Objective: To investigate the reliability for fast estimation of Michaelis-Menten constant (Km) with calibrated specific activity at only two medium concentrations of substrate by both simulation and experimentation w...Objective: To investigate the reliability for fast estimation of Michaelis-Menten constant (Km) with calibrated specific activity at only two medium concentrations of substrate by both simulation and experimentation with arylesterase (ArE)as model. Methods: Initial rates were simulated by randomly inserting uniform absolute error, and the experimental initial rates of ArE were determined by measuring the increaser of product absorbance. Calibrated specific activities at two substrate concentrations were obtained by regression analysis, and Km was calculated according to Michaelis-Menten equation. Results: By simulation with calibrated specific activities at two medium substrate concentrations, Km could be calculated according to Michaelis-Menten equation with reasonable precision and accuracy. By experimentation with substrates of 2-naphthyl acetate, phenyl acetate, and p-nitrophenyl acetate, there were no differences between the mean and SD of Km of ArE for either substrate by this linear kinetic method and the Lineweaver-Burk plot. Conclusion: This linear kinetic method was reliable for fast estimation of the Km of some specified enzyme on its substrate of lower solubility or lower sensitivity for quantification by common methods.展开更多
In order to improve the substrate diffusion properties and stability of an immobilized enzyme alginate microgels modified with TiO2 nanoparticles were employed as the enzyme immobilizing support.Ionotropic gelation wa...In order to improve the substrate diffusion properties and stability of an immobilized enzyme alginate microgels modified with TiO2 nanoparticles were employed as the enzyme immobilizing support.Ionotropic gelation was applied for the preparation of hybrid gels while Ca2＋ Ce3＋ Ni2＋Cu2＋and Fe3＋were employed as the crosslinkers.Papain was selected as the model enzyme. UV-Vis spectroscopy was employed to investigate the activity of papain to evaluate kinetics and stability.Analysis results show that the highest affinity the lowest Michaelis-Menten constant Km =11.0 mg/mL and the highest stability are obtained when using Cu2＋as the crosslinker.The effect of the mass ratio of TiO2 to papain on the stability and leakage of papain is also investigated and the results show that 10∶1 TiO2∶papain is optimal because the proper use of TiO2 can reduce enzyme leakage and ensure enzyme stability.Preparing Cu/alginate/TiO2 hybrid gels via ionotropic gelation can provide a satisfactory diffusion capability and enzyme stability.展开更多
Eucalyptus clones are selected according to productivity,wood quality,rooting capacity,and resistance to drought,frost and diseases.However,kinetic and morphological parameters that determine the absorption efficiency...Eucalyptus clones are selected according to productivity,wood quality,rooting capacity,and resistance to drought,frost and diseases.However,kinetic and morphological parameters that determine the absorption efficiency of nutrients such as nitrate(NO_(3)^(-)) and ammonium(NH_(4)^(+))are often not considered in breeding programs.The objective of this study was to evaluate the morphological,physiological and kinetic parameters of nitrogen uptake by clones of Eucalyptus saligna(32,864) and Eucalyptus grandis(GPC23).Morphological parameters in shoot and root systems,biomass and N concentrations in different organs,photosynthetic pigment concentrations,parameters of chlorophyll a fluorescence and photosynthetic rates were evaluated.Kinetic parameters,maximum absorption velocity(V_(max)),Michaelis-Menten constant(K_(m)),minimum concentration(C_(min)) and influx(I) were calculated for NO_(3)^(-)and NH_(4)^(+) in the two clones.E.granais clone was more efficient in the uptake of NO_(3)^(-)and NH_(4)^(+),and showed lower K_(m) and C_(min)values,allowing for the absorption of nitrogen at low concentrations due to the high affinity of the absorption sites of clone roots to NO_(3)^(-)and NH_(4)^(+).Higher root lengths,area and volume helped the E.grandis clone in absorption efficiency and consequently,resulted in higher root and shoot biomass.The E.saligna clone had higher K_(m) and Cmin for NO_(3)^(-)and NH_(4)^(+),indicating adaptation to environments with higher N availability.The results of NO_(3)^(-)and NH_(4)^(+) kinetic parameters indicate that they can be used in Eucalyptus clone selection and breeding programs as they can predict the ability of clones to absorb NO_(3)^(-)and NH_(4)^(+) at different concentrations.展开更多
文摘Objective: To investigate the reliability for fast estimation of Michaelis-Menten constant (Km) with calibrated specific activity at only two medium concentrations of substrate by both simulation and experimentation with arylesterase (ArE)as model. Methods: Initial rates were simulated by randomly inserting uniform absolute error, and the experimental initial rates of ArE were determined by measuring the increaser of product absorbance. Calibrated specific activities at two substrate concentrations were obtained by regression analysis, and Km was calculated according to Michaelis-Menten equation. Results: By simulation with calibrated specific activities at two medium substrate concentrations, Km could be calculated according to Michaelis-Menten equation with reasonable precision and accuracy. By experimentation with substrates of 2-naphthyl acetate, phenyl acetate, and p-nitrophenyl acetate, there were no differences between the mean and SD of Km of ArE for either substrate by this linear kinetic method and the Lineweaver-Burk plot. Conclusion: This linear kinetic method was reliable for fast estimation of the Km of some specified enzyme on its substrate of lower solubility or lower sensitivity for quantification by common methods.
基金The National Natural Science Foundation of China(No.21005016)the Foundation of Educational Commission of Jiangsu Province(No.JHB2011-2)
文摘In order to improve the substrate diffusion properties and stability of an immobilized enzyme alginate microgels modified with TiO2 nanoparticles were employed as the enzyme immobilizing support.Ionotropic gelation was applied for the preparation of hybrid gels while Ca2＋ Ce3＋ Ni2＋Cu2＋and Fe3＋were employed as the crosslinkers.Papain was selected as the model enzyme. UV-Vis spectroscopy was employed to investigate the activity of papain to evaluate kinetics and stability.Analysis results show that the highest affinity the lowest Michaelis-Menten constant Km =11.0 mg/mL and the highest stability are obtained when using Cu2＋as the crosslinker.The effect of the mass ratio of TiO2 to papain on the stability and leakage of papain is also investigated and the results show that 10∶1 TiO2∶papain is optimal because the proper use of TiO2 can reduce enzyme leakage and ensure enzyme stability.Preparing Cu/alginate/TiO2 hybrid gels via ionotropic gelation can provide a satisfactory diffusion capability and enzyme stability.
基金funded partly by the Conselho Nacional de Desenvolvimento Científico and Tecnológico(CNPq)。
文摘Eucalyptus clones are selected according to productivity,wood quality,rooting capacity,and resistance to drought,frost and diseases.However,kinetic and morphological parameters that determine the absorption efficiency of nutrients such as nitrate(NO_(3)^(-)) and ammonium(NH_(4)^(+))are often not considered in breeding programs.The objective of this study was to evaluate the morphological,physiological and kinetic parameters of nitrogen uptake by clones of Eucalyptus saligna(32,864) and Eucalyptus grandis(GPC23).Morphological parameters in shoot and root systems,biomass and N concentrations in different organs,photosynthetic pigment concentrations,parameters of chlorophyll a fluorescence and photosynthetic rates were evaluated.Kinetic parameters,maximum absorption velocity(V_(max)),Michaelis-Menten constant(K_(m)),minimum concentration(C_(min)) and influx(I) were calculated for NO_(3)^(-)and NH_(4)^(+) in the two clones.E.granais clone was more efficient in the uptake of NO_(3)^(-)and NH_(4)^(+),and showed lower K_(m) and C_(min)values,allowing for the absorption of nitrogen at low concentrations due to the high affinity of the absorption sites of clone roots to NO_(3)^(-)and NH_(4)^(+).Higher root lengths,area and volume helped the E.grandis clone in absorption efficiency and consequently,resulted in higher root and shoot biomass.The E.saligna clone had higher K_(m) and Cmin for NO_(3)^(-)and NH_(4)^(+),indicating adaptation to environments with higher N availability.The results of NO_(3)^(-)and NH_(4)^(+) kinetic parameters indicate that they can be used in Eucalyptus clone selection and breeding programs as they can predict the ability of clones to absorb NO_(3)^(-)and NH_(4)^(+) at different concentrations.
