水相双相系统中细菌菊粉酶的纯化
Purification of bacterial inulinase in aqueous two-phase systems.
作者信息
Temkov Mishela, Strinska Hristina, Dobrev Georgi, Velickova Elena, Muresan Vlad, Krastanov Albert
机构信息
Department of Food Technology and Biotechnology Faculty of Technology and Metallurgy Ss. Cyril and Methodius University in Skopje Skopje Republic of Macedonia.
Department of Biotechnology and Molecular Biology Faculty of Technology University of Food Technologies Plovdiv Bulgaria.
出版信息
Eng Life Sci. 2018 Mar 30;18(11):840-850. doi: 10.1002/elsc.201700174. eCollection 2018 Nov.
In this study, extracellular inulinase from . 11/3 was partially purified and concentrated using aqueous two-phase system (ATPS). Two different phase forming salts and four types of polyethylene glycol (PEG) were used. Binodal curves and tie-length lines (TLLs) for eight ATPS were developed. For inulinase purification, concentrations of PEG and salt according to binodal curves (between 17 and 26%) were chosen. All ATPSs for inulinase purification were characterized. An ATPS consisted of 26% PEG1000 and 26% MgSO was found to be the most suitable for inulinase purification. This ATPS has 28.47% TLL, 1.03 of volume ratio, purification factor of 4.65 fold and recovery yield of 66.17%. On the SDS-PAGE electrophoresis two protein bands with molecular weight of around 24 and 56 kDa were observed. The partially purified enzymes had optimal activity at pH 8.0 and 6.5, optimal temperature at 30 and 70°C and kinetic parameters K = 26.32 mmol and V = 526 mmol/min.
在本研究中，使用双水相系统（ATPS）对来自... 11/3的胞外菊粉酶进行了部分纯化和浓缩。使用了两种不同的成相盐和四种聚乙二醇（PEG）。绘制了八种ATPS的双节线曲线和系线长度（TLL）。对于菊粉酶的纯化，根据双节线曲线（17%至26%之间）选择了PEG和盐的浓度。对所有用于菊粉酶纯化的ATPS进行了表征。发现由26% PEG1000和26% MgSO组成的ATPS最适合菊粉酶的纯化。该ATPS的TLL为28.47%，体积比为1.03，纯化倍数为4.65倍，回收率为66.17%。在SDS-PAGE电泳上观察到两条分子量约为24 kDa和56 kDa的蛋白条带。部分纯化的酶在pH 8.0和6.5时具有最佳活性，最佳温度为30°C和70°C，动力学参数K = 26.32 mmol，V = 526 mmol/min。
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