磷酸缓冲盐和硫酸钠对洋葱假单胞菌脂肪酶的非水相激活

研究了kosmotropic型磷酸缓冲盐和硫酸钠对洋葱假单胞菌脂肪酶(Pseudomonas cepacia lipase, PCL)非水相催化性能的影响.以往磷酸缓冲盐被用来调控体系的pH值,其掺杂量对酶的催化活性无明显影响,而适量硫酸钠的掺杂则可有效提高酶在非水相的催化活性.本文研究发现,通过精确调控冻干过程,磷酸缓冲盐掺杂能够将PCL在有机相中的转酯化活性提高近10倍,达到其水相本征活性的50%,这一激活效果甚至高于硫酸钠掺杂.利用热重方法分析了盐掺杂PCL的含水量和蛋白结构,并将失重结果同其在有机相中的催化活性相关联,发现PCL在磷酸缓冲盐和硫酸钠掺杂下的催化构型与蛋白含水量及其周围盐环境具有不同的依赖关系.利用2-(4’-氨基-2’-羟基苯基)苯并恶唑作为荧光探针,研究了磷酸缓冲盐和硫酸钠掺杂的PCL悬浮于有机相时对荧光探针发射光谱的影响,发现盐掺杂酶制剂的存在能够大大增加荧光探针稳定于极性溶剂的构型含量,这可能与蛋白周围掺杂盐键和的水分子有关.如果用探针分子稳定于极性溶剂和非极性溶剂的构型比值间接表示悬浮酶制剂的极性结构,在正己烷体系中硫酸钠掺杂的PCL具有比磷酸缓冲盐掺杂的PCL大得多的极性,且酶制剂的极性大小与其非水相转酯化活性之间具有相似的变化趋势.上述研究结果表明,掺杂盐对粗PCL酶制剂的激活可能部分归因于掺杂盐键和的水分子在蛋白周围构筑的极性环境.

Comparison of the salt-induced activation of Pseudomonas cepacia lipase in organic media by phosphate buffer and sodium sulfate

The effects of a kosmotropic phosphate buffer (PB) or sodium sulfate (SS) on the enzymatic activity of crude Pseudomonas cepacia lipase (PCL) in organic media were explored. Previously, it was believed that SS acts as an effective enzyme activator, and PB was only used to adjust the solution pH. The present work showed PB-induced activation of PCL in the transesterification activity, where an increase of nearly 10-fold was achieved by carefully controlling the lyophilization condition. This activation, which gave about one-half of the native catalytic efficiency of PCL in aqueous media, was even more effective than the SS-induced activation. Thermogravimetric analysis of PCL preparations showed that different local environments surrounded PCL due to the presence of PB or SS. The local polarity of PCL preparations suspended in n-hexane was investigated by fluorescence spectroscopy using 2-(4’-amino-2’-hydroxy phenyl)benzoxazole as the probe. SS-incorporated PCL showed a much stronger local polarity than PB-incorporated and salt-free PCL. A correlation was found between the transesterification activity and local polarity of the enzyme preparation as a function of salt content. The data confirmed that the salt-induced activation of PCL in organic media can be attributed in part to the polar environment constituted by the salt-bonded water surrounding the PCL.