The bifunctional fusion protein systems consisting of Rhodococcus erythropolis chiral alcohol dehydrogenase(READH),Candida boidinii formate dehydrogenase(CbFDH) or maltose binding protein(MBP) were constructed to rege...The bifunctional fusion protein systems consisting of Rhodococcus erythropolis chiral alcohol dehydrogenase(READH),Candida boidinii formate dehydrogenase(CbFDH) or maltose binding protein(MBP) were constructed to regenerate the cofactors for biocatalysis.READH originated from Rhodococcus erythropolis is an(S)-specific nicotinamide adenine dinucleotide(NADH)-dependent alcohol dehydrogenase,meanwhile,CbFDH originated from Candida boidinii is an NADH-dependent formate dehydrogenase.The strategies of the different fusion protein systems included:(1) fusion of the N terminus of READH to the C terminus of MBP,(2) fusion of the N terminus of CbFDH to the C terminus of MBP,(3) fusion of the N terminus of READH to the C terminus of CbFDH,(4) fusion of the C terminus of READH to the N terminus of CbFDH.The activities of READHs were depressed in all fusion strategies.When the N terminus of READH was fused to the C terminus of CbFDH,READH reached the highest activity,but CbFDH had no activity.In contrast,when the C terminus of READH was fused to the N terminus of CbFDH,CbFDH showed the highest activity,and both moieties displayed activities.From this study,the authors suggest that the rational design of the bifunctional fusion protein system may improve the biocatalysis efficiency by the simultaneous cofactor regeneration.展开更多
使用两步发酵法培养博伊丁假丝酵母(Candida boidinii),菌株细胞经破碎与分离所得粗酶液经DEAE SepHaroses Fast Flow层析快速纯化获得NAD+依赖型的甲酸脱氢酶,酶的比活从0.83U/mg提高到2.67U/mg,纯化倍数和回收率分别为3.22倍和29.7%...使用两步发酵法培养博伊丁假丝酵母(Candida boidinii),菌株细胞经破碎与分离所得粗酶液经DEAE SepHaroses Fast Flow层析快速纯化获得NAD+依赖型的甲酸脱氢酶,酶的比活从0.83U/mg提高到2.67U/mg,纯化倍数和回收率分别为3.22倍和29.7%。研究了从反应物消耗到产物生成之间的酶反应历程,确定了甲酸脱氢酶的酶促反应为有序BiBi反应机制,其中NAD+是第一底物,HCOONa是第二底物,NADH是首先释放的第一产物,HCO3ˉ是随后释放的第二产物;二次作图法求解出Vmax、KiS1、KmS2、KmS1等动力学参数,确定甲酸脱氢酶有序BiBi反应速度方程为r=(2.3*10-4[S1][S2])/(1.123*10-2[S1]+1.91*10-6+[S1][S2])。展开更多
甲酸脱氢酶(formate dehydrogenase,FDH)属于D-2-羟基酸脱氢酶类,能催化甲酸氧化生成二氧化碳,同时能将氧化型辅酶I(Oxdized form of nicotinamide adenine dinucleotide,NAD+)还原成还原型辅酶I(Redued form of nicotinamide adenine d...甲酸脱氢酶(formate dehydrogenase,FDH)属于D-2-羟基酸脱氢酶类,能催化甲酸氧化生成二氧化碳,同时能将氧化型辅酶I(Oxdized form of nicotinamide adenine dinucleotide,NAD+)还原成还原型辅酶I(Redued form of nicotinamide adenine dinucleotide,NADH),在NADH的再生中起重要作用。为了获得高活性的甲酸脱氢酶突变体,本研究以博伊丁假丝酵母甲酸脱氢酶(Candida boidinii formate dehydrogenases,CbFDH)突变体(CbFDHC23S)为亲本,进行了2轮定向进化,获得了一个比酶活性约为亲本4倍,且更适合于在生理条件下进行辅酶再生的突变体M2。然后,利用计算机辅助的手段初步阐明了其温度特性和催化效率改变的分子机制。最后,借助共表达策略进一步提高了突变体M2在大肠杆菌中的表达水平,超声裂解液中的甲酸脱氢酶活性达到45.85 U/mL,远远高于亲本单拷贝表达水平。本研究为增强NADH再生能力、降低NADH的再生成本,实现FDH偶联催化的手性醇及氨基酸衍生物等食品添加剂高效、廉价的绿色生物合成奠定理论基础。展开更多
文摘The bifunctional fusion protein systems consisting of Rhodococcus erythropolis chiral alcohol dehydrogenase(READH),Candida boidinii formate dehydrogenase(CbFDH) or maltose binding protein(MBP) were constructed to regenerate the cofactors for biocatalysis.READH originated from Rhodococcus erythropolis is an(S)-specific nicotinamide adenine dinucleotide(NADH)-dependent alcohol dehydrogenase,meanwhile,CbFDH originated from Candida boidinii is an NADH-dependent formate dehydrogenase.The strategies of the different fusion protein systems included:(1) fusion of the N terminus of READH to the C terminus of MBP,(2) fusion of the N terminus of CbFDH to the C terminus of MBP,(3) fusion of the N terminus of READH to the C terminus of CbFDH,(4) fusion of the C terminus of READH to the N terminus of CbFDH.The activities of READHs were depressed in all fusion strategies.When the N terminus of READH was fused to the C terminus of CbFDH,READH reached the highest activity,but CbFDH had no activity.In contrast,when the C terminus of READH was fused to the N terminus of CbFDH,CbFDH showed the highest activity,and both moieties displayed activities.From this study,the authors suggest that the rational design of the bifunctional fusion protein system may improve the biocatalysis efficiency by the simultaneous cofactor regeneration.
基金the National Natural Science Foundation of China(30270852,30471116)Program for New Century Excellent Talents in University(NCET-2004-0913)+1 种基金Special Project of Development of Animal and Plant Varieties in Guizhou Province([2007]026)Mega-projects of China (2006BAD02B06)
文摘使用两步发酵法培养博伊丁假丝酵母(Candida boidinii),菌株细胞经破碎与分离所得粗酶液经DEAE SepHaroses Fast Flow层析快速纯化获得NAD+依赖型的甲酸脱氢酶,酶的比活从0.83U/mg提高到2.67U/mg,纯化倍数和回收率分别为3.22倍和29.7%。研究了从反应物消耗到产物生成之间的酶反应历程,确定了甲酸脱氢酶的酶促反应为有序BiBi反应机制,其中NAD+是第一底物,HCOONa是第二底物,NADH是首先释放的第一产物,HCO3ˉ是随后释放的第二产物;二次作图法求解出Vmax、KiS1、KmS2、KmS1等动力学参数,确定甲酸脱氢酶有序BiBi反应速度方程为r=(2.3*10-4[S1][S2])/(1.123*10-2[S1]+1.91*10-6+[S1][S2])。
文摘甲酸脱氢酶(formate dehydrogenase,FDH)属于D-2-羟基酸脱氢酶类,能催化甲酸氧化生成二氧化碳,同时能将氧化型辅酶I(Oxdized form of nicotinamide adenine dinucleotide,NAD+)还原成还原型辅酶I(Redued form of nicotinamide adenine dinucleotide,NADH),在NADH的再生中起重要作用。为了获得高活性的甲酸脱氢酶突变体,本研究以博伊丁假丝酵母甲酸脱氢酶(Candida boidinii formate dehydrogenases,CbFDH)突变体(CbFDHC23S)为亲本,进行了2轮定向进化,获得了一个比酶活性约为亲本4倍,且更适合于在生理条件下进行辅酶再生的突变体M2。然后,利用计算机辅助的手段初步阐明了其温度特性和催化效率改变的分子机制。最后,借助共表达策略进一步提高了突变体M2在大肠杆菌中的表达水平,超声裂解液中的甲酸脱氢酶活性达到45.85 U/mL,远远高于亲本单拷贝表达水平。本研究为增强NADH再生能力、降低NADH的再生成本,实现FDH偶联催化的手性醇及氨基酸衍生物等食品添加剂高效、廉价的绿色生物合成奠定理论基础。
