Lytic polysaccharide monooxygenases(LPMOs)are copper enzymes that boost the degradation of different polysaccharides and play important roles in the sustainable production of biofuels,in human and plant pathogens,and ...Lytic polysaccharide monooxygenases(LPMOs)are copper enzymes that boost the degradation of different polysaccharides and play important roles in the sustainable production of biofuels,in human and plant pathogens,and potentially also in plastic degradation.Their activity depends on a co-substrate,where recent results show that hydrogen peroxide is the preferred co-substrate.Under typical experimental conditions,no hydrogen peroxide is added and it is instead produced in situ by LPMOs themselves,which could be the rate-limiting step.Previous theoretical investigations of the oxidase reaction have been highly inhomogeneous and focused on different aspects of LPMO reactivity.In this paper,we systematically investigate how LPMOs generate hydrogen peroxide using accurate quantum mechanics/molecular mechanics(QM/MM)hybrid methods with extended QM regions.We find that the reaction of the reduced LPMO active site with O₂ yields a superoxide coordinated to Cu(Ⅱ),from which[Cu(Ⅱ)–OOH⁻]⁺ can be formed via a proton-coupled electron transfer,using a second-coordination-sphere histidine as the proton donor.Either OOH⁻ dissociates from this species(while abstracting a proton from a water molecule)or[Cu(Ⅱ)–OOH⁻]⁺ reacts in a second protonation from the second-sphere histidine,yielding[Cu(Ⅱ)–H₂O₂]²⁺,followed by dissociation of H₂O₂.Energetically,all three oxygen species can dissociate into solution,but the dissociation of H₂O₂ from the Cu(Ⅱ)active site is the most favorable while the dissociation of O₂•⁻ is least favorable.展开更多
Lytic polysaccharide monooxygenases(LPMOs)are copper-dependent enzymes that have fueled the hope for sustainable biofuel production since they enhance the breakdown of recalcitrant polysaccharides like cellulose.In th...Lytic polysaccharide monooxygenases(LPMOs)are copper-dependent enzymes that have fueled the hope for sustainable biofuel production since they enhance the breakdown of recalcitrant polysaccharides like cellulose.In the consensus mechanism,their catalytic activity relies on forming an‘oxyl’,[CuO^(·-)]^(+),species at the active site,followed by subsequent hydrogen atom abstraction(HAA)from the substrate.Some studies report rather high barriers for this reaction,identifying it as the rate-limiting step in the oxidation process,whereas other investigations have reported significantly lower barriers.In this study,we have constructed a force field for the active site and show through extensive sampling from molecular dynamics simulations that the QM/MM reaction barrier depends critically on the underlying structural conformations of the enzyme-substrate complex.The results support low-energy barriers for the HAA step and help to explain previous discrepancies in the literature,which may be attributed to insufficient conformational sampling.展开更多
This study reports the development of a novel and cost-effective cellulolytic enzyme cocktail,named Remzyme,using Rasamsonia emersonii.By supplementing the heterologously expressed carbohydrate-active enzymes(CAZymes)...This study reports the development of a novel and cost-effective cellulolytic enzyme cocktail,named Remzyme,using Rasamsonia emersonii.By supplementing the heterologously expressed carbohydrate-active enzymes(CAZymes)such as lytic polysaccharide monooxygenase(Rem_LPMO1,Rem_GH7CBHI),and xylanase(Malci_GH10xyl),the cocktail was optimized using a Simplex lattice mixture design.This innovative blend achieved a saccharification efficiency of 98.59%when applied to unwashed,acid/steam-pretreated rice straw slurry sourced from an industrial-scale 2G ethanol plant.The process was conducted under industrially relevant conditions with 15%substrate loading and protein loading of 8 mg/g dry substrate.Remarkably,the Remzyme cocktails was comparable to the leading commercial enzyme mix,CellicCTec3,at equivalent protein loadings,underscoring its potential as a cost-effective alternative in enzymatic saccharification.The study demonstrates the synergistic efficacy of accessory enzymes and core cellulases,offering significant advancements in enzyme technology for biorefinery applications.展开更多
基金the Villum Foundation,Young Investigator Program(grant no.29412)the Swedish Research Council(grants no.2019-04205 and 2022-04978+2 种基金the Independent Research Fund Denmark(grant no.2064-00002B)for supportthe European Commission(LyticPol projectproject 101106997)for support。
文摘Lytic polysaccharide monooxygenases(LPMOs)are copper enzymes that boost the degradation of different polysaccharides and play important roles in the sustainable production of biofuels,in human and plant pathogens,and potentially also in plastic degradation.Their activity depends on a co-substrate,where recent results show that hydrogen peroxide is the preferred co-substrate.Under typical experimental conditions,no hydrogen peroxide is added and it is instead produced in situ by LPMOs themselves,which could be the rate-limiting step.Previous theoretical investigations of the oxidase reaction have been highly inhomogeneous and focused on different aspects of LPMO reactivity.In this paper,we systematically investigate how LPMOs generate hydrogen peroxide using accurate quantum mechanics/molecular mechanics(QM/MM)hybrid methods with extended QM regions.We find that the reaction of the reduced LPMO active site with O₂ yields a superoxide coordinated to Cu(Ⅱ),from which[Cu(Ⅱ)–OOH⁻]⁺ can be formed via a proton-coupled electron transfer,using a second-coordination-sphere histidine as the proton donor.Either OOH⁻ dissociates from this species(while abstracting a proton from a water molecule)or[Cu(Ⅱ)–OOH⁻]⁺ reacts in a second protonation from the second-sphere histidine,yielding[Cu(Ⅱ)–H₂O₂]²⁺,followed by dissociation of H₂O₂.Energetically,all three oxygen species can dissociate into solution,but the dissociation of H₂O₂ from the Cu(Ⅱ)active site is the most favorable while the dissociation of O₂•⁻ is least favorable.
基金The Villum Foundation,Young Investigator Program(Grant No.29412)the Swedish Research Council(Grants No.2019-04205 and 2022-04978)Independent Research Fund Denmark(Grant No.2064-00002B)for support。
文摘Lytic polysaccharide monooxygenases(LPMOs)are copper-dependent enzymes that have fueled the hope for sustainable biofuel production since they enhance the breakdown of recalcitrant polysaccharides like cellulose.In the consensus mechanism,their catalytic activity relies on forming an‘oxyl’,[CuO^(·-)]^(+),species at the active site,followed by subsequent hydrogen atom abstraction(HAA)from the substrate.Some studies report rather high barriers for this reaction,identifying it as the rate-limiting step in the oxidation process,whereas other investigations have reported significantly lower barriers.In this study,we have constructed a force field for the active site and show through extensive sampling from molecular dynamics simulations that the QM/MM reaction barrier depends critically on the underlying structural conformations of the enzyme-substrate complex.The results support low-energy barriers for the HAA step and help to explain previous discrepancies in the literature,which may be attributed to insufficient conformational sampling.
文摘This study reports the development of a novel and cost-effective cellulolytic enzyme cocktail,named Remzyme,using Rasamsonia emersonii.By supplementing the heterologously expressed carbohydrate-active enzymes(CAZymes)such as lytic polysaccharide monooxygenase(Rem_LPMO1,Rem_GH7CBHI),and xylanase(Malci_GH10xyl),the cocktail was optimized using a Simplex lattice mixture design.This innovative blend achieved a saccharification efficiency of 98.59%when applied to unwashed,acid/steam-pretreated rice straw slurry sourced from an industrial-scale 2G ethanol plant.The process was conducted under industrially relevant conditions with 15%substrate loading and protein loading of 8 mg/g dry substrate.Remarkably,the Remzyme cocktails was comparable to the leading commercial enzyme mix,CellicCTec3,at equivalent protein loadings,underscoring its potential as a cost-effective alternative in enzymatic saccharification.The study demonstrates the synergistic efficacy of accessory enzymes and core cellulases,offering significant advancements in enzyme technology for biorefinery applications.
