Extraction, partial purification and characterization of peroxidase from Malva neglecta

Other Title(s)

استخلاص، تنقية جزئية و توصيف أنزيم البيروكسيديز من نبات الخيار

Dissertant

al-Badri, Rihab Hamid Saghir

Thesis advisor

Aziz, Ghazi Munim

University

University of Baghdad

Faculty

College of Science

Department

Biotechnology Department

University Country

Iraq

Degree

Master

Degree Date

2012

English Abstract

Peroxidase has been detected in crude of plant Malva neglecta extract in each part of plant parts such as roots, leaves, stems as well as whole plant.

The enzymatic activity was found to be higher in the whole plant than other parts.

The guaiacol is used as substrate in the detection of enzymatic activity of peroxidase.

The optimization of extraction process was done by controlling the type and concentration of buffer, pH of the buffer used, and the ratio of extraction.

The Sodium acetate buffer with 0.2mM and pH 5.0 was found to be the best buffer for extraction of peroxidase.

By using the extraction ratio for plant tissue of 1:10 (W/V), the specific activity was 10.7 U/mg protein.

peroxidase has been purified from plant Malva neglecta following three purification steps.

These steps include: Ammonium sulfate precipitation with 70% saturation, followed by Ion exchange chromatography using DEAE- Cellulose and Gel filtration chromatography using Sephadex G-100.

These three purification steps raised the specific activity to 23U/mg protein in the precipitation step with purification fold 2.1 and enzyme recovery 46.7%, the specific activity was increased to 40.3U/mg protein in Ion exchange step with purification fold 3.7 and enzyme recovery 25%, also the specific activity doubled after gel filtration step to 67.5U/mg protein with purification fold 6.3 and enzyme recovery 18.2%.

Characterization results demonstrated that, the optimal pH for activity and stability was 7 and 7-7.5 respectively, the optimal temperature for activity and stability was 45 and 30-40˚C respectively.

The activation energy was calculated and it was 14Kcal/mol, The kinetics of peroxidase such as maximum velocity Vmax and Michalis constant Km were also determined using four different methods including (Linweaver- Burk plot, Woolf- Augustenson plot, Hanes- Woolf plot and Eadie- Scatchard plot )using guaiacol and hydrogen peroxide as substrates, the Km and Vmax were Summary II measured to be 1.4× 10-2 mM towards Hydrogen peroxide and 3.2 × 10-2 mM towards guaiacol, and 2.5× 10-1 mM/min and1.6× 10-1 mM/min respectively.

The effect of some activators such as metallic chlorides (sodium chloride (NaCl), potassium chloride (KCl), copper chloride (CuCl2), Calcium chloride (CaCl2), and magnesium chloride (MgCl2)) on peroxidase activity was estimated.

Reagents such as EDTA, 2-mercaptoethanol, and Urea are also used as inhibitors of peroxidase activity.

It was observed that the peroxidase activity increased when incubated with metallic chlorides.

The highest remaining activity was 212% treated with 20mM CaCl2, while the specific activity of peroxidase decreased if treated with 20mM of Urea and losses 80% of its activity, and the peroxidase loses its complete activity when it treated with 0.25M Na2So4.

The purified peroxidase was used in preparation of glucose determination kit and proved its efficiency in achieving of linear relationship between glucose concentrations; the absorbance was at 460nm when using reaction substances such as guaiacol and O-dinizidine.

Main Subjects

Biology

No. of Pages

108

Table of Contents

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Language

English

Data Type

Arab Theses

Record ID

BIM-600943