发酵乳杆菌CGMCC2921来源的L-阿拉伯糖异构酶的蛋白质纯化、结晶及初步X射线衍射分析
Protein purification, crystallization and preliminary X-ray diffraction analysis of L-arabinose isomerase from Lactobacillus fermentum CGMCC2921.
作者信息
Xu Zheng, Li Sha, Liang Jinfeng, Feng Xiaohai, Xu Hong
机构信息
State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing 210009, People's Republic of China.
出版信息
Acta Crystallogr F Struct Biol Commun. 2015 Jan 1;71(Pt 1):28-33. doi: 10.1107/S2053230X14025321.
L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose, as well as that of D-galactose to D-tagatose. A thermophilic AI derived from Lactobacillus fermentum CGMCC2921 (LFAI) was overexpressed in Escherichia coli BL21 (DE3). This enzyme was purified to over 95% purity by nickel affinity, Mono-Q ion-exchange and size-exclusion chromatography. The LFAI protein was crystallized from either 0.1 M bis-tris pH 6.5, 23% PEG 3350, 0.3 M NaCl (form 1 crystals) or 0.1 M bis-tris pH 6.0, 25% PEG monomethyl ether 5000 (form 2 crystals). Diffraction data from form 1 LFAI crystals were collected to 2.80 Å resolution using synchrotron radiation. The form 1 crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=85.11, b=184.57, c=186.26 Å, α=β=γ=90°. The asymmetric unit contained six LFAI subunits, corresponding to a calculated Matthews coefficient of 2.29 Å3 Da(-1) and a solvent content of 46.22%.
L-阿拉伯糖异构酶（AI）催化L-阿拉伯糖异构化为L-核酮糖，以及D-半乳糖异构化为D-塔格糖。来源于发酵乳杆菌CGMCC2921的嗜热AI（LFAI）在大肠杆菌BL21（DE3）中过表达。该酶通过镍亲和、Mono-Q离子交换和尺寸排阻色谱法纯化至纯度超过95%。LFAI蛋白从0.1 M双三羟甲基氨基甲烷pH 6.5、23%聚乙二醇3350、0.3 M氯化钠（晶型1晶体）或0.1 M双三羟甲基氨基甲烷pH 6.0、25%聚乙二醇单甲醚5000（晶型2晶体）中结晶。使用同步辐射收集晶型1 LFAI晶体的衍射数据至2.80 Å分辨率。晶型1晶体属于正交空间群P2(1)2(1)2(1)，晶胞参数a = 85.11、b = 184.57、c = 186.26 Å，α = β = γ = 90°。不对称单元包含六个LFAI亚基，对应于计算得到的马修斯系数为2.29 Å3 Da(-1)，溶剂含量为46.22%。
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