Isolation and separation of α-amylase inhibitors I-1 and I-2 from seeds of ragi (Indian finger millet, Eleusine coracana) by metal chelate affinity precipitation


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Abstract

The concept of immobilized metal affinity chromatography (IMAC) was integrated with affinity precipitation for the single step isolation of α-amylase inhibitors I-1 and 1-2 from the seeds of ragi (Indian finger millet, Eleusine coracana).α-Amylase inhibitor I-1 was purified 13-fold with a yield of 84%, using Cu(II) loaded thermosensitive metal chelate copolymer of N-isopropylacrylamide (NIPAM) and 1-vinyl imidazole (VI). The protein also showed trypsin inhibitory activity. The binding of the protein to the copolymer was strongly pH dependent. α-Amylase inhibitor I-2 was recovered in the supernatant as unprecipitated protein with significant purification and constituted 27% of the total inhibitor power. The yield with respect to inhibitor I-2 was around 85%. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed significant purification of inhibitor I-1 and indicated evident separation of the two proteins on metal chelate affinity precipitation.

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