Download The nature of mycelial lipolytic enzymes in filamentous fungi

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.