丁二酸酐修饰对枯草芽孢杆菌氨肽酶结构及酶学特性的影响

Chemical Modification of the Bacillus subtilis Aminopeptidase by Succinic Anhydride and Its Enzyme Properties

DOI：10.3969/j.issn.1673-1689.2013.06.011

中文关键词: 氨肽酶 丁二酸酐 化学修饰 稳定性 动力学

英文关键词: aminopeptidase succinic anhydride chemical modification stability kinetics

基金项目:国家863计划项目(2011AA100905)

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中文摘要:

采用丁二酸酐(SA)对经硫酸铵盐析和截留相对分子质量30 000的超滤膜分离提取后的氨肽酶(AE)在优化条件下进行化学修饰,SA对AE的平均氨基修饰率为55.86%,酶活保留率为105.81%;修饰后的氨肽酶紫外吸收光谱发生一定红移,圆二色谱分析其二级结构中无规卷曲比例有一定的增加,α螺旋、β折叠以及β转角的含量有所下降,说明修饰后的氨肽酶空间构象有一定改变;修饰后的氨肽酶,70℃保温5 h后仍然能保留70%以上的活性,与修饰前同等条件下酶活残留35%相比,其热稳定性显著提高。修饰后的氨肽酶动力学参数Km从1.0 mmol/L降低到0.75 mmol/L,表明其对底物的亲和力有明显的提高。此外,修饰后的氨肽酶最适温度和最适pH值也有一定的变化。结果表明SA化学修饰是一种可明显提高枯草芽孢杆菌氨肽酶与底物的亲和力及其热稳定性的有效方法。

英文摘要:

Aminopeptidase which was isolated from Bacillus subtilis was purified by ammonium sulfate precipitation and ultrafiltration using of a 30 kDa MWCO polyether sulfone(PES) membrane.Succinic anhydride(SA) was used as a modifier for the chemical modification of the purified Aminopeptidase.The effects of modification were characterized using the 6-trinitrobenzene sulfonic acid method,ultraviolet spectroscopy,and CD spectroscopy.The results showed the structure of native and modified Aminopeptidase is different.The average amino modification yield of the modified Aminopeptidase was 55.86% and the enzymatic acticity increased to 105.81%.Compared with the native Aminopeptidase,the modified Aminopeptidase exhibited apparently higher thermal stability.The Km of the modified Aminopeptidase(Km =0.75 mmol/L) was lower than native Aminopeptidase(Km =1.0 mmol/L).The result indicated that the chemical modification of Aminopeptidase increased the affinity.In addition,enzyme properties have been changed after modification.

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