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<p>Three phase partitioning (TPP) technique was employed to purify the milk coagulating enzyme (bromelain) from pineapple fruit. Optimization of three TPP parameters (the concentration of ammonium sulfate salt, the ratio of crude dialyzed pineapple extract to tert-butanol solvent, and the pH level) was carried out. 50% (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> concentration (w/v), 1:1 crude pineapple extract to tert-butanol ratio (v/v) and 7 pH yielded the highest activity recovery of 126.15% and purification fold of 3.31. The enzyme recovered only in the interfacial phase exhibited milk clotting activity (MCA). The SDS-PAGE analysis revealed 24.5&#xa0;kDa molecular mass of purified bromelain. The enzyme exhibited its peak activity at 50ºC and pH 7. Bromelain activity was enhanced by Ca<sup>2+</sup> and Mg<sup>2+</sup> ions and inhibited by iodoacetamide, confirming its nature as a cysteine protease. Its V<sub>max</sub> and K<sub>m</sub> values were recorded as 55 U/min and 3.4&#xa0;mg/mL respectively. Its optimum conditions for maximum MCA (1142.86 U/mL) were 6 milk pH and 55ºC milk temperature. This enzyme retained a maximum MCA at -20&#xa0;°C. Hence, this study demonstrated that the pineapple fruit bromelain could be purified effectively and economically by single step non-chromatographic TPP protocol, and it could function as a clotting agent.</p>

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Single step three phase partitioning for the purification of bromelain enzyme from pineapple (Ananas comosus) fruit

  • 期刊论文

  • 发表日期：

    2025年6月4日

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  • Bunty Maskey,
  • Sira Shrestha,
  • Alisha Shrestha,
  • Pradeep Sangroula,
  • Dhan Bahadur Karki,
  • Nabindra Kumar Shrestha

摘要

Three phase partitioning (TPP) technique was employed to purify the milk coagulating enzyme (bromelain) from pineapple fruit. Optimization of three TPP parameters (the concentration of ammonium sulfate salt, the ratio of crude dialyzed pineapple extract to tert-butanol solvent, and the pH level) was carried out. 50% (NH4)2SO4 concentration (w/v), 1:1 crude pineapple extract to tert-butanol ratio (v/v) and 7 pH yielded the highest activity recovery of 126.15% and purification fold of 3.31. The enzyme recovered only in the interfacial phase exhibited milk clotting activity (MCA). The SDS-PAGE analysis revealed 24.5 kDa molecular mass of purified bromelain. The enzyme exhibited its peak activity at 50ºC and pH 7. Bromelain activity was enhanced by Ca2+ and Mg2+ ions and inhibited by iodoacetamide, confirming its nature as a cysteine protease. Its Vmax and Km values were recorded as 55 U/min and 3.4 mg/mL respectively. Its optimum conditions for maximum MCA (1142.86 U/mL) were 6 milk pH and 55ºC milk temperature. This enzyme retained a maximum MCA at -20 °C. Hence, this study demonstrated that the pineapple fruit bromelain could be purified effectively and economically by single step non-chromatographic TPP protocol, and it could function as a clotting agent.