Extra tyrosine in the carbohydrate-binding module of Irpex lacteus Xyn10B enhances its cellulose-binding ability

The xylanase (Xyn10B) that strongly adsorbs on microcrystalline cellulose was isolated from Driselase. The Xyn10B contains a Carbohydrate-binding module family 1 (CBM1) (IrpCBM_Xyn10B) at N-terminus. The canonical essential aromatic residues required for cellulose binding were conserved in IrpCBM_Xyn10B; however, its adsorption ability was markedly higher than that typically observed for the CBM1 of an endoglucanase from Trametes hirsuta (ThCBM_EG1). An analysis of the CBM-GFP fusion proteins revealed that the binding capacity to cellulose (7.8 μmol/g) and distribution coefficient (2.0 L/μmol) of IrpCBM_Xyn10B-GFP were twofold higher than those of ThCBM_EG1-GFP (3.4 μmol/g and 1.2 L/μmol, respectively), used as a reference structure. Besides the canonical aromatic residues (W24-Y50-Y51) of typical CBM1-containing proteins, IrpCBM_Xyn10B had an additional aromatic residue (Y52). The mutation of Y52 to Ser (IrpCBM_Y52S-GFP) reduced these adsorption parameters to 4.4 μmol/g and 1.5 L/μmol, which were similar to those of ThCBM_EG1-GFP. These results indicate that Y52 plays a crucial role in strong cellulose binding.     

体外多酶分子机器的现状和最新进展

体外多酶分子机器遵循所设计的多酶催化路径,将若干种纯化或部分纯化的酶元件进行合理的优化与适配,高效地在体外将特定的底物转化为目标化合物。体外多酶分子机器反应系统呈现元件化和模块化的特点,在设计、组装和调控方面具有较高的自由度。近年来,体外多酶分子机器在实现反应过程的精准调控和提高产品得率方面的优势逐渐体现,展示了其在生物制造领域重要的应用潜力。对体外多酶分子机器的相关研究已成为合成生物学的一个重要分支领域,日益受到广泛的关注。文中系统地综述了基于酶元件/模块的体外多酶分子机器的构建策略,以及改善该分子机器中酶元件/模块之间适配性的研究进展,并分析了该生物制造平台的发展前景与挑战。     

Extensive unfolding of the C-LytA choline-binding module by submicellar concentrations of sodium dodecyl sulphate

We have investigated the stability of the choline-binding module C-LytA against sodium dodecyl sulphate (SDS)-induced unfolding at pH 7.0 and 20 degrees C. A major intermediate with an unfolded N-terminal region accumulates at around 0.75 mM SDS, whereas 2.0 mM SDS was sufficient for a complete unfolding. This might be the first report of a protein being extensively unfolded by submicellar concentrations of SDS, occurring through formation of detergent clusters on the protein surface. All transitions were reversible upon SDS complexation with beta-cyclodextrin, allowing the calculation of thermodynamic parameters. A model for the unfolding of C-LytA by SDS is presented and compared to a previous denaturation scheme by guanidine hydrochloride.