ENZYMOLOGICAL AND BIOTECHNOLOGICAL PROSPECTS IN
LIGNINOLYTIC SYSTEM OF WHITE ROT FUNGI
M.Sc DISSERTATION SUBMITTED TO THE DEPT. OF BOTANY
SIDHO-KANHO-BIRSHA UNIVERSITY, PURULIA
Submitted by payel karmakar
Under the supervision of Professor (Dr.) Subrata Raha
REG NO: 012664 of 2020-21
ROLL: NO: 0013
INTRODUCTION
Enzyme application in biotechnological and environmental process is of increasing interest
owing to their efficiency, selectivity and importantly their being environmental friendly health
wise, but very expensive and application requires large volume of enzymes
Ligninolytic enzymes finds wide commercial applications within food industry, pulp and
paper industries, textile industry, synthetic chemistry, cosmetics, soil bioremediation and
biodegradation of environmental phenolic pollutants.
The production of ligninoenzymes by macrofungi is very interesting due to combind
production of enzyme which help in the degradation of lignocellulosic materials.
The current study was to screen locally for highly efficient LiP and MnP producing fungi
with ability to secrete copious amounts of the enzyme to reduce the cost of production.
What is Ligninolytic system ?
The ligninolytic system is an extracellular enzymatic complex that includes peroxidase ,
laccases and oxidases responsible for the production of extracellular hydrogen peroxide
Structure of lignin
Enzyme involve in the degradation of lignin
Ligninolysis
Peroxidase Laccases
Lignin Manganese Aryl alcohol
peroxidase dependent peroxidase
peroxidase
Current status on the research of macrofungal ligninolytic enzyme
LITERATURE AUTHORS YEAR OF PUBLICATION
Molecular biology of the lignin MH Gold and M Alic September 1993
degrading basidiomycetes
Phanerochaete chysosporium
Experimental recreation of the I Aysuo- Fernandez 2017
evolution of Lignin
Lignin degrading enzyme S Camareo 1997
produce by Pleurotus species
Lignin degradation G Janusz 2017
microorganisms, enzymes
Fungal biodegradation and Mehdi Dashtban, Heidi May 2010
enzymatic modification of lignin Schraft,Tarannum A Syed,
Wensheng Qin
Biodegradation of lignin I D Reid 1995
AIM AND OBJECTIVES
Aim
Study and production of extracellular Ligninolytic enzymes from basidiomycetes fungi.
Objectives:
▪ Collection of fungal species and optimization of growth condition.
▪ Quantitative and qualitative production of extracellular Ligninolytic enzymes.
▪ Study and degradation of lignin by basidiomycetes.
Collection of samples
Fungal samples were collected from rotting wood of forest and agricultural field
Kingdom: Fungi Kingdom: Fungi
Basidiomycota Division: Basidiomycota
Division:
Class: Agaricomyccet
Class: Agaricomycet
es
es
Order: Agaricales
Order: Polyporales
Family: Hydnangiaceae
Family: Meruliaceae
Genus: Ganoderma Genus: Microporus
Species: G. lucidum Species: M. xanthopus
Binomial name Binomial name
Ganoderma lucidum
Microporus Xanthopus
(scop.) Cooke (Berk.) Pat.(1903)
Ganoderma lucidum Microporus xanthopus
Isolation of macro fungi
Methodology for isolation of macro
fungi
Fungal samples cut into small pieces
Surface sterilization
Placed on PDA plates, incubated at 25ºC
Fungal sample surface Emerging Fungi
After 5-7 days Mycelia grow on PDA plate sterilized & placed on PDA
Colony morphology of isolates observed and
noted
Slides mounted with isolated fungi observed
under microscope
Microscopic view Light microscope Pure culture of
Identification using standard manuals. analysis fungus
Observation of fungal growth and enzyme activity
in different Incubation period
The test fungi were inoculated in 100
ml sterilized screening medium.
The growth measurement was done by
estimating the dry weight of the Growth in liquid media
fungus.
The inoculated flask containing the
medium was taken out for dry weight
Dry mycelia
estimation at every 2 days time
interval.
Enzyme Activity
After incubation the fungal mycelia
were separated by filtration.
The best production was confirmed by
Incubation of the fungal Filtrate as a crude
the estimation of the enzyme activity mycelium enzyme source
using indicators as a substrate and the
Reaction mixture for LiP Reaction mixture for MnP
filtrates were used as a crude enzyme 0.2% Azure B (1.0 ml) 0.2% MnSO4 (1.0 ml)
+ +
source. Sodium Tartarate buffer Sodium Malonate buffer
Enzyme activity was spectro- of different pH (3.0 ml) of different pH (3.0 ml)
+ +
photometrically determined at different crude enzyme (0.5 ml) crude enzyme (0.5 ml)
+ +
wavelength. H2O2 (0.5 ml) Phenol red (0.5 ml)
Calculations for enzyme activity
Enzyme activity in the sample was spectrophotometrically determined by
monitoring the rate of product (dark brown color) formation (De Souza-Ticlo
2008, Setti et al. 1998, Xiao et al.2022).
The enzyme activity was calculated by using the formula:
Vol. activity (U/ml) = [ ΔA/T х Vt х dil. Factor х106 ]/1000
ε х Vs
ΔA=Increase in Absorbance at 430 nm
T= Time of observation
Vt = Final vol. of reaction mixture= 5.00
Vs = Sample volume= 1.00
ε= extinction coefficient of the product= 27.75
The enzyme activity was expressed in U/ml and it is defined as an increase in
absorbance spectrophotometrically per minute at 430 nm under specified
conditions.
Optimization
Effect of
nitrogen Effect of
sources carbon Thermo
sources stability set
Growth of fungus on Crude enzyme Time Effect of
different media course Effect of
pH
study Different
Solvent Filtrate as a crude
enzyme source
Enzyme Assay
Results & Discussion
Microscopic Images of mycelia of isolated macro
Fungal mycelia
fungi
S-3 S-4 S-3 S-4
Optimization of physical parameters(LiP activity of Ganoderma lucidum)
Study of time course Effect of temperature Effect of pH
Effect of nitrogen sources Effect of carbon sources Effect of different solvents
MnP activity of Ganoderma lucidum
Study of time course Effect of temperature Effect of pH
Effect of nitrogen sources Effect of carbon sources Effect of different solvents
LiP activity of Microporus xanthopus
Study of time course Effect of temperature Effect of pH
Effect of nitrogen sources Effect of carbon sources Effect of different solvents
MnP activity of Microporus xanthopus
Study of time course Effect of temperature Effect of pH
Effect of nitrogen sources Effect of carbon sources Effect of different solvents
The degradation of dyes by ligninolytic enzymes
Malachite green Enzyme Degradation of Malachite Degradation of Congo red
green
Block of
fungi
Malachite green Degradation of Malachite
Degradation of Nigrosine
containing plate green on solid media
Screening of the best dye decolorizing isolates based on degradation rate
Average
Degradation rate Duration of
Isolates Initial OD Final OD degradation
(%) observation
rate (%)
1.41 0.05 96.45
MG–S3 1.41 0.05 96.45 96.47 72 h
1.43 0.05 96.5
1.52 0.07 95.4
MG–S4 1.52 0.07 95.4 95.4 72 h
1.52 0.07 95.4
1.39 0.07 94.96
CR–S3 1.40 0.07 95 94.98 72 h
1.39 0.07 94.96
Initial OD – Final OD
Dye Degradation(%)= 100
Initial OD
Average
Degradation rate Duration of
Isolates Initial OD Final OD degradation
(%) observation
rate (%)
1.19 0.07 94.17
CR-S4 1.19 0.07 94.17 94.17 72 h
1.19 0.07 94.17
0.84 0.08 90.47
NS–S3 0.84 0.08 90.47 90.47 72 h
0.84 0.08 90.47
1.31 0.08 93.89
NS–S4 1.31 0.08 93.89 93.89 72 h
1.31 0.08 93.89
Lignin determination ( Klason method )
0.2gm of ground wood powder stir for 2mins
with glass rod then stand for 2hrs
Dilute the acid conc. to 3% with 112ml hot water Treatment of wood blocks with fungus
and seal them with aluminium cap
Tray containing bottles were placed inside
autoclave to process at 120C for 1hr
Cool it until room temp.
Saw dust(sal) control Treated saw dust(sal)
The mass of glass filter was obtained
Collection of precipitate by vaccum filtration Sample Lignin Content (%)
Control 45Days 90Days 120Days
Cover with aluminium foil for avoiding contamination
Place in the oven at 75C for drying overnight S3 26.4 17.9 11.6 9.7
Take filter outside and record the mass of glass filter with lignin
S4 34.5 18.8 12.4 11.9
Conclusion
❖ The isolated macrofungi contains LiP and MnP enzymes
❖ After optimization, we can conclude that the optimum pH for best enzyme activity is ranging from 6-6.5,
incubation periods 5-7 days and optimum temperature is 40-50℃ for all the test samples.
❖ The synthetic dyes were also screened for decolorization by the crude enzyme for which 3 majorly employed
dyes were selected and first investigated using solid plate assay. After seven days, Malachite green and congo
red completely decolorized, as revealed by the > 80 mm diameter of the decolorized zone. Decolorization of
nigrosine was less extensive with a diameter of decolorized zone between 40 to 60 mm. But in liquid media
we found 96.47% degradation of malachite green followed by congo red 94.98% and nigrosine 90.47% in 72
hours.
❖ The results show a significant reduction in lignin content in the wood block after fungal treatment. The lignin
content decreased by 7.7% (from 30.2% to 22.5%) after fungal treatment, indicating that the fungus
effectively degraded lignin in the wood block. This reduction in lignin content can be attributed to the
enzymatic activity of the fungus, which breaks down lignin into smaller fragments. The fungus used in this
study, likely a white rot fungus, produces enzymes such as manganese peroxidase, and lignin peroxidase,
which are known to degrade lignin
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Acknowledgement
My sincere regards to all teachers, staffs and research scholars of Dept. of Botany
for providing the necessary facilities.
I express my sincere respect to the honourable V.C Prof. Pabitra Kumar Chakrabarti
and officers of SKBU for providing the necessary facilities.
Special thanks to my classmates for their support and help during the collection of
samples.
