The goal of this study was to determine whether mutation of the Mn-binding site of wild-type recombinant Phlebia radiata manganese peroxidase 3 affected the pH-dependence kinetic parameters. pH range investigated was ...The goal of this study was to determine whether mutation of the Mn-binding site of wild-type recombinant Phlebia radiata manganese peroxidase 3 affected the pH-dependence kinetic parameters. pH range investigated was 2.5 – 12.0. The catalytic efficiency of the mutant enzymes at high and low pH in comparison to the wild-type was investigated using standard rPr-MnP3 protocol. Wild-type recombinant Phlebia radiata MnP3 enzyme showed optimal activity with Mn (II) as substrate at pH 5.0 and remained moderately active (approximately 40%) in the pH range of 6.0 - 9.0. The rPr-MnP3 mutants’ maximum activity ranged between 5.5 and 8.0. Wild-type and mutants rPr-MnP3 enzymes exhibited a similar pH profile with optimum pH of 3.0 for ABTS oxidation. Mutation has severely decreased the catalytic efficiency for Mn (II) oxidation at pH 5.0. The rPr-MnP3 enzymes showed enhanced affinity for Mn (II) at alkaline pH and a more alkaline range for catalysis than ever reported for any Manganese Peroxidase. This study reveals that at higher pH, rPr-MnP3 can function with alternative ligands in the Mn (II) site and does not have an absolutely obligate requirement for an all carboxylate ligand set. These results further strongly confirm that Mn<sup>2+</sup> binding site is the only productive catalytic site for Mn (II) oxidation.展开更多
This investigation is aimed at understanding the specific role of pH and calcium ions on the activity and stability of wild-type recombinant Phlebia radiata manganese peroxidase 3 (rPr-MnP3). The pH-dependent cycle of...This investigation is aimed at understanding the specific role of pH and calcium ions on the activity and stability of wild-type recombinant Phlebia radiata manganese peroxidase 3 (rPr-MnP3). The pH-dependent cycle of reactions for rPr-MnP3 was evaluated by investigating time-dependent changes in the activity and electronic absorption spectrum of rPr-MnP3.The rPr-MnP3 had maximum efficacy (kcat/Km) for Mn (II) oxidation at pH 5.0 and 3.0 for oxidation of ABTS. Raising the pH of a solution of resting rPr-MnP3 from pH 6.7 (form XH) to pH 8.6 (form X<sup>−</sup>), a rapid alkaline transition occurs. Leaving the X<sup>−</sup> form of the enzyme at pH 8.6, it slowly becomes converted to a third form of the enzyme Y<sup>−</sup>, which returned to the original XH form of the enzyme at pH 6.7. Recovery of form XH from form Y<sup>−</sup> occurred through an intermediate Z form. The pH inactivation of rPr-MnP3 followed first-order kinetics. The rate of formation of XH from Z is pH-dependent and biphasic in nature, with measured rate constants (k) = 0.25 min<sup>−1</sup>, and half-life (T<sub>1/2</sub>) = 2.8 min. The pH-dependent properties observed may be indicative of a greater degree of conformational flexibility at rPr-MnP3 active site due to disruption of the haem-linked hydrogen-bonding network in the distal haem pocket. Calcium ions were observed to significantly stabilised the enzyme’s spectral features and reduce the loss of activity during the alkaline pH transition. Calcium ions enhance the recovery of the initial activity but cannot prevent the final time-dependent irreversible denaturation and aggregation.展开更多
Manganese peroxidases(MnP)from Phanerochaete chrysosporium were adsorbed onto multi-walled carbon nanotubes(MWNT).Four different loadings of MnP on MWNTs were investigated,and the maximum enzyme loading of 47.5µg...Manganese peroxidases(MnP)from Phanerochaete chrysosporium were adsorbed onto multi-walled carbon nanotubes(MWNT).Four different loadings of MnP on MWNTs were investigated,and the maximum enzyme loading of 47.5µg/mg of MWNTs was obtained in 12 h.The adsorbed MnP showed a catalytic activity of up to 0.1 U/mg of the weight of the system of MnP/MWNTs,with 23%of its original activity retained.The AFM image of the adsorbed enzymes indicated that a layer of MnP covered the surface of the MWNTs and retained its original three-dimensional shape.Amino-based nonspecific interactions may play the dominant role in the adsorption of MnP on MWNTs.展开更多
Endophytic fungi are being investigated for their ability to create industrially relevant secondary metabolites.In recent years,there has been a surge in interest in these fungi as a source of novel enzymes,particular...Endophytic fungi are being investigated for their ability to create industrially relevant secondary metabolites.In recent years,there has been a surge in interest in these fungi as a source of novel enzymes,particularly hydrolytic enzymes.The present study investigated the effect of different carbon,organic and inorganic nitrogens on the growth and ligninolytic enzyme production by the endophytic fungus Diaporthe phaeolorum.The fungus was isolated from the leaves of Dillenia indica and analyzed by morpho-molecular basis.The fungus showed promising results for in vitro production of ligninolytic enzymes.Sucrose was the most favorable carbon compound for growth among all the carbon compounds tested.It displayed maximum lignin peroxidase(Lip)activity in fructose(3.5 U/ml),followed by pectin(2.60 U/ml)and glucose(2.53 U/ml).Glucose gave the highest manganese peroxidase(MnP)activity i.e.,6.88 U/ml followed by starch,sucrose and raffinose.Similarly,the maximum laccase activity was 44.5 U/ml in pectin.Potassium nitrate and L-asparagine were the best inorganic and organic nitrogen for growth.In the case of ligninolytic enzyme production,ammonium acetate and ammonium phosphate were the best media for LiP and MnP,whereas laccase production was highest in ammonium nitrate supplemented medium.In organic nitrogen sources,medium supplemented with DL-tryptophan gave the highest Lip production,whereas MnP and laccase production was observed in the medium containing L-tyrosine and L-asparagine.To the best of our knowledge,this is the first report related to the growth and ligninolytic enzyme production by Diaporthe phaseolorum.The findings from the study will assist researchers in improving the production of ligninolytic enzymes by this fungus under in vitro conditions on an industrial scale.展开更多
Spent mushroom substrate(SMS)is an abundant residue generated by the mushroom industry,which is mostly discarded.In this study,the SMS of four cultivated mushrooms were evaluated as potential sources of ligninolytic e...Spent mushroom substrate(SMS)is an abundant residue generated by the mushroom industry,which is mostly discarded.In this study,the SMS of four cultivated mushrooms were evaluated as potential sources of ligninolytic enzymes.Pleurotus ostreatus,P.eryngii,P.djamor and Ganoderma lucidum were cultivated in two different substrates to determine growth,mushroom yields and ligninolytic activity of laccase(Lac)and manganese peroxidase(MnP).Maximum activities in the SMS were 3.731 U/g Lac and 0.191 U/g MnP,both for P.eryngii.Colonized mushroom substrates were also analyzed,and higher activities were found in almost all samples.A correlation test between mushroom yields and ligninolytic activities of each culture was made,and we found that high enzymatic activity in the substrate did not result in high mushroom productivity or vice versa.The SMS was a good source of ligninolytic enzymes.Enzymatic activity depended on the mushroom species and its affinity to the substrate,and it was not influenced by the mushroom yields.Production of mushrooms in diverse lignocellulosic substrates is gaining interest,and ob-taining ligninolytic enzymes from residual SMS is an excellent alternative in the circular economy concept.展开更多
文摘The goal of this study was to determine whether mutation of the Mn-binding site of wild-type recombinant Phlebia radiata manganese peroxidase 3 affected the pH-dependence kinetic parameters. pH range investigated was 2.5 – 12.0. The catalytic efficiency of the mutant enzymes at high and low pH in comparison to the wild-type was investigated using standard rPr-MnP3 protocol. Wild-type recombinant Phlebia radiata MnP3 enzyme showed optimal activity with Mn (II) as substrate at pH 5.0 and remained moderately active (approximately 40%) in the pH range of 6.0 - 9.0. The rPr-MnP3 mutants’ maximum activity ranged between 5.5 and 8.0. Wild-type and mutants rPr-MnP3 enzymes exhibited a similar pH profile with optimum pH of 3.0 for ABTS oxidation. Mutation has severely decreased the catalytic efficiency for Mn (II) oxidation at pH 5.0. The rPr-MnP3 enzymes showed enhanced affinity for Mn (II) at alkaline pH and a more alkaline range for catalysis than ever reported for any Manganese Peroxidase. This study reveals that at higher pH, rPr-MnP3 can function with alternative ligands in the Mn (II) site and does not have an absolutely obligate requirement for an all carboxylate ligand set. These results further strongly confirm that Mn<sup>2+</sup> binding site is the only productive catalytic site for Mn (II) oxidation.
文摘This investigation is aimed at understanding the specific role of pH and calcium ions on the activity and stability of wild-type recombinant Phlebia radiata manganese peroxidase 3 (rPr-MnP3). The pH-dependent cycle of reactions for rPr-MnP3 was evaluated by investigating time-dependent changes in the activity and electronic absorption spectrum of rPr-MnP3.The rPr-MnP3 had maximum efficacy (kcat/Km) for Mn (II) oxidation at pH 5.0 and 3.0 for oxidation of ABTS. Raising the pH of a solution of resting rPr-MnP3 from pH 6.7 (form XH) to pH 8.6 (form X<sup>−</sup>), a rapid alkaline transition occurs. Leaving the X<sup>−</sup> form of the enzyme at pH 8.6, it slowly becomes converted to a third form of the enzyme Y<sup>−</sup>, which returned to the original XH form of the enzyme at pH 6.7. Recovery of form XH from form Y<sup>−</sup> occurred through an intermediate Z form. The pH inactivation of rPr-MnP3 followed first-order kinetics. The rate of formation of XH from Z is pH-dependent and biphasic in nature, with measured rate constants (k) = 0.25 min<sup>−1</sup>, and half-life (T<sub>1/2</sub>) = 2.8 min. The pH-dependent properties observed may be indicative of a greater degree of conformational flexibility at rPr-MnP3 active site due to disruption of the haem-linked hydrogen-bonding network in the distal haem pocket. Calcium ions were observed to significantly stabilised the enzyme’s spectral features and reduce the loss of activity during the alkaline pH transition. Calcium ions enhance the recovery of the initial activity but cannot prevent the final time-dependent irreversible denaturation and aggregation.
基金the National Natural Science Foundation of China(Grant No.20677033)the special fund of State Key Joint Laboratory of Environment Simulation and Pollution Control,China(08Z01ESPCT).
文摘Manganese peroxidases(MnP)from Phanerochaete chrysosporium were adsorbed onto multi-walled carbon nanotubes(MWNT).Four different loadings of MnP on MWNTs were investigated,and the maximum enzyme loading of 47.5µg/mg of MWNTs was obtained in 12 h.The adsorbed MnP showed a catalytic activity of up to 0.1 U/mg of the weight of the system of MnP/MWNTs,with 23%of its original activity retained.The AFM image of the adsorbed enzymes indicated that a layer of MnP covered the surface of the MWNTs and retained its original three-dimensional shape.Amino-based nonspecific interactions may play the dominant role in the adsorption of MnP on MWNTs.
基金Acknowledgments The authors acknowledge the Department of Botany,Panjab University Chandigarh,India,for providing infrastructure and instrumentation.Vijay Kumar is also thankful for Senior Research Fellowship(File No.09/135(0854)/2019-EMR-I)the Council of Scientific and Industrial Research(CSIR),India,during research work.
文摘Endophytic fungi are being investigated for their ability to create industrially relevant secondary metabolites.In recent years,there has been a surge in interest in these fungi as a source of novel enzymes,particularly hydrolytic enzymes.The present study investigated the effect of different carbon,organic and inorganic nitrogens on the growth and ligninolytic enzyme production by the endophytic fungus Diaporthe phaeolorum.The fungus was isolated from the leaves of Dillenia indica and analyzed by morpho-molecular basis.The fungus showed promising results for in vitro production of ligninolytic enzymes.Sucrose was the most favorable carbon compound for growth among all the carbon compounds tested.It displayed maximum lignin peroxidase(Lip)activity in fructose(3.5 U/ml),followed by pectin(2.60 U/ml)and glucose(2.53 U/ml).Glucose gave the highest manganese peroxidase(MnP)activity i.e.,6.88 U/ml followed by starch,sucrose and raffinose.Similarly,the maximum laccase activity was 44.5 U/ml in pectin.Potassium nitrate and L-asparagine were the best inorganic and organic nitrogen for growth.In the case of ligninolytic enzyme production,ammonium acetate and ammonium phosphate were the best media for LiP and MnP,whereas laccase production was highest in ammonium nitrate supplemented medium.In organic nitrogen sources,medium supplemented with DL-tryptophan gave the highest Lip production,whereas MnP and laccase production was observed in the medium containing L-tyrosine and L-asparagine.To the best of our knowledge,this is the first report related to the growth and ligninolytic enzyme production by Diaporthe phaseolorum.The findings from the study will assist researchers in improving the production of ligninolytic enzymes by this fungus under in vitro conditions on an industrial scale.
基金funded by Consejo Nacional de Investigaciones Científicas y Técnicas(CONICET)in Argentina(No.PIP 2021-1292).
文摘Spent mushroom substrate(SMS)is an abundant residue generated by the mushroom industry,which is mostly discarded.In this study,the SMS of four cultivated mushrooms were evaluated as potential sources of ligninolytic enzymes.Pleurotus ostreatus,P.eryngii,P.djamor and Ganoderma lucidum were cultivated in two different substrates to determine growth,mushroom yields and ligninolytic activity of laccase(Lac)and manganese peroxidase(MnP).Maximum activities in the SMS were 3.731 U/g Lac and 0.191 U/g MnP,both for P.eryngii.Colonized mushroom substrates were also analyzed,and higher activities were found in almost all samples.A correlation test between mushroom yields and ligninolytic activities of each culture was made,and we found that high enzymatic activity in the substrate did not result in high mushroom productivity or vice versa.The SMS was a good source of ligninolytic enzymes.Enzymatic activity depended on the mushroom species and its affinity to the substrate,and it was not influenced by the mushroom yields.Production of mushrooms in diverse lignocellulosic substrates is gaining interest,and ob-taining ligninolytic enzymes from residual SMS is an excellent alternative in the circular economy concept.
