Purification and characterisation of alliinase produced by Cupriavidus necator and its application for generation of cytotoxic agent: Allicin
| Autor: | Krutika Desai, Sagar V. Chhabria |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
0301 basic medicine Cupriavidus necator Alliin 01 natural sciences Article 03 medical and health sciences chemistry.chemical_compound 010608 biotechnology Alliinase MTT assay MIA PaCa-2 cells lcsh:QH301-705.5 Purification chemistry.chemical_classification Agricultural and Biological Sciences(all) Allicin biology Characterisation Substrate (chemistry) biology.organism_classification 030104 developmental biology Enzyme chemistry Biochemistry lcsh:Biology (General) Sephadex biology.protein General Agricultural and Biological Sciences |
| Zdroj: | Saudi Journal of Biological Sciences, Vol 25, Iss 7, Pp 1429-1438 (2018) |
| Popis: | Allicin, an extremely active constituent of freshly crushed garlic, is produced upon reaction of alliin with the enzyme alliinase (EC 4.4.1.4). A bacterium Cupriavidus necator with the ability of alliinase production was isolated from a soil sample and was identified by morphological, biochemical and 16S rRNA sequence. Alliinase production was optimised and it was further purified to apparent homogeneity with 103-fold purification and specific activity of 209 U/mg of protein by using DEAE Cellulose and Sephadex G-100 chromatography. The enzyme is a homodimer of molecular weight 110 kDa with two subunits of molecular weight 55 kDa each. The optimum activity of the purified enzyme was found at pH 7 and the optimum temperature was 35 °C. The enzyme exhibited maximum reaction rate (Vmax) at 74.65 U/mg and Michaelis–Menten constant (Km) was determined to be 0.83 mM when alliin was used as a substrate. The cytotoxic activity of in-situ generated allicin using purified alliinase and alliin was assessed on MIA PaCa-2 cell line using MTT assay and Acridine orange–ethidium bromide staining. This approach of in-situ allicin generation suggests a novel therapeutic strategy wherein alliin and alliinase work together synergistically to produce cytotoxic agent allicin. Keywords: Alliinase, Characterisation, Cupriavidus necator, Purification, MIA PaCa-2 cells |
| Databáze: | OpenAIRE |
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