目的研究三种模拟结肠发酵培养基和粪菌固定化对体外结肠发酵体系菌群构成的影响,为建立高度模拟结肠体外结肠发酵提供参考。方法采集健康女性成人粪便,粪便制备悬液或用结冷胶-黄原胶固定化粪菌接种于模拟结肠发酵反应器,分别用三种培...目的研究三种模拟结肠发酵培养基和粪菌固定化对体外结肠发酵体系菌群构成的影响,为建立高度模拟结肠体外结肠发酵提供参考。方法采集健康女性成人粪便,粪便制备悬液或用结冷胶-黄原胶固定化粪菌接种于模拟结肠发酵反应器,分别用三种培养基按0.07 m L/h稀释率进行连续发酵10 d,用末端限制性片段长度多态性(T-RFLP)与高通量测序技术分析菌群多样性,并分析发酵液短链脂肪酸含量。结果 T-RFLP分析显示,培养基类型主要影响粪菌固定体系菌群α多样性达到稳定的时间和多样性。β多样性分析显示,第10天时培养基Ⅲ悬液培养和培养基I固定化培养系统的菌群构成与粪便菌群最相近。而培养基Ⅲ在第10天达到较高而稳定的丁酸浓度。结论由于比粪菌固定培养更易操作,培养基Ⅲ悬液培养适于模拟人结肠发酵。展开更多
The binding of Endonuclease colicin 9 (E9) by Immunity protein 9 (Im9) was found to involve some hotspots from helix III of Im9 on protein-protein interface that contribute the dominant binding energy to the complex.I...The binding of Endonuclease colicin 9 (E9) by Immunity protein 9 (Im9) was found to involve some hotspots from helix III of Im9 on protein-protein interface that contribute the dominant binding energy to the complex.In the current work,MD simulations of the WT and three hotspot mutants (D51A,Y54A and Y55A of Im9) of the E9-Im9 complexes were carried out to investigate specific interaction mechanisms of these three hotspot residues.The changes of binding energy between the WT and mutants of the complex were computed by the MM/PBSA method using a polarized force field and were in excellent agreement with experiment values,verifying that these three residues were indeed hotspots of the binding complex.Energy decomposition analysis revealed that binding by D51 to E9 was dominated by electrostatic interaction due to the presence of the carboxyl group of Asp51 which hydrogen bonds to K89.For binding by hotspots Y54 and Y55,van der Waals interaction from the aromatic side chain of tyrosine provided the dominant interaction.For comparison,calculation by using the standard (nonpolarizable) AMBER99SB force field produced binding energy changes from these mutations in opposite direction to the experimental observation.Dynamic hydrogen bond analysis showed that conformations sampled from MD simulation in the standard AMBER force field were distorted from the native state and they disrupted the inter-protein hydrogen bond network of the protein-protein complex.The current work further demonstrated that electrostatic polarization plays a critical role in modulating protein-protein binding.展开更多
文摘目的研究三种模拟结肠发酵培养基和粪菌固定化对体外结肠发酵体系菌群构成的影响,为建立高度模拟结肠体外结肠发酵提供参考。方法采集健康女性成人粪便,粪便制备悬液或用结冷胶-黄原胶固定化粪菌接种于模拟结肠发酵反应器,分别用三种培养基按0.07 m L/h稀释率进行连续发酵10 d,用末端限制性片段长度多态性(T-RFLP)与高通量测序技术分析菌群多样性,并分析发酵液短链脂肪酸含量。结果 T-RFLP分析显示,培养基类型主要影响粪菌固定体系菌群α多样性达到稳定的时间和多样性。β多样性分析显示,第10天时培养基Ⅲ悬液培养和培养基I固定化培养系统的菌群构成与粪便菌群最相近。而培养基Ⅲ在第10天达到较高而稳定的丁酸浓度。结论由于比粪菌固定培养更易操作,培养基Ⅲ悬液培养适于模拟人结肠发酵。
基金the National Natural Science Foundation of China(21003048,10974054,and 20933002)Shanghai PuJiang Program (09PJ1404000) for financial support XXY is also supported by "Scientific Research Foundation for Agricultural Machinery Bureau of Jiangsu Province (gxz10008)"CGJ is also supported by "the Fundamental Research Funds for the Central Universities"
文摘The binding of Endonuclease colicin 9 (E9) by Immunity protein 9 (Im9) was found to involve some hotspots from helix III of Im9 on protein-protein interface that contribute the dominant binding energy to the complex.In the current work,MD simulations of the WT and three hotspot mutants (D51A,Y54A and Y55A of Im9) of the E9-Im9 complexes were carried out to investigate specific interaction mechanisms of these three hotspot residues.The changes of binding energy between the WT and mutants of the complex were computed by the MM/PBSA method using a polarized force field and were in excellent agreement with experiment values,verifying that these three residues were indeed hotspots of the binding complex.Energy decomposition analysis revealed that binding by D51 to E9 was dominated by electrostatic interaction due to the presence of the carboxyl group of Asp51 which hydrogen bonds to K89.For binding by hotspots Y54 and Y55,van der Waals interaction from the aromatic side chain of tyrosine provided the dominant interaction.For comparison,calculation by using the standard (nonpolarizable) AMBER99SB force field produced binding energy changes from these mutations in opposite direction to the experimental observation.Dynamic hydrogen bond analysis showed that conformations sampled from MD simulation in the standard AMBER force field were distorted from the native state and they disrupted the inter-protein hydrogen bond network of the protein-protein complex.The current work further demonstrated that electrostatic polarization plays a critical role in modulating protein-protein binding.
