粘质沙雷氏菌磷脂酶A1辅助蛋白对磷脂酶A1活性的调控机制

Phospholipase A1 has absolute advantage when used in vegetable oil degumming compared with the traditional method.The lysophospholipids produced by the hydrolysis of PLA1 was widely applied in various industial areas as excellent emulsifier, food additives and antibacterial agent. The application of bacterial PLA1 was limited by the low enzyme activity. An accessory protein located downstream of PLA1 gene could greatly promote the activity of the heterologous expression in Serratia marcescens, but the accessory protein showed inhibition on the growth of the host cell simultaneously. In this project, the fusion expression stain of phospholipase and the accessory protein with the fluorescent protein was constructed, the interaction between phospholipase A1 and the accessory protein in vivo was revealed by Fluorescence resonance energy transfer (FRET). The phospholiapse A1 and the accessory protein was purified, and the interaction between phospholipase A1 and the accessory protein in vitro would be clarified by the surface plasmon resonance technology（SPR）.In order to obtain the related information of high activity recombinant phospholipaseA1,the key binding sites between phospholipase A1 and the accessory protein were also being analyzed and alteration by directed molecular evolution and site directed mutagenesis. Verify the property of vegetable oil degumming by ecombinant phospholipaseA1.This project can reveal the interaction mechanisms between the accessory protein and phospholipase A1 from the molecular level and provide a scientific basis for the study of phospholipase A1 and other phospholipases.

磷脂酶A1应用于植物油脱胶相对于传统方法具有绝对优势，其水解产物溶血磷脂是优良的乳化剂、食品添加剂和抗菌剂,具有广泛的工业用途。细菌磷脂酶A1的低活性限制了它的应用。粘质沙雷氏菌磷脂酶A1基因下游的一段辅助蛋白可以极大促进其异源表达活性，但也对宿主细胞也表现较强的抑制作用。为揭示辅助蛋白对磷脂酶A1的调控机制，本研究拟分别构建磷脂酶A1和辅助蛋白与荧光蛋白融合表达菌株，利用荧光共振能量转移技术（FRET）揭示磷脂酶A1与辅助蛋白体内相互作用，并通过表面等离子共振技术（SPR）解析二者的体外互作特性，进而阐明辅助蛋白与磷脂酶A1相互作用机制。同时结合分子定向进化和定点突变，对磷脂酶A1与辅助蛋白的关键结合位点进行分析和改造，以期获得高活性重组A1的相关信息。验证重组酶A1植物油脱胶性能。本项目从分子水平对辅助蛋白与磷脂酶A1的互作机制进行解析，为磷脂酶A1及其他磷脂酶的活性研究提供科学依据。

磷脂酶A1应用于植物油脱胶相对于传统方法具有绝对优势，其水解产物溶血磷脂是优良的乳化剂、食品添加剂和抗菌剂,具有广泛的工业用途。细菌磷脂酶 A1的低活性限制了它的应用。粘质沙雷氏菌磷脂酶A1基因下游的一段辅助蛋白可以极大促进其异源表达活性，但也对宿主细胞也表现较强的抑制作用。为揭示辅助蛋白对磷脂酶A1的调控机制，本研究拟分别构建磷脂酶A1和辅助蛋白与荧光蛋白融合表达菌株，利用荧光共振能量转移技术（FRET）揭示磷脂酶A1与辅助蛋白体内相互作用，并通过表面等离子共振技术（SPR）解析二者的体外互作特性，进而阐明辅助蛋白与磷脂酶 A1 相互作用机制。同时结合分子定向进化和定点突变，对磷脂酶A1与辅助蛋白的关键结合位点进行分析和改造，以期获得高活性重组A1的相关信息。验证重组酶A1植物油脱胶性能。研究发现：PlaA与辅助蛋白PlaS能够特异结合，并呈现很好的浓度依赖，两蛋白间的结合速率ka为9223Ms-1，解离速率kd为1.054E-5s-1,平衡亲和力KD为1.143E-9 M。辅助蛋白PlaS的N端的一段信号肽序列，不利于磷脂酶A1的胞外分泌；并且通过分析高低产菌的磷脂酶A1的基因序列发现PlaA的第98个氨基酸序列发生了突变。因此，构建了PlaS的N端截短突变株，并定点突变磷脂酶A1 P98位点为S，成功将磷脂酶A1的脱胶性能提高11倍。本项目从分子水平对辅助蛋白与磷脂酶A1的互作机制进行解析，并为磷脂酶 A1及其他磷脂酶的活性研究提供科学依据。