was added to 1.0 ml of the sample. To the same mixture
2.5 ml of concentrated sulphuric acid was added and kept at
25 °C for 10 min. Absorbance was read at 489 run [18].
2.2.6. Protein estimation
Protein was estimated according to the dye binding
method using bovine serum albumin as the standard protein
[19]. These estimations were done by withdrawing aliquots
from various systems in the range of 100-500 \xl.
2.2.7. Binding of phospholipase D from peanut and
carrot with alginate as macroaffinity ligand in aqueous
two-phase system
Aqueous two-phase separation using the PEG-salt system
was tried for phospholipase D after incorporating 0.2% (w/v)
alginate solution. The crude preparations of peanut (2.0 ml
containing 10 U) and carrot (2.0 ml containing 35 U) were
added to the respective systems. These two systems were
prepared as described earlier. During the purification work,
unclarified crude extracts were used. In such cases, PEG-
and salt phases were separated by an interface consisting of
cell debris and other insoluble matter. After vortexing, the
phases were separated after 30 min. The volume of the total
system before adding the crude extracts were 5.0 ml (with-
out alginate) and 6.0 ml (with alginate), respectively. Algi-
nate distribution was restricted to the PEG phase with less
than 5% (of the initially added amount) partitioning to the
bottom phase. Alginate concentration in the two phases was
estimated by phenol sulphuric acid method [18]. The top
phase containing the enzyme bound alginate was removed
with a pipette. The alginate was precipitated in the presence
of 70 mM Ca2+ [20,21] by incubating for 20 min at 25 °C.
The precipitate was centrifuged at 8000 x g for 10 min at
25 °C. The supernatant and subsequent washings with buffer
(till no enzyme activity was detected in the washings) were
collected. Bound phospholipase D was calculated by the dif-
ference of initial activities loaded on alginate and recovered
in supernatant and washings. The elution of the bound en-
zyme was tried using 2.0 ml of 0.5 M NaCl and were kept
at 4 °C for overnight incubation. Polymer was recovered by
precipitation with CaCl2, followed by centrifugation and en-
zyme activity was estimated in the supernatant [20,21]. After
removal of the top phase, the lower aqueous phase was sepa-
rated by using a pipette which pierced through the interface.
The interface left was also analyzed for protein and activity.
2.2.8. Polyacrylamide gel electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophore-
sis (SDS-PAGE) of the protein samples was performed
using 12% gel according to Hames [22] on a Genei gel
electrophoresis unit (Bangalore Genei, Bangalore, India).
3. Results
Earlier work has shown that alginate could be incorpo-
rated in the PEG-salt two-phase system [8,9]. Fig 1a shows
the variation in extent of activity bound and eluted (to algi-
nate in PEG-salt two-phase systems) with the different start-
ing amounts of peanut phospholipase D activity in the crude
extract. As number of enzyme molecules increase, they start
occupying available affinity sites on the incorporated algi-
nate molecules. As the extent of occupancy increases, the
"crowding effect" prevents easy access to incoming enzyme
molecules. At the point, the available affinity sites are mostly
occupied. This gets reflected in the "approach to saturation"
phase with decreased extent of binding of initial activity
load. About 10 U activity as starting load was found to be op-
timum. The elution throughout was in the range of 89-94%
of the bound activity. Table 1 shows that peanut PLD activ-
ity (10 U) got distributed more or less evenly in PEG-salt

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299
Table 1
Purification of peanut phospholipase D using PEG-alginate-salt two-phase system
Steps
Activity (U)Protein (mg) Specific activity (Umg ')Yield (%) Fold purification
Unclarified crude extract 10
Elution8.2
15
0.7
Lower phase (salt)
No alginate
+Alginate
Interface
No alginate
+Alginate
Upper phase (PEG)
No alginate
+Alginate (supernatant + washing)
4.5
0.6
1.5
0.3
3.5
0.4
7.3
7.5
3.2
2.7
2.5
1.0
0.6
0.1
0.5
0.1
1.4
0.15 56
100
45
1
15
3
35
4
82
1
-
2
-
78
Purification was carried out using 0.2% sodium alginate solution. The elution was carried out using 2.0ml of 0.5 M NaCl at 4°C for overnight. The
reaction velocity was measured titrimetrically using 0.02 M NaOH by measuring the rate of proton liberation during hydrolysis of soybean lecithin. All
the experiments were performed in duplicate and the difference in the readings in the duplicates was less than ±5%.
two-phase system. However, incorporation of alginate in the
PEG phase dramatically improved the partition of the en-
zyme activity in the PEG phase. The alginate-enzyme com-
plex could be separated from PEG phase by precipitation
with Ca2+ [20,21] and the enzyme activity was recovered
by dissolving the precipitate in 0.5 M NaCl and precipitating
alginate alone by adding Ca2+ [20]. 82% enzyme activity
could be recovered with 78-fold purification. Fig. 2a shows
the SDS-PAGE of the purified enzyme which appeared as a
single band at molecular mass position of 22 000 Da. This
molecular mass is in agreement with what has been reported
earlier for this enzyme [13,23].
Similar results were obtained in the case of carrot PLD.
Fig. 1b shows that 39U of enzyme activity was optimum
load in this case for binding maximum amount of activity.
Again, extent of bound activity which got eluted with dif-
ferent starting activity loads did not show much variation.
It was in the range of 92-96% of bound activity. Table 2
shows that incorporation of alginate again led to partition of
90% of applied activity (35 U) to the PEG-alginate phase.
Following similar procedure as in the case of peanut PLD,
85% of initial activity could be recovered with 17-fold pu-
rification. SDS-PAGE (Fig. 2b) shows a single band for the
purified preparation in this case as well. Again the molec-
ular mass obtained (60 000 Da) was in agreement with the
value reported earlier [24].
4. Discussion
The various purification protocols described for PLD gen-
erally consists of multi-step procedures [25,26]. In the case
of PLD from peanut and carrot, simpler and shorter purifi-
cation procedures have been developed in this laboratory
[13,24]. Thus, it may be worthwhile to compare the re-
sults obtained here with those described earlier [13,24]. PLD
Table 2
Purification of carrot phospholipase D using PEG-alginate-salt two-phase system
Steps
Activity (U)Protein (mg)Specific activity (Umg ') Yield (%) Fold Purification
Unclarified crude extract 35
Elution29.8
500
70
100
Lower phase (salt)
No alginate
+Alginate
Interface
No alginate
+Alginate
Upper phase (PEG)
No alginate
+Alginate (supernatant +f- washing)
14
1.8
3.8
0.7
15.8
1.4
200
220
100
140
110
70
70
8
39
5
143
20
40
5
11
2
45
4
1
-
1
-
2
3
2511908517
Purification was carried out using 0.2% sodium alginate solution. The elution was carried out using 2.0ml of 0.5 M NaCl at 4°C for overnight. The
reaction velocity was measured titrimetrically using 0.02 M NaOH by measuring the rate of proton liberation during hydrolysis of soybean lecithin. All
the experiments were performed in duplicate and the difference in the readings in the duplicates was less than ±5%.

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S. Teotia, M.N. Gupta/Journal of Chromatography A, 1025 (2004) 297-301
100 j
90 -
80 -
70 -
60 -
50 -
40 -
30 -
20 - -
10 -
Bound activity (%)
EZ3Eluted activity (%)
—•— Bound activity (U)
15
13
9
7
5
3
1
re
•D
C
3o
m
(a)
0 | I I -i | I I - I [ I I - - I | I I - ' - I | I I I | I I . J | I I I | . - I
2.5 5 7.5 10 15 20 30
Peanut phospholipase D activity loaded (U)
i
EZSEIuted activity (%)
- • - Bound activity (U)
i Bound activity (%)
100 T
tlVI
o
re
•a
lute
o
•a
c
re
•a
c
3o
m
90 -
80 -
70 -
60 -
50 -
40 -
30 -
20 --
10 -
60
50
40
30
20 g
0
00
10
0
(b)
9.75
Carrot phospholipase D activity loaded (U)
19.5 39 58.5 78 97.5
Fig. 1. (a) Effect of enzyme activity loaded (2.5-20 U) on the binding
and elution of peanut phospholipase D to alginate (0.2%) solution. The
binding of phospholipase D was carried out as described in Section 2. For
the optimization experiment, the clear crude extract after centrifugation
(15000 x g, 30min, 10°C) was used. Bound phospholipase D activity
was calculated by the difference of initial activity loaded and the activ-
ities of the supernatant and washing. Phospholipase D activity was de-
termined titrimetrically using soybean lecithin as substrate. (b) Effect of
enzyme activity loaded (9.75-78 U) on the binding and elution of carrot
phospholipase D to alginate (0.2%) solution. For the optimization exper-
iment, the clear crude extract after centrifugation (15 000 x g, 30min,
10 °C) was used. The binding and elution of phospholipase D was car-
ried out as described in Section 2. Bound phospholipase D activity was
calculated by the difference of initial activity loaded and the activities of
the supernatant and washing. Phospholipase D activity was determined
titrimetrically using soybean lecithin as substrate.
from peanut has been purified earlier by affinity precipitation
with alginate itself [13]. The yield as well as fold purifica-
tion in that case were lower (55% and 34-fold, respectively)
than those obtained in the present work (82% and 78-fold).
kDa
97
66
45
21
18
16
Fig. 2. (a) SDS-PAGE pattern of peanut phospholipase D. Lane 1: Crude
peanut phospholipase D; lane 2: marker proteins; lane 3: purified prepa-
ration; (b) SDS-PAGE pattern of carrot phospholipase D. Lane 1: crude
carrot phospholipase D; lane 2: marker proteins; lane 3: purified prepa-
ration.
Besides, in the case of affinity precipitation approach, the
starting material had to be centrifuged before carrying out
the process. In the present work, no such step was used/
required.
PLD from carrot has been purified earlier by three-phase
partitioning (TPP) with an activity recovery of 72% and
13-fold purification [24]. Again, better recovery of activity
and fold purification were obtained in the present case. In
the case of this enzyme as well, the TPP work was carried
out with the crude extract after centrifugation.
The results described here show that PLD from peanut and
carrot can be purified without any preprocessing/clarification
step by two-phase affinity extraction. Apart from Kami-
hira et al. [4], polymer-dye conjugates have also been used
as smart macroaffinity ligand in two-phase system [27]. In
both cases, the two-phase system is of polymer-polymer
systems. In the present work, more economical PEG-salt
system has been used. The second important aspect of the
present approach is that inherent affinity of a naturally oc-
curring polysaccharide has been used. Thus, no conjugation

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301
step of crafting a polymer-affinity ligand was involved. The
usefulness of exploiting this unexpected affinity of both nat-
urally occurring and synthetic polymer in bioseparation has
been discussed earlier [28,29]. Thus, the present approach
can be extended to use of various economical and easily
available polymers for purification of large number of en-
zymes/proteins.
Acknowledgements
This work was supported by Council for Scientific and In-
dustrial Research (Technology Mission on Oilseeds, Pulses
and Maize), India. The financial support of Department of
Science and Technology (DST) to S.T. is also acknowledged.
References
[1] B.A. Andrews, J.A. Asenjo, in: E.L.V. Harris, S. Angel (Eds.), Protein
Purification Methods, IRL Press, Oxford, 1989, p. 161.
[2] J.G. Huddleston, A. Lyddiatt, in: M. Verrall (Ed.), Downstream
Processing of Natural Products, Wiley, Chichester, 1996, p. 53.
[3] R. Hatti-Kaul (Ed.), Aqueous Two Phase Systems—Methods and
Protocols, Humana Press, NJ, 2000.
[4] M. Kamihira, R. Kaul, B. Mattiasson, Biotechnol. Bioeng. 40 (1992)
1381.
[5] I. Roy, M.N. Gupta, in: M.N. Gupta (Ed.), Methods for Affinity-Based
Separation of Proteins/Enzymes, Birkhäuser, Basel, 2002, p. 130.
[6] A. Sharma, M.N. Gupta, Biotechnol. Bioeng. 80 (2002) 228.
[7] A. Sharma, K. Mondal, M.N. Gupta, J. Chromatogr. A 995 (2003)
127.
[8] S. Teotia, M.N. Gupta, J. Chromatogr. A 923 (2001) 275.
[9] S. Teotia, M.N. Gupta, Protein Exp. Purif. 22 (2001) 484.
[10] S. Teotia, R. Lata, S.K. Khare, M.N. Gupta, J. Mol. Recognit. 14
(2001) 295.
[11] S. Teotia, S.K. Khare, M.N. Gupta, Enzyme Microb. Technol. 28
(2001) 792.
[12] I. Roy, M.N. Gupta, J. Chromatogr. A 950 (2002) 131.
[13] S. Sharma, A. Sharma, M.N. Gupta, Bioseparation 9 (2000) 92.
[14] A.J. Morris, M.A. Frohman, J. Engebrecht, Anal. Biochem. 252
(1997) 1.
[15] K. Pappan, S. Zheng, X. Wang, J. Biol. Chem. 272 (1997) 7048.
[16] S. Servi, in: W.D. Fessner (Ed.), Topics in Current Chemistry, vol.
200, Springer-Verlag, Berlin, 1999, p. 127.
[17] L.A. Decker, Worthington Enzyme Manual, Worthington Biochemi-
cal Corp., Freehold, NJ, 1977, p. 173.
[18] C.H.W. Hirs (Ed.), Methods Enzymol. 11 (1967) 411.
[19] M.M. Bradford, Anal. Biochem. 72 (1976) 248.
[20] A. Sharma, S. Sharma, M.N. Gupta, Protein Exp. Purif. 18 (2000)
111.
[21] O. Smidsrod, G. Skjak-Braek, Trends Biotechnol. 8 (1990) 71.
[22] B.D. Hames, in: B.D. Hames, D. Rickwood (Eds.), Gel Electrophore-
sis of Proteins. A Practical Approach, IRL Press, Oxford, 1986, p. 1.
[23] M. Heller, N. Mozes, I. Peri, E. Maes, Biochim. Biophys. Acta 369
(1974) 397.
[24] S. Sharma, M.N. Gupta, Protein Exp. Purif. 21 (2001) 310.
[25] M. Heller, N. Mozes, I. Peri, E. Maes, Methods Enzymol. 14 (1969)
226.
[26] S. Imamura, Y. Horiuti, J. Biochem. (Tokyo) 85 (1979) 79.
[27] D. Guoqiang, R. Kaul-Hatti, B. Mattiasson, J. Chromatogr. A 668
(1994) 145.
[28] I. Roy, M.N. Gupta, Enzyme Microb. Technol. 27 (2000) 53.
[29] I. Roy, M.N. Gupta, Sep. Sci. Technol. 37 (2002) 1591.