辣木籽凝乳酶的提取分离条件优化Optimization of extraction and separation conditions of milk-clotting protease from Moringa oleifera seeds

Abstract

为给辣木籽凝乳酶的开发利用提供理论依据，以脱脂辣木籽粉为原料，采用盐法提取、硫酸铵（AS）分级沉淀和离子交换色谱法提取分离凝乳酶。通过单因素试验和正交试验对辣木籽凝乳酶的盐法提取条件进行优化，采用单因素试验对离子交换色谱法分离纯化条件进行优化，并采用SDS-PAGE和RP-HPLC对分离得到的辣木籽凝乳酶的分子质量和纯度进行分析。结果表明：盐法提取的最佳条件为料液比1∶ 10、提取温度30 ℃、pH 7.15、NaCl浓度0.3 mol/L、提取时间0.5 h；在20%~40% AS饱和度下的沉淀蛋白凝乳活性最好且蛋白质总占有率达6171%；离子交换色谱法的最佳分离条件为进样质量浓度30 mg/mL、流动相pH 5.15、流速1.68 mL/min、流动相组成为0.05 mol/L 醋酸钠+0.05 mol/L氯化钠，在此条件下辣木籽凝乳酶的凝乳比活力值达到24 SU/mg；该凝乳酶的分子质量主要分布在35～45 kDa，纯度达到90%以上。采用该方法能有效提取分离辣木籽凝乳酶，且能很好地保持其凝乳活性。 To provide theoretical basis for the development and utilization of milk-clotting protease from Moringa oleifera seeds, the milk-clotting protease were extracted from defatted Moringa oleifera seed powder by salt method, and separated by ammonium sulfate (AS) fractional precipitation and ion exchange chromatography. The salt extraction conditions of milk-clotting protease from Moringa oleifera seeds were determined by single factor experiment and orthogonal experiment, and the ion exchange chromatography separation conditions were optimized by single factor experiment. The molecular weight and purity of the milk-clotting protease from Moringa oleifera seeds were analyzed using SDS-PAGE and RP-HPLC. The results showed that the optimal extraction conditions of salt method were obtained as follows: solid-liquid ratio 1∶ 10, extraction temperature 30 ℃, pH 7.15, sodium chloride concentration 0.3 mol/L and extraction time 0.5 h. Under 20%-40% AS saturation, the precipitated protein had the best milk coagulating activity and the total percentage of extracted protein reached 61.71%. The optimal separation conditions of ion exchange chromatography were obtained as follows: injection mass concentration 30 mg/mL, mobile phase pH 5.15, flow rate 1.68 mL/min, and mobile phase consisted of 0.05 mol/L sodium acetate and 0.05 mol/L sodium chloride. Under these conditions, the milk coagulating specific activity value of the milk-clotting from Moringa oleifera seeds reached 24 SU/mg.The molecular weight of milk-clotting protease was mainly distributed in the range of 35-45 kDa, and the purity was over 90%. This method can effectively extract and separate milk-clotting protease from Moringa oleifera seeds, and maintain its milk coagulating activity well.

Keywords

• 辣木籽；凝乳酶；硫酸铵分级沉淀；离子交换色谱法；凝乳活性；sds-page
• moringa oleifera seeds; milk-clotting protease; ammonium sulfate fractional precipitation; ion exchange chromatography; milk coagulating activity; sds-page