肉品风味与肌内磷脂的水解密切相关。申请者前期研究肌内磷脂水解时发现Patatin样磷脂酶3（PNPLA3），相较于其它磷脂酶在肌肉中表达量最高，且在加工过程中保持较高含量和活力，在肌内磷脂水解中发挥重要作用。但PNPLA3如何在肉品中发挥水解作用未见报道。本项目首先通过体外表达纯化，制备PNPLA3纯品，分析其活性和影响因素；采用表面等离子共振和红外光谱分析PNPLA3结合磷脂的常数，确定其与磷脂的结合位点；运用液质联用和气相色谱分析PNPLA3水解磷脂的产物，推断其水解位点；通过X射线衍射解析PNPLA3-磷脂复合物的晶体结构，确定其磷脂结合结构域；通过结构比较和序列比对，设计突变体并用等温滴定量热法检测，明确PNPLA3的催化结构域；融合表达PNPLA3的2个功能结构域，进行功能验证，阐明其水解肌内磷脂的分子机制，为肉品风味控制提供新思路和理论依据。

Flavor of meat is closely correlated with lipolysis of intramuscular phospholipids. Our previous studies on intramuscular phospholipids hydrolyzed by phospholipases discovered the patatin like phospholipase 3 (PNPLA3). The expression level of PNPLA3 in meat was found highest among kinds of phospholipases. In the process of meat processing, PNPLA3 maintained high concentration and showed stable activity, suggesting its important role in lipolysis of intramuscular phospholipids. However, the lipolysis mechanism of PNPLA3 on phospholipids remains unknown. In the present project, PNPLA3 with high purity will be prepared in vitro, and its activity and the optimal conditions will be determined. The phospholipid binding site will be identified with the aid of surface plasmon resonance (SPR) and Fourier Transform Infrared Spectrometer (FITR). The phospholipid cleavage site will also be determined by analyzing the hydrolysis products of PNPLA3 using HPLC-MS and gas chromatography. The three-dimensional structure of PNPLA3 and phospholipid complex will be determined by X ray diffraction analysis; with the structural data and SPR analysis, the substrate-binding domain of PNPLA3 and its allosteric activation mechanism will be determined; the catalytic domain will be confirmed by the site-specific mutagenesis based on the structure and sequence analysis, and following biochemistry experiments such as ITC. Subsequently, co-expression of active domains of PNPLA3 will be employed to verify the enzymatic activity. The mechanism will be further validated in intramuscular environment. The result will provide a new approach for studies on meat flavor, as well as a theoretical basis for the regulation of meat flavor.