摘要
N-乙酰鸟氨酸脱乙酰基酶(NAO)具有广泛的底物选择性,可用于多种活性氨基酸的酶法拆分,具有广阔的工业应用前景。文中采用多种洗涤剂对重组N-乙酰鸟氨酸脱乙酰基酶包涵体进行洗涤去杂,并用DEAE Sepharose Fast Flow层析柱和三缓冲体系作为复性系统对重组N-乙酰鸟氨酸脱乙酰基酶包涵体进行复性实验。结果表明,4mol/L尿素与0.5%Triton X-100联合洗涤可以大量去除杂质;三缓冲体系能有效地实现重组N-乙酰鸟氨酸脱乙酰基酶包涵体的柱上复性。在流速为0.5 mL/min,尿素梯度为21 mL,尿素终浓度为1.8mol/L,蛋白质上样量为0.99 mg的实验条件下,蛋白回收率和复性酶比活分别达到52%和10.27 U/mg。
N-acetylornithine deacetylase （NAO） is for separation of chiral Amino Acids. The recombinant a prospective industrial enzyme, which could be used N-acetylornithine deacetylase （NAO） was overexpressed as inclusion bodies in Escherichia coli. The insoluble fractions were separated from cellular debris by centrifugation. Different washing buffers were used to wash the inclusion bodies. The results showed that the washing buffer with 4 mol/L urea and 0.5% Triton X--100 is the best buffer, which can significantly reduce the contaminant level. With an on-column refolding procedure using DEAE Sepharose Fast Flow resin and three--buffer refolding system, the active NAO protein was recovered effectively from inclusion bodies. 0.99 mg samples were loaded to the column and with the flow rate of 0.5 mL/min with 21 mL urea gradient elution volume at the final urea concentration of 1.8 mol/L, the protein yield and specific activity of the NAO were up to 52% and 10. 27U/mg
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2008年第2期11-15,共5页
Food and Fermentation Industries
基金
江苏省高校"青蓝工程"项目资助
南京工业大学国家自然科学基金预研项目
关键词
离子交换
重组N-乙酰鸟氨酸脱乙酰基酶
包涵体
复性
ion-exchange chromatography, N-acetylornithine deacetylase, inclusion body, refolding
作者简介
硕士研究生（
姚忠为通讯作者
