Unspecific peroxygenases(UPOs, EC 1.11.2.1) is a kind of thioheme enzyme capable of catalyzing various oxidations of inert C–H bonds using H_(2)O_(2) as an oxygen donor without cofactors. However, the enhancement of ...Unspecific peroxygenases(UPOs, EC 1.11.2.1) is a kind of thioheme enzyme capable of catalyzing various oxidations of inert C–H bonds using H_(2)O_(2) as an oxygen donor without cofactors. However, the enhancement of the H_(2)O_(2) tolerance of UPOs is always challenging. In this study, the A161C mutant of r Dca UPO,which originates from Daldinia caldariorum, was found to be highly H_(2)O_(2)-resistant. Compared with the wild type, the mutant r Dca UPO-A161C showed a 10-h prolonged half-life and a 64% improved enzyme activity when incubated in 10 mmol/L H_(2)O_(2). The crystal structure analysis at 1.47 A showed that r Dca UPOA161C exhibited 10 α-helixes(cyan) and a series of ordered rings, forming a single asymmetric spherical structure. The two conserved domains near heme formed an active site with the catalytic PCP and EHD regions(Glu86, His87, Asp88 residues). The H_(2)O_(2) tolerance of r Dca UPO-A161C was preliminarily explored by comparing its structure with the wild type. Notably, r Dca UPO-A161C showed significantly higher catalytic efficiency than the wild type for the production of hydroxyl fatty acids. This study is anticipated to provide an insight into the structure-function relationship and expand potential applications of UPOs.展开更多
Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterolog...Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterologous expression has hindered their practical application.Here,a novel UPO from Marasmius fiardii PR910(MfiUPO)was identified and heterologously expressed in Pichia pastoris.By employing a two-copy expression cassette,the protein titer reached 1.18 g L−1 in a 5 L bioreactor,marking the highest record.The glycoprotein rMfiUPO exhib-ited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation,epoxidation and alcohol oxidation.Moreover,the peroxidative activity was enhanced by 150%after exposure to 50%(v/v)acetone for 40 h.A semipreparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2%isolated yield with 95%purity.With its high expression level,rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution,thereby meeting the necessary criteria for practical application.展开更多
Heme peroxygenases exhibit remarkable catalytic versatility in facilitating a wide array of oxidative reactions under mild conditions,eliminating the need for coenzymes and intricate electron transport systems.This un...Heme peroxygenases exhibit remarkable catalytic versatility in facilitating a wide array of oxidative reactions under mild conditions,eliminating the need for coenzymes and intricate electron transport systems.This unique character underscores their essentiality and potential as promising tools in synthetic biology.Recent advancements in enzyme engineering have significantly enhanced the catalytic performance of both natural and artificial peroxygenases.Extensive engineering efforts have been directed towards unspecific peroxygenases and fatty acid peroxygenases,aiming to expand their substrate specificities,and enhance reaction selectivities,as well as increase enzyme stability.Furthermore,innovative strategies such as dual-functional small molecule-assisted systems and H_(2)O_(2) tunnel engineering have been harnessed to transform P450 monooxygenases into highly efficient peroxygenases,capable of catalyzing reactions with a variety of unnatural substrates.This review consolidates the latest progress in the engineered and artificial heme peroxygenases,emphasizing their catalytic performances as potent biocatalysts for sustainable organic synthesis.展开更多
Musk macrolides are a kind of rare flavors structurally featuring 14-16 membered rings in the flavour and fragrance industry.While various chemical synthesis routes for musk macrolides exist,there is a growing interes...Musk macrolides are a kind of rare flavors structurally featuring 14-16 membered rings in the flavour and fragrance industry.While various chemical synthesis routes for musk macrolides exist,there is a growing interest in biocatalytic approaches.In this study,a new biocatalytic approach is proposed for the synthesis of musk macrolides from fatty acids using unspecific peroxygenases(UPOs)in combination with lipase.Specifically,the UPO from Marasmius rotula(MroUPO)was utilized as an oxidation catalyst together with the choline oxidase from Arthrobacter nicotinate(AnChOx)for the in situ generation of H_(2)O_(2).The enzymatic cascade process involved the hydroxylation of fatty acids using MroUPO as a catalyst to prepare terminal hydroxylated fatty acids.Under the optimized conditions,14-hydroxy tetradecanoic acid,15-hydroxy pentadecanoic acid and 16-hydroxy hexadecanoic acid were obtained at concentrations of 11.1,18.2 and17.1 mg/L in a 50-mL reaction system,respectively.These hydroxylated fatty acids were then separated and esterified by lipase Novozymes 435 to produce the desired macrolides.Variousω-hydroxy fatty acids were successfully converted into macrolides,with the concentrations of cyclotetradecanolide,cyclopentadecanolide and cyclohexadecanolide reaching 11,29 and 23 mg/L,respectively,in a 10-mL reaction system.This new biocatalytic approach represents a promising method for synthesizing valuable natural flavour compounds.展开更多
Unspecific peroxygenases exhibit high activity for the selective oxyfunctionalization of inert C(sp3)–H bonds using only H_(2)O_(2) as a clean oxidant,while also exhibiting sensitivity to H_(2)O_(2) concentration.CdS...Unspecific peroxygenases exhibit high activity for the selective oxyfunctionalization of inert C(sp3)–H bonds using only H_(2)O_(2) as a clean oxidant,while also exhibiting sensitivity to H_(2)O_(2) concentration.CdS-based semiconductors are promising for the photosynthesis of H_(2)O_(2) owing to their adequately negative potential for oxygen reduction reaction via a proton-coupled electron transfer process,however,they suffer from fast H_(2)O_(2) decomposition on the surface of pristine CdS.Therefore,[Cp*Rh(bpy)H_(2)O]2+,a highly selective proton-coupled electron transfer catalyst,was anchored onto a supramolecular polymer-grafted CdS nanoflower to construct an efficient integrated photocatalyst for generating H_(2)O_(2),mitigating the surface issue of pristine CdS,increasing light absorption,accelerating photonic carrier separation,and enhancing oxygen reduction reaction selectivity to H_(2)O_(2).This photocatalyst promoted the light driven H_(2)O_(2) generation rate up to 1345μmol·L^(-1)·g^(-1)·h^(-1),which was 2.4 times that of pristine CdS.The constructed heterojunction photocatalyst could supply H_(2)O_(2) in situ for nonspecific peroxygenases to catalyze the C–H oxyfunctionalization of ethylbenzene,achieving a yield of 81%and an ee value of 99%under optimum conditions.A wide range of substrates were converted to the corresponding chiral alcohols using this photo-enzyme catalytic system,achieving the corresponding chiral alcohols in good yield(51%–88%)and excellent enantioselectivity(90%–99%ee).展开更多
基金supported by the National Natural Science Foundation of China (No.32001633)the Key Program of Natural Science Foundation of China (No.31930084)Guangzhou Science and technology planning project (No.202102020370)。
文摘Unspecific peroxygenases(UPOs, EC 1.11.2.1) is a kind of thioheme enzyme capable of catalyzing various oxidations of inert C–H bonds using H_(2)O_(2) as an oxygen donor without cofactors. However, the enhancement of the H_(2)O_(2) tolerance of UPOs is always challenging. In this study, the A161C mutant of r Dca UPO,which originates from Daldinia caldariorum, was found to be highly H_(2)O_(2)-resistant. Compared with the wild type, the mutant r Dca UPO-A161C showed a 10-h prolonged half-life and a 64% improved enzyme activity when incubated in 10 mmol/L H_(2)O_(2). The crystal structure analysis at 1.47 A showed that r Dca UPOA161C exhibited 10 α-helixes(cyan) and a series of ordered rings, forming a single asymmetric spherical structure. The two conserved domains near heme formed an active site with the catalytic PCP and EHD regions(Glu86, His87, Asp88 residues). The H_(2)O_(2) tolerance of r Dca UPO-A161C was preliminarily explored by comparing its structure with the wild type. Notably, r Dca UPO-A161C showed significantly higher catalytic efficiency than the wild type for the production of hydroxyl fatty acids. This study is anticipated to provide an insight into the structure-function relationship and expand potential applications of UPOs.
基金funded by the National Key Research and Development Program of China(2021YFC2102700)was Supported by the Key Science and Technology Innovation Project of Hubei Province(2021BAD001)as well as the Research Program of State Key Laboratory of Biocatalysis and Enzyme Engineering。
文摘Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterologous expression has hindered their practical application.Here,a novel UPO from Marasmius fiardii PR910(MfiUPO)was identified and heterologously expressed in Pichia pastoris.By employing a two-copy expression cassette,the protein titer reached 1.18 g L−1 in a 5 L bioreactor,marking the highest record.The glycoprotein rMfiUPO exhib-ited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation,epoxidation and alcohol oxidation.Moreover,the peroxidative activity was enhanced by 150%after exposure to 50%(v/v)acetone for 40 h.A semipreparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2%isolated yield with 95%purity.With its high expression level,rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution,thereby meeting the necessary criteria for practical application.
文摘Heme peroxygenases exhibit remarkable catalytic versatility in facilitating a wide array of oxidative reactions under mild conditions,eliminating the need for coenzymes and intricate electron transport systems.This unique character underscores their essentiality and potential as promising tools in synthetic biology.Recent advancements in enzyme engineering have significantly enhanced the catalytic performance of both natural and artificial peroxygenases.Extensive engineering efforts have been directed towards unspecific peroxygenases and fatty acid peroxygenases,aiming to expand their substrate specificities,and enhance reaction selectivities,as well as increase enzyme stability.Furthermore,innovative strategies such as dual-functional small molecule-assisted systems and H_(2)O_(2) tunnel engineering have been harnessed to transform P450 monooxygenases into highly efficient peroxygenases,capable of catalyzing reactions with a variety of unnatural substrates.This review consolidates the latest progress in the engineered and artificial heme peroxygenases,emphasizing their catalytic performances as potent biocatalysts for sustainable organic synthesis.
基金supported by the Key Realm R&D Program of Guangdong Province(2022B0202050003)National Natural Science Foundation of China(31930084,32001633)。
文摘Musk macrolides are a kind of rare flavors structurally featuring 14-16 membered rings in the flavour and fragrance industry.While various chemical synthesis routes for musk macrolides exist,there is a growing interest in biocatalytic approaches.In this study,a new biocatalytic approach is proposed for the synthesis of musk macrolides from fatty acids using unspecific peroxygenases(UPOs)in combination with lipase.Specifically,the UPO from Marasmius rotula(MroUPO)was utilized as an oxidation catalyst together with the choline oxidase from Arthrobacter nicotinate(AnChOx)for the in situ generation of H_(2)O_(2).The enzymatic cascade process involved the hydroxylation of fatty acids using MroUPO as a catalyst to prepare terminal hydroxylated fatty acids.Under the optimized conditions,14-hydroxy tetradecanoic acid,15-hydroxy pentadecanoic acid and 16-hydroxy hexadecanoic acid were obtained at concentrations of 11.1,18.2 and17.1 mg/L in a 50-mL reaction system,respectively.These hydroxylated fatty acids were then separated and esterified by lipase Novozymes 435 to produce the desired macrolides.Variousω-hydroxy fatty acids were successfully converted into macrolides,with the concentrations of cyclotetradecanolide,cyclopentadecanolide and cyclohexadecanolide reaching 11,29 and 23 mg/L,respectively,in a 10-mL reaction system.This new biocatalytic approach represents a promising method for synthesizing valuable natural flavour compounds.
基金supported by the National Natural Science Foundation of China(Grant No.22378096)the Natural Science Foundation of Hebei Province(Grant No.B2023202014)+1 种基金the Science Technology Research Project of Higher Education of Hebei Province(Grant Nos.QN2021045,and QN2023207)the Tianjin Science and Technology Project(Grant No.22KPHDRC00260).
文摘Unspecific peroxygenases exhibit high activity for the selective oxyfunctionalization of inert C(sp3)–H bonds using only H_(2)O_(2) as a clean oxidant,while also exhibiting sensitivity to H_(2)O_(2) concentration.CdS-based semiconductors are promising for the photosynthesis of H_(2)O_(2) owing to their adequately negative potential for oxygen reduction reaction via a proton-coupled electron transfer process,however,they suffer from fast H_(2)O_(2) decomposition on the surface of pristine CdS.Therefore,[Cp*Rh(bpy)H_(2)O]2+,a highly selective proton-coupled electron transfer catalyst,was anchored onto a supramolecular polymer-grafted CdS nanoflower to construct an efficient integrated photocatalyst for generating H_(2)O_(2),mitigating the surface issue of pristine CdS,increasing light absorption,accelerating photonic carrier separation,and enhancing oxygen reduction reaction selectivity to H_(2)O_(2).This photocatalyst promoted the light driven H_(2)O_(2) generation rate up to 1345μmol·L^(-1)·g^(-1)·h^(-1),which was 2.4 times that of pristine CdS.The constructed heterojunction photocatalyst could supply H_(2)O_(2) in situ for nonspecific peroxygenases to catalyze the C–H oxyfunctionalization of ethylbenzene,achieving a yield of 81%and an ee value of 99%under optimum conditions.A wide range of substrates were converted to the corresponding chiral alcohols using this photo-enzyme catalytic system,achieving the corresponding chiral alcohols in good yield(51%–88%)and excellent enantioselectivity(90%–99%ee).
