漆酶淀粉样融合酶聚集体的形成机制研究

Amyloid fused enzyme aggregates (AFEAs) are newly developed and high efficient carrier-free immobilization methods. However, the formation mechanisms of AFEAs have not been reported systematically yet, which hinders their high efficient construction, performance improvement, and widespread use. To solve the problem, laccase, a lignolytic enzyme, is selected as a model and the amyloid fused enzyme (AFE) of laccase and amyloid protein Sup35pNM linked with Gly-Ser (GS) linker is expressed in E. coli. By utilizing Fourier transform-infrared spectroscopy (FT-IR), X-ray scattering (XS), atomic force microscopy (AFM), etc., the influences of amyloid protein length, AFE / amyloid protein monomer (APM) molar ratio, as well as GS linker length on laccase AFEAs will be analyzed to investigate the self-assembly of AFE as well as the structure-activity relationship and stability of laccase AFEAs. On the basis of the experimental data, we will reveal the formation mechanisms of laccase AFEAs and establish a technology platform of AFEAs for Sup35pNM. The findings would provide theoretical basis and technology support for the extensive use of laccase and the construction of AFEAs for other enzymes.

淀粉样融合酶聚集体（AFEAs）是一种新型的高效无载体酶固定化方法，然而其形成机制的研究目前尚无系统报道，阻碍了AFEAs的高效构建、性能提升和广泛应用。为此本项目拟以木质素降解酶漆酶为研究对象，通过在大肠杆菌中构建甘氨酸-丝氨酸（GS）间隔臂连接的漆酶和淀粉样蛋白Sup35pNM的漆酶淀粉样融合酶（AFE），利用傅里叶红外光谱FT-IR、X射线衍射XS、原子力显微镜AFM等现代分析技术，系统阐述关键因素淀粉样蛋白长度、漆酶AFE与淀粉样蛋白单体APM的摩尔比（AFE/APM摩尔比）以及GS间隔臂长度对漆酶AFE的自组装动力学以及漆酶AFEAs构效关系和稳定性的影响，从而揭示漆酶AFEAs的形成机制，建立Sup35pNM型AFEAs构建的技术平台，为漆酶的广泛应用、以及其它AFEAs的构建奠定理论基础和技术支撑。

本项目首先将漆酶、淀粉样蛋白Sup35pNM和连接臂在酵母体系中融合表达出漆酶淀粉样融合酶（AFE），并通过优化自组装条件，构建出AFEAs。考察了AFE/APM摩尔比、温度以及pH等对漆酶AFE的自组装动力学以及漆酶AFEAs构效关系和稳定性的影响，揭示了漆酶AFEAs的形成机制，建立了Sup35pNM型AFEAs构建的技术平台。第二，本项目分离纯化出一株高产漆酶的细菌，并将其鉴定为Bacillus sp. A4。考察了该菌株生产漆酶的最适条件为37 °C、pH6、2%接种量。漆酶分子量为33 kDa，最适温度和pH为40 °C和pH4.6。铜离子能够显著提升漆酶活性。第三，本项目利用GO对酵母细胞进行了包封，并对包封条件进行了优化，发现包封后的酵母细胞呈现更高的活性，且对pH和温度的耐受性能均有提升，这对于提升AFE的表达量、对抗外界不良环境具有重要意义。本项目研究结果为漆酶的广泛应用、以及其它AFEAs的构建奠定了理论基础和技术支撑。