Preliminary preparation of peroxidase from leaves of castor plant (Ricinus communis L.) using ammonium sulfate precipitation and aqueous two-phase systems

Name: Phuwanat Kettsap

Organization : Chiang Mai University. Faculty of Science

Name: Sunutcha Piyaming

Organization : Chiang Mai University. Faculty of Science

Name: Apinun Kunpienjai

Organization : Chiang Mai University. Faculty of Science

Name: Piyarat Nimmanpipug

Organization : Chiang Mai University. Faculty of Science

Name: Lalida Shank

Organization : Chiang Mai University. Faculty of Science

Email : lalida.shank@cmu.ac.th

MeSH: Peroxidases

MeSH: Plant leaves

LCSH: Ricinus communis

LCSH: Precipitation (Chemistry)

Abstract: Peroxidase, an oxido-reductase commonly used in analytical test-kits, was prepared from the leaves of castor plant (Ricinus communis L.) in this study. Preparative methods of the enzyme of this source were explored to compare their purification efficiencies. The enzyme was first precipitated out from crude leaf extract using ammonium sulfate (NH4)2SO4) in 0-20%, 20-40%, 40-60%, and 60-80% saturation fractions. Aqueous two-phase systems (ATPs) were then employed. Different molecular weights of polyethylene glycol (PEG) of 1500, 4000, and 6000 Da at 14-18% (w/W) in combination with (NH4)2SO4, 8-12% (w/w) were used in the ATPs for comparison. The supernatant from the fraction of 60-80% saturation resulted in the highest specific activity of 5.41+0.71 U/mg. Castor peroxidase mostly partitioned into the bottom phase of ATPs at PH 6.0 for the majority of the systems tested. The bottom phase of 14% PEG 6000 with a 12% (NH.)SO4 combination showed the highest specific activity of 18.78t8.51 U/mg. The bottom phase of 14% PEG 4000 with a 10% (NH4)2SO4 combination with slightly higher activity had lower specific activity of 16.21-2.30 U/mg. Zymogram using semi-native polyacrylamide gel electrophoresis revealed that both the 60-80% fraction and its supernatant contained peroxidase displaying intense orange bands of tetraguaiacol, a catalytic product. The ATPs with the highest specific activity also showed distinct bands of proteins with peroxidase- like activity. The presence of proteins in the samples was investigated using sodium dodecy1 sulfate polyacrylamide gel electrophoresis. The 14% PEG 6000 with 12% (NH4)2SO4 combination was chosen for the preparation of this enzyme for future purification and characterization

King Mongkut's University of Technology North Bangkok. Central Library

Address: BANGKOK

Email: library@kmutnb.ac.th

Created: 2023

Modified: 2023-06-26

Issued: 2023-06-26

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BibliograpyCitation : In Mae Fah Luang University. School of Science. The Pure and Applied Chemistry International Conference 2023 (PACCON 2023) (pp.711-716). Chiang Rai : Mae Fah Luang University

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