Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido 099-2493, Japan.
Appl Biochem Biotechnol. 2011 Dec;165(7-8):1494-506. doi: 10.1007/s12010-011-9370-4. Epub 2011 Sep 21.
Phospholipase D (PLD) catalyzes transphosphatidylation, causing inter-conversion of the polar head group of phospholipids and phospholipid hydrolysis. Previously, we cloned PLD103, a PLD with high transphosphatidylation activity, from Streptomyces racemochromogenes strain 10-3. Here, we report the construction of an expression system for the PLD103 gene using Streptomyces lividans as the host bacterium to achieve large-scale production. The phosphatidylcholine (PC) hydrolysis activity of S. lividans transformed with the expression plasmid containing the PLD103 gene was approximately 90-fold higher than that of the original strain. The recombinant PLD103 (rPLD103) found in the supernatant of the transformant culture medium was close to homogeneous. The rPLD103 was indistinguishable from the native enzyme in molecular mass and enzymatic properties. Additionally, rPLD103 had high transphosphatidylation activity on PC as a substrate in a simple aqueous one-phase reaction system and was able to modify the phospholipid content of soybean lecithin. Consequently, the expression system produces a stable supply of PLD, which can then be used in the production of phosphatidyl derivatives from lecithin.
磷脂酶 D（PLD）催化转磷酸化，导致磷脂的极性头基的相互转化和磷脂水解。此前，我们从链霉菌属 10-3 菌株中克隆了具有高转磷酸化活性的 PLD103。在这里，我们报告了使用链霉菌作为宿主菌构建 PLD103 基因表达系统以实现大规模生产的方法。含有 PLD103 基因的表达质粒转化的链霉菌的卵磷脂（PC）水解活性约比原始菌株高 90 倍。转化体培养基上清液中的重组 PLD103（rPLD103）接近均一。rPLD103 在分子量和酶学性质上与天然酶相同。此外，rPLD103 在简单的单相水相反应体系中作为底物对 PC 具有高的转磷酸化活性，并能够修饰大豆卵磷脂中的磷脂含量。因此，该表达系统可稳定供应 PLD，可用于从卵磷脂生产磷脂衍生物。
