G.Narendrakumaret al.

/ Journal of Pharmacy Research 2012,5(8),4021-4023

Research Article
ISSN: 0974-6943

Available online through

www.jpronline.info

A Comparative Study of the Levels of Antioxidant Scavenger Enzymes Catalase and

Superoxide Dismutase in Escherichia coli and Pseudomonas aeruginosa during Exogenous
Hydrogen Peroxide Stress
G.Narendrakumar, V.Rameshkumar
Department of Biotechnology, Sathyabama University, Chennai 600119.

Received on:09-05-2012; Revised on: 14-06-2012; Accepted on:22-07-2012

ABSTRACT
The present investigation was carried out in Escherichia coli and Pseudomonas aeruginosa. Hydrogen peroxide (H2 O2 ) was selected for the stress
conditions because it is central to the cytotoxic action of many oxidative agents and there is also a routine generation of it as a by-product of oxidative
metabolism. Further on the efficiency of these enzymes under oxidative stress was analyzed. The significant rise of catalases in the organisms under study
is evident from the results. When bacteria treated with low doses of oxidants adapted well and subsequent treatment which leads to high doses of these
oxidants results in the expression of numerous genes. During second day the nutrient depletion could have also resulted in the exorbitant rise of proteins.
The increase in protein levels could be because of over-expression of regular proteins found in the cell or might also be because of expression of novel
proteins in the cells. The experimental values indicated that cultures of Pseudomonas aeruginosa had comparatively higher levels of proteins and
antioxidants scavengers enzymes than E.coli.
Key words: Hydrogen peroxide, E.coli, Pseudomonas aeruginosa, Catalase, Superoxide dismutase (SOD).

INTRODUCTION
Optimal growth conditions in their natural environments for a microorganism
are uncertain because they are exposed to stress in all means. Many bacteria
have numerous different single stress-induced responses at their disposal,
that allow them to cope with specific stress situations by eliminating the
stress agent and repairing actual damage. By contrast, the general stress
response can be triggered by many different stress conditions and renders
bacteria broadly stress resistant, even against stresses that they have not
yet experienced. This means that damage is prevented rather than repaired.
Oxidative stress can be functionally defined as an excess of reactive oxygen
species in the cell. Under certain conditions, production of reactive oxygen
species overwhelms the scavenging capacity of the cells, giving rise to
oxidative stress. In case of severe stress, vital cellular functions are damaged.
There are many enzymatic defenses in bacteria against oxidative stress like
superoxide dismutase, catalase etc.

broth were divided into four groups. One group consisted of control and the
rest of the three flasks were treated with different concentration of H2 O2 .
The same was executed for two days each for Escherichia coli and
Pseudomonas aeruginosa. A viable count was performed and growth of the
bacteria were compared by plotting a growth curve of control and stressed
bacterial cultures.

MATERIALS AND METHOD

CELL DISRUPTION
Cells from Day-1 cultures of Escherichia coli and Pseudomonas aeruginosa
were centrifuged at 7000 rpm for 10 minutes. Cells were then washed with
0.1 M potassium phosphate of pH 7.0. Washed cells were suspended in
0.05 M potassium phosphate and disrupted by sonication. Cell debris were
removed by centrifugation at 20,000 rpm for 30 minutes in a refrigerated
centrifuge. Supernatant was used directly for protein estimation and enzyme
assays. Protein assay was done by Lowry method using Bovine serum
albumin as standard.

BACTERIAL CULTURES
Escherichia coli and Pseudomonas aeruginosa were purchased from MTCC,
Chandigarh, India and maintained in Eosine Methylene Blue agar and
Pseudomonas isolation agar respectively.
BACTERIAL GROWTH UNDER H2 O 2 STRESS CONDITION
About 25ml fresh nutrient broth (Himedia) were prepared in 16 Erlenmeyer
flasks. The prepared broth were inoculated with 0.1ml Escherichia coli in 8
flasks and 0.1ml Pseudomonas aeruginosa culture in 8 flasks. The inoculated

*Corresponding author.
G. Narendrakumar
Assistant Professor
Department of Biotechnology,
Sathyabama University,
Chennai 600119,India

HYDROGEN PEROXIDE STRESS CONDITIONS

Hydrogen peroxide solution (Qualigens) was taken in three concentrations
(3mM, 6mM, 9mM) and was used to induce stress in Escherichia coli and
Pseudomonas aeruginosa cultures, with one flask from each group being
maintained as control without adding H2 O2 . The flasks were incubated at
37o C and one set Escherichia coli and Pseudomonas aeruginosa of were
analysed in Day -1 and another on Day-2.

ASSAY OF ANTIOXIDANT ENZYME

SUPEROXIDE DISMUTASE (SOD)
SOD was assayed in the cell lysate according to Misra and Fridovich (1972),
based on the inhibition of the oxidation of epinephrine to adenochrome by
superoxide. A 50% inhibition of epinephrine to adrenochrome transition by
the enzyme was taken as one enzyme unit. SOD activity was expressed as
units per mg.

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CATALASE
This enzyme was assayed according to the method of Arsu K.Sinha (1972).
Un-reacted H2 O2 was measured by its ability to reduce dichromate in acetic
acid solution. Catalase activity was expressed in mmol/min/mg protein.
RESULTS AND DISCUSSION
Growth analysis of the bacterial cultures shows that there is a marked
decrease in the cell density when exposed to hydrogen peroxide stress. This
also indicates that stress has made a significant effect on the physiology of
the cell. This observation stands in line with what the human immune
system uses (Respiratory burst) against bacterial infection. In general due
to hydrogen peroxide stress a set of proteins are known to be expressed in
bacterial cells. This response is defined as the peroxide mediated stress
response. The protein levels in E.coli (Bin Rui et al., 2010), increase with
increase in hydrogen peroxide concentration. This indicates that E.coli
responds to stress and has made peroxide stress responsive proteins. It can
also be seen that E.coli has more tolerance to hydrogen peroxide as even the
higher end concentration of hydrogen peroxide only increased stress
responsive protein levels. The growth curve also substantiates the tolerance
of E.coli towards stress.

Figure III SOD levels expressed in E.coli on Day 1 & 2.

Figure IV SOD levels expressed in P.aeruginosa on Day 1& 2.

Figure I Protein levels expressed in E.coli on Day-1 & Day-2.

Superoxidase dismutase was also found to be induced to a considerable

extent under stress condition. Induction of SOD was also dependent on
soxRS regulon and likely to be induced by preoxidase stress (Manchodo
and Michan, 2000). Although E.coli was sustaining stress environment by
producing more proteins, the levels of SOD in both the organisms under
study indicate that Pseudomonas aeruginosa made more amount of SOD,
the free radical scavenger enzyme. (Seon Joo Yoon et al., 2002).

Figure II Protein levels expressed in Ps.aeruginosa on Day-1 & Day-2.

Stress had a significant effect on Pseudomonas aeruginosa. The gradual

increase in protein levels in correlation with increasing hydrogen peroxide
concentration indicates that Pseudomonas aeruginosa also produces
peroxide stress responsive proteins. However the percentage of stress
proteins induced in Pseudomonas aeruginosa is considerably low with
respect to E.coli. This indicates that Pseudomonas aeruginosa is more
susceptible to hydrogen peroxide stress compared to E.coli.

Figure - V Catalase levels expressed in E.coli on Day 1& 2.

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survive in a wide variety of environments makes it a ubiquitous bacterium
in nature and likely contributes to its opportunistic behavior.

Figure VI Catalase levels expressed in Ps.aeruginosa on Day 1& 2.

The significant rise of catalases in the organisms under study is evident

from the results. To counteract toxic oxidants, the cells have induced these
enzymes to the required levels. In connection with these organisms, the
levels of both proteins and antioxidant scavenger enzymes were found to be
higher in Day-2 culture that Day-1. This is because that when bacteria
treated with low doses of oxidants adapted well and subsequent treatment
which leads to high doses of these oxidants results in the expression of
numerous genes. During second day the nutrient depletion could have also
resulted in the exorbitant rise of proteins. (Spencer et al., 2002). The increase
in protein levels could be because of over-expression of regular proteins
found in the cell or might also be because of expression of novel proteins in
the cells. The experimental values indicated that cultures of Pseudomonas
aeruginosa had comparatively higher levels of proteins and antioxidants
scavengers enzymes than E.coli. The ability of E.coli to resist, adapt and

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Source of support: Nil, Conflict of interest: None Declared

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