School of Biology and Biological Engineering South China University of Technology 382 East Outer Loop Rd, University Park, Guangzhou 510006, China.
School of Electronic Science and Engineering, University of Electronic Science and Technology of China, Chengdu 610054, China.
Biomolecules. 2020 Feb 4;10(2):231. doi: 10.3390/biom10020231.
Phospholipase B (EC 3.1.1.5) are a distinctive group of enzymes that catalyzes the hydrolysis of fatty acids esterified at the and positions forming free fatty acids and lysophospholipids. The structural information and catalytic mechanism of phospholipase B are still not clear. Herein, we reported a putative phospholipase B (TmPLB1) from GD-0079 synthesized by genome mining library. The gene (TmPlb1) was expressed and the TmPLB1 was purified using shuffle T7 expression system. The putative TmPLB1 was purified by affinity chromatography with a yield of 13.5%. The TmPLB1 showed optimum activity at 35 °C and pH 7.0. The TmPLB1 showed enzymatic activity using Lecithin (soybean > 98% pure), and the hydrolysis of TmPLB1 by P NMR showed phosphatidylcholine (PC) as a major phospholipid along with lyso-phospholipids (1-LPC and 2-LPC) and some minor phospholipids. The molecular modeling studies indicate that its active site pocket contains Ser125, Asp183 and His215 as the catalytic triad. The structure dynamics and simulations results explained the conformational changes associated with different environmental conditions. This is the first report on biochemical characterization and structure dynamics of TmPLB1 enzyme. The present study could be helpful to utilize TmPLB1 in food industry for the determination of food components containing phosphorus. Additionally, such enzyme could also be useful in Industry for the modifications of phospholipids.
磷脂酶 B（EC 3.1.1.5）是一组独特的酶，能够催化酯化在 和 位置的脂肪酸水解，形成游离脂肪酸和溶血磷脂。磷脂酶 B 的结构信息和催化机制尚不清楚。本文报道了一种来源于 GD-0079 的假定磷脂酶 B（TmPLB1），通过基因组挖掘文库合成。通过 shuffle T7 表达系统表达基因（TmPlb1）并纯化 TmPLB1。通过亲和层析纯化假定的 TmPLB1，产率为 13.5%。TmPLB1 在 35°C 和 pH 7.0 时表现出最佳活性。TmPLB1 对大豆卵磷脂（Lecithin，纯度>98%）具有酶活性，并且 P NMR 显示 TmPLB1 水解生成的主要磷脂是磷脂酰胆碱（PC），同时还有溶血磷脂（1-LPC 和 2-LPC）和一些少量的磷脂。分子建模研究表明，其活性位点口袋包含 Ser125、Asp183 和 His215 作为催化三联体。结构动力学和模拟结果解释了与不同环境条件相关的构象变化。这是首次报道 TmPLB1 酶的生化特性和结构动力学。本研究可以帮助在食品工业中利用 TmPLB1 来确定含磷的食品成分。此外，这种酶在工业中也可以用于修饰磷脂。
