Multifunctional endoglucanase, RfGH5_4 from Ruminococcus flavefaciens showed (β/α)₈-TIM barrel structure by homology modeling. Glu168 and Glu292 residues acted as general acid and base during catalysis. Circular Dichroism results, 40.83 % α-helices, 13.84 % β-strands and 45 % random turns-coils for RfGH5_4 corroborated with predictions by PSIPRED and SOPMA. Molecular Dynamic simulation of RfGH5_4 for 100 ns showed RMSD, 0.71 nm while for RfGH5_4-Cellopentaose complex was 0.55 nm, confirming that the binding of cellulosic ligand stabilizes its structural fold. RfGH5_4 showed strong affinity towards cellulosic ligands having higher degree of polymerization such as cellohexaose (−11.70 kcal/mol) and cellodecaose (−12.64 kcal/mol). Interestingly, complex hemicellulosic ligands such as XLLG of xyloglucan also showed higher affinity (−13.2 kcal/mol) and accommodated at RfGH5_4 active-site. Its catalytic cleft was broad enough to accommodate and hydrolyse various cellulosic and hemicellulosic ligands like XLLG of xyloglucan setting the basis of multifunctionality of RfGH5_4. Loops L2, L3 and L4 having Trp58 formed barrier at active-site of RfGH5_4 were responsible for processivity. RfGH5_4 showed monodispersed state at 2.5 mg/mL and a rattle-toy shape by SAXS. Zeta potential, −16 mV of RfGH5_4 by indicated its higher stability. Multifunctional RfGH5_4 endoglucanase could be beneficial for generation lignocellulosic bioethanol and in health, prebiotic and food sector.

来自 Ruminococcus flavefaciens 的多功能内切葡聚糖酶 Rf GH5_4通过同源建模显示 (β/α) ₈ -TIM 桶结构。Glu168 和 Glu292 残基在催化过程中充当一般酸和碱。圆二色性结果、 Rf GH5_4的 40.83% α-螺旋、13.84% β-链和 45% 随机匝圈证实了 PSIPRED 和 SOPMA 的预测。Rf GH5_4 100 ns的分子动力学模拟显示 RMSD 为 0.71 nm，而Rf GH5_4 - Cellopentaose 复合物为 0.55 nm，证实纤维素配体的结合稳定了其结构折叠。射频GH5_4 对聚合度较高的纤维素配体表现出很强的亲和力，例如纤维六糖 (-11.70 kcal/mol) 和纤维十糖 (-12.64 kcal/mol)。有趣的是，复杂的半纤维素配体如木葡聚糖的 XLLG 也显示出更高的亲和力 (−13.2 kcal/mol) 并容纳在Rf GH5_4 活性位点。它的催化裂缝足够宽，可以容纳和水解各种纤维素和半纤维素配体，如木葡聚糖的 XLLG，为Rf GH5_4 的多功能性奠定了基础。具有 Trp58 的环 L2、L3 和 L4 在Rf GH5_4 的活性位点形成屏障，负责持续合成能力。Rf GH5_4 在 2.5 mg/mL 时呈单分散状态，SAXS 呈拨浪鼓玩具形状。Zeta 电位，-16 mV 的Rf GH5_4 by 表明其较高的稳定性。多功能Rf GH5_4 内切葡聚糖酶可能有益于木质纤维素生物乙醇的产生以及健康、益生元和食品领域。