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Activity of laccase and the fruiting body formation of Pleurotus spp. Presented By Mr. Suparat Laohapitakvorn Mentor Dr. Charida Pukahuta Assist. Prof. Dr. Weerasuk Suraruengchai
Activity of laccase and the fruiting body formation of Pleurotus spp.
Presented ByMr. Suparat Laohapitakvorn
Mentor
Dr. Charida PukahutaAssist. Prof. Dr. Weerasuk Suraruengchai
Most mushroom which are cultivated in sawdust do not yield desirable amount
of produce. This become major problem for mushroom farmers.
Why I did project ?
Bhutanese oyster mushroom - Pleurotus sajor-caju
Introduction
Oyster Mushroom - Pleurotus ostreatus
Edible mushrooms are always grown in sawdust with
supplements.
• Be able to decompose lignocellulose
• Enzymes involved in lignocellulolytic degradation are cellulase, hemi-cellulase, xylanase, phenoloxidases example laccase
• Laccase relates in
– Decomposition of lignocellulose
– Stage of fruiting body formation
– Be able to detoxify organic wastwater
Objective
To study relationship between laccase activities and fruiting body production
Mushroom culture
• stock culture in PDA• In Potato Dextrose Agar (PDA) • Aseptic technique • incubate at room temperature for 7 days
Measure mycelial growth in PDA
• Grow in PDA each 5 replication• Incubate at room temperature for 9 days• Measure colony diameter by x-axis and y-
axis daily• Record and calculate average growth of
each culture
Laccase assay in PDA
• 3 replication of each mushroom culture on PDA for 4 days at room temperature.
• Assay with o-tolidine solution by pouring on mushroom mycelia.
• Observe the color changes of solution to dark blue and record data.
Cultivation in PDB
• Potato Dextrose Broth (PDB)• 10 pieces of inoculums/flask,
4replications/strain• Incubation 150 rpm., 26oC• Laccase assay at day 4, 6 and 8 by
measurement of color changes of o-tolidine and PDB, using Spectrophotomiter at 590 nm.
Culture in sawdust bag
• Fully grow mushroom culture in sorghum seed (2 weeks)
• Inoculate to sawdust with supplements in plastic bags. Incubate at room temperature (1 month)
• Move to fruiting house• Watering daily• Harvest the fruiting body and record
the weight.
Result : 1
• Measurement of mycelial growth in PDA • Pleurotus sajor-caju (No. 1-3) showed dense
mycelia in horizontal growth (substrate mycelia) and vertical growth (aerial mycelia)
• Pleurotus ostreatus (No. 4) showed dense mycelia in vertical growth
• Pleurotus sajor-caju Number 1-3 grew about 12 – 13 mm/day but Pleurotus ostreatus Number 4 grew 9 mm/day
1
0
10
20
30
40
50
60
70
80
90
1 2 3 4 5 6 7 8 9
day
co
lon
y d
iam
ete
r (m
m)
1
2
3
4
Result : 2
NumberColor change ( 3
replications)
1 ++++ center of colony
2 + all area of colony
3 +++ center of colony
4 +++ all area of colony
+ mean dark
Result : 3
• The activity of No. 1, 2 and 3 increased daily in day 4, 6 and 8
• The activity of No.4 increased in day 4 and 6 but decreased in day 8
0
200
400
600
800
1000
1200
1400
4 6 8
day
un
it
blank
1
2
3
4
Laccase acting in sawdust bag
specific activity = laccase activity (Unit)
protein content ( Lowry method)
0
0.5
1
1.5
2
2.5
3
3.5
4
16 30 48 62
day
spec
ific
activ
ity (U
nit/m
in/m
l)
1
2
3
4
Mushroom Production
• The data of weight of each strain and amount of fruiting body was record for 2 months
• The weight of production was high during day 16-30
• The weight of production was stable during day 48-62
Pleurotus sajor-cajuPleurotus sajor-caju Number Number 1 1Pleurotus ostreatusPleurotus ostreatus Number Number 4 4
Production Mushroom
Mushroom
No.
AmOu-nt of
Bags
Total production Average/bag
Flush
Total weight
(g)
Amount of
fruiting body
Flush
Total Weig
ht (g)
Amount
of fruiting body
1 63 200 7943 826 3.17 126.08 13.11
2 59 186 7350 1340 3.15 124.58 22.71
3 56 173 6812 1134 3.09 121.64 20.25
4 60 91 5767 1672 1.52 96.12 27.87
Conclusions
1. Correlation was found between laccase in PDB (8th day mycelia) and total fruiting body production.
2. The mushroom No.1,2 and 3 had high laccase activity and high fruiting body production.
3. The mushroom No. 4 had lower laccase activity and lower fruiting body production than the others.
0
200
400
600
800
1000
1200
1400
4 6 8
day
un
it
blank
1
2
3
4
Flush/bag Total weight (g)/bag
In PDB
0
0.5
1
1.5
2
2.5
3
3.5
1 2 3 4
number
tim
e
0
20
40
60
80
100
120
140
1 2 3 4
number
gam
e
0
100
200
300
400
500
600
700
800
900
16 30 48 62
day
tota
l w
eig
ht
(g)
1
2
3
4
0
0.5
1
1.5
2
2.5
3
3.5
4
16 30 48 62
day
spec
ific
activ
ity (U
nit/m
in/m
l)
1
2
3
4
0
5
10
15
20
25
30
16 30 48 62
day
Am
ou
nt
of
bag
s
1
2
3
4
0102030405060708090
100
16 30 48 62
day
amo
un
t o
f fr
uit
ng
bo
dy
1
2
3
4
Discussions
• Laccase activity (in PDB) and sawdust correlates with number of flushing bag, total weight and total amount of fruiting body
• The kit to test the fruiting body may be invented by using this correlation
Acknowledgement
• Junior Science Talent Project• Dr. Charida Pukahuta• Assist. Prof. Dr. Weerasuk Suraruengchai• Teachers from Mahidol Wittayanusorn School
