The ability of catalyzing indole into indigo of gene engineering strain expressing P450 BM3 immobi- lized by entrapment in calcium-alginate gel capsules was examined,and various characteristics of immobilized cells we...The ability of catalyzing indole into indigo of gene engineering strain expressing P450 BM3 immobi- lized by entrapment in calcium-alginate gel capsules was examined,and various characteristics of immobilized cells were assessed.Optimum conditions for cells activity were not affected after immobilization,and pH and tempera- ture for both free and immobilized cells were found to be pH 7.5 and 35℃,respectively.The immobilized cells ex- hibited a markedly improved thermal stability than free cells.After five repeated experiments,the yield of indigo with the immobilized cells retained over 94%of their original activity,which indicated that the operational stability for recycling in batch processes was improved.展开更多
Cytochrome P450s(CYPs)are ubiquitously found in all kingdoms of life,playing important role in various biosynthetic pathways as well as degradative pathways;accordingly find applications in a vast variety of areas fro...Cytochrome P450s(CYPs)are ubiquitously found in all kingdoms of life,playing important role in various biosynthetic pathways as well as degradative pathways;accordingly find applications in a vast variety of areas from organic synthesis and drug metabolite production to modification of biomaterials and bioremediation.Significantly,CYPs catalyze chemically challenging CAH and CAC activation reactions using a reactive high-valent iron-oxo intermediate generated upon dioxygen activation at their heme center,while the other oxygen atom is reduced to the level of water by electrons provided through a reductase partner protein.Self-sufficient CYPs,encoding their heme domain and reductase protein in a single polypeptide,facilitate increased catalytic efficiency and render a less complicated system to work with.The self-sufficient CYP enzyme from CYP102A family(CYP102A1,BM3)is among the earliest and most-investigated model enzymes for mechanistic and structural studies as well as for biotechnological applications.An increasing number of self-sufficient CYPs from the same CYP102 family and from other families have also been reported in last decade.In this review,we introduce chemistry and biology of CYPs,followed by an overview of the characteristics of self-sufficient CYPs and representative reactions.Enzyme engineering efforts leading to novel self-sufficient CYP variants that can catalyze synthetically useful natural and non-natural(nature-mimicking)reactions are highlighted.Lastly,the strategy and efforts that aim to circumvent the challenges for improved thermostability,regio-and enantioselectivity,and total turnover number;associated with practical use of self-sufficient CYPs are reviewed.展开更多
Indigoids,a class of bis-indoles,have long been applied in dyeing,food,and pharmaceutical industries.Recently,interest in these‘old’molecules has been renewed in the field of organic semiconductors as functional bui...Indigoids,a class of bis-indoles,have long been applied in dyeing,food,and pharmaceutical industries.Recently,interest in these‘old’molecules has been renewed in the field of organic semiconductors as functional building blocks for organic electronics due to their excellent chemical and physical properties.However,these indigo derivatives are difficult to access through chemical synthesis.In this study,we engineer cytochrome P450 BM3 from an NADPH-dependent monooxygenase to peroxygenases through directed evolution.A select number of P450 BM3 variants are used for the selective oxidation of indole derivatives to form different indigoid pigments with a spectrum of colors.Among the prepared indigoid organic photocatalysts,a majority of indigoids demonstrate a reduced band gap than indigo due to the increased light capture and improved charge separation,making them promising candidates for the development of new organic electronic devices.Thus,we present a useful enzymatic approach with broad substrate scope and cost-effectiveness by using low-cost H2O2 as a cofactor for the preparation of diversified indigoids,offering versatility in designing and manufacturing new dyestuff and electronic/sensor components.展开更多
Fatty acid binding and oxidation kinetics for wild type P450_(BM3)(CYP102A1)from Bacillus megaterium have been found to display chain length-dependent homotropic behavior.Laurate and 13-methyl-myristate display Michae...Fatty acid binding and oxidation kinetics for wild type P450_(BM3)(CYP102A1)from Bacillus megaterium have been found to display chain length-dependent homotropic behavior.Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths.Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate,pH 7.4,but sigmoidal kinetics(with an apparent intercept)in low ionic strength buffers and at physiological phosphate concentrations.In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites,with high cooperativity for the binding of the fourth palmitate molecule,and the full-length enzyme showing tighter palmitate binding than the heme domain.The first flavin-to-heme electron transfer is faster for laurate,myristate and palmitate in 100 mmol/L phosphate than in 50mmol/L Tris(pH 7.4),yet each substrate induces similar high-spin heme content.For palmitate in low phosphate buffer concentrations,the rate constant of the first electron transfer is much larger than kcat.The results suggest that phosphate has a specific effect in promoting the first electron transfer step,and that P450_(BM3) could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.展开更多
基金Supported by the National Natural Science Foundation of China （No.30570411）. The authors wish to thank Prof R. D. Schmid （Institute of Technical Biochemistry, University of Stuttgart, Germany）, as he gave us some materials and much help.
文摘The ability of catalyzing indole into indigo of gene engineering strain expressing P450 BM3 immobi- lized by entrapment in calcium-alginate gel capsules was examined,and various characteristics of immobilized cells were assessed.Optimum conditions for cells activity were not affected after immobilization,and pH and tempera- ture for both free and immobilized cells were found to be pH 7.5 and 35℃,respectively.The immobilized cells ex- hibited a markedly improved thermal stability than free cells.After five repeated experiments,the yield of indigo with the immobilized cells retained over 94%of their original activity,which indicated that the operational stability for recycling in batch processes was improved.
基金Financial supports from Novo Nordisk Foundation(NNF16OC0021740)Aarhus Universitets Forskningsfond AUFFNOVA(AUFF-E-2015-FLS-9-12)Danmarks Frie Forskningsfond(DFF Technology and Production,0136-00206B)are greatly acknowledged.
文摘Cytochrome P450s(CYPs)are ubiquitously found in all kingdoms of life,playing important role in various biosynthetic pathways as well as degradative pathways;accordingly find applications in a vast variety of areas from organic synthesis and drug metabolite production to modification of biomaterials and bioremediation.Significantly,CYPs catalyze chemically challenging CAH and CAC activation reactions using a reactive high-valent iron-oxo intermediate generated upon dioxygen activation at their heme center,while the other oxygen atom is reduced to the level of water by electrons provided through a reductase partner protein.Self-sufficient CYPs,encoding their heme domain and reductase protein in a single polypeptide,facilitate increased catalytic efficiency and render a less complicated system to work with.The self-sufficient CYP enzyme from CYP102A family(CYP102A1,BM3)is among the earliest and most-investigated model enzymes for mechanistic and structural studies as well as for biotechnological applications.An increasing number of self-sufficient CYPs from the same CYP102 family and from other families have also been reported in last decade.In this review,we introduce chemistry and biology of CYPs,followed by an overview of the characteristics of self-sufficient CYPs and representative reactions.Enzyme engineering efforts leading to novel self-sufficient CYP variants that can catalyze synthetically useful natural and non-natural(nature-mimicking)reactions are highlighted.Lastly,the strategy and efforts that aim to circumvent the challenges for improved thermostability,regio-and enantioselectivity,and total turnover number;associated with practical use of self-sufficient CYPs are reviewed.
文摘细胞色素P450s是用于生物合成的多功能催化剂.在P450催化循环中,需要两个电子来还原血红素铁,并通过电子转移途径(eTPs)激活随后的还原,该步骤是反应的限速步骤.本文重新设计了巨大芽孢杆菌P450 BM3的e TPs,大幅提高了其催化性能.通过在P450 BM3的e TP中引入芳香族氨基酸,“最佳”变体P2H02(A399Y/Q403F)的催化效率比P450 BM3野生型催化效率提高了12.9倍(kcat/KM从65.8 L mol^(-1)s^(-1)提高到913.5 L mol^(-1)s^(-1)).分子动力学模拟和电子传递分析表明,在辅因子FMN和血红素之间引入的芳香族氨基酸可以显著提高电子转移速率和酶催化性能.同时,在电子传递途径中引入酪氨酸可以保护P450的催化中心,使其避免被氧化性中间产物所破坏,从而提高其催化效率.此外,引入芳香族氨基酸的策略被证明对其他P450(如CYP116B3)同样有效,改造后的酶表现出显著提高的催化效率.综上,本文策略有望拓展到其他带有长程电子传递链的氧化还原酶上,成为改造的通用策略.
基金the National Key Research and Development Program of China(2019YFA0905100)the National Natural Science Foundation of China(32025001,32071266 and 32170088)+1 种基金the Shandong Provincial Natural Science Foundation(ZR2019ZD20)the State Key Laboratory of Microbial Technology Open Projects Fund(Project NO.M2022-01).
文摘Indigoids,a class of bis-indoles,have long been applied in dyeing,food,and pharmaceutical industries.Recently,interest in these‘old’molecules has been renewed in the field of organic semiconductors as functional building blocks for organic electronics due to their excellent chemical and physical properties.However,these indigo derivatives are difficult to access through chemical synthesis.In this study,we engineer cytochrome P450 BM3 from an NADPH-dependent monooxygenase to peroxygenases through directed evolution.A select number of P450 BM3 variants are used for the selective oxidation of indole derivatives to form different indigoid pigments with a spectrum of colors.Among the prepared indigoid organic photocatalysts,a majority of indigoids demonstrate a reduced band gap than indigo due to the increased light capture and improved charge separation,making them promising candidates for the development of new organic electronic devices.Thus,we present a useful enzymatic approach with broad substrate scope and cost-effectiveness by using low-cost H2O2 as a cofactor for the preparation of diversified indigoids,offering versatility in designing and manufacturing new dyestuff and electronic/sensor components.
基金supported by grants from the Natural Sciences and Engineering Research Council(NSERC)Canada,and the Rhodes Trust(JY)the Higher Educational Funding Council for England.
文摘Fatty acid binding and oxidation kinetics for wild type P450_(BM3)(CYP102A1)from Bacillus megaterium have been found to display chain length-dependent homotropic behavior.Laurate and 13-methyl-myristate display Michaelis-Menten behavior while there are slight deviations with myristate at low ionic strengths.Palmitate shows Michaelis-Menten kinetics and hyperbolic binding behavior in 100 mmol/L phosphate,pH 7.4,but sigmoidal kinetics(with an apparent intercept)in low ionic strength buffers and at physiological phosphate concentrations.In low ionic strength buffers both the heme domain and the full-length enzyme show complex palmitate binding behavior that indicates a minimum of four fatty acid binding sites,with high cooperativity for the binding of the fourth palmitate molecule,and the full-length enzyme showing tighter palmitate binding than the heme domain.The first flavin-to-heme electron transfer is faster for laurate,myristate and palmitate in 100 mmol/L phosphate than in 50mmol/L Tris(pH 7.4),yet each substrate induces similar high-spin heme content.For palmitate in low phosphate buffer concentrations,the rate constant of the first electron transfer is much larger than kcat.The results suggest that phosphate has a specific effect in promoting the first electron transfer step,and that P450_(BM3) could modulate Bacillus membrane morphology and fluidity via palmitate oxidation in response to the external phosphate concentration.
