毛竹脱氧辛糖酸-8-磷酸合酶的原核表达、纯化及酶的特性

Expression, purification and characterization of 3-Deoxy-D-manno-octulosonate 8-phosphate synthase from Phyllostachys edulis

摘要:

脱氧辛糖酸-8-磷酸合酶 (3-Deoxy-D-manno-octulosonate 8-phosphate synthase，KDO8PS) 是一种参与细胞壁八碳糖 (KDO) 合成代谢的关键酶之一。为了解该酶催化特性和功能，本实验采用逆转录聚合酶链式反应 (RT-PCR) 方法首次分离得到毛竹PeKDO8PS基因。序列分析表明该基因CDS区全长876 bp，编码291个氨基酸。通过IPTG诱导，含有该基因的原核表达载体在大肠杆菌中获得了大量可溶性活性蛋白表达；经Ni-NTA亲和层析和分子筛层析 (SEC) 方法进行酶蛋白的两步分离纯化，得到纯度大于90%以上的高纯度酶；SEC结果发现，纯化后目的蛋白KDO8PS在溶液中主要以二聚体形式存在。戊二醛交联实验证实该酶具有形成二聚体/四聚体的可能性，进一步通过超速离心 (AUC) 分析，发现水溶液中KDO8PS在高浓度时二聚体会聚合转化形成四聚体。推测形成二聚体/四聚体可能是保持酶稳定性的一种机制。通过测定毛竹KDO8PS酶学性质，发现该酶催化反应的pH值范围在4.0?9.0，最适pH值为8.0；热稳定性范围 25?65 ℃，最适作用温度为55 ℃；各种二价金属阳离子在低浓度下均对酶活性存在不同程度的抑制作用，其中以Fe3+对酶活性的抑制作用最强，而低浓度EDTA可通过螯合作用消除金属离子的抑制作用。以上研究结果为植物KDO8PS蛋白结构与功能及其在新型抗生素领域中的工业化应用提供了重要理论基础。

Abstract:

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) is the key enzyme to synthesize eight-carbon sugar in plant and gram-negative bacterial cell wall. To analyze the polymerization and characterization in plant KDO8PS, the candidate gene was cloned from fresh Phyllostachys edulis seedling by RT-PCR. The open reading frame of PeKDO8PS is 876 bp deduced into 291 amino acid residues. The target protein was overexpressed in Escherichia?coli induced by IPTG and then lager amount of fusion protein was purified through two-step methods with affinity chromatography and size-exclusion chromatography (SEC). SEC analysis shows that PeKDO8PS protein existed mainly in the form of dime in solution. Glutaraldehyde cross-linking experiments confirmed that the enzyme could form dimers. Further we identified that KDO8PS at high concentration two dimers could form tetramer in aqueous solution by analytical ultracentrifuge (AUC) analysis. The pH of the catalytic reaction was between 4.0 and 9.0, the optimum pH value was 8, the thermal stability range was between 25 and 65 ℃, and the optimum temperature was about 55 ℃. The enzyme activity was inhibited by some metal ions at lower concentrations, especially in the presence of Fe3+metal ion and activated by metal protease inhibitor EDTA at low concentration.