从克氏苦木（Streblus asper）叶中纯化丝氨酸蛋白酶的热处理双水相体系。
'Heat-treatment aqueous two phase system' for purification of serine protease from Kesinai (Streblus asper) leaves.
机构信息
Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
出版信息
Molecules. 2011 Dec 8;16(12):10202-13. doi: 10.3390/molecules161210202.
A 'Heat treatment aqueous two phase system' was employed for the first time to purify serine protease from kesinai (Streblus asper) leaves. In this study, introduction of heat treatment procedure in serine protease purification was investigated. In addition, the effects of different molecular weights of polyethylene glycol (PEG 4000, 6000 and 8000) at concentrations of 8, 16 and 21% (w/w) as well as salts (Na-citrate, MgSO₄ and K₂HPO₄) at concentrations of 12, 15, 18% (w/w) on serine protease partition behavior were studied. Optimum conditions for serine protease purification were achieved in the PEG-rich phase with composition of 16% PEG6000-15% MgSO₄. Also, thermal treatment of kesinai leaves at 55 °C for 15 min resulted in higher purity and recovery yield compared to the non-heat treatment sample. Furthermore, this study investigated the effects of various concentrations of NaCl addition (2, 4, 6 and 8% w/w) and different pH (4, 7 and 9) on the optimization of the system to obtain high yields of the enzyme. The recovery of serine protease was significantly enhanced in the presence of 4% (w/w) of NaCl at pH 7.0. Based on this system, the purification factor was increased 14.4 fold and achieved a high yield of 96.7%.
首次采用“热处理双水相体系”从苦丁茶叶片中纯化丝氨酸蛋白酶。本研究考察了热处理程序在丝氨酸蛋白酶纯化中的引入。此外，还研究了不同分子量的聚乙二醇（PEG 4000、6000 和 8000）在浓度为 8、16 和 21%（w/w）以及盐（柠檬酸钠、MgSO₄和 K₂HPO₄）在浓度为 12、15、18%（w/w）对丝氨酸蛋白酶分配行为的影响。在 PEG 丰富相（16%PEG6000-15%MgSO₄）中实现了丝氨酸蛋白酶的最佳纯化条件。此外，与未经热处理的样品相比，苦丁茶叶片在 55°C 下热处理 15 分钟可获得更高的纯度和回收率。此外，本研究还考察了不同浓度的 NaCl 添加（2、4、6 和 8%w/w）和不同 pH（4、7 和 9）对该体系进行优化以获得高酶产量的影响。在 pH 7.0 时存在 4%（w/w）的 NaCl 可显著提高丝氨酸蛋白酶的回收率。基于该体系，纯化因子提高了 14.4 倍，达到了 96.7%的高产率。
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