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Purification and Characterization of Peroxidase from Anthracnose Disease Infected Papaya (carica Papaya L.)

L Bari
Department of Food Technology & Nutritional Science, Mawlana Bhasani Science & Technology University, Tangail

P Hassan
Molecular Biology Laboratory, Institute of Biological Science, Rajshahi University, Rajshahi;

N Absar
Department of Biochemistry & Biotechnology, University of Science & Technology, Chittagong

S Khatun
Department of Biochemistry & Molecular Biology, Rajshahi University, Rajshahi;

MI Hossain
Department of Biotechnology & Genetic Engineering, Mawlana Bhasani Science & Technology University, Tangail, Bangladesh

Abstract/ Executive Summary:

Peroxidase enzyme was isolated and purified from the pulp of disease infected ripen papaya of local variety by 90% ammonium sulphate precipitation, chromatography on DEAE cellulose followed by hydrophobic chromatography on Phenyl Sepharose CL-4B and the purifications achieved was about 7.2 fold with 2.5% recovery. The purified enzyme was homogeneous as judged by polyacrylamide slab gel electrophoresis. The purified enzyme had a Mr of about 55,000 and 50 000 as determined by gel filtration on Sephadex G-100 and SDS-PAGE, respectively. The molecular mass of the enzyme was found to be very similar under both reducing and non-reducing conditions indicating that the enzyme contains no subunit. The enzyme has the following characteristics: pH optima at 6.0, temperature optima around 38°C, enzyme activity was found to be strongly inhibited in the presence of potassium cyanide and Fe+2 while the activity was found to be remarkably increased in the presence of ammonium sulphate. The Km value for the peroxidase obtained with pyrogallol as substrate was 0.027 mM.

Keywords: Papaya, Anthracnose disease, Peroxidase purification

Location:

Start Date:

End Date:

Program Area: Pest Management

Commodity: Papaya

Objectives:

This paper describes the purification and characterization of peroxidase from rotten stage of papaya fruit infected with Colletotrichum gloeosporiodes.

Methodology:

Chemicals and gel matrices All chemicals used were of analytical grade. DEAE-cellulose was purchased from Pharmacia Fine Chemical Co. Ltd. Upsala, Sweden. SDSPAGE chemicals were from Sigma Chemical Co. Ltd. USA. Phenyl Sepharose CL-4B was the product of Aldrich-Sigma Chemical Co. Ltd. USA and Sephadex G-100 from Sigma Chemical Co. Ltd. USA. Plant material: Local variety of papaya containing the highest amount of peroxidase activity as reported in our study was used for enzyme purification and characterization. Preparation of crude enzyme extract: Rotten papaya pulp (250 g) were cut into small pieces and homogenized to fine paste in cold 0.1 M sodium phosphate buffer, pH 6.0, using a mortar and pestle. The suspension was kept for 2 hrs at 4°C in a refrigerator and filtered through double layer of cheese cloth. The filtrate was collected and centrifuged at 8,000 g for 20 min at 4°C. The total volume of clear supernatant was recorded and used as crude enzyme extract. All operations were performed at 4°C unless otherwise indicated. Purification of Peroxidase from Fruit Disease Infected Papaya Pulp Ammonium sulphate fractionation: The enzyme were precipitated from the supernatant by the addition of solid (NH4)2SO4 (90% saturation) under constant and gentle stirring at 4°C and left overnight in a refrigerator. The resulting precipitate was collected by centrifugation at 8,000 g for 20 min and dissolved in a minimum volume of pre-cold Tris-HCl buffer, pH 8.2 and dialyzed overnight against 2 x 5 L of distilled water at 4°C. The dialyzed solution was then centrifuged in a refrigerated centrifuge at 8,000 g for 15 min. to remove insoluble materials. The clear supernatant was used as a source of peroxidase designated as "crude enzyme solution". DEAE-cellulose ion-exchange chromatography: The soluble enzyme extract was immediately applied to a column of DEAE-cellulose (2.1 x 20 cm) previously equilibrated with 10 mM Tris-HCl buffer, pH 8.2. After sample application, the column was washed with 10 mM Tris-HCl buffer, pH 8.2 to wash out the unbound proteins. The column bound proteins were eluted stepwise from the column with same buffer containing different concentrations of NaCl. The elutes from both the unbound and bound proteins were collected as 3 ml/tube fractions on an automatic fraction collector and monitored for protein at 280 nm and assayed for peroxidase activity.18 Hydrophobic chromatography on phenylsepharose CL-4B: The separation using hydrophobic chromatography on Phenyl Sepharose column CL-4B was same as in the purification of peroxidase/catalase from Pseudomonas aeruginosa.19 The unbound fraction from DEAE-cellulose column possessing peroxidase activity was pooled together and dialyzed against deionized water for 12 hrs followed by dialysis against the wash buffer (50 mM Tris-HCl + 200 mM AmSO4, pH 7.5.) at 4ºC for 8 hrs. A 10 ml column containing Phenyl Sepharose CL-4B was packed and washed with 10 times the column volume with the wash buffer (50 mM Tris-HCl + 200 mM AmSO4, pH 7.5). The column was equilibrated with the equilibration buffer (50 mM Tris-HCl + 200 mM AmSO4, pH 7.5). After column equilibration, the enzymatically active fractions were applied to the column. The column bound proteins were eluted from the column using gradient buffer (50 mM Tris-HCl, 200 mM AmSO4, pH 7.5) and 3 ml fractions were collected on an automatic fraction collector and monitored for protein at 280 nm and peroxidase activity were assayed following the method described earlier. Protein assay: The protein content of different fractions was measured using Bovine serum albumin (BSA) as the standard and the protein in column elute fractions was also monitored spectrophotometrically at 280 nm.20 Test of protein homogeneity The homogeneity of purified protein was judged by SDS-PAGE on 7.5% gel on a Bio-Rad mini electrophoresis system.21 Characterization of Purified Protein Determination of relative molecular mass (Mr) a) Determination of relative molecular mass (Mr) by gel filtration: The relative molecular mass (Mr) of purified peroxidase was determined by Gel filtration on Sephadex G-100 column (100 X 0.85 cm) equilibrated with 10 mM Tris-HCl buffer, pH 8.2.22 The marker proteins used were Trypsin inhibitor (12.028 kD), carbonic anhydrase (29 kD), a-amylase (58 kD), bovine serum albumin (68 kD), phosphorylase-b (97.4 kD) and e-galactosidase (116 kD). The relative molecular mass (Mr) was estimated using a plot of molecular weight versus elution volume. b) Determination of relative molecular mass (Mr) by SDS-PAGE: The relative molecular mass (Mr) of purified papaya peroxidase was also determined by SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) on a Bio-Rad mini electrophoresis system.23 The standard proteins used were the same as those used in determination of the relative molecular mass (Mr) by Gel filtration on Sephadex G-100 column mentioned above. The gels were stained with Coomassie Brilliant Blue, CBB R-250 for 1 hr and destaining was performed in 7% acetic acid (v/v). Determination of subunit structure of the purified enzyme: In order to determine subunit structure SDS-PAGE of purified enzyme under reducing and non-reducing conditions was performed.22 Dissociation and reduction of protein was performed by heating for 5 min at 100° C in 0.1 % SDS with or without 0.1% of 2-mercapto ethanol.

Source of Information: Bangladesh J Med Biochem 2013; 6(2): 49-57

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Achievement/Findings:

The present study revealed that the ripe papaya pulp infected by Colletotrichum gloeosporioides, is a rich source of peroxidase having high pH stability and thermostability, so might be considered for use industrially by large scale production.

Knowledge Management: Journal