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85 FEMS MicrobiologyLetters 3 (1978) 85-87 © Copyright Federation of European MicrobiologicalSocieties Published by Elsevier/North-HollandBiomedicalPress THE N A T U R E OF MYCELIAL LIPOLYTIC ENZYMES IN FILAMENTOUS F U N G I J.A. BLAIN, J.D.E. PATTERSON and C.E.L. SHAW Department of Biochemistry, Universityof Strathclyde, The Todd Centre, 31 TaylorStreet, Glasgow, G40NR, Scotland Received 10 November1977 1. Introduction It has been shown previously [1] that both Mucor ]avanicus and Rhizopus arrhizus mycelia exhibit strong phospholipase activity of the At type (EC 3.1.1.32). In the present paper, some indication has been sought as to whether phosphoUpase A1 activity is the predominant type in the filamentous fungi, twelve species (including two different strains of Aspergillus niger) being examined. The assay method used involved reaction of solid phase enzyme with phosphatide substrate dissolved in diisopropyl ether [1 ] and information on the presence of lysophospholipase (EC 3.1.1.5), phospholipase B activity, phospholipase D (EC 3.1.4.4) and lipase was also obtained. 2. Materials and Methods 2.1. Organisms, medium and growth conditions The organisms examined in this survey were: Aspergillus nidulans (CMI 38576), Aspergillus oryzae (CMI 44242), Penicillium notatum (CMI 15378), P. roquefortii (CMI 24313), Rhizopus stolonifer (CMI 57761), Rhizopus japonicus (CMI 21600), Geotrichum candidum (CMI 50119), Neurospora crassa (CMI 53238), Mucor subtilissimus (CMI 45618), Mucor mucedo (CMI 122485), and Mucor pusillus (CMI 57407), cultures of which were obtained from the Commonwealth Mycological Institute (Surrey, England). The two strains of A. niger examined were A. niger (strain No. A-1-233 of Clark [2]) and A. niger (B) (strain 627; Department of Microbiology, University of Strathclyde). The culture medium employed contained 1% glucose, 2% neutralised bacteriological peptone (Oxoid Ltd., London), 0.2% KH2PO,, 0.05% KC1, 0.05% NaNO3 and 0.05% MgSO4.7H20 (all w/v). Inoculation was carried out by the addition of 2 ml of a 2 • 106/ml spore suspension to an erlenmeyer flask (2 litres) containing 400 ml medium and incubation was as described elsewhere [I]. The mycelia were harvested by filtration, washing with distilled water and freeze-drying. The mycelial powder was sieved through No. 16 (0.05") mesh and stored over P2Os. 2.2. Assay methods Reaction of dry mycelia with phosphatide substrate in diisopropyl ether, separation and analysis of reaction substrates and products were as described previously [1]. The substrate used contained a small quantity of lysophosphatidylcholine (LPC) as well as phosphatidylcholine (PC). Lipase activity was measured by substitution of olive oil for phosphatide as substrate and estimation of fatty acid liberated [1 ]. 3. Results and Discussion The dried mycelia, obtained at 24-h intervals from the second to seventh day of growth, were assayed for phospholipase and lipase activities using reaction periods of 2, 6 and 24 h. The amounts of PC and fatty acid present after 86 TABLE 1 Hydrolysis of phosphatidylcholine by mycelia PC destroyed #M LPC #M Fatty acid #M S/U (F.A.) a Reaction time (h) 2 6 24 0 2 6 24 2 6 24 2 24 Organism A. nidulans A. niger A. niger (B) A. oryzae G. candidum M. subtilissimus N. crassa R. ]aponicus R. stolonifer M. mucedo M. pusillus P. notatum P. roquefortii 45 87 61 70 38 52 44 36 50 38 68 74 42 63 135 116 104 63 66 54 38 78 47 101 127 50 87 139 131 137 68 118 82 59 95 65 114 135 68 27 25 28 25 25 27 27 27 27 27 27 27 27 27 32 18 25 ll 36 15 27 36 30 38 16 23 23 8 13 21 11 44 28 38 38 38 85 8 15 23 4 13 9 13 38 21 46 51 61 59 6 15 12 116 86 118 62 20 26 14 6 22 54 150 42 53 287 240 172 137 62 44 40 32 31 77 251 53 142 310 307 288 180 118 116 71 72 46 132 288 122 1.4 0.9 1.0 1.1 1.2 4.3 6.2 4.4 2.1 2.1 5.7 1.0 1.7 0.9 1.0 1.0 1.0 1.0 4.3 3.1 2.4 2.9 1.9 7.6 1.0 1.1 s/u (LPC) a * * * * * 0.4 0.3 0.4 0.5 0.7 0.2 * * Predominant phospholipase activity B B B B B AI, L c AI, L c AI A1 At A1 B B a Ratio of saturated to unsaturated fatty acids liberated. b Ratio of saturated to unsaturated fatty acids remaining esterified to LPC. c L, lysophospholipase. * Insufficient LPC produced for analysis. each r e a c t i o n p e r i o d are s h o w n in Table 1 for the 13 to hydrolyse PC) was chosen for inclusion in the Table. The ratio of saturated to unsaturated fatty acid strains e x a m i n e d . In each case t h e day o f highest mycelial p h o s p h o l i p a s e activity (in t e r m s o f c a p a c i t y TABLE 2 Relative lipase and phospholipase activities of mycelial samples Organism Lipase: fatty acid (uM)/2 h Day 2 A. nidulans A. niger A. niger (B) A. oryzae G. candidum M. subtilissimus N. crassa R. ]aponicus R. stolonifer M. mucedo M. pusillus P. notatum P. roquefortii 24 0 0 38 8 0 2 22 26 38 0 0 2 Phospholipase: fatty acid (uM)/2 h 3 4 5 6 7 2 3 4 5 6 7 10 0 0 28 22 4 0 40 28 22 0 0 4 8 0 0 10 0 0 0 0 8 0 0 0 8 4 0 0 32 18 0 0 2 4 0 10 0 6 0 6 2 2 8 4 0 8 0 4 0 0 0 8 19 0 12 16 0 0 4 0 2 0 0 2 10 140 89 104 24 6 6 4 0 9 32 9 30 10 166 86 116 45 1 33 14 17 10 26 17 8 12 183 85 118 38 20 26 0 6 22 47 17 27 24 148 98 112 56 20 21 0 4 0 54 21 42 24 192 105 87 62 14 20 0 8 16 32 46 32 24 195 73 128 67 8 34 0 11 6 10 74 39 87 liberated is indicative of the positional specificity of the hydrolysis. According to the data of Kuksis and Marai [3], a ratio of up to 8 would imply phospholipase A1 attack whereas A2 attack would be indicated by a ratio as low as 0.02. A ratio approximating to unity would be obtained following phospholipase Btype attack. Similarly, further evidence pertaining to positional specificity can be obtained from the ratio found for the LPC remaining after hydrolysis. The final column in Table 1 indicates the predominant type of phospholipase activity associated with each organism. Since apparent phospholipase At activities of fungal preparations have been attributed to lipases [4,5] it was of interest to compare the relative lipase and phospholipase activities of the mycelial samples. Table 2 shows the variation in lipase and phospholipase activities of each organism over the growth period examined. It can be seen that in many samples the mycelia exhibit only phospholipase activities. In others, the day of growth on which the highest phospholipase activity occurs differs from that on which optimum lipase activity is observed. Such variations in the relative phospholipase and lipase activities suggest that separate enzymes are involved. In addition to the activities already mentioned, some mycelial samples hydrolysed the substrate to phosphatidic acid. Results are reported in Table 3. This is in conformity with a previous observation [1] of the presence of phospholipase D in filamentous fungi. The results of this survey would suggest that phospholipase At and lysophospholipase are of common occurrence among filamentous fungi whereas phospholipase B type of activity is dominant in some organisms. Phospholipase B activity in P. notatum was first reported by Dawson in 1958 [6]. It is of course recognised that the phospholipase B type of activity could be due to the action of phospholipase A1 and highly active lysophospholipase. No evidence for the presence of phospholipase A2 was found associated with any of the organism examined. Acknowledgements We thank Mr. S. Adams for technical assistance. TABLE 3 Phospholipase D activity of some mycelial preparations Organism Day of growth References Phosphatidic acid I~M Reaction time (h) A. niger (B) R. ]aponicus R. stolonifer R. stolonifer 3 3 3 6 2 6 24 15 17 32 32 15 17 28 28 19 15 23 21 [1] Blain, J.A., Patterson, J.D.E., Shaw, C.E.L. and Akhtar, W.A. (1976) Lipids 11,553-560. [2] Clark, D.S. (1962) Canad. J. Microbiol. 8,587-588 [3] Kuksis, A. and Marai, L. (1967) Lipids 2, 217-224 [4] Slotboom, A.J., de Haas, G.H., Bonsen, P.P. BurbackWesterhuis, G.J. and van Deenen, L.L.M. (1970) Chem. Phys. Lipids 4, 15-29. [5] Ishihara, B.H., Okuyama, H., Ikezawa, H. and Tejima, S. (1975) Biochim. Biophys. Acta 388,413-422. [6] Dawson, R.M.C. (1958) Biochem.J. 70,559-570.