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Role of enzymes in arsenite tolerance in bacterial isolate Wng-1 full report
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Role of enzymes in arsenite tolerance in bacterial isolate Wng-1
A project report submitted for the partial fulfillment of Master of Science in Biotechnology

Submitted by:
Sanjay Kumar 07-BT-09
Department of Biotechnology University of Pune Pune-411007
Under the guidance of:
Dr. W. N. Gade
Prof. and Head Department of Biotechnology University of Pune Pune-411007
INTRODUCTION:
Bacteria are simple cells with simple organization of cellular structure and limited compartmentalization as compared to eukaryotes. They are found in very diverse range of geographical locations and environmental conditions. This is a way of natural selection through which they have developed various strategies to combat the unfavorable conditions like high and low temperature, different extremes of living conditions. They are also found at the location where heavy metals concentration is extremely high enough to sustain the life, because heavy metals are well known toxic forms to living cells. Survival of micro organism facing heavy metal stress environment is enhanced by physiological changes at the biochemical level that enable the organism to overcome different types of stress. However metals are important cellular components for all organisms, some of which are cofactors of enzymes involved in the key cellular processes where as others are needed to maintain both inter and intracellular ionic balance. Heavy metals on the other hand are normally toxic to cells even at very low concentration, thus presents a serious threat to life forms.
Most heavy metals are transition elements, these includes Ag, Bi, Cd, Co, Cu, Ge, Hg, Pd, Ni, Tl or Zn cations and the oxyanions of As, Cr, Sb, Te or W etc .Transition elements have incompletely filled d-orbitals that provide heavy metals cations, the ability to form complex compounds, which may be redox active or not .These redox active complexes having free electrons that impart them the tendency to bind with important biomolecules like proteins, nucleic acid, enzymes and phospholipids etc[1].
Heavy metals are also known to generate reactive oxygen species (ROS). ROS have antibacterial effects mainly attributed to DNA and protein damage by the Fenton reaction. Mechanism of protection does not depend on transcriptional gene induction but rather suppression of DNA damage by preventing Fenton reaction[2].
Fe2+ + H202 Fe3+ + OH + OH -
OH+DNA H20 + Oxidized DNA
FADH2+Fe3+ * FADH +Fe2+ + H+
Arsenic, a naturally occurring element is one of the most prevalent toxic metals in the environment; mainly of geochemical origin (rocks and minerals) and also derives from anthropogenic sources as Industrial operations include the production of antimicrobial drug . Wood preservative chromated copper arsenate, arsenic pesticides, glass production, pharmaceuticals, non-ferrous alloys and wastes of leather industries. Inorganic arsenic is still used as an anti-parasitic agent in veterinary medicine and in homeopathic and flock remedies. This leads to ecological problems and it is frequently present in high concentrations in drinking water. Non-biodegradability of this heavy metal ion is responsible for its bioaccumulation in the food chain. Although arsenic is generally toxic to life, it has been demonstrated that microorganisms can use arsenic compounds as electron donors or electron acceptors, and that they can possess arsenic detoxification mechanisms [3-6].
Arsenic is found in mainly in two forms: arsenite and arsenate. Both forms are toxic: arsenite disrupts sulfhydryl groups of proteins and interferes with enzyme function, whereas arsenate acts as a phosphate analog and can interfere with phosphate uptake and transport. Arsenic, like other heavy metals, cannot be destroyed once it has entered the environment [7]. Microorganisms have evolved a variety of mechanisms for coping with arsenic toxicity, including minimizing the amount of arsenic that enters the cell (e.g., through increased specificity of phosphate uptake), oxidizing the arsenite (through the activity of arsenite oxidase), or arsenite peroxidation with membrane lipids. Resistance to arsenic species in both Gram-positive and Gram-negative organisms results from energy-dependent efflux of either arsenate or arsenite from the cell, mediated by the ars operon [4-6]. Our earlier studies
confirmed the existence of a bacterium with an arsC gene that is responsible for the
[8]
conversion of As(V) to As(II) which may be either extruded from the cells or sequestered in the intracellular compartment in its free form and/or in conjugation with glutathione (GSH) or other thiols [9].
The elevated expression of stress proteins is considered to be a universal response to adverse conditions, representing a potential mechanism of cellular defense against disease and a potential target for novel therapeutics. The stress response is a phenomenon of adaptation of organisms. Altered patterns of protein synthesis, including stress proteins (SP), may serve to monitor the impact of exposure to natural and anthropogenic stressors. This response is also metal specific and it is related to factors such as mode of metal uptake, distribution and accumulation among tissues, subcellular distribution within a tissue, and secondary generation of molecular stressors'101 .
Exposure to arsenicals either in vitro or in vivo in a variety of model systems has been shown to cause the induction of a number of the major stress protein families such as heat shock proteins (Hsp), Superoxide dismutase, Catalase, Glutathione Reductase, Glutathione Peroxidase, Arsenite oxidase enzyme. Superoxide dismutases are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. Catalase is a common enzyme found in nearly all living organisms which are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen. Glutathione peroxidase, whose main biological role is to protect the organism from oxidative damage. The biochemical function of glutathione peroxidase is to reduce free hydrogen peroxide to water. Glutathione reductase, which reduces glutathione disulfide (GSSG) to the sulfhydryl form GSH, which is an important cellular antioxidant[11].
Removals of heavy metals from the soil and water or their remediation from the waste streams at source have been a long-term challenge. Although the removal of the toxic heavy metals from industrial waste water has been practiced for several decades, the cost effectiveness of the most common physico-chemical processes such as oxidation and reduction, chemical precipitation, filtration, electrochemical treatment, evaporation, reverse osmosis processes etc has been matter of debate.
Biological approaches on the other hand, offer the potential for the highly selective removal of toxic metals coupled with considerable operational flexibility; they can be used either in situ or ex situ in a range of bioreactor configurations. Many such processes utilize microorganisms that have key roles in the biochemical cycling of toxic metals and radio nucleotides. Advances in understanding the roles of microorganisms in such processes, together with the ability to fine tune their activities using the tools of microbiology, has led to the development of noval or improved metal remediation processes. The use microbes for the removal and recovery of toxic metals from industrial effluents can be economical and effective methods for metal
removal. The heavy metal removal capacities of microbes are higher than those conventional methods and the up take of heavy metal can be selective [12].
Understanding of the arsenite tolerance mechanism in microorganism will give a potent approach for the use of microorganism in bioremediation of toxic arsenic as well as their use in bioleaching of arsenic from its ore. It will also help us to develop an recombinant approach of bioremediation using genetically engineered bacterial strain having overexpressed genes responsible for arsenite tolerance. This type of approach has already been used for some other heavy metals by over expressing metal binding peptides or proteins such as poly histidines [13].
LITERATURE REVIEW:
The metalloid arsenic (As) is a member of group V of the periodic table and is thus classified as a heavy metal [14]. The characteristics of As salts' reactivity and toxicity depend on their oxidative states, the trivalent form being the most reactive. In nature, As is found as oxides or sulfur compounds and it is mainly distributed throughout the environment by water. Among the chemical species of As present in the environment, inorganic arsenic (iAs) is generally considered the most hazardous. The presence of As in ground water is largely the result of minerals being dissolved naturally from weathered rocks and soils. Drinking water contaminated with iAs, along with industrial emissions, are the major sources of human exposure worldwide. Exposure to iAs causes many adverse human health effects, including cardiovascular, hepatic, and renal diseases, in addition to cancer in kidney, liver, lung, urinary bladder, and skin [15]. Currently, the estimation of cancer risk due to the presence of iAs in drinking water, generally obtained from deep wells, is of great concern since more than 100,000,000 individuals worldwide are considered to be exposed to excessive amounts of iAs [16]. The toxicity of As compounds highly depends on the oxidation state and chemical composition of the arsenical. Traditionally, inorganic arsenicals have been considered more toxic than organoarsenicals. Comparative toxicity between inorganic arsenicals and pentavalent organoarsenicals showed that inorganic arsenicals are the most toxic As species [17]
Thus, regardless of being inorganic or organic, trivalent As species appear to be the most toxic. Trivalent As toxicity could be carried out either directly, by attacking -SH groups, or indirectly, through the generation of reactive oxygen species (ROS) [18]. The toxicity of iAs(V) appears to be mediated through its ability to substitute phosphate groups, affecting enzymes that depend on this group for their activity (e.g., interfering in the synthesis of ATP and DNA synthesis). The As-binding site facilitates the oxidative damage to proteins,in particular that of the amino acid residues involved. Among the amino acid residues normally found in proteins, methionine (Met) is one of the most readily oxidized at or near the cation binding site of the protein ' 1 converting Met to methionylsulfoxide. The site-specific alteration of an amino acid usually inactivates the enzyme by destruction of the cation binding site. A critical understanding of the capacity of arsenicals to modulate the expression and/or accumulation of stress proteins will definetly help to understand the exact mechanism of arsenic tolerance machinery in microorganism. The physiological consequences of the arsenic-induced stress and its usefulness in monitoring effects resulting from arsenic exposure in humans and other organisms are discussed.
The effect of Arsenite (II) stress on redox enzyme(s) activities [20]
Superoxide is an anion with the chemical formula O2-. It is important as the product of the one-electron reduction of dioxygen O2, which occurs widely in nature. With one unpaired electron, the superoxide ion is a free radical, and, like dioxygen, it is paramagnetic.
The enzyme superoxide dismutase (EC. 1.15.1.1), catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, it is an important antioxidant defense in nearly all cells exposed to oxygen. Superoxide is one of the main reactive oxygen species (ROS) in the cell and as such, SOD serves a key antioxidant role. The SOD-catalyzed dismutation of superoxide may be written with the following half-reactions:
M(n+1)+ - SOD + O2- Mn+ - SOD + O2
Mn+ - SOD + O2- + 2H+ M(n+1)+ - SOD + H2O2
Where M =Heavy Metals (As)
In this reaction the oxidation state of the metal cation oscillates between n and n+1.
The enzyme Catalase (EC. 1.11.1.6) is a common enzyme found in nearly all living organisms which are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen.
Catalase has one of the highest turnover numbers of all enzymes; one molecule of catalase can convert millions of molecules of hydrogen peroxide to water and oxygen per second. Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long. It contains four porphyrin heme (iron) groups that allow the enzyme to react with the hydrogen peroxide.
While the complete mechanism of catalase is not currently known, the reaction is believed to occur in two stages:
H2O2 + As (II)-E H2O2 + O=As(V)-E(.+)
H2O + O=As(V)-E(.+) H2O + As(II)-E + O2
Any heavy metal ion will act as a noncompetitive inhibitor of catalase. The poison cyanide is a competitive inhibitor of catalase, strongly binding to the heme of catalase and stopping the enzyme's action.
Hydrogen peroxide is a harmful by-product of many normal metabolic processes: To prevent damage, it must be quickly converted into other, less dangerous substances. Catalase is frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide into less reactive gaseous oxygen and water molecules. It works at an optimum temperature of 37 C, which is approximately the temperature of the human body. It is usually located in a cellular organelle called the peroxisome. Peroxisomes in plant cells are involved in photorespiration (the use of oxygen and production of carbon dioxide) and symbiotic nitrogen fixation (the breaking apart of diatomic nitrogen (N2) to reactive nitrogen atoms (RNS).
The enzyme Glutathione reductase (GR) (EC. 1.6.4.2) is an enzyme that reduces glutathione disulfide (GSSG) to the sulfhydryl form GSH, which is an important cellular antioxidant.
For every mole of oxidized glutathione (GSSG) one mole of NADPH is required to reduce GSSG to GSH. NADPH reduces FAD present in GSR to produce a transient FADH- anion. This anion then quickly breaks a disulfide bond (Cys58 - Cys63) and leads to Cys63 nucleophilically attacking the nearest sulfide unit in the GSSG molecule (promoted by His467) which creates a mixed disulfide bond (GS-Cys58) and a GS- anion. His467 of GSR then protonates the GS- anion to form the first GSH. Next, Cys63 nucleophilically attacks the sulfide of Cys58 releasing a GS- anion which in turn picks up a solvent proton and is released from the enzyme, thereby creating the second GSH. So, for every GSSG and NADPH you gain two reduced GSH molecules that can again act as antioxidants scavenging reactive oxygen species in the cell.
The enzyme Glutathione peroxidase (EC 1.11.1.9) is the general name of an enzyme family with peroxidase activity whose main biological role is to protect the organism from oxidative damage. The biochemical function of glutathione peroxidase is to reduce lipid hydroperoxides to their corresponding alcohols and to reduce free hydrogen peroxide to water.
An example reaction that glutathione peroxidase catalyzes is:
2GSH + H2O2 GS-SG + 2H2O
Where GSH represents reduced monomelic glutathione and GS-SG represents glutathione disulfide. Glutathione reductase then reduces the oxidized glutathione to complete the cycle.
GS-SG + NADPH + H+ 2 GSH + NADP+
The mechanism is at the Selenocystein site, which is in a Se (-) form as resting state. This is oxidized by the peroxide to SeOH which is then trapped by a GSH molecule to Se-SG and by another GSH molecule to Se (-) again, releasing a GS-SG by product. Glutathione peroxidase is a selenium-containing tetrameric glycoprotein, that is, a molecule with four selenocysteine amino acid residues. As the integrity of the cellular and subcellular membranes depends heavily on glutathione peroxidase, the antioxidative protective system of glutathione peroxidase itself depends heavily on the presence of selenium.
The enzyme Arsenite Oxidase (EC 1.20.98.1) This enzyme belongs to the family of oxidoreductases, specifically those acting on phosphorus or arsenic in donor with other, known, acceptors.
The phrase "eating and breathing" minerals in the context of seeking evidence of inorganic signatures for life on other planets in contrast to earlier searches for life on Mars that emphasized organic carbon- containing signatures. For arsenic, "eating" means arsenite functioning as an electron donor at the start of a membrane\ respiratory chain (Fig. 1A), and "breathing" means arsenate functioning as a terminal electron acceptor for an anaerobic respiratory chain (Fig. 1B), as do other minerals, such as Fe and Mn with some microbes'21-25' .
Figure 1: Cellular location and function of bacterial respiratory arsenite oxidase and respiratory arsenite reductase.
Thus understanding the regulation of heavy metal resistance could be useful for biological waste treatment and to estimate the impact that industrial activity may have on natural ecosystems. In our laboratory a bacterial strain has been isolated, named as Wng-1, which has been found to withstand high concentration of arsenite. This project makes small efforts at exploring the mechanism of this metal tolerance. The details of aims and objectives are given below.
AIMS & OBJECTIVES:
1) To study the growth profile of Wng-1 in presence and absence of arsenite.
2) To determine Minimum Inhibitory Concentration of Wng-1.
3) To assay the redox enzymes of Wng-1 in the presence and absence of arsenite.
A) B)
C)
D)
Superoxide dismutase
Catalase
Glutathione Reductase
Glutathione Peroxidase
4) To assay the arsenite oxidase enzyme of Wng-1 in presence and absence of arsenite.
MATERIALS AND METHODS:
A) Study the growth profile of Wng-1:-
To determine the growth characteristics of the strain Wng-1, bacteria were grown in 148.5 ml LB flasks containing 1.5 mL of LB medium inoculated with cells to obtain an initial cellular concentration of about (O.D=0.02 at 600 nm). It was distributed as 5ml aliquots in each tube, and was incubated at 280C with continuous shaking at 180 rpm. and taking optical density at 600 nm after every hour till a constant O.D. was observed'26' .
B) Minimum inhibitory concentrations (MIC):-
The liquid media previously described (LB), non-amended (controls) or amended with the arsenite (Sodium arsenite) at different concentrations from stock solutions, were inoculated with cell suspensions from precultures (O.D=0.02 at 600 nm). The following concentrations, of metal was tested: (0.0,1.0,1.5,2.0,2.5,3.0, 3.5,4.0,4.5,5.0, 6.0 7.0 mM). Absorbance of the culture was taken after 20 hours where late log phase of bacteria was started (known from the growth profile study of Wng-1 '27' .
C) Growth profile of bacterial isolate Wng-1 in 3.5mM arsenite:-
The growth pattern of the bacterium was also studied in Arsenite by maintaining the culture in LB medium amended with Arsenite. It was distributed as 5ml aliquots in each tube, and was incubated at 280C with continuous shaking at 180 rpm. and taking optical density at 600 nm after every hour till a constant O.D. was observed'26'
D) Protein Extraction from Wng-1:-
Wng-1 was inoculated in 100 ml of non-amended and LB amended with arsenite and was incubated at 280C for 20 hours; cells were pellet down from both culture and were resuspended in lysis buffer. Cells were sonicated for and were centrifuged. Supernatant was aliquoted and aliquots were stored at -800C for further analysis.
E) Enzyme Assays 1
1. Determination of Specific activity of Superoxide Dismutase (SOD): Protocol:
Superoxide dismutase was estimated according to the methodology of Kono (1978). Reagents:
COMPONENTS CONCENTRATION
Sodium carbonate buffer 50 mM, pH 10.0
Nitroblue tetrazolium (NBT) 96 mM
Triton X-100 0.6%
Hydroxylamine hydrochloride 20mM pH 6.0
In the test cuvette, the reaction mixture containing: 1.8 ml sodium carbonate buffer, 750 ml NBT and 150 ml Triton X-100. The reaction was initiated by the addition of 150 ml hydroxylamine hydrochloride. After 2 minutes, 20 ml of the enzyme extract was added. The percentage inhibition in the rate of NBT reduction was recorded as increase in absorbance at 540nm.
2. Determination of Specific activity of Catalase: Protocol:
Catalase activity was determined as per the method of Aebi (1974).
Reagents:
COMPONENTS CONCENTRATION
Phosphate buffer 50 mM pH 7.0
Hydrogen peroxide (H2O2) 30 mM
The reaction mixture contained 1.5 ml potassium phosphate buffer (100 mM, pH 7.0), 1.2 ml H2O2 (150 mM) and 100 ml of enzyme extract. The decrease in absorbance/minute was recorded at 240nm. Enzyme activity was determined using the extinction coefficient of 6.93 x 10" mM- cm- .
3. Determination of Specific activity of Glutathione reductase (GR): Protocol:
The activity of glutathione reductase (GR) was measured by the method of Carlberg and Mannervik (1975).
The reaction mixture contained 1.8 ml potassium phosphate buffer (50 mM, pH 7.6), 300 ml each of EDTA (3 mM), NADPH (0.1 mM), oxidized glutathione (GSSG) (1 mM) and enzyme extract. Enzyme activity was determined using the extinction coefficient of 6.22 mM-1cm-1.
4. Determination of Specific activity of Glutathione Peroxidase:
Protocol:
The activity of glutathione Peroxidase (GR) was measured by the method of
Carlberg and Mannervik (1975).
Reagents:
COMPONENTS CONCENTRATION
Phosphate buffer 50 mM pH 7.6
Ethylenediamine tetra acetic acid (disodium salt) (EDTA) 3 mM
NADPH (Nicotinamide adenine dinucleotide phosphate) 0.1 mM
Oxidized glutathione (GSSG) 1 mM
The reaction mixture contained 1.8 ml potassium phosphate buffer (50 mM, pH 7.6), 300 ml each of EDTA (3 mM), NADPH (0.1 mM), oxidized glutathione (GSSG) (1 mM) and enzyme extract. Enzyme activity was determined using the extinction coefficient of 6.22 mM-1cm-1.
5) Determination of Specific activity of Arsenite Oxidase: Protocol:
Anderson and colleagues (1992).
Reagents:
COMPONENTS CONCENTRATION
Arsenite Metal As (II) 50mM
2,4 - dichlorophenolindophenol (DCPIP) 60 mM
Marpholinoethane Sulfonic Buffer (MES) 50 mM pH 6
Phenazine methosulfate (PMS) 2 mM
Arsenite oxidase activity was determined by following the transfer of reducing equivalents from As(II) 200 mM to 60 mM 2,4-dichlorophenolindophenol (DCPIP) in 50 mM MES (pH 6) with 2 mM phenazine methosulfate (PMS),absorbance taken at 600nm for 3 minutes according to Anderson and colleagues (1992).
Extinction coefficient = 6.22mM-1cm-1
RESULTS:
1. Study of growth curve of Wng-1:
Growth analysis of Wng-1 was done in non-amended LB media as well as in LB containing 3.5 mM Arsenite at which growth was inhibited by 50%. The absorbance at 600nm was plotted against time to obtain a growth curve.
Growth Curve of Given Bacterium
2.5
2
Abs. at 600 nm 1.5
1 -0.5
0 i L

i i i i
0 5 10 15 20 25
Time (hours)
.- Wng- 1 in plain LB media " Wng- 1 in plain LB media
With arsenite
Figure 1: Growth curve of Wng-1 in plain LB (Blue) and LB containing arsenite
(Pink).
2. Determination of MIC of arsenite for Wng-1 :-
Minimal Inhibitory Concentration (MIC) of arsenite was calculated for Wng-1. Bacterial culture was grown in various concentration of arsenite. The absorbance of culture was measured at 600nm after 20 hours and graph was plotted between absorbance and different concentration of arsenite. The MIC of arsenite was found to be 7 mM for Wng-1.
Figure 2: MIC of arsenite was determined. Curve showing absorbance of culture at different concentration of arsenite.
Arsenite metal cone. (mM)
Figure 3: % Inhibition of Growth in presence of arsenite was determined. Curve showing absorbance of culture at different concentration of arsenite.
3. Protein Extraction from Wng-1:
During MIC determination, the MIC of arsenite for Wng-1 was found to be 7mM, however at 3.5mM conc. 50 % inhibition of growth has occurred. This conc. (3.5mM) was used for extraction of protein to assay redox enzymes and arsenite oxidase. Protein was extracted in mid stationary phase. Protein concentration was estimated by Bradford Method (Appendix-I).
4. Enzyme assay:
A)
SUPEROXIDE DISMUTASE ASSAY:
The specific activity of the superoxide dismutase was calculated from the linear portion of the graph, and was found to be more in metal amended culture that non-amended culture, i.e., 3443.78 and 1092.7 respectively (as shown below).
Hydroxylamine hydrochloride is autoxidized by superoxide radicals to nitrite. The addition of NBT induces an increase in absorbance at 540 nm due to the accumulation of blue formazon. With the addition of enzyme SOD, superoxide radicals get trapped and hence there is an inhibition of reduction of NBT to blue formazon formation. The percent inhibition of NBT reduction is calculated as below:
AA(BLANK) - AA(TEST) X 100 = UNITS AA(BLANK)
Unit Activity (Units/min)
Specific Activity =
(IU/ mg protein) Protein Content (mg)
Change in abs./ min in control = 0.1376
Thus, there was 1.81 fold increase in the specific activity in the superoxide dismutase enzymes in metal-amended culture over non-metal amended.
B) CATALASE ASSAY
Specific activity of catalase was calculated by using the linear part of graph and compared with control and arsenite stress condition. As can be seen form figs below, the specific activity of the catalase has increased by 2.3 fold in metal amended culture over non--metal amended culture.
Figure 5: Catalase assay: Graph showing the progress of reaction at specific concentration over a period of time.
Calculation of specific activity of catalase in control and arsenite stress condition:
One unit of the enzyme activity was calculated from the following equation:
Change in abs./minute x Total volume Unit Activity (Units/min) =
Ext. coefficient x Vol. of sample
3 11
Where, Extinction coefficient = 6.93 x 10- mM- cm-
Unit Activity (Units/min)
Specific Activity =
(IU/ mg protein) Protein Content (mg)
C) GLUTATHIONE REDUCTASE ASSAY
Glutathione reductase assay was done for the extracted protein. Specific activity of glutathione reductase was calculated and compared in control and arsenite stress condition.
Glutathione Reductase
0.32
0.31 A
i
0.3
0.29
0.28
y = -0.0002x + 0.314 R2 = 0.9669
In LB
y = -0.0003x + 0.2704 R2 = 0.9883
In LB + Arsenite
0.27 A
0.26
0
2
4 6 8
Time (seconds)
10
12
Figure 6: Glutathione reductase: Graph showing the absorbance of sample at 340nm during the glutathione reductase assay.
One unit of the enzyme activity is defined as the amount of enzyme required to oxidize 1 uM of NADPH/minute/g tissue. The enzyme activity was calculated from the equation given above in catalase.
As can be seen glutathione reeducates has decreased in metal-amended culture over non-metal amended, by 5.29 fold.
D) GLUTATHIONE PEROXIDASE ASSAY:
Glutathione peroxidase assay was done for the extracted protein. Specific activity was calculated by using the linear part of graph and compared with control and arsenite stress condition. The specific activity of the glutathione peroxidase was more by 1.70 fold in metal amended-culture over non-metal amended culture.
Glutathione Peroxidase Assay
1.4
1.2
1
1 08
JS
< 0.6
0.4 -\
y = -0.0058x + 0.7443 R2 = 0.9944
In LB
0.2
0
0
5
10 15 20
Time (Second)
25
30
35
Figure 7: Glutathione peroxidase assay: Graph showing the progress of reaction at specific concentration over a period of time.
One unit of the enzyme activity is defined as the amount of enzyme required to oxidize 1 \lM of NADPH/minute/g tissue. The enzyme activity was calculated from the equation given above in catalase.
E) ARSENITE OXIDASE ASSAY
Arsenite oxidase assay was done for the extracted protein. Specific activity was calculated by using the linear part of graph and compared with control and arsenite stress condition.
As can be seen, arsenite oxidase assay has increased in metal amended culture (0.4677, specific activity)) by 5.14 fold over non-metal culture (0.0909, specific activity).
DISCUSSION:
Bacteria are cells with simple organization of cellular structure and limited compartmentalization as compared to eukaryotes. They are found in very diverse range of geographical locations and environmental conditions. Through a process of natural selection they have developed various strategies to combat the unfavorable conditions like high and low temperature, heavy metal stress and similarly other different extremes of living conditions. Survival of microorganisms facing heavy metal stress is enhanced by physiological changes at the biochemical level that enable the organism to overcome different types of stress.
The elevated expression of stress proteins is considered to be a universal response to adverse conditions, representing a potential mechanism of cellular defense against disease and a potential target for novel therapeutics. The stress response is a phenomenon of adaptation of organisms. Altered patterns of protein synthesis, including stress proteins (SP), may serve to monitor the impact of exposure to natural and anthropogenic stressors [10].
Exposure to arsenicals either in vitro or in vivo in a variety of model systems has been shown to cause the induction of a number of the major stress protein families such as heat shock proteins (Hsp), Superoxide dismutase, Catalase, Glutathione Reductase, Glutathione Peroxidase, Arsenite oxidase enzyme. Superoxide dismutases are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. Catalase is a common enzyme found in nearly all living organisms which are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen. Glutathione peroxidase, whose main biological role is to protect the organism from oxidative damage, the biochemical function of glutathione peroxidase is to reduce free hydrogen peroxide to water. Glutathione reductase, which reduces glutathione disulfide (GSSG) to the
sulfuhydryl form GSH, which is an important cellular antioxidant[11] .
[28]
From the previous study it was enlighted that during oxidative stress condition the
activity of antioxidant enzyme increases to combat that unfavorable condition. Arsenite and arsenate; both forms are toxic, arsenite disrupts sulfhydryl groups of proteins and interferes with enzyme function, whereas arsenate acts as a phosphate analog and can interfere with phosphate uptake and transport. Arsenic, like other heavy metals, cannot be destroyed once it has entered the environment [7]. Microorganisms have evolved a variety of mechanisms for coping with arsenic toxicity, including minimizing the amount of arsenic that enters the cell (e.g., through increased specificity of phosphate uptake), oxidizing the arsenite (through the activity of arsenite oxidase), or arsenite peroxidation with membrane lipids[29].
During Arsenite metal stress condition, superoxide radicals are generated by the autooxidation of hydroxylamine hydrochloride (HH). From the previous studies[30], different bacteria were shown to have higher activity of superoxide dismutase under stressed conditions. Similar results were shown by the Wng-1 strain. Thus there is an increase (1.81 fold) in specific activity of superoxide dismutase, which showed that during stress conditions superoxide dismutase results in increased survival of that organism due to its antioxidant activity.
Catalase catalyses the decomposition of H2O2 to water and oxygen. The rate of decomposition of H2O2 was followed by decrease in absorbance at 240 nm in a reaction mixture. During Arsenite metal stress conditions, there is increase (2.3 fold) in specific activity of catalase, which showed that during stress condition catalase results in increased survival of that organism due to its antioxidant activity.
Glutathione reductase activity was determined by measuring the rate of NADPH oxidation as the decrease in absorbance at 340 nm. Glutathione reductase catalyzes the reduction of glutathione disulphide (GSSG) involving the oxidation of NADPH.
Glutathione reductase could play an important role in the control of endogenous H2O2 content through an oxido-reduction cycle involving glutathione. Glutathione reductase's specific activity decreases (5.2 fold) under arsenite metal stress condition.
[31-321
This was varying result from the others
Glutathione peroxidase specific activity was found to be more in arsenite stress
condition (1.7 fold) which was similar to others work so the organism Wng-1
showed more glutathione peroxidase activity during stress to combat from that unfavorable condition.
[351
Arsenite oxidase activity was found to be significantly increased during Arsenite oxidase assays. There was 5.14 fold increases in specific activity which was varied during the stress condition of arsenite for Wng-1 bacterium.Which showed that during stress conditions arsenite oxidase results in increased survival of that organism due to its antioxidant activity.
CONCLUSION:
The specific activity of Superoxide dismutase, Catalase, Glutathione peroxidase and Arsenite oxidase was increased which showed during stress condition the antioxidant enzymes as mentioned above help to combat that unfavorable condition. However, the specific activity of Glutathione reductase was decreased which was not expected because Glutathione reductase is also a redox enzyme which help to combat in case of oxidative stress condition, so its expression level should be increased. So this result can not be interpreted in accordance to previous known facts and so it needs further validation.
These whole study want further validation to conform these results by proteomic approaches such as 2-D Polyacryl amide Gel Electrophoresis'36-371 followed by analytical tools like MALDI-TOF Mass Spectrophotometery'38-401.
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to get information about the topic"Role of enzymes in arsenite tolerance in bacterial isolate Wng-1 full report" refer the page link bellow

http://seminarsprojects.in/t-role-of-enz...5#pid59085
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can u provide the exact details for enzyme extraction and the composition of lysis buffer
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