Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN 968-878-105-3
DETECTION AND ANALYSIS OF EXTRACELLULAR ENZYME ACTIVITIES IN
AGROCYBE AEGERITA STRAINS
N. Wang, F. Shen, Q. Tan, M. J. Chen and Y. J. Pan
Key Lab for Genetics and Breeding of Edible Fungi, Agricultural Ministry of China
Key Lab for Agricultural Genetics and Breeding, Edible Fungi Institute,
Shanghai Academy of Agricultural Sciences
Shanghai 201106, Peoples’ Republic of China
< nwang999@hotmail.com >
ABSTRACT
The activities of nine extracellular enzymes were detected and analyzed at six different
developmental stages of A. aegerita. The three A. aegerita strains under study utilized
non-lignin-cellulose more efficiently than the rest, and their cellulose-degrading activity was slightly
lower. These strains displayed very low activity in the degradation of lignin. We therefore concluded
that A. aegerita is a brown rot fungus.
INTRODUCTION
As a new kind of edible mushroom, A. aegerita has received much attention because of its unique
flavor, rich nutrition and high medicinal value. However, large-scale cultivation of A. aegerita has
been limited (Wang et al. 1998) because yields are lower than those of other mushrooms. Until now,
the major factors limiting yields have not been understood. In this study, three strains of A. aegerita of
different origins were investigated for their ability to utilize lignin, cellulose, hemicelluloses and
other components of the substrate. The activities of nine extracellular enzymes were analyzed at
different developmental stages. A cultivation method for A. aegerita is then further discussed.
MATERIALS AND METHODS
Strains and culture conditions
The three strains of Agrocybe aegerita used in this work were from the collection of the Edible Fungi
Institute, Shanghai Academy of Agricultural Sciences, and were originally collected from Taiwan (T),
Guizhou (Ag9), China, and Greece (Ag8), respectively. The stock culture medium was PDA. The
composition of the substrate was: sawdust 390g, cotton seed hull 390g, wheat bran 100g, corn meal 100g,
sugar 10g, gypsum 10g and water 1000ml. The culturing temperature for mycelia growth, 25C and for
fruit body formation and production, 22C. Plastic bottles were used for cultivation.
197
Extraction of extracellular enzymes
Samples were taken from the cultures at various stages of development as shown in Figure 1.
Extracellular enzymes were extracted from the fresh samples with distilled water for four hours. The
crude enzyme solution was harvested after centrifugation at 1,5000g for 10 min. Substrates were then
added into individual tubes containing the enzyme solutions and the activities of the enzymes were
detected using colorimetry. All the extracting steps were carried out at 4C .
Analyses of enzyme activity
Analyses of carboxymethylcellulases (CMCases), hemicellulases (HCases), filter paper cellulases
(FPases), pectases and amylases were carried out as described by Wang and Xu (1989). The activities
of laccases and tyrosinases were analyzed using the protocol of Pan et al. (1991). Peroxidases and
polyphenol oxidases were measured as by Pan (1993).
RESULTS
Changes in pectase activitiy during mycelial growth and fruiting
Pectase activities(u)
The pattern of pectase activity was unique to each of the three A. aegerita strains (T, Ag8, and Ag9)
at each developmental stage (Figure 1). However, as shown by the following results, the average
activity of pectase (9-27u) was higher than for other enzyme activities. The results indicated that A.
aegerita strains could degrade and utilize pectic substrates very efficiently. Furthermore the pectase
activity appeared in the early stages of mycelial growth and had no direct relation with fruit body
formation.
30
25
20
15
10
A g9
A g8
5
0
T
1
2
3
4
5
6
Stages of development
Figure 1. Pectase activity of three A. aegerita strains (T, Ag8 and Ag9) at the following stages of
development: 1. Mycelia fully colonized, 2. Appearance of primordial, 3. First flush, 4. One week after first
flush, 5. Second flush, 6. One week after second flush.
198
Changes in amylolytic enzyme activity during mycelial growth and fruiting.
In strains Ag9 and T, amylolytic enzyme activities were relatively higher when primordia appeared,
(15u and 19u respectively) and then fell to a low level (0-5u) (Figure 2). Ag8 had a high level of
amylolytic enzyme activity during mycelial growth (8u). The activity dropped to a low level (0-5u)
in later stages. The results indicate that amyloid materials were largely degraded and utilized before
the first flush. Thus, no starch in the cultivating media could be utilized by A. aegerita. Similar
results were reported by Ni (1991) for Lentinula edodes.
Amylolytic enzymes
activities(u)
20
A g9
A g8
15
T
10
5
0
1
2
3
4
5
6
Stages of development
Figure 2. Amylolytic enzyme activity of three Agrocybe aegerita strains (T, Ag8 and Ag9) at the
same stages of development as described in Figure 1.
Changes in HCase activity during mycelial growth and fruiting
Again, the pattern of HCase activity was unique to each of the three A. aegerita strains (T, Ag8, and
Ag9) at each developmental stage (Figure 3). Enzyme activity (0-2.5u) was lower than CMCase
activity.
3
A g9
HCase activities(u)
2 .5
A g8
T
2
1 .5
1
0 .5
0
1
2
3
4
5
6
Stages of development
Figure 3. Hemicellulase activity in three Agrocybe aegerita strains (T, Ag8, Ag9) at the same stages
of development as shown in Figure 1.
199
Changes in CMCase activity during mycelial growth and fruiting.
CMCase activities(u)
As seen in Figure 4, all three strains had a high level (14-25u) of CMCase activity during mycelial
growth, and the activity then dropped to 0-5u. Results show that cellulose had been largely degraded
and utilized in the early stage of mycelial growth.
30
Ag9
25
Ag8
20
T
15
10
5
0
1
2
3
4
5
6
Different stages of development
Figure 4. carboxymethylcellulase activity of three Agrocybe aegerita strains (T, Ag8, and Ag9) at the
same stages of development as shown in Figure 1.
Changes in FPase activity during mycelial growth and fruiting.
FPase activity was higher during mycelial growth and dropped slightly when primordia appeared
(9-14u) (Figure 5). The results indicate that A. aegerita degraded cellulose efficiently at an earlier
stage of development.
FPase activies(u)
15
A g9
A g8
T
10
5
0
1
2
3
4
5
6
Stages of development
Figure 5. The filter paper cellulase activity of three Agrocybe aegerita strains (T, Ag8, and Ag9) at
the same stages of development as shown in Figure 1.
200
Changes in laccase activities during mycelial growth and fruiting.
Laccase activity of all three strains was very low (0-4.8u) in the development of A. aegerita (Figure
6).
Laccase activities(u)
5
A g9
4
A g8
3
T
2
1
0
1
2
3
4
5
6
Stages of development
Figure 6. Laccase activity of three Agrocybe aegerita strains (T, Ag8, and Ag9) at the same stages
of development as shown in Figure 1.
Changes in peroxidase activity during mycelial growth and fruiting.
Peroxidase activity in the three A. aegerita strains ranged from 0 to 1u, and all had the lowest level
of activity during the first flush.
Peroxidase activities(u)
1
A g9
A g8
T
0 .8
0 .6
0 .4
0 .2
0
1
2
3
4
5
6
Stages of development
Figure 7. Peroxidase activities of three Agrocybe aegerita strains (T, Ag8 and Ag9) at the same
stages of development shown in Figure 1.
201
Changes in polyphenol oxidase activity during mycelial growth and fruiting.
Polyphend oxidase activities(u)
Polyphenol oxidase in the three A. aegerita strains ranged from 0.1 to 1.5u (Figure 8), and was at
its lowest during the first flush.
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Ag9
Ag8
T
1
2
3
4
5
6
Different stages of development
Figure 8. The polyphenol oxidase activity of three Agrocybe aegerita strains (T, Ag8 and Ag9) at
the same stages of development as shown in Figure 1.
Changes in tyrosinases activity during mycelium growth and fruiting.
Tyrosinase activities(u)
Tyrosinase activity was in the range of 0 to 1.7u (Figure 9). As in the cases of polyphenol oxidase
and peroxidase, the lowest tyrosinase activity was during first flush.
2
Ag9
1.5
Ag8
T
1
0.5
0
1
2
3
4
5
6
Different stages of development
Figure 9. Tyrosinases activity of three Agrocybe aegerita strains (T, Ag8 and Ag9) at the same
stages of development as shown in Figure 1.
202
DISCUSSION
Laccase, peroxidase, polyphenol oxidase and tyrosinase are all involved in the metabolism of
phenolic compounds and the degradation of lignin ( Wei 1990, Ni and Pan 1998, Pan et al. 1991,
Wood 1998). Our results showed that activities of these enzymes were all very low in the three
strains of A. Aegerita. However, these strains displayed high activity in the utilization of cellulose
and non-lignin-cellulose, suggesting that A. aegerita is a brown rot fungi.
It has been reported that sawdust contains 20% lignin wrapping its cellulose (Mandels 1974, Johne
1983, Zeikns 1981, Tien and Kirk 1983). White rot fungi can degrade lignin effectively and then
utilize cellulose further. Brown rot fungi cannot utilize lignin-cellulose easily (Wang and Shi 1990).
Agrocybe aegerita has very low activity in the utilization of lignin and other phenolic compounds;
therefore we classify A. aegerita as a brown rot fungus. This may explain the low yield of A.
aegerita grown on sawdust media. We suggest the use of cotton seed hulls or cotton wastes instead
of 100% sawdust, in order to increase A. aegerita production and more effectively utilize the
substrate.
ACKNOWLEDGMENTS
The authors would like to thank those who contributed in carrying out the research and writing this
manuscript. N. Wang especially thanks Dr. S. T. Chang, Dr. Jose E. Sanchez-Vazquez and Dr. R. C.
Ma for their kind help. This work was supported by Foundation of Science and Technology
Commission of the Municipality of Shanghai , China.
REFERENCES
Johne, E. 1983. The filamentous fungi, Fungal technology 4:265
Mandels, M. 1974. Enzymatic hydrolysis of waste cellulose, Biotech. 16:1471-1493
Ni, X. J. 1991. Study on the Variation Law of physiology and biochemistry during growth and fruiting in
Lentinus edodes. Nanjing Agricultural University Master Degree Dissertation.
Ni, X. J. and Y. J. Pan,1998. The Degradation of the Lignocellulose and the Changes of Relative Enzyme
Activity at Different Layers of the Compost in Plastic-Bag during the Growth of Letinula edodes. Acta
Edulis Fungi. 5(1):13-17
Pan, L.P. 1993. The Application of Genetic Distance and Clustering Analysis in the Evalution of
Germaplasm Bank in Lentinus edodes. Nanjing Agricultural University Master Degree Dissertation. 13-17
Pan, Y. J., M. J. Chen, H. G. Zheng, 1991. Activity Chenges of Laccase, Tyrosinase and Cellulose in Lentinus
edodes and Pleurotus ostreatus During Their Growth. Acta Agriculturae Shanghai. 7(2):21 - 26
Tien, M. And T. K. Kirk, 1983. Lignin-Degrading Enzyme From the Hymenomycete phauevochaete
chrysosporium Burds. Science, 221(4): 661-662
Wang, N., Q. Tan, M. J. Chen, 1998. Advance in Agrocybe aegerita Research. Acta Edulis Fungi. 5(4):56-60
Wang, Y. W. and W. Y. Xu, 1989. Degradation of Lignocellulose and Changes in Activities of Some
203
Polysaccharidases During Growth of Flammulina velutipes. Microbiology. 16(3):137-140
Wang, Y. W., and Y. J. Shi,1990. Variation Law of enzymes activities during growth and fruiting in Flammulina
velutipes. Handbook of new technology on mushroom researching. Da Lian University of Science and
Engineering Press. 24
Wei, X. Y. 1990. Study on the Application of Mushroom Enzymes. Edible Fungi of China. 9(5):15-16
Wood, D. 1998, Substrate bioconversion by Agricus bisporus, Proceedings of 1998 Nanjing International
Symposium. 17-23
Zeikns, J.G. 1981. Advances in microb—Lignin Metabolism and the carbon cycle polymer biosynthesis,
biodegradation, and environmental recalcitrance. Ecology. (5): 211
204