冷适应海洋假交替单胞菌α-半乳糖苷酶催化水解反应的立体化学过程。

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences Vladivostok, Russia.

Front Chem. 2014 Oct 13;2:89. doi: 10.3389/fchem.2014.00089. eCollection 2014.

The recombinant α-galactosidase of the marine bacterium (α-PsGal) was synthesized with the use of the plasmid 40Gal, consisting of plasmid pET-40b (+) (Novagen) and the gene corresponding to the open reading frame of the mature α-galactosidase of marine bacterium Pseudoalteromonas sp. KMM 701, transformed into the Escherichia coli Rosetta(DE3) cells. In order to understand the mechanism of action, the stereochemistry of hydrolysis of 4-nitrophenyl α-D-galactopyranoside (4-NPGP) by α-PsGal was measured by (1)H NMR spectroscopy. The kinetics of formation of α- and β-anomer of galactose showed that α-anomer initially formed and accumulated, and then an appreciable amount of β-anomer appeared as a result of mutarotation. The data clearly show that the enzymatic hydrolysis of 4-NPGP proceeds with the retention of anomeric configuration, probably, due to a double displacement mechanism of reaction.

利用质粒 40Gal（由质粒 pET-40b（+）（Novagen）和海洋细菌 Pseudoalteromonas sp. KMM 701 成熟α-半乳糖苷酶的开放阅读框对应的基因组成），将海洋细菌的重组α-半乳糖苷酶（α-PsGal）合成到大肠杆菌 Rosetta（DE3）细胞中。为了了解作用机制，通过（1）H NMR 光谱测量了α-PsGal 水解 4-硝基苯-α-D-半乳糖吡喃糖苷（4-NPGP）的立体化学。α-和β-半乳糖苷的形成动力学表明，α-端首先形成并积累，然后由于差向异构化作用出现相当数量的β-端。数据清楚地表明，4-NPGP 的酶促水解是通过保留端基构型进行的，可能是由于反应的双取代机制。