重组N-乙酰鸟氨酸脱乙酰基酶包涵体的稀释复性

Dilution renaturation of recombinant N-acetylornithine deacetylase inclusion bodies

N-乙酰鸟氨酸脱乙酰基酶(N-Acetylornithine deacetylase,NAO)是一种重要的用于手性拆分的酶,具有广泛的底物选择性,常用于多种活性氨基酸的酶法拆分。采用稀释复性法研究了重组NAO包涵体的复性条件,如蛋白浓度、复性液中尿素浓度、pH、GSH浓度及c(GSH)/c(GSSG)比例,同时对稀释操作方式进行了考察,得到了较为适宜的复性条件。结果表明,尿素能有效抑制复性过程中蛋白质的聚集,随着蛋白质浓度的增加,复性效果变差。当复性缓冲液中尿素浓度为2 mol/L,GSH浓度为5 mmol/L,c(GSH)/c(GSSG)为2.5,pH为8.5,在4℃下进行分批稀释复性操作,复性后重组NAO的活性为1.077 U/mL,比酶活达到14.943 U/mg,与可溶性表达的NAO比较,复性率达到21.48%。

Recombinant N-acetylornithine deacetylase （NAO） inclusion bodies were renatured by dilution method. Factors influencing the dilution process, which include denatured NAO concentration, Urea concentration, pH, glutathione （GSH） concentration and the ratio of GSH to oxidized glutathione （GSSG） were investigated. At the conditions of 4℃, pH 8.5, Urea concentration at 2.0 mol/L, GSH concentration at 5 mmol/L, and the ratio of GSH to GSSG is 2.5, the apparent activity and specific activity of NAO were 4. 077 U/mL and 14.94 U/mg, respectively. The renaturation ratio of NAO was 21.48% of the total soluble expressed NAO.