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Journal of General Microbiology (1980), 118, 59-65.
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59
Purification and Partial Characterization of an Acid Phosphatase
(EC 3.1.3.2) Produced by Pvopionibacterium acnes
By E I L E E N INGHAM,* l K. T. H O L L A N D , 2 G. G O W L A N D l
A N D w. J. CUNLIFFE3
University Departments of Immunology1, Microbiology2 and Dermatology3,
The General Infirmary, Leeds LS1 3EX
(Received 28 August 1979; revised 21 November 1979)
A strain of Propionibacterium acnes (type I; Marples & McGinley, 1974), isolated from a
blackhead acne lesion, produced an acid phosphatase which was present in the culture
supernatant in the late-exponential and early-stationary phases of growth. This acid
phosphatase was purified more than 45000-fold (4.5 yo yield). The purified enzyme gave
two protein bands on sodium dodecyl sulphate-polyacrylamide gel electrophoresis corresponding to molecular weights of 155000 and 87 100. The enzyme had a single peak of
activity on Sephadex G-100, with a molecular weight corresponding to 93000. The highly
purified acid phosphatase had an optimum activity a t pH 5.8, was stable from pH 4.0 to
5.5 and was totally inactivated after 30 min at 55 "C.The enzyme did not show an absolute
requirement for metal ions, but was stimulated by Mg2+, Ca2+,Zn2+and K+ at concentrations between 0.1 and 1 mM. The acid phosphatase was active against a number of monophosphate esters.
INTRODUCTION
Propionibacterium acnes produces at least three extracellular enzymes ; a lipase (Reisner
et al., 1968), a protease (Marples & McGinley, 1974) and a hyaluronidase (Puhvel & Reisner,
1972). The production of 'extracellular' phosphatase by P. acnes has not been reported
previously. Two independent investigations showed P. acnes to have no or weak phosphatase activity. Porschen & Spaulding (1974) found no phosphatase activity in four strains
of P. acnes, using whole cell suspensions and assaying the release of free phthalein from
phenolphthalein monophosphate and testing a t pH values between 4.0 and 8.6. Hoeffler
(1977) used a plate method for screening 40 P. acnes strains for phosphatase production and
found that 21 of them were weakly positive.
This communication describes the production of an ' extracellular ' acid phosphatase produced by a strain of P. acnes in the late-exponential and early-stationary phases of growth
in batch culture. The enzyme was isolated and several of its properties are described.
METHODS
Reagents. Most biochemicals were obtained from Sigma. 4-Nitrophenyl disodium orthophosphate and the
zwitterionic buffers 2-(N-morpholino)ethanesulphonic acid (MES) and 3-(N-morpholino)propanesulphonic
acid (MOPS) were obtained from BDH. Alcohol dehydrogenase (yeast) was from Boehringer. Sephadex
G-100, CM-Sephadex (2-50 and Blue Dextran 2000 were from Pharmacia.
Bacterial strain. A laboratory strain of P. acnes (type I; Marples & McGinley, 1974) isolated from a blackhead lesion on a patient in Leeds General Infirmary, and designated P-37, was used throughout this study.
Stock cultures were maintained in 40 % (w/v) glycerol in 0.1 M-potassium phosphate-buffered saline (PBS,
pH 7.3) at - 20 "C.
Batch culture studies. Three litres of Brain Heart Infusion broth (Difco) supplemented with 0-3:;(w/v)
0022-1287/80/0000-8933 $02.00 @ 1980 SGM
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60
E . I N G H A M A N D OTHERS
glucose was inoculated with 0.5 % (v/v) exponential phase culture of strain P-37 in the same medium. The
culture was grown at 37 "C and stirred at 500 rev. min-'. Anaerobiosis was maintained by a continuous flow
of nitrogen at 150 ml min-l with vortex mixing.
Dry weight detennination. A 10 ml culture sample was centrifuged at 3000 g for 10 min. The cells were
resuspended in PBS and then deposited on pre-weighed Nuflow membrane filters (0.45 pm pore size) which
were dried at 85 "C and reweighed.
Acid phosphatase assay. Acid phosphatase (EC 3 . 1 . 3 . 2 ) was assayed using 4-nitrophenyl disodium
orthophosphate (PNP-P) as substrate (Bessey et al., 1946). The reaction mixture contained 2.5 ml 0.1 Msodium acetate buffer (pH 5.8), 1 mM-MgCl,, 0.5 ml 1 (w/v) PNP-P and 0-5 ml of a suitable dilution of
enzyme preparation. One ml of the reaction mixture was transferred to 2 ml 0.2 M-NaOH before and after
15 min incubation at 37 "C.The amount of 4-nitrophenol (PNP) liberated was measured by recording the
absorbance at 420 nmusinganappropriatecalibration curve. Activity is expressed aspmol PNP liberated min-'.
Hyaluronate lyase (hyaluronidase) assay. Hyaluronidase (EC 4.2.2.1) was assayed by the appearance
of N-acetyl-D-glucosamine end-group colour with umbilical cord hyaluronic acid as substrate. To 2.5 ml
0.1 M-sodium acetate buffer (pH 6.4) was added 0-5 m l O . 1 ~
(w/v) hyaluronic acid and 0.5 ml of a suitable
dilution of enzyme in the same buffer. After incubation at 37 "C for 15 min, 0.5 ml of the reaction mixture
was added to 0.1 ml 0.8 M-potassium tetraborate (pH 9.2). N-Acetylglucosamine end-group colour was
determined by the method of Reissig et al. (1955) using N-acetylglucosaniineas the standard. No breakdown of hyaluronic acid occurred at pH 6.4 in the absence of the enzyme. Substrate blank controls were
included. One unit of enzyme activity is equivalent to 1 pmol N-acetylglucosamine formed min-l (limit of
sensitivity of 0.001 pmol N-acetylglucosamine formed ml-l min-I).
Lipuse assay. Lipase (EC 3.1.1.3) was assayed using triolein as substrate. The reaction mixture contained 0.5 ml triolein emulsion [lo % (w/v) triolein emulsified with 5 % (w/v) gum acacia at top speed on a
Polytron homogenizer for 1 min], 0.5 ml enzyme solution and 2-5 ml 0.1 M-citrate/phosphate buffer
(pH 6.5). One ml of reaction mixture was transferred to 5 ml Dole's reagent before and after 1 h incubation
at 37 "C.The amount of oleic acid released was determined by the method of Dole & Meinertz (1960) using
oleic acid as the standard. Activity is expressed as pmol oleic acid formed min-l (limit of sensitivity of 0.01
pmol oleic acid formed ml-l min-l).
Protease assay. Protease was determined by the method of Millet (1970) using azocasein as substrate.
The reaction mixture contained 1 ml 1 % (w/v) azocasein in 0.1 M-MES(pH 6.5) and 1 ml enzyme solution.
Trichloroacetic acid (2 ml; lo%, w/v) was added to control tubes at time zero and to assay tubes after up
to 16 h incubation at 37 "C. Enzyme blanks were included. After centrifugation and filtration, the absorbances at 440 nm were recorded.
Glucose assay. Glucose was assayed according to the method of Hugget & Nixon (1957).
Phosphatase activity of P . acnes isolates. Twenty-four isolates of P. acnes (type I; Marples & McGinIey,
1974) from foreheads were tested for the production of extracellular phosphatase activity in vitro. Portions
(10 ml) of Reinforced Clostridial Medium (Oxoid; filtered to remove the agar) were inoculated with 1 yo (v/v)
exponential phase cultures of the isolates in the same medium, incubated at 37 "C in an atmosphere of
HJCO, (90: 10, v/v) for 5 to 7d (stationary phase) and then harvested by centrifugation. The cell-free
supernatants were assayed for phosphatase activity as described above.
Purification of acid phosphatase. Strain P-37 was grown as above, except in static batch culture. Bottles
containing 400 ml culture were maintained in an atmosphere of H,/CO, (90: 10, v/v) in cold catalyst
anaerobic jars at 37 "C for 6 to 7 d. Cultures were then centrifuged at 3000 g for 1 h. Solid (NH,),SO, was
added to the supernatant to give 50 % saturation, the solution was stirred for 2 h at 4 "C and then centrifuged.
The supernatant was brought to 60(% saturation with (NH4),S04, stirred for 2 h at 4 "C, centrifuged at
3000 g for 30 min and the precipitate was redissolved in a minimum volume of 0.1 M-sodium acetate buffer
(pH 5.5). The precipitate was desalted by ultrafiltration at 4 "C (Amicon Diaflo cell fitted with a PMlO
membrane, mol. wt cut-off 10000) and then passed through a Sephadex G-100 column ( 2 . 4 ~31 cm) preequilibrated with 0.1 M-sodium acetate buffer (pH 5.5). Fractions (4.5 ml) were collected at 10 ml h l. Those
containing high enzyme activity were dialysed against 50 mwsodium citrate buffer (pH 6) and loaded on to
a CM-Sephadex (2-50 column (2.5 x 31 cm) pre-equilibrated with the same buffer. The column was washed
with 56 ml of the buffer before applying a 400 ml linear gradient from 0 to 0.5 M-NaCI in 50 mM-sodium
citrate buffer (pH 6). Fractions (6 ml) were collected at 12 ml h-l. Fractions with constant specific activity
(per mg protein) were combined and either used for studies on the properties of the enzyme or desalted and
lyophilized for sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis.
Protein estimations. The elution of protein from the various columns was monitored on an LKB Uvicord
or by recording the absorbance at 280 nm on a UV SP1800 Pye Unicam spectrophotometer. Protein determinations were made by the method of Lowry using bovine serum albumin as the standard.
Sodium dodecyl sulphate-polyacrylamidu gel electrophoresis. This was carried out according to Weber et al.
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P. acnes acid phosphatase
61
(1972) in 7.5 % gels. Samples (10 to 20 pg protein in 50 p l ) were prepared by heating to 100 "C for 2 min in
10 mM-sodium phosphate buffer (pH 7) containing 1% (w/v) SDS and 1% (w/v) 2-mercaptoethanol.
Electrophoresis was for 2.5 h at 8 mA per tube. Reference proteins were: phosphorylase a (mol. wt 94000),
pyruvate kinase (rnol. wt 57000), alcohol dehydrogenase (mol. wt 37000) and cytochrome c (mol. wt
12 384).
Sephadex G-100gelfiltration.A 2 ml sample of fraction V enzyme (see Table 1) was loaded on to a Sephadex G-100 column (2.4 x 36 cm). Fractions (3.5 ml) were collected at 10 ml h-l, with 0.1 M-sodium acetate
buffer (pH 5.5) as eluant. The column was calibrated with Blue Dextran 2000, P-galactosidase (rnol. wt
130000), ovalbumin (mol. wt 43000) and ribonuclease A (mol. wt 13700).
pHprofife. The pH profile of the purified acid phosphatase was determined by assaying 0.5 ml samples
of fraction VI enzyme (see Table 1) at 37 "C for 30 min in 0.1 M-sodium acetate buffer (pH 5 to 5.6), 0.1 MMES (pH 5.8 to 6.2) and 0.1 M-MOPS(pH 6.4 to 8.0), at increments of 0.2 pH units.
Sirbsrrate specificity. The range of substrates hydrolysed by the purified acid phosphatase was determined
semi-quantitatively by incubating 1 ml samples of fraction VI enzyme (see Table 1) with 2 ml 0.1 M-sodium
acetate buffer (pH 5.8), 1 m-MgC1, and 0.5 ml of a 0-5% (w/v) solution of substrate in the same buffer.
Enzyme blanks were included for each substrate. Reaction mixture/blank (1 ml) was transferred to 2 ml
0.2M-NaOH before and after 30 min incubation at 37 "C.The amount of inorganic phosphate released was
determined by the method of Chen et af. (1956).
RESULTS
Growth and phosphatase production by P. acnes
The growth of strain P-37 in the stirred batch fermenter is shown in Fig. 1. Extracellular
phosphatase was produced late in the exponential phase of growth and in the earlystationary phase. Extracellular phosphatase activity decreased after about 30 h of stationary
phase under these conditions. The production of extracellular acid phosphatase occurred
when the glucose in the medium was less than 2 mg ml-1 and the pH was below 6. At no
time was the cell-bound acid phosphatase activity greater than 10% of the extracellular
activity.
Protease was produced in the exponential phase of growth and no activity was detectable
in the culture supernatant after 40 h. Lipase and hyaluronidase production roughly paralleled the production of extracellular phosphatase.
When strain P-37 was grown in static batch culture for enzyme purification studies, the
growth curve was similar, with the exception that growth was slower. The stationary phase
was not reached until 4 to 5 d under these conditions. These cultures were therefore harvested
after 6 to 7 d incubation for maximum acid phosphatase activity.
All of the 24 P. acnes isolates tested (see Methods) produced phosphatase activity which
was present in the cell-free supernatants of stationary phase cultures grown in Reinforced
Clostridial Medium.
Purllfcation of acid phosphatase
The purification of extracellular acid phosphatase is summarized in Table 1. The most
reliable method of obtaining concentrated acid phosphatase for purification was by 50 to
60 (NH,),SO, precipitation of the culture supernatant. The yield varied between batches
from 76 to 95% and resulted in greater than 100-fold purification of the enzyme.
Chromatography on Sephadex G-100 of 8 ml (NH&SO,-precipitated enzyme after dialysis against 0.1 M-sodium acetate buffer (pH 5.5) resulted in a single peak of enzyme activity
eluting after the void volume and beforc the major protein peak. The enzyme from Sephadex
G-100 was reprecipitated by 50 to 60 yo (NH,),SO, saturation and re-chromatographed on
Sephadex G-100 until no further increase in specific activity was obtained, after which the
peak of enzyme activity corresponded directly to the protein peak. This enzyme preparation
(fraction V, Table 1) generally had a specific activity of greater than 3 pmol PNP formed
min-l (mg protein)-l. It was dialysed against 50 mM-citrate buffer (pH 6) and chromatographed on CM-Sephadex C-50. A major peak of anionic protein emerged without retarda-
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62
E. I N G H A M AND OTHERS
0
'0
40
00
Tinic of' incuhation (11)
130
Fig. 1. Growth of P.acnes strain P-37 in batch culture. The conditions for growth are described in
Metnods. Extracellular phosphatase was determined after removal of the cells by centrifugation at
3000 g for 10 min. Phosphatase activity associated with the cells was determined by washing the
cells from a 5 ml sample in PBS (pH 7.3), resuspending them in 0.5 ml PBS (pH 7.3) and assaying
directly as described in Methods. --, Dry weight of cells; - - -, pH of medium; ..., glucose concentration in medium ; 0,
extracellular acid phosphatase; 0 , cell-bound acid phosphatase.
Table I. PuriJcation of P. acnes strain P-37 acid phosphatase
I.
11.
111.
IV.
V.
VI.
Fraction
Crude culture
supernatant
50 to 60% satn
(NH4)2S04
Sephadex G-100
50 to 60% satn
(NH4)2S04
Sephadex G -100
(second step)
CM-Sephadex C-50
*
Volume
(m0
1500
Total
activity*
21.1
Total
protein
(mg)
33000
Specific
activity?
0.00064
8
19.89
204
0-0975
152
94
31.5
6
15-02
11-26
47.25
10.5
0.318
1 -072
497
1675
71
53
27
5.4
1.51
3.58
5594
26
78
0-95
0.033
pmol PNP formed min-'.
28.8
Purification
factor
-
45016
Yield
(70)
-
4.5
-f pmol PNP formed min-l (mg protein)-l.
tion but the acid phosphatase was retarded and eluted as a single peak of activity with
325 mM-NaC1. This activity corresponded to a minor protein peak. Fractions with constant
specific activity were combined (fraction VI, Table 1).
The above procedure, summarized in Table 1, resulted in greater than 45 000-fold purification for a 4.5 yo yield; 33 ,ug enzyme with a specific activity 28.8 pmol PNP (mg protein)-l
was obtained from 1.5 1 of culture supernatant. The results obtained using various batches
were reproducible with overall yields of enzyme from 3 to 8 %, the purification factors being
dependent on the initial specific activity of the culture supernatant.
Lipase and hyaluronidase activities of fractions obtained during the purijication
of acid phosphatase
The total activities of hyaluronidase and lipase in the fractions are given in Table-2.
Hyaluronidase was separated from the acid phosphatase by (NH,),SO, precipitation
(fractions I1 and IV ;Table 2) ;most of the hyaluronidase was precipitated at 50 yb (NH,),SO,
saturation. Lipase was separated by a combination of gel filtration and (NH,),S04 precipitation (fractions 11, I11 and IV; Table 2). Neither enzyme was detectable in fraction VI
acid phosphatase.
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63
P. acnes acid phosphatase
Table 2. Lipase and hyaluronidase activities of the fractions obtained during
the purijication of acid phosphatase
Lipase and hyaluronidase activities were determined as described in Methods.
Total activity
Fraction
I
I1
111
IV
V
VI
Lipase*
30.1
7.10
0.85
0.19
0
0
Hyaluronidase?
19-31
2.0
1-51
0.135
0-102
0
0, No detectable activity.
* pmol oleic acid formed min-l.
pmol N-acetylglucosamineformed min-I.
SDS-polyacrylamide gel electrophoresis and molecular weight of the
acid phosphatase
SDS-gel electrophoresis of the purified acid phosphatase revealed two protein bands, a
slow moving component with a molecular weight of about 155000 and a second protein
band of molecular weight 87 100.
The molecular weight of the acid phosphatase was estimated to be 93000 by gel filtration
on Sephadex G-100. This suggested that the faster moving component on SDS-polyacrylamide gel electrophoresis represented the acid phosphatase. It was unlikely that the slow
moving component represented a contaminating protein, as the enzyme had been repeatedly
chromatographed on Sephadex G-100 which should have removed proteins in that molecular weight range. It was possible that the 155000 component was an artifact produced in
the system by incomplete reduction by 2-mercaptoethanol or insufficient SDS binding.
Properties of the purified acid phosphatase
pHproJile. Purified acid phosphatase had high activity between pH 5.0 and 5.8, with
optimal activity at pH 5.8. Above pH 6.0 there was a rapid decrease in activity with no
detectable activity at pH 6.8.
Eflect of metal ions. The acid phosphatase did not have a specific metal ion requirement.
In general, the ions tested (Mg2+, Ca2+,Zn2+and K+) inhibited the enzyme at concentrations of 100 mM, but stimulated its activity at 0.1 and 1 mM (Table 3). Maximum activity was
observed with MgCI,.
Substrate speczjicity. Purified acid phosphatase had high activity against AMP and
P-glycerophosphate (96 to 98% of the activity against PNP-P) and was moderately active
against 0-phosphorylethanolamine, 0-phospho-1-serine, D-glucose 6-phosphate, D-glucose
1-phosphate and phosvitin (18 to 66% of the activity against PNP-P). No activity was
demonstrated with phosphatidylethanolamine. Conflicting results were obtained when
different enzyme preparations were assayed against ATP. Fraction V (semi-pure) acid phosphatase showed 27 to 100% activity. Highly purified fraction VI acid phosphatase had no
or low activity against ATP. It is possible that there was a second phosphatase (in fractionv)
with ATPase activity which contaminated some of the enzyme preparations. Further studies
will be necessary to determine if this is the case.
Stability. Purified enzyme retained more than 90% activity from pH 4 to pH 5.5 when
incubated in 0.1 M-citrate buffer at 37 "C for 1 h. The enzyme was stable at pH 4.5 to 5.5
in 0.1 M-citrate buffer at 4 "C for 7 d. The temperature stability of the purified enzyme was
studied by incubating 1 ml of the enzyme at temperatures between 4 and 70 "C in sodium
5
MIC
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XI8
64
E. INGHAM AND OTHERS
Table 3. Eflect of metal ions on acid phosphatase activity
Fraction VI enzyme (0.5 ml) was assayed in the presence of the metal ions indicated (added as
chlorides) as described in Methods. Activities are expressed as a percentage of the activity in
controls with no added metal ions.
Acid phosphatase activity (76)
r
Metal ion
MgWCa2+
Zn2
K+
1
-
-
10-l~M
22
76
0
98
-
-
-
-
lo-'
~
-
~
A
~
Metal ion concn
M
122
123
34
110
-
-
10-3 M
123
119
110
118
~
-
~
7-
M
123
116
119
112
citrate buffer (pH 5-5) and assaying the residual activity at 37 "C against PNP-P. The enzyme
had lost 25 % activity after 30 min at 40 "C and was completely inactivated after 30 min at
55 "C.
DISCUSSION
Acid phosphatase was present in the culture supernatant in the late-exponential and earlystationary phases of growth. At no time during the growth of the strain P-37 in batch
culture was more than 10% of the activity associated with the cells.
According to Priest (1 977), ' extracellular or exoenzymes are those enzymes that are
completely dissociated from the cell and found free in the surrounding medium'; however,
he does point out that the division between extracellular enzymes and cell wall- or membrane-bound enzymes is often narrow.
Acid phosphatases produced by Gram-positive organisms are generally bound to the cell
surface (Weimberg & Orton, 1965; Malveaux & San Clemente, 1969) or are membranebound (Kniittila & Makinen, 1972). These cell-bound phosphatases are removed by washing
the cells in highly concentrated salt solutions (Weimberg & Orton, 1965; Malveaux & San
Clemente, 1969). The nature of the binding has been suggested to be, in part, due to electrostatic forces. When Saccharomyces rneIlis was grown in medium with a high salt concentration the otherwise cell-bound enzyme was released into the surrounding medium (Weimberg
& Orton, 1965).
The presence of the P. acnes acid phosphatase in the culture supernatant could be due
either to the truly extracellular production of this enzyme, or to the release of a loosely
bound enzyme from the cell surface under the conditions of growth (Brain Heart lnfusion
contains 0.5% NaC1). From the foregoing discussion, P. acnes acid phosphatase may be
regarded as extracellular.
That Porschen & Spaulding (1974) and Hoeffler (1977) found no, or only weakly positive,
phosphatase-producing strains of P. acnes could be explained if only a few strains of P. acnes
produce this enzyme or if the growth conditions used in their investigations were not
suitable for the production of measurable amounts of acid phosphatase by the P. acnes
strains tested. In both investigations phosphatase production was measured after only 48 h
incubation. Studies in this laboratory have indicated that 24/24 P. acnes isolates produced
an acid phosphatase when grown in liquid culture for 5 to 7 d at 37 "C.
An interesting question is the possible role of this enzyme in the physiology of P. acnes.
Propionibacteria are the major inhabitants of the face and back of man (Marples & McGinley, 1974). It is possible that, in vivo, free carbon energy sources are limiting and that an
acid phosphatase may play a ' scavenger ' role in rendering phosphorylated sugars available
as sugars for subsequent transport into the cell (Hamilton, 1975). Further investigations will
be necessary to determine whether the acid phosphatase is produced by P. acnes in vivo.
The range of pH stability and pH optimum for activity of the enzyme suggest that it would
be stable and highly active at the normal p H range of the skin (Holland et al., 1978).
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P . acnes acid phosphatase
65
This work was generously supported by a grant from the A. H. Robins Co., and we wish
to thank Dr J. S. Templeton and Mr F. S. Walker of that company for their interest
throughout.
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