A Project report on
PRO D U CTIO N O F LA CCA SE
BY CURVULARIA SPECIES
Completed at
M IT C ON B IO P HAR M A , PUN E
Submitted by
VANDANA KUMARI
DECLARATION
I hereby declare that this project report is my own account of work. The content of
this project work has not previously been submitted for degree at any tertiary
education institution.
Place : Pune
Date: 18/01/11
Vandana kumari
ACKNOWLEDGEMENT
First I express my hearted Thanks to Rituraj Patil (HOD-MITCON) for his kind
permission and inspiration to carry out this Project Work.
I express my deep sense of Gratitude of the Guide Ms. Priya Bande and Ms.
A.Pakhale who have been a great source of Inspiration and Guidance and
Encouragement to successful completion of my Project Work.
I express my sincere Thanks to HOD Dr. Jeyanti Rebecca Dept. of Industrial
Biotechnology of Bharath University for her whole hearted support
I express my hearted Thanks to my parents Mr. B.B.Singh & Mrs. Archana
Singh for their whole hearted support and encouragement.
I will be failing in my duty if I do not acknowledge with grateful thanks to the
author of references and other literature refers to this Project Work.
Last but not least I convey my sincere thanks to all staff members of MITCON
for their support, guidance and encouragement.
LIST OF ABBREVIATION
1. PDA :-Potato dextrose agar
2. YEPD:-Yeast extract peptone
3. MAX :- Maximum
4. C:-Celcius
5. ML :-Mililitre
6. M/ML :-Milli mole per litre
7. PCM :POTATO carrot medium
8. CD medium :Czpack dox medium
ABSTRACT
The project work focuses on the production of Laccase by curvularia sp.There are
following steps involved in this project work. First is selection of organism,
isolation of organism (curvularia lunata) from soil sample. Potato Dextrose Agar
is used as media for growth of curvularia. After 3 days of inoculation colonies of
fungus curvularia is observed. Next step is preparation of fermentation media.
Fermentation media is prepared by YEPD(Yeast Extract Peptone Dextrose) in
respective amount. Mycelium is obtained from colonies of fungus. Myceliun is
introduced into YEPD medium. Culture is kept on rotary shaker incubator for five
days.
Laccase (Benzenodial Oxygen Oxidoreductase) activity test is done. ABTS method
is used for detection of Laccase. Purification and characterization of Laccase is
also performed by column chromatography. Sephadex column is prepared to purify
the enzyme.
Initial medium PH 4.0 is and cultivation temperature 30 C were found to be most
suitable for maximum enzyme production. The optimum temperature for Laccase
activity were found to 5.2 and 50 C respectively under optimum condition. Some
divalent metal ions inhibited Laccase activity at very low concentration.
INTRODUCTION
The Tricopper site found in many laccases, notice that each copper center is bound
to imidazole (color code: copper is brown, nitrogen is blue).
Laccases (EC 1.10.3.2) are copper-containing oxidase enzymes that are found in
many plants, fungi, and microorganisms. The copper is bound in several sites;
Type 1, Type 2, and/or Type 3. The ensemble of types 2 and 3 copper is called a
trinuclear cluster. Type 1 copper is available to action of solvents, such as water. It
can be displaced by mercury, substituted by cobalt or removed via a copper
complexone. Removal of type 1 copper causes a decrease in laccase activity.
Cyanide can remove all copper from the enzyme however re-embedding with type
1 and type 2 copper has been shown to be impossible. Type 3 copper however can
be embed back into the enzyme.
Laccases
act
on phenols and
similar
molecules,
performing
a
one-
electron oxidations, which remain poorly defined. It is proposed that laccases play
a role in the formation of lignin by promoting the oxidative coupling of lignols, a
family of naturally occurring phenols. Laccases can be polymeric, and the
enzymatically active form can be a dimer or trimer. Other laccases, such as ones
produced by the fungus Pleurotus ostreatus, play a role in the degradation of
lignin, and can therefore be included in the broad category of ligninases.
Because laccase belongs to the oxidase enzyme family it requires oxygen as a
second substrate for the enzymatic action.
Spectrophotometry can be used to detect laccases, using the substrates ABTS,
syringaldazine, 2,6-dimethoxyphenol, and dimethyl-p-phenylenediamine. Activity
can also be monitored with an oxygen sensor, as the oxidation of the substrate is
paired with the reduction of oxygen to water.
Laccase Activity
Laccases do not release toxic peroxide intermediates. The four copper centers
creates four monoelectronic oxidations of a substrate which is catalyzed by type 1
copper. From type 1 copper, the electrons are transferred to the trinuclear cluster of
type 2 and type 3 copper. This is where the reduction of oxygen and release of
water takes place. It is because of the various copper centers that allow the
reduction of substrate to oxygen without generation of toxic intermediates. The
products of oxidation can undergo non-enzymatic reactions such as cross linking of
monomers, degradation of polymers and ring cleavage of aromatics.
Crosslinking of monomers
Both phenolic compounds and anilines generate radicals when oxidized by laccase.
These radicals can form dimers, oligomers or polymers via a covalent bond
between C-C, C-O or C-N.
Degradation of polymers
Laccase can degrade complex polymers such as lignin or humic acids. Reactive
radicals generate cause cleavage of covalent bonds and formation of monomers.
Because complex polymers are large it may create steric hindrance and prevent
direct enzyme contact. Laccase can also oxidize small organic compounds or
metals. These compounds once oxidized can cause radical degradation and
depolymerization of larger polymers.
Ring cleavage of aromatics
Laccase is able to catalyze ring cleavage of aromatic compounds.
Inhibition
Laccase can be inhibited by small anions such as: azide, halides, cyanide, and
fluoride. These anions bind to type 2 and type 3 copper and disrupts electron
transfer via copper centers, therefore reduces activity. Metal ions, fatty acids,
hydroxyglycine, and kojic acid can also inhibit laccase by causing amino acid
residue changes, conformational changes or copper chelation.
Applications and potential utility
Laccases have been examined as the cathode in enzymatic biofuel cells. They can
be paired with an electron mediator to facilitate electron transfer to a solid
electrode wire. Laccases are some of the few oxidoreductases commercialized as
industrial catalysts. The enzymes can be used for textile dyeing/textile finishing,
wine cork making, teeth whitening, and many other industrial, environmental,
diagnostic, and synthetic uses. Laccases can be used in bioremediation. Protein
ligand docking can be used to predict the putative pollutants that can be degraded
by laccase.
Activity of Laccase in Wheat Dough
Laccases have the potential to cross link food polymers such as proteins and
nonstarch polysaccharides in dough. In non starch polysaccharides, such as
arabinoxylans (AX), laccase catalyzes the oxidative gelation of feruloylated
arabinoxylans by dimerization of their ferulic esters. These cross links have been
found to greatly increased the maximum resistance and decreased extensibility of
the dough. The resistance was increased due to the crosslinking of AX via ferulic
acid and resulting in a strong AX and gluten network. Although laccase is known
to cross link AX, under the microscope it was found that the laccase also acted on
the flour proteins. Oxidation of the ferulic acid on AX to form ferulic acid radicals
increased the oxidation rate of free SH groups on the gluten proteins and thus
influenced the formation of S-S bonds between gluten polymers. Laccase is also
able to oxidize peptide bound tyrosine, but very poorly. Because of the increased
strength of the dough, it formed irregular bubble formation during proofing. This
was a result of the gas (carbon dioxide) becoming trapped within the crust and
could not diffuse out (like it would have normally) and causing abnormal pore size.
Resistance and extensibility was a function of dosage, but at very high dosage the
dough showed contradictory results: max resistance was reduced drastically. The
high dosage may have caused extreme changes in structure of dough, resulting in
incomplete gluten formation. Another reason is that it may mimic overmixing,
causing negative effects on gluten structure. Laccase treated dough had low
stability over prolonged storage. The dough became softer and this is related to
laccase mediation. The laccase mediated radical mechanism creates secondary
reactions of FA-dervived radicals that result in breaking of covalent linkages in
AX and weakening of the AX gel.
Use in Food Industry
The hazing effect is a quality defect in beer. It is characterized by “cloudiness” in
the final product. Laccase can be added to the wort or at the end of the process to
remove the polyphenols that may still remain in beer. The polyphenol complexes,
formed by laccases, can be separated via filtration and removes probability of the
hazing effect from occurring.
Because oxygen is not desired in beer, laccase can also remove excess oxygen and
increase the storage life of beer.
In fruit juices such as apple and grape, excess oxidation of phenolics causes
negative effects on the taste, color, odour and mouthfeel. Laccasae has been
proposed to delay the oxidation of polyphenols and stabiliz. Curvularia is a
hyphomycete (mold) fungus which is a facultative pathogen of many plant species
and of the soil. Most Curvularia are found in tropical regions, though a few are
found in temperate zones.
Curvularia defined by the type species C. lunata (Wakker) Boedijn. Curvularia
lunata appears as shiny velvety-black, fluffy growth on the colony surface. C.
lunata is distinguished by septate, dematiaceous hyphae producing brown,
geniculate conidiophores. The poroconidiaare curved slightly to distinctly,
transversely septate, with an expanded third cell from the pore end of the
conidium. Curvularia can be easily distinguished from Bipolaris and Drechslera
spp. since the conidia are non-distoseptate, that is, septate from edge to edge of the
conidial
wall.
The teleomorphic state
of
the type
species Curvularia
lunata is Cochliobolus lunatus (Fam. Pleosporaceae, Ord. Pleosporales, Cla.
Loculoascomycetes, Phy. Ascomycota).
Curvularia is a fungal genus primarily native to tropical and subtropical regions,
although some species live in temperate zones. These fungi are primarily of
interest in their role as plant pathogens, as they can cause severe damage to a
variety of crops. In some cases, Curvularia can also cause disease in humans or
animals.
These fungi particularly enjoy cellulose as a substrate, and they can be found
growing on wood, decaying plant matter, and living plants. Some colonize grasses
and cereal grains, and others can live in the soil. When a Curvularia colony
develops, the fungi mass together to create a mat with a slightly woolly texture.
These fungi are gray to black in color, typically darkening with age, and the
underside of the colony will be a more pale white to gray.
In the tropics and subtropics, Curvularia are endemic to soil and crops, and they
can be difficult to eradicate. Some antifungal applications appear to be effective,
but because the fungi can lie dormant in the soil, it may reappear in the future. In
temperate zones, the fungi are often found indoors, where they like to grow on
wood walls, and near hot springs, where they enjoy the warm temperatures. In fact,
in some cases Curvularia can form a symbiotic relationship with plants, allowing
the plants to grow closer to hot springs than they would be able to on their own.
MATERIALS & METHODOLOGY
Techniques used in production of Laccase :
1. ISOLATION OF CURVULARIA LUNATA
Procedure
Preparation of Media
 Natural Media
 Semisynthetic Media
 Liquid Media
2. NATURAL MEDIA:PDA(Potato Dextrose Agar)
Components:
i. Potato
ii. Dextrose
iii. Agar
iv. Distilled Water
v. Soil Sample
3. EQUIPMENT USED:
i. Autoclave
ii. Incubator
iii. Weighing Machines
iv. Measuring Cylinder
v. Flask
4. PREPARATION OF SEMISYNTHETIC MEDIA:
SB Broth
Ingradient (Gram per Litre)
i. Casein Enzymatic hydrolysate:5.0 gram
ii. Peptic digest of animal tissue:5.0 gram
iii. Dextrose: 40gram
iv. Chloramphenicol: 0.05 gram
Equipment Used
i. Autoclave
ii. 250 ml flask
5. PREPARATION OF LIQUID MEDIA:
Components :
i. 5gram of Carbon source
ii. 1gram of KH2PO4
iii. 0.5gram MgSO4.7H2O
iv. 0.2 gram NH4NO3
v. 0.1gram YEAST EXTRACT
vi. 0.01 gram CaCl2
vii. 1mg CuSO4.5H20
viii.1 mg FeSO4.7H20
ix. 1mg MnSO4
x. Water:1 Litre
PREPARATION OF FERMENTATION MEDIA:
Fermentation media
is required for growth of Curvularia lunata as well as
production of extracellular enzyme Laccase.
Components of Fermentation Media:
YEPD(Yeast Extract Peptone Dextrose)
1,Yeast Etract:1%(m/v)
2 Peptone:2%
3 Dextrose: 1%
4 Distilled Water
EQUIPMENT USED
1 Autoclame
2 Measuring cylinder
3 Weighing Machine
4 250 ml Flask
PROCEDURE:
 Fementation Media is prepared.
 YEPD is autoclaved at 30 minutes.
 Laminar flow is used for inoculation of mycelium of Curvularia into the
respective media.
 After a 5 day of incubation at 30 degree in a reciprocal shaker with an
 agitation frequency of 4.2Hz .
 The mycelium were harvested and supernatant was checked with Laccase
activity .extracellular protein.
All experiment were conducted in triplicate.
PREPARATION OF LACCASE :
 The fungal culture was filtered through aWhatman filter paper no.1 to
remove mycelia.
 The filtrate was passed through a sephadex column .
 A column is preequilibrated with phosphate buffer (10m mol/litre.PH 6.5)
and Laccase was eluted from the column with the same buffer.
 5 ml sample were collected and assayed for Laccase activity.
ENZYME ASSAY :
 Laccase activity was assayed with guaicol as substrate.
 The reaction mixture contained 3.9 ml acetate buffer (10 m mol /L,PH
5.0),1ml guiacol (2 m mol/L) and 1.0 ml proprerly.
 Properly diluted enzyme solution was incubated at 50 C for 30 minutes.
 Absorbance was read at 450 nm.
 Guaicol was replaced with buffer in the blank.
 Enzyme activity was expressed in terms of unit (CU /ML)
OBSERVATION
Colonies of Curvularia on myceliumn disks plated on water agar and incubated at different
temperature for 7 days
Sporulation of colonies of Curvularia grown from spores shown on malt extract agar and
incubated at different temperatures for 7 days
Sporulation of mycelia disks of Curvularia plated on different agar media grown and incubated
at 15o C temperatures for 7 days
Sporulation of colonies of Curvularia grown from spores shown on different agar media and
incubated at 24oC temperatures for 7 days
Growth on different Nitrogen Sources
Germination of spores harvested at weekly intervals from culture of Curvunaria store on water
agar at 10oC temperatures for 7 days
Germination of spores harvested on weekly intervals from culture of Curvunaria stored on Malt
extract agar at 10oC temperatures for 7 days
Colonies of Curvularia lunata grown after incubation of 3 days
Growth was best in organic nitrogen sources with Glycine.
RESULT AND DISCUSSION
Growth Measurement :