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Journal of General Microbiology (1980), 118, 59-65. Printed in Great Britain 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 03:44:47 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 03:44:47 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- Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 03:44:47 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 03:44:47 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 03:44:47 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). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 03:44:47 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. REFERENCES BESSEY, 0. A., LOWRY, 0. H. & BROCK,M. J. (1946). MARPLES, R. R. & MCGINLEY, K. J. (1974). Corynebacterium acnes and other anaerobic diphtheroids A method for the rapid determination of alkaline from human skin. Journal of Medical Microbiophosphatase with five cubic millimeters of serum. Journal of Biological Chemistry 164, 321-329. logy 7, 349-357. CHEN,P. S., TORIBARA, T. Y.& WARNER, H. (1956). MILLET,J. (1970). Characterization of proteinases Microdetermination of phosphorus. Analytical secreted by Bacillus subtilis Marburg strain during sporulation. Journal of Applied Bacteriology 33, Chemistry 28, 1756-1758. 207-21 9. DOLE,V. P. & MEINERTZ, H. (1960). MicrodeterR. K. & SPAULDING, E. H. (1974). Phosmination of long chain fatty acids in plasma and PORSCHEN, phatase activity of anaerobic organisms. Applied tissues. Journal of Biological Chemistry 235, 2595-2599. Microbiology 27, 744-747. HAMILTON, W. A. (1975). Energy coupling in micro- PRIEST,F. G. (1977). Extracellular enzyme synthesis in the genus Bacillus. Bacteriological Reviews 41, bial transport. Advances in Microbial Physiology 71 1-753. 12, 2-48. S. M. & REISNER, M. D. (1972). The proHOEFFLER, U. (1977). Enzymatic and hemolytic PUHVEL, duction of hyaluronidase (hyaluronate lyase) by properties of Propionibacterium acnes and related Corynebacterium acnes. Journal of Investigative bacteria. Journal of Clinical Microbiology 6, Dermatology 58, 66-70. 55 5-558, HOLLAND,K. T., CUNLIFFE,W. J. & ROBERTS, REISNER,R. M., SILVER,D. Z., PLTHvEL, S. M. & STERNBERG, T. H. (1968). Lipolytic activity of C. D. (1978). The role of bacteria in acne - a new Corynebacterium acnes. Journal of Investigative approach. Clinical and Experimental DermatoDermatology 51, 190-196. logy 3, 235-257. J. L. & LELOIR,L. F. HUGGETT, A. ST. G. & NIXON,D. A. (1957). Enzymic REISSIG,J. L., STROMINGER, (1955). A modified colorimetric method for the determination of blood glucose. Biochemical estimation of N-acetyl amino sugars. Journal of Journal 66, 12P. Biological Chemistry 217, 959-966. KNUTTILA,M. L. E. & MAKINEN,K. K. (1972). M. (1972). Purification and characterization of a phosphatase WEBER,K., PRINGLE,J. R. & OYBORN, Measurement of molecular weights by electrospecifically hydrolysing p-nitrophenyl phosphate phoresis on SDS-acrylamide gel. Methods in from an oral strain of Streptococcus mutans. Enzymology 26, 3-27. Archives of Biochemistry and Biophysics 152, 685WEIMBERG, R. & ORTON,W. L. (1965). Elution of 701. acid phosphatase from the cell surface of SaccharoMALVEAUX, F. J. & SAN CLEMENTE, C. L. (1969). myces mellis by potassium chloride. Journal of Staphylococcal acid phosphatase : extensive puriBacteriology 90, 82-94. fication and characterisation of the loosely bound enzyme. Journal of Bacteriology 97, 1209-1 214. 5-2 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 12 Jun 2017 03:44:47