野油菜黄单胞菌8004甘天丝亮特征序列酯酶及其酯酶结构域在大肠杆菌中的表达，包涵体复性及性质

GDSL esterase Xcc_est from Xanthomonas campestris pv. campestris 8004 and its esterase domain: gene expression in Escherichia coli，refolding and characterization

摘要:

【目的】了解野油菜黄单胞菌（Xanthomonas campestris pv. campestris）8004 GDSL(蛋白序列中甘氨酸、天冬氨酸、丝氨酸和亮氨酸特征序列)酯酶的性质。【方法】利用PCR方法扩增Xcc_est及其不同结构域的基因，这些基因以组氨酸标签融合蛋白的形式在大肠杆菌中获得表达。融合蛋白通过镍亲和色谱纯化。【结果】部分纯化的Xcc_est 在催化对硝基苯丁酸酯时，最适pH值为8.0，最适温度为52 °C。 Xcc_est 对于对硝基苯丁酸酯的Km值和Vmax值分别是47.6 ± 4.6 μmol/L, 和67.6 ± 7.8 U/mg，Xcc_est的酯酶结构域(Xcc_estN1-334)对于同一底物的Km值和Vmax值分别是 469.4 ± 9.8 μmol/L和2.5 ±0.9 U/mg。Xcc_est的成熟结构域(Xcc_estN26-606)可以获得成功复性，但是成熟酯酶结构域(Xcc_estN26-334)不能获得复性。复性后的Xcc_estN26-606底物谱较广，在室温下具有较高稳定性。【结论】复性的成熟结构域蛋白(Xcc_estN26-606)具有一定的生物转化应用前景。

Abstract:

Abstract: [Objective] To characterize the GDSL (glycine, asparticacid, serine and leucine motif in protein sequence) esterase Xcc_est from Xanthomonas campestris pv. campestris (Xcc) 8004. [Methods] Xcc_est gene and different domains of Xcc_est gene were PCR amplified and expressed in Escherichia coli, the HIS-Tagged fusion proteins were purified by Ni-NTA chromatography. [Results] The optimum pH and temperature of partly purified Xcc_est were pH 8.0 and 52 °C when pNPB (4- nitrophenylbutyrate) was used as substrate. The Km and Vmax value of Xcc_est and the passenger domain (Xcc_estN1-334) for pNPB were 47.6 ± 4.6 μmol/L, 67.6 ± 7.8 U/mg and 469.4 ± 9.8 μmol/L, 2.5 ±0.9 U/mg respectively. Inclusion bodies of mature domain Xcc_est (Xcc_estN26-606) could be refolded but inclusion bodies of the passenger domain (Xcc_estN26-334) could not be refolded. Refolded mature domain had broad substrate spectrum and showed higher stability than Xcc_est when stored at 25 °C. [Conclusions] To some extend, refolded Xcc_estN26-606 is a candidate for biotransformation application.