Purification and characterisation of alliinase produced by Cupriavidus necator and its application for generation of cytotoxic agent: Allicin |
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Authors: | Sagar Chhabria Krutika Desai |
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Institution: | 1. Dept. of Biological Sciences, Sunandan Divatia School of Science, SVKM’s NMIMS University, Vile Parle (W), Mumbai 400056, India;2. Dept. of Microbiology, SVKM’s Mithibai College of Arts, Chauhan Institute of Science & Amrutben Jivanlal College of Commerce & Economics, Vile Parle (W), Mumbai 400056, India |
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Abstract: | 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. |
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Keywords: | DEAE diethylaminoethyl β-ME β-mercaptoethanol PLP Pyridoxal 5′ Phosphate Alliinase Characterisation Purification MIA PaCa-2 cells |
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