5/20/2018 A Simple Technique For Rapid Detection of Fungal Catalases by Visualization of Agar gel entrapped microbubbles

1/1

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 largenumber 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 h ydrogen

peroxide, a powerful and potentially harmful oxidizing agent to water and oxygen thereby protecting

cells from the toxic effects of hydrogen peroxide. They have the unique catalytic capacity t o 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 stabilit y 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 evolutio n, its rapid and effective removal by variousoxidoreductases 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 -haem peroxidases,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

from studies on bacterial and archaeal species. Eukaryotic KatGs, abund ant mainly among fungi and

protists, have hardly been described (Marcel et al,2009).

Much of the hydrogen peroxide that is produced during oxi dative 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 from milk 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 biodi versity 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 Oxygenthe gaseous product of the Catalase reaction. The Catalase assay involved formulation of a so ft 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 i n 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 , fi ve 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 mycelialfungi 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 grownmycelial colonies were overlaid with the medium.

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

In 3 cultures vigorous submerged growth was also observed indicating good tolerance forH2O2.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 thistechnique were clear indicators of catalase activity from the innoculated cultures.

The assay helped to detect satisfactory the catalase activity in 4 species namelyMacrolepiota procera, Termitomyces globulus, Volvariella bombycina, Bovista plumbearesulting in 40% success rate for screening the culture for biotechnologically importantenzyme. More test are in progress to validate the results and further refine the techniquewith respect to viscocity of the medium, biomass, size of the culture plug, concentration ofH2O2and the temperature of incubation.After this test the refined technique could be usedfor large scale screening of fungal cultures for catalase activity.It would be easier toefficiently determine superior strains which could be further researched for biotechnological

purpose.

ACKNOWLEDGEMENTS:-

We wish to acknowledge the support under UGC Sap Phase II program onBiodiversity.Bioprospecting and Biotechnology and all the facilites granted to work in thedepartment.

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 OblingerChristian (2009). Occurrence, phylogeny, structure, and function of catalases andperoxidases in cyanobacteria Journal of Experimental Botany, Vol. 60, No. 2, 423440

Zamocky Marcel, Paul G. Furtmuller and Oblinger Christian(2009).Two distinct groups offungal 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 ofBiosciences 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 t igr i nus

Termitomyces globulu s Termitomyces petaloides Termitomyces sp.

Bov is ta plumbea

Macrolepiota procera

Volvariella bombycina Pleurotus cystidiosus

Scores:-Size of bubbles Density of bubbles

Less than 1-2mm + Low

1-2mm ++ Moderate

Greater than 2m +++ High

Pleurotus pulmonar ius

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 globulusR.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

S r.n o. S pe ci es H ab it at L oc at io n

1 AgaricuscampestrisL. Lawn Porvorim, Bardez2 Macrolepiota procera(Scop.ex.Fr.)Kumm Litterrich soil GoaUniversity, Taleigao,

Tiswadi

3 TermitomycesglobulusR.Heim &Gooss Soil Santa Cruz, Tiswadi

4 Termitomycespetaloides Soil Santa Cruz, Tiswadi

5 Termitomyces Heim

sp.

Soil Santa Cruz, Tiswadi

6 Volvariella bombycina(Schaeff.)Singer Soil GoaUniversity, Taleigao,

Tiswadi

7 Bovista plumbeaP er s. S oil Go aUn iv er si ty, Ta le ig ao ,

Tiswadi

8 Panustigrinus(Bull.)Fr. LogofMangifera indica Raia, Salcete

9 Pleurotuspulmonarius (Fr .) D eca ye d wo od Go aUn iv er si ty, Ta le ig ao ,Tiswadi

10 PleurotuscystidiosusO.K.Mill. TreeofAverrhoabilimbi Nuvem. Salcete

Fig 1 Fig 2

Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7

Fig. 8 Fig.10 Fig. 11Fig. 9 Fig. 12

Fig. 14a Fig. 14b Fig. 14c Fig. 15bFig. 13

Control

Vigorous

react ion

in T.

globulusM. procera

Detai ls of

macrobuubles in

T. globulus

Lower sec tion of

tube ind ica ting

v igorous reac tion

in T. globulus M. proceraM. 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 gelEntrapped Oxygen Microbubbles

Devika Sinai Priolkar and Nandkumar M Kamat*Department of Botany,Goa University,Taleigao Plateau,Goa- 403 206

*email: nandkamat@gmail.com

Testtubeno.

Species Treatment

1 Agaricuscampestris L. 1mls teriledistilled waterwithmycelial biomass +2ml autoclavedPDA media+7mlsyringefi ltered

30% H2O2

2 Macrolepiotaprocera (Scop.ex.Fr.)Kumm 1mls teriledistilled waterwithmycelial biomass +2ml autoclavedPDA media+7mlsyringefi ltered30% H2O2

3 TermitomycesglobulusR.Heim &Gooss 1mls teriledistilled waterwithmycelial biomass +2ml autoclavedPDA media+7mlsyringefil tered

30% H2O2

4 Termitomycespetaloides 1mls teriledistilled waterwithmycelial biomass +2ml autoclavedPDA media+7mlsyringefi ltered30% H2O2

5 Termitomyces Heim

sp.

1mls teriledistilled waterwithmycelial biomass +2ml autoclavedPDA media+7mlsyringefi ltered

30% H2O2

6 Volvariellabombycina(Schaeff.)Singer 1mls teriledistilled waterwithmycelial biomass um +2mlautoclaved PDA media+7mlsyringefiltered

30% H2O2

7 Bovista plumbeaPers. 1mls teriledistilled waterwithmycelial biomass +2ml autoclavedPDA media+7mlsyringefi ltered30% H2O2

8 Panustigrinus (Bull.)Fr. 1mls teriledistilled waterwithmycelial biomass +2ml autoclavedPDA media+7mlsyringefi ltered30% H2O2

9 Pleurotuspulmonarius (Fr.) 1mls teriledistilled waterwithmycelial biomass +2ml autoclavedPDA media+7mlsyringefi ltered

30% H2O2

10 Pleurotuscystidiosus O.K.Mill. 1mls teriledistilled waterwithmycelial biomass +2ml autoclavedPDA media+7mlsyringefi ltered

30% H2O2

Control - 1 m l s t er i le d i s ti l l ed w a te r + 2 m l au t oc l a ve d P DA m e di a +7 m l s yr i ng e f il t e re d 3 0% H2O2

Assay for detection of catalase activity