Benoit Isabelle, Coutard Bruno, Oubelaid Rachid, Asther Marcel, Bignon Christophe
UMR 1163 INRA de Biotechnologie des Champignons Filamenteux, IFR86-BAIM, Université de Provence et de la Méditerranée, ESIL, 163 avenue de Luminy CP 925, 13288 Marseille cedex 09, France.
Protein Expr Purif. 2007 Sep;55(1):166-74. doi: 10.1016/j.pep.2007.04.001. Epub 2007 Apr 10.
Hydrolysis of plant biomass is achieved by the combined action of enzymes secreted by microorganisms and directed against the backbone and the side chains of plant cell wall polysaccharides. Among side chains degrading enzymes, the feruloyl esterase A (FAEA) specifically removes feruloyl residues. Thus, FAEA has potential applications in a wide range of industrial processes such as paper bleaching or bio-ethanol production. To gain insight into FAEA hydrolysis activity, we solved its crystal structure. In this paper, we report how the use of four consecutive factorial approaches (two incomplete factorials, one sparse matrix, and one full factorial) allowed expressing in Escherichia coli, refolding and then crystallizing Aspergillus niger FAEA in 6 weeks. Culture conditions providing the highest expression level were determined using an incomplete factorial approach made of 12 combinations of four E. coli strains, three culture media and three temperatures (full factorial: 36 combinations). Aspergillus niger FAEA was expressed in the form of inclusion bodies. These were dissolved using a chaotropic agent, and the protein was purified by affinity chromatography on Ni column under denaturing conditions. A suitable buffer for refolding the protein eluted from the Ni column was found using a second incomplete factorial approach made of 96 buffers (full factorial: 3840 combinations). After refolding, the enzyme was further purified by gel filtration, and then crystallized following a standard protocol: initial crystallization conditions were found using commercial crystallization screens based on a sparse matrix. Crystals were then optimized using a full factorial screen.
植物生物质的水解是通过微生物分泌的、作用于植物细胞壁多糖主链和侧链的酶的联合作用实现的。在降解侧链的酶中，阿魏酸酯酶A（FAEA）能特异性去除阿魏酸残基。因此，FAEA在造纸漂白或生物乙醇生产等广泛的工业过程中具有潜在应用。为深入了解FAEA的水解活性，我们解析了其晶体结构。在本文中，我们报告了如何使用四种连续的析因方法（两种不完全析因法、一种稀疏矩阵法和一种全析因法），在6周内实现黑曲霉FAEA在大肠杆菌中的表达、重折叠并结晶。使用由四种大肠杆菌菌株、三种培养基和三种温度的12种组合构成的不完全析因法（全析因：36种组合）确定了提供最高表达水平的培养条件。黑曲霉FAEA以包涵体形式表达。使用离液剂溶解这些包涵体，并在变性条件下通过镍柱亲和层析纯化蛋白质。使用由96种缓冲液构成的第二种不完全析因法（全析因：3840种组合）找到了适合重折叠从镍柱洗脱的蛋白质的缓冲液。重折叠后，通过凝胶过滤进一步纯化该酶，然后按照标准方案进行结晶：使用基于稀疏矩阵的商业结晶筛选找到初始结晶条件。然后使用全析因筛选优化晶体。
