Reaction mechanism based on X-ray crystallography at atomic resolution of endopolygalacturonase

〇紫胃内聚半乳糖醛酸酶I在大肠杆菌中的表达、纯化和结晶。我们利用大肠杆菌构建了表达体系，并采用纯化方法制备了具有功能活性的endoPG I，用于突变和晶体学研究。在大肠杆菌Origami （DE3）中表达的酶具有可溶性和活性，具有合适的二硫键，而在BL21（DE3）中表达的酶则定位在包涵体中。Origami （DE3）产生的足够量的重组endoPG I通过阳离子交换色谱法一步纯化。〇紫荆多聚半乳糖醛酸内聚酶IVa和IVb的纯化、鉴定及氨基酸序列分析。为了分析内聚半乳糖醛酸酶I的功能，我们对同一种真菌Stereum purpureum产生的具有不同等电点的内聚半乳糖醛酸酶IV进行了纯化、鉴定和氨基酸序列分析。从培养滤液中通过三步柱层析纯化两种EndoPG IV、VIa和VIb至均质。通过ESI-MS测定了EndoPG VIa和VIb的分子质量分别为36017 da和37266 da。这些pg具有共同的一级结构和糖链的1和2。通过克隆5′小种和3′小种cDNA，确定了氨基酸序列。在这个EndoPG IV中，未发现成熟的EndoPG i的c端区（44个氨基酸残基）缺失，EndoPG IV与成熟的EndoPG i的氨基酸序列同源性为72%。pKa_1和pKa_2的测定值分别为4.35和5.3。因此，我们尝试用x射线晶体学在pH2.5下测定一般碱性催化。结果表明，Asp153似乎是碱性催化剂的候选物。

○Expresion, purification and crystallization of Sterum purpureum endopolygalacturonase I in Escherichia coli.We constructed an expression system, using Escherichia coli, and a purification method to prepare functionally active endoPG I for the mutation and crystallographic studies. Expression in E.coli strain Origami (DE3) provided soluble and active enzyme with proper disulfide bonds, whereas the enzyme expressed in BL21(DE3) was localized in inclusion bodies. Sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography.○Purification, characterization and amino acid sequence of endopolygalacturonase IVa and IVb from Sterum purpureum.To analysisi function of endoplygalacrturonase I we traied purification, characterization and amino acid sequence analysis of endopolygalacturonase IV, which have difference isoelectoric point and is produced by the same fungus Stereum purpureum. Two EndoPG IV, VIa and VIb were purified to homogeneity by three steps of column chromatography from the culture filtrate. The molecular masses of the EndoPG VIa and VIb were determined to be 36,017Da and 37,266Da using ESI-MS, respectively. These PGs have the common primary structure and one and two of sugar chain, respectively. By cloning of the cDNA with 5'-race and 3'race, the ammo acid sequence was decided. In this EndoPG IV, there are no deletion of C-terminal region (44 amino acid residues) observed in mature EndoPG I. The EndoPG IV was 72 % homology for amino acid sequences of mature EndoPG I.○Determination of general basic catalysis by X ray crystallography at pH 2.5.The pKa_1 and pKa_2 of the enzyme were determined at 4.35 and 5.3, respectively. Therefore, we tried to determine general basic catalysis by Xray crystallography at pH2.5 by for low. As the results, Asp153 seems to be the base catalyst candidate.