Abstract:-Catalase is an ubiquitous, efficient enzyme which catalyzes the decomposition of hydrogen peroxide to water and oxygen.

As part of our bioprospecting work aimed to detect industrially useful hydrolytic enzyme activities, we report a simple, rapid and reliable technique to screen a large
number of fungal cultures for catalase activity based on visualization of the microbubbles of Oxygen trapped in the agar gel matrix. Catalases are usedin industries in conjunction with glucose oxidase, for treatment of food wrappers to prevent the deterioration of food and also to remove traces of
hydrogen peroxide in the process of cold sterilization of milk and cheese. Promising catalase producing cultures would be tested further for identifying biotechnologically useful strain.

INTRODUCTION:-
Catalase (EC 1.11.1.6) is a haem containing enzyme which catalyzes the conversion of hydrogen
peroxide, a powerful and potentially harmful oxidizing agent to water and oxygen thereby protecting
cells fromthe toxic effects of hydrogen peroxide. They have the unique catalytic capacity to dismutate
hydrogen peroxide by their striking ability to evolve molecular oxygen (O2) by oxidation of H2O2. They
are produced by Bacteria, Archaea, and Eukarya (Margit et al,2009). Their stability and resistance to
proteolysis is an evolutionary advantage, especially since they are produced during the stationary phase
of cell growth when levels of proteases are high and there is a rapid rate of protein turnover.H2O2 is a
harmful metabolic by-product of aerobic life hat also acts as second messenger in signal messenger in
signal transduction pathways. During cellular evolution, its rapid and effective removal by various
oxidoreductases was of essential importance. Cells evolved not only enzymes capable of efficient
dismutation of H2O2 , but also enzymes that reduce hydrogen peroxide with the help of various organic
and inorganic one-and two-electron donors (haem peroxidases and non-haemperoxidases,e.g.
peroxiredoxins)
KatGs (Catalase/Peroxidase)represent one of the most abundant families of Class I of the non-
animal haem peroxidase superfamily. They are unique in accomplishing efficiently both catalytic and
peroxidatic activity with various substrates. Most currently known KatG representatives are encoded in
bacterial genomes, and mechanistic knowledge about these peculiar bifunctional peroxidases has derived
fromstudies on bacterial and archaeal species. Eukaryotic KatGs, abundant mainly among fungi and
protists, have hardly been described (Marcel et al,2009).
Much of the hydrogen peroxide that is produced during oxidative cellular metabolism comes from
the breakdown of one of the most damaging ROS, namely the superoxide anion radical
(O2-). Superoxide is broken down by superoxide dismutases into hydrogen peroxide and oxygen.
Fungal catalases are the enzymes produced by fungi which catalyze the decomposition of
hydrogen peroxide to water and oxygen.
Catalase is used in the food industry for removing hydrogen peroxide frommilk prior to cheese
production. Another use is in food wrappers where it prevents food from oxidizing.
As compared to Bacteria, Archaea there is scanty work on fungal catalases. Fungi are reported to
be high producers of catalases. However most of the work is restricted to a small number of microfungi
(Isobe et al., 2006). There is a need for efficient and rapid detection of catalase from fungal sources. In
our laboratory efforts have been focused on biodiversity survey, bioprospecting of microorganisms
(actinomycetes, yeasts and mycelial fungi) and identification of industrially/biotechnologically useful
strains. An efficient and easily reproducible technique was developed for rapid detection of yeast
catalases (DSilva and Kamat,2008; also see figures 1 and 2) based on the release of Molecular Oxygen
the gaseous product of the Catalase reaction. The Catalase assay involved formulation of a soft medium
to trap the evolving microbubbles of Oxygen released in the reaction. The visualization of number, size,
density and distribution of the microbubbles indicated the relative intensity of Catalase activity. This
permitted primary selection of superior catalase producers.
Considering the large number of promising and underexplored cultures of basidiomycetes in our
collection, as a part of systematic screening for industrial enzymes from fungi, the present work was
aimed to modify the technique employed for Yeast catalase for detection of fungal catalases and in the
present work specifically to screen basidiomycetes cultures.

MATERIALS AND METHODS:-
Source of cultures
Basidiome context tissue cultures were obtained using standard isolation techniques from
mushrooms collected during June-November 2009 and identified in our lab using several keys
(Matchmaker, Kendrick, 2002, Singer,1986).
Maintenance of the cultures
The purified cultures were maintained on MEA slants and labeled. These were subcultured on
MEA slants after every month to ensure viability. All the cultures obtained were deposited in Goa
University Fungus Culture Collection (GUFCC)
Selection of cultures
Basidiomycetes cultures belonging to three different orders , five families, seven genera and 10
species were used in the present study as depicted in Table 1.








DISCUSSION:-
The technique used earlier was successful since the yeast cells used to get easily dispersed
in the media. Compared to unicellular yeast it is difficult to disperse filamentous mycelial
fungi in the medium, however, positive results were obtained with viable basidiomycetes
cultures in the work. This was possible because small plugs harvested from the well grown
mycelial colonies were overlaid with the medium.

Since fragmented innoculum was used it was thought that the smaller fragments could float
up and disperse in the medium and act as small colony forming units capable of utilizing the
dissolved hydrogen peroxide incorporated in the medium. The surface growth seen in all the
tubes clearly showed that mycelogenic units had reached the surface to allow such colony
formation-an event which took sometime (upto 7 days).

In 3 cultures vigorous submerged growth was also observed indicating good tolerance for
H
2
O
2
.Visualization of microbubbles permitted scoring of the intensity of catalase activity.
Since no bubble formation was noticed in control tubes the positive results obtained for this
technique were clear indicators of catalase activity from the innoculated cultures.

The assay helped to detect satisfactory the catalase activity in 4 species namely
Macrolepiota procera, Termitomyces globulus, Volvariella bombycina, Bovista plumbea
resulting in 40% success rate for screening the culture for biotechnologically important
enzyme. More test are in progress to validate the results and further refine the technique
with respect to viscocity of the medium, biomass, size of the culture plug, concentration of
H
2
O
2
and the temperature of incubation.After this test the refined technique could be used
for large scale screening of fungal cultures for catalase activity.It would be easier to
efficiently determine superior strains which could be further researched for biotechnological
purpose.

ACKNOWLEDGEMENTS:-
We wish to acknowledge the support under UGC Sap Phase II program on
Biodiversity.Bioprospecting and Biotechnology and all the facilites granted to work in the
department.

REFERENCES:-
DSilva, N.V. and Kamat, N.M. (2008).Simple Techniques for studying angiosperm nectar
ecology and microbiology. In: Novel Techniques and Ideas in Mycology (eds. K.R. Sridhar,
F. Barlocher, & K.D. Hyde).Fungal Diversity Research Series 20:183-201
Bernroitner Margit, Zamocky Marcel , Paul G. Furtmuller, Gunter A. Peschek and Oblinger
Christian (2009). Occurrence, phylogeny, structure, and function of catalases and
peroxidases in cyanobacteria Journal of Experimental Botany, Vol. 60, No. 2, 423440
Zamocky Marcel, Paul G. Furtmuller and Oblinger Christian(2009).Two distinct groups of
fungal catalaseBiochemical Society Transaction Vol.37.772-777
Isobe Kimiyasu, Inoue Noubaki, Takamatsu Yuuki, Kamada Kiyohiro and Wakao Norio
(2006).Production of Catalase by Fungi Growing at low pH and high temperature, Journal of
Biosciences and Bioengineering,Vol.101,73-76
















































Results:-
Colony morphology of selected basidiomycetes cultures. (The colony morphology of 7-14 days old cultures selected for the catalase assay is depicted in
figures-3-12)
Agaricus campestris
Panus tigrinus
Termitomyces globulus Termitomyces petaloides Termitomyces sp.
Bovista plumbea
Macrolepiotaprocera
Volvariellabombycina Pleurotus cystidiosus
Scores:-
Size of bubbles Density of bubbles
Less than 1-2mm + Low

1-2mm ++ Moderate

Greater than 2m +++ High
Pleurotus pulmonarius
Sr.no. Culture designation Species Size of bubbles Density of bubbles Colony development
1 GUFCC-9091 Agaricus campestris L.

++ Surface Growth
2 GUFCC-9092

Macrolepiota procera (Scop.ex.Fr.)Kumm

+++ Surface Growth

3 GUFCC-9093

Termitomyces globulus R.Heim & Gooss +++ Surface and
Submerged
4 GUFCC-9094 Termitomyces petaloides

+ Surface
5 GUFCC-9095 Termitomyces Heim
sp.
+ Surface
6 GUFCC-9096 Volvariella bombycina (Schaeff.) Singer +++ Surface and
Submerged
7 GUFCC-9097 Bovista plumbea Pers.

+++ Surface
8 GUFCC-9098 Panus tigrinus (Bull.)Fr.

+ Surface and
Submerged
9 GUFCC-9099 Pleurotus pulmonarius (Fr.)

+ Surface
10 GUFCC-9100 Pleurotus cystidiosus O.K.Mill.

+ Surface
Sr.no. Species Habitat Location
1 Agaricus campestris L. Lawn Porvorim, Bardez
2 Macrolepiota procera (Scop.ex.Fr.)Kumm Litter rich soil Goa University, Taleigao,
Tiswadi
3 Termitomyces globulus R.Heim & Gooss Soil Santa Cruz, Tiswadi
4 Termitomyces petaloides Soil Santa Cruz, Tiswadi
5 Termitomyces Heim
sp.
Soil Santa Cruz, Tiswadi

6 Volvariella bombycina (Schaeff.) Singer Soil Goa University, Taleigao,
Tiswadi
7 Bovista plumbea Pers. Soil Goa University, Taleigao,
Tiswadi
8 Panus tigrinus (Bull.)Fr. Log of Mangifera indica Raia, Salcete
9



Pleurotus pulmonarius (Fr.) Decayed wood Goa University, Taleigao,
Tiswadi
10 Pleurotus cystidiosus O.K.Mill. Tree of Averrhoa bilimbi Nuvem. Salcete
Fig 1 Fig 2
Fig. 3 Fig. 4
Fig. 5 Fig. 6 Fig. 7
Fig. 8

Fig.10 Fig. 11 Fig. 9 Fig. 12
Fig. 14a Fig. 14b Fig. 14c Fig. 15b
Fig. 13
Control
Vigorous
reaction
in T.
globulus
M. procera
Details of
macrobuubles in
T. globulus
Lower section of
tube indicating
vigorous reaction
in T. globulus M. procera
M. procera
Vigorous Catalase reaction in yeast
cultures (Desilva & Kamat, 2008)
An array of tubes used in Yeast catalase
detection (Desilva and Kamat, 2008)
Table 3 Scoring of catalase activity by visualization of microbubbles of molecular oxygen.
Table 2 Dilution reaction carried out to prepare a series of test tubes for the detection of catalase activity
Evolution of Macrobubbles of molecular O2
Fig. 15a
A Simple Technique For Rapid Detection of Fungal Catalases by Visualization of Agar gel
Entrapped Oxygen Microbubbles

Devika Sinai Priolkar and Nandkumar M Kamat*
Department of Botany,Goa University,Taleigao Plateau,Goa- 403 206
*email: nandkamat@gmail.com

Test tube
no.
Species Treatment
1 Agaricus campestris L. 1 ml sterile distilled water with mycelial biomass +2 ml autoclaved PDA media+7ml syringe filtered
30% H2O2
2 Macrolepiota procera (Scop.ex.Fr.)Kumm 1 ml sterile distilled water with mycelial biomass +2 ml autoclaved PDA media+7ml syringe filtered
30% H2O2
3 Termitomyces globulus R.Heim & Gooss 1 ml sterile distilled water with mycelial biomass +2 ml autoclaved PDA media+7ml syringe filtered
30% H2O2
4 Termitomyces petaloides 1 ml sterile distilled water with mycelial biomass +2 ml autoclaved PDA media+7ml syringe filtered
30% H2O2
5 Termitomyces Heim
sp.
1 ml sterile distilled water with mycelial biomass +2 ml autoclaved PDA media+7ml syringe filtered
30% H2O2
6 Volvariella bombycina (Schaeff.) Singer 1 ml sterile distilled water with mycelial biomass um +2 ml autoclaved PDA media+7ml syringe filtered
30% H2O2
7 Bovista plumbea Pers. 1 ml sterile distilled water with mycelial biomass +2 ml autoclaved PDA media+7ml syringe filtered
30% H2O2
8 Panus tigrinus (Bull.)Fr. 1 ml sterile distilled water with mycelial biomass +2 ml autoclaved PDA media+7ml syringe filtered
30% H2O2
9



Pleurotus pulmonarius (Fr.) 1 ml sterile distilled water with mycelial biomass +2 ml autoclaved PDA media+7ml syringe filtered
30% H2O2
10 Pleurotus cystidiosus O.K.Mill. 1 ml sterile distilled water with mycelial biomass +2 ml autoclaved PDA media+7ml syringe filtered
30% H2O2
Control - 1 ml sterile distilled water +2 ml autoclaved PDA media+7ml syringe filtered 30% H2O2
Assay for detection of catalase activity