目的探究96孔板高通量筛选方法在TrxA-溶菌酶热稳定性改造中的可行性。方法通过易错聚合酶链式反应建立高质量突变体文库,待单克隆长出后利用96孔板进行微量表达,之后采用冷热循环裂解的方法破坏菌体,离心取上清后检测不同温度下的杀菌...目的探究96孔板高通量筛选方法在TrxA-溶菌酶热稳定性改造中的可行性。方法通过易错聚合酶链式反应建立高质量突变体文库,待单克隆长出后利用96孔板进行微量表达,之后采用冷热循环裂解的方法破坏菌体,离心取上清后检测不同温度下的杀菌活性,借此实现高通量筛选,最后通过摇瓶表达验证初筛结果。结果在终质量浓度20μg/mL条件下,突变体Y262M的完全失活温度从野生型的48℃提高到52℃,分子动力学模拟结果显示Y262M的均方根偏差(root mean square deviation,RMSD)及均方根波动(root mean square fluctuation,RMSF)值均低于野生型,疏水簇分析显示Y262M在野生型基础上将细胞壁结合域的2个单独疏水簇融合成一个较大的疏水簇,总面积增加了约25%。结论本研究采用96孔板高通量筛选方法成功筛选出热稳定性提高的突变体Y262M,证实了该方法在溶菌酶热稳定性改造中的可行性。此方法不需要深入了解目标蛋白质的空间结构,实验操作相对简单,且随机突变也使得改造结果具有多样性,同时进一步的结构分析也为了解溶菌酶结构与功能关系提供有益参考。展开更多
Kinesin-8 motors can move with a high processivity on microtubule lattices toward the plus end.After reaching the plus end,the kinesin-8 motors can pause for a long time and promote the microtubule depolymerization.He...Kinesin-8 motors can move with a high processivity on microtubule lattices toward the plus end.After reaching the plus end,the kinesin-8 motors can pause for a long time and promote the microtubule depolymerization.Here,using atomistic molecular dynamics simulations we studied the structural changes of the kinesin-8 head in different nucleotide states bound to the straight and curved tubulins and the corresponding interactions between them.We found that the kinesin-8 head in ATP and/or ADP–Pi state has the similar strong affinity while in ADP state has the similar weak affinity to both the straight and curved tubulins,which is strongly implicated in the mechanism of the long but very different residence times of the kinesin-8 motor on the microtubule lattice and at the end.Moreover,we found that loop-2 of the kinesin-8 head bound strongly to the curved tubulin in the stable state has a large interference with its neck linker pulled in the minusended orientation.This is contrary to the case of the head bound strongly to the straight tubulin,where loop-2 has little interference with its neck linker pulled in the minus-ended orientation.The large interference can induce a larger internal force between the two heads and thus can induce the two curved tubulins bound strongly by the two heads to be more curved relative to each other.This is strongly implicated in the mechanism of the depolymerase activity of the kinesin-8motors and explains the origin of loop-2 playing a facilitating role in the depolymerase activity.展开更多
胆盐水解酶(bile salt hydrolase,BSH)是由肠道菌群中部分微生物产生并且广泛分布于动物胃肠道中的一种胞内酶,具有优良的胆酸盐水解活性。该研究旨在克隆并原核表达植物乳植杆菌胆盐水解酶(Lactobacillus plantarumbile bile salt hydr...胆盐水解酶(bile salt hydrolase,BSH)是由肠道菌群中部分微生物产生并且广泛分布于动物胃肠道中的一种胞内酶,具有优良的胆酸盐水解活性。该研究旨在克隆并原核表达植物乳植杆菌胆盐水解酶(Lactobacillus plantarumbile bile salt hydrolase,LpBSH),系统研究其酶学特性和结构特征,为开发新型降脂酶制剂提供理论基础。通过基因工程技术构建重组LpBSH表达系统,测定其酶学性质并进行动力学分析。结合生物信息学方法,对LpBSH的三维结构、活性位点及底物结合口袋进行预测和分析。结果表明,原核表达的LpBSH在最适条件下活性为88.7 U/mL,显著高于野生型。重组LpBSH在pH为6.5时活性最高,并在pH_(4).5~6.5范围内保持较高活性;K+对LpBSH水解活性有轻微促进作用,而重金属离子普遍抑制活性;动力学分析显示,LpBSH催化反应符合Hill方程,表现出底物正协同效应;通过生物信息学分析LpBSH的氨基酸序列并预测其三维结构特征,研究了关于其氨基酸序列、活性位点、底物结合口袋及底物偏好性的关系。该研究为深入理解LpBSH的结构与功能关系及其在动物胆固醇代谢中的作用奠定了基础,并为LpBSH酶制剂的开发提供了科学依据。展开更多
文摘目的探究96孔板高通量筛选方法在TrxA-溶菌酶热稳定性改造中的可行性。方法通过易错聚合酶链式反应建立高质量突变体文库,待单克隆长出后利用96孔板进行微量表达,之后采用冷热循环裂解的方法破坏菌体,离心取上清后检测不同温度下的杀菌活性,借此实现高通量筛选,最后通过摇瓶表达验证初筛结果。结果在终质量浓度20μg/mL条件下,突变体Y262M的完全失活温度从野生型的48℃提高到52℃,分子动力学模拟结果显示Y262M的均方根偏差(root mean square deviation,RMSD)及均方根波动(root mean square fluctuation,RMSF)值均低于野生型,疏水簇分析显示Y262M在野生型基础上将细胞壁结合域的2个单独疏水簇融合成一个较大的疏水簇,总面积增加了约25%。结论本研究采用96孔板高通量筛选方法成功筛选出热稳定性提高的突变体Y262M,证实了该方法在溶菌酶热稳定性改造中的可行性。此方法不需要深入了解目标蛋白质的空间结构,实验操作相对简单,且随机突变也使得改造结果具有多样性,同时进一步的结构分析也为了解溶菌酶结构与功能关系提供有益参考。
基金Project supported by the Science and Technology Research Program of Chongqing Municipal Education Commission(Grant No.KJQN202504529)the General Program of Chongqing Natural Science Foundation(Grant No.CSTB2025NSCQ-GPX0833)。
文摘Kinesin-8 motors can move with a high processivity on microtubule lattices toward the plus end.After reaching the plus end,the kinesin-8 motors can pause for a long time and promote the microtubule depolymerization.Here,using atomistic molecular dynamics simulations we studied the structural changes of the kinesin-8 head in different nucleotide states bound to the straight and curved tubulins and the corresponding interactions between them.We found that the kinesin-8 head in ATP and/or ADP–Pi state has the similar strong affinity while in ADP state has the similar weak affinity to both the straight and curved tubulins,which is strongly implicated in the mechanism of the long but very different residence times of the kinesin-8 motor on the microtubule lattice and at the end.Moreover,we found that loop-2 of the kinesin-8 head bound strongly to the curved tubulin in the stable state has a large interference with its neck linker pulled in the minusended orientation.This is contrary to the case of the head bound strongly to the straight tubulin,where loop-2 has little interference with its neck linker pulled in the minus-ended orientation.The large interference can induce a larger internal force between the two heads and thus can induce the two curved tubulins bound strongly by the two heads to be more curved relative to each other.This is strongly implicated in the mechanism of the depolymerase activity of the kinesin-8motors and explains the origin of loop-2 playing a facilitating role in the depolymerase activity.
文摘胆盐水解酶(bile salt hydrolase,BSH)是由肠道菌群中部分微生物产生并且广泛分布于动物胃肠道中的一种胞内酶,具有优良的胆酸盐水解活性。该研究旨在克隆并原核表达植物乳植杆菌胆盐水解酶(Lactobacillus plantarumbile bile salt hydrolase,LpBSH),系统研究其酶学特性和结构特征,为开发新型降脂酶制剂提供理论基础。通过基因工程技术构建重组LpBSH表达系统,测定其酶学性质并进行动力学分析。结合生物信息学方法,对LpBSH的三维结构、活性位点及底物结合口袋进行预测和分析。结果表明,原核表达的LpBSH在最适条件下活性为88.7 U/mL,显著高于野生型。重组LpBSH在pH为6.5时活性最高,并在pH_(4).5~6.5范围内保持较高活性;K+对LpBSH水解活性有轻微促进作用,而重金属离子普遍抑制活性;动力学分析显示,LpBSH催化反应符合Hill方程,表现出底物正协同效应;通过生物信息学分析LpBSH的氨基酸序列并预测其三维结构特征,研究了关于其氨基酸序列、活性位点、底物结合口袋及底物偏好性的关系。该研究为深入理解LpBSH的结构与功能关系及其在动物胆固醇代谢中的作用奠定了基础,并为LpBSH酶制剂的开发提供了科学依据。
