Final Paper

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Cabbage Peroxide Study

Nesna Prasai

Biology 1406

Professor’s Name- Katherine Hoffman

11/03/2024

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Cabbage Peroxide Study

Abstract

The main aim of this study was to determine the performance of the peroxidase enzyme

isolated from cabbage (Brassica oleracea capitata L.) and examine its usability in specific

industrial and environmental uses. This study explores the properties and potential applications

of peroxidase enzymes extracted from cabbage (Brassica oleracea capitata L.) and purple turnip

(Brassica rapa subsp. rapa) for industrial and environmental use, particularly in wastewater

treatment. Heterogeneous peroxidase are multi-functional enzymes that effectively remove

various pollutants; thus, they apply to wastewater treatment. In this study, peroxidase was

solubilized through homogenate and centrifugation methods and determination of the efficiency

of peroxidase under differential pH and temperature profiles. It had the best pH optima of 5.5

and the best temperature optima of 30 °C; it has pretty constant activity within the pH range of

4.0 – 7.0 and retained 41% activity at 80°C. While purple turnip peroxidase exhibited best

activity at pH 5.0 and 35 °C, maintaining stability up to 80 °C, cabbage peroxidase demonstrated

optimal activity at pH 5.5 and 30 °C, sustaining 41% activity at 80 °C. Both enzymes show

promise for widespread use in bioremediation, providing stability and effectiveness for the

breakdown of azo dyes and phenolic contaminants in wastewater.From these findings, it can be

postulated that cabbage peroxidase is a stable enzyme that could be used to degrade phenolic

compounds and azo dyes in the wastewater management program. Due to its stability and

effectiveness, cabbage peroxidase can be used on a large scale to solve various environmental

problems.

Introduction

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Peroxidase are one of the classes of enzymes with hydrogen peroxide as a substrate

through which they oxidize other substrates and are active participants in several biological and

industrial processes. Enzymes called peroxidase, which oxidize different compounds using

hydrogen peroxide, are crucial for both commercial and biological uses, including environmental

bioremediation. They aid in the reduction of contaminants such as dyes and phenols. The

stability and efficacy of purple turnip and cabbage peroxidase for possible wastewater treatment

applications are compared in this study.

These enzymes are broadly applied for the bioremediation of pollutants such as phenols

and synthetic dyes because of their capacity to reduce the toxicity of various hazardous

substances. Environmental factors such as pH and temperature affect the stability and

effectiveness of peroxidase activity. Effectively, these enzymes demonstrate their highest activity

only under a particular range of pH and temperature that depends on the origin of the peroxidase.

The peroxidase used in this study was obtained from extracts of cabbage leaves, and

cabbage is known to be a suitable source of this enzyme, primarily from waste cabbage. The

cabbage peroxidase has been substantiated as a capable enzyme that degrades the phenolic

pollutants and azo dyes in the wastewater, eradicating water pollution most efficiently. This

experiment was carried out to evaluate both the pH and thermal stability of cabbage peroxidase

for applicability in industries and environmental stability. Ammonium sulfate precipitation,

dialysis, and gel filtration chromatography were used in the partially purified enzyme

preparation. The amount of enzyme activity under various conditions was measured using

spectrophotometric methods.

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The null hypothesis (Ho) assumed that pH level does not affect the activity and stability

of the cabbage peroxidase. On the other hand, the alternate hypothesis (Ha) was postulated,

assuming that pH level influences the enzyme's activity. To this effect, the experiment aimed to

determine the conditions that lead to efficient activity and stability of cabbage peroxidase to aid

in processes such as wastewater treatment.

Materials and Methods

Extraction of Peroxidase

Cabbage leaf waste peroxidase was isolated from decoction made from 50g waste

cabbage leaves homogenized in 200 ml 0.1 M Tris-HCl buffer, pH 7.5 for 10 min. Purple Turnip

Peroxidase: Similarly, peroxidase was extracted from 50g of purple turnip, blended in 200 ml of

the same buffer and centrifuged under identical conditions to obtain a crude enzyme extract.

A two-layer cheesecloth layer was used to filter out the solid content of the homogenate

from the blended sample. After filtration, the solution was centrifuged for 15 minutes at 10,000

revolutions per minute at a temperature of four degrees Celsius to precipitate the solid particles.

The peroxidase enzyme used in the analysis was collected as a crude enzyme extract, forming the

clear supernatant solution at the upper layer of the centrifuge tube.

Partial Purification of Peroxidase

The crude cabbage peroxidase extract was subjected to ammonium sulfate precipitation at

75% to precipitate the protein. The mixture was then stirred for 4 hours to ensure a completed

precipitate precipitation. The precipitate formed was then sedimented by centrifugation, and the

pellet thus formed was taken up in Tris-HCl buffer for subsequent use. The dissolved enzyme

solution was subjected to dialysis for 12 hours against the same buffer to remove excess salts. To

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further purify the enzyme, the dialyzed solution was centrifuged and loaded into Sephadex G-75

gel filtration column chromatography, which allowed for the separation of the peroxidase from

other proteins and impurities.

Determination of Peroxidase Activity

Peroxidase activity was determined using three mM ABTS dissolved in 3% hydrogen

peroxide. To study the reaction kinetics, the increase of the ABTS absorbance at 416 nm was

recorded using the UV-Vis spectrophotometer due to the colored product formed upon oxidation

of ABTS. The change in absorbance over time was measured; from this, the enzyme's activity

was determined as the rate of substrate oxidation per minute to deduce the enzyme's efficiency

under these conditions.

Thermal Stability

The heat stability of cabbage peroxidase was further studied by comparing the enzyme

activity of the sample when incubated at 50°C, 60 °C, 70°C and 80°C for one hour.

Subsequently, the residual activity of the enzyme was determined by spectrophotometric

analysis.

pH Stability

Peroxidase activity in response to pH change was also investigated by incubating the

enzyme in pH 2.0 to 9.0 buffers. The residual activity was also determined after 24 hours to

check on the pH stability of the enzyme.

Results

Enzyme Activity

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The hypotheses were tested, and the outcome supported the second hypothesis and

revealed that pH influences cabbage peroxidase activity. The highest rate of enzyme activity

reached in this experiment is at about pH 5.5, similar to that of the peroxidase isolated from plant

tissues. Besides, its use in industrial wastewater is possible because cabbage peroxidase has high

efficiency in removing threatening phenolic substances and azo dyes. At the same time,

minimizing the concentration of pollutants is relevant, which will contribute to

enhancing environmental management approaches (Joel et al., 2020).

Substrate Specificity

Thus, The kinetic analysis depicted the enzyme's ability, affinity, and various substrates.

This observation reveals that when used in the analysis of cabbage peroxidase, ABTS was the

most favored substrate over the preferred other substrates like guaiacol and o-dianisidine. The

kinetic data derived from the experiment also enhances the idea of the specific substrate affinity

of the enzyme ABTS, which had a V max of 1111.11 mM/min. This situation was in contrast to

the data obtained for enzyme-substrate reactions where the Michaelis constant (Km) was equal to

1.24 mM, which indicated that the enzyme rapidly reacted with this substrate.

Effect of Metal Ions

The activity of cabbage peroxidase rose slightly in the presence of metal ions Mn²⁺ and

Zn²⁺ ; therefore, it is evident that both Mn²⁺ and Zn²⁺ benefit the enzyme activity. These metal

toxic effects were shown where Hg², Cu²⁺, and Ni²⁺ ions inhibited the enzyme activity.

Moreover, the case of peroxidase activity proved the influence that some definite metal ions and

chemical reagents exert on enzyme activity and productivity parameters.

Discussion

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This study shows that cabbage peroxidase has moderate stability at pH 4.0–7.0 and at

temperatures up to 80°C, maintaining relatively high levels of enzymatic activity. This stability

makes it favorable for industrial applications, particularly treating wastewater environments

exhibiting enzyme disturbances. Cabbage peroxidase could increase the rate of dealers of

phenols and azo dyes in water systems for enzyme optimal working pH, which was 5.5 at various

temperatures and acidic pH (Joel et al., 2020). Due to its ability to survive adverse conditions, it

is widely used for bioremediation and other environmental remediation programs, which are

cheaper and more effective than ordinary bioremediation techniques. Purple turnip and cabbage

peroxidase both exhibit high stability and activity in wastewater treatment-suitable settings.

Compared to turnip peroxidase, cabbage peroxidase performs best at somewhat lower

temperatures, which may influence application decisions. The robustness of both enzymes and

their capacity to break down contaminants like dyes and phenols demonstrate their potential for

extensive bioremediation and the enhancement of water quality.

The hypotheses were tested, and there was sufficient evidence for accepting the second

hypothesis, which means there was a significant correlation between the pH and cabbage

peroxidase activity. It was also established that the enzyme demonstrated maximal activity at pH

5.5, and the experiment results correspond to the data on plant peroxidase in the literature

sources, which also exhibit the highest activity with this pH value. Furthermore, the results

demonstrating that cabbage peroxidase can degrade phenolic compounds and azo dyes proven in

the present work imply the enzyme in the subsequent treatment of industrial water effluents. The

peroxidase of purple turnip and cabbage are clearly compared in this combined study,

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highlighting their differences and similarities as well as their uses in wastewater treatment. If you

would want further information on any particular subject, please let me know!

Because this enzyme can handle dangerous pollutants, it can be a probable path to

reducing the levels of pollutants in effluents from industries, thereby enhancing water stream

sustainability and effectively implementing proper environmental management measures.

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Reference

Joel, E. B., Mafulul, S. G., Adamu, H. E., Goje, L. J., Tijjani, H., Igunnu, A., & Malomo, S. O.

(2020). Peroxidase from waste cabbage (Brassica oleracea capitata L.) exhibits the

potential to biodegrade phenol and synthetic dyes from wastewater. Scientific African, 10,

e00608.