Kinetic behaviour of alkaline phosphatase desorbed from different clay minerals

The kinetic (K_m, V_max) of alkaline phosphatase (AP) desorbed from different Ca-homoionic clay minerals (montmorillonite, illite, and kaolinite) by extraction with Tris-Malate-Citrate buffer solution (pH 9.6) was studied in model experiments. After extraction (shaking for 15 min.) the K_m and V_max were measured in the extract, the remaining sediment and in the whole set-up. With kaolinite and illite, V_max of the desorbed AP was lower than that of the sediment. However, with montmorillonite, V_max of AP in the extract and whole system increased if compared to the control, but decreased in the sediment. The K_m of desorbed AP increased from 4.3 × 10^?3 (control) to 5.0 × 10^?3 M (illite), 5.4 × 10^?3M (kaolinite), and 5.5 × 10^?3M (montmorillonite). These values were lower than those obtained with the various sediments and whole experimental systems. An aberrant behaviour was recorded with the illite sorbed AP which showed an increase in affinity towards the substrate. Generally speaking, AP desorbed from clays may be reduced in its affinity towards the substrate p-nitrophenylphosphate by residual inhibitor and/or conformational change of the enzyme.     

Arylsulphatase activity in soils amended with crop residues: Kinetic and thermodynamic parameters

Laboratory experiments were performed to determine the Michaelis-constants (K_m values) and maximum reaction velocities (V_max values) of arylsulphatase in a clay-loam and a sandy clay-loam soil. When crop residues were buried in either soil, the K_m and V_max values were altered markedly; the changes depended not only on the soil properties but also on the type of crop residue incorporated in the soils. This was confirmed at various temperatures, and data obtained were used to calculate the activation energies (E_a), and changes in enthalpy (ΔH_a) and entropy (ΔS_a) of activation. Whereas these properties were also affected by incorporating crop residues, the free energies of activation (ΔG_a) values, were similar in both soils and derived soil samples.     

旱地长期培肥土壤脲酶和碱性磷酸酶动力学及热力学特征研究

研究了连续25年长期培肥试验条件下土壤脲酶和碱性磷酸酶酶促反应的动力学和热力学特征,从酶学角度揭示长期培肥的效应。结果表明,长期培肥增加了脲酶和碱性磷酸酶酶促反应的Vmax、Vmax/Km和k值;降低了Ea、△G、△H和△S值,说明培肥能提高酶促反应速度、减小活化自由能、加快土壤中物质的生物循环过程。酶促反应动力学参数和热力学参数与土壤性质相关分析表明,酶促反应动力学参数大多依赖于土壤化学性状,基于动力学参数的土壤肥力指标体系可评价土壤肥力水平,且U-Vmax、P-Km、P-Vmax可作为土壤肥力的重要指标。     

Extraction,purification and properties of soil hydrolases

Invertase, cellulase, phosphatases, protease and β-glucosidase were extracted from permanent pasture soil with 0.2 M phosphate buffer (pH 8) in the presence of 0.2 M EDTA. This extract was further treated with ammonium and salmine sulphates. Attempts were made to fractionate these enzyme activities by gel and anion-exchange chromatography. Specific activities were estimated in all fractions and some characteristics of the purified enzymes (optimum pH, temperature and substrate concentration, and K_m and V_max) were investigated. The results indicated that extracted enzyme activities occurred partly in soil as a carbohydrate-enzyme complex and partly as a humo-carbohydrate complex.     

Adsorption,activity, and kinetics of acid phosphatase as influenced by selected oxides and clay minerals

Abstrac

The effects of 3 oxides (Fe, Al, and Mn oxides) and 3 clay minerals (kaolin, montmorillonite, and allophane) on the adsorption and subsequent kinetic properties of acid phosphatase were compared. The amount of enzyme adsorbed by the oxides and clay minerals followed the order: montmorillonite ? kaolin > Mn oxide > Fe oxide > Al oxide ? allophane. The adsorption isotherms of the enzyme on the oxides and clay minerals, except for montmorillonite and allophane, fitted the Langmuir equation. The activity of the enzyme immobilized by the inorganic components studied was in the order of allophane > kaolin > Fe oxide > montmorillonite > Al oxide ≒ Mn oxide. Compared to the free enzyme, the V _max, K_m, and V _max / K _m values of the immobilized enzyme decreased, increased, and decreased, respectively. Among the oxides or clay minerals, the higher the ability of the inorganic components to adsorb the enzyme, the lower the value of the V _max / K _m ratio of the immobilized enzyme. These findings suggest that the catalytic efficiency of the enzyme complexes formed is determined by the adsorbability of the inorganic components for the enzyme.     

Characterization of extracellular phosphatase activities in periphytic biofilm from paddy field

Periphytic biofilms exist widely in paddy fields, but their influences on the hydrolysis of organic phosphorus(P) have rarely been investigated. In this study,a periphytic biofilm was incubated in a paddy soil solution, and hydrolysis kinetic parameters(half-saturation constant(Km) and maximum catalytic reaction rate(Vmax)), optimal environmental conditions, substrate specificity, and response to different P regimes of the phosphatase activities in the periphytic biofilm were determined, in order to characterize extracellular phosphatase activities in periphytic biofilms from paddy fields. The results indicated that the periphytic biofilm could produce an acid phosphomonoesterase(PMEase), an alkaline PMEases, and a phosphodiesterase(PDEase). These three phosphatases displayed high substrate affinity, with Km values ranging from 141.03 to 212.96 μmol L^-1. The Vmax/Km ratios for the phosphatases followed the order of alkaline PMEase > acid PMEase > PDEase, which suggested that the PMEases, especially the alkaline PMEase, had higher catalytic efficiency. The optimal pH was 6.0 for the acid PMEase activity and 8.0 for the PDEase activity, and the alkaline PMEase activity increased with a pH increase from 7.0 to 12.0. The optimal temperature was 50℃ for the PMEases and 60℃ for the PDEase. The phosphatases showed high catalytic efficiency for condensed P over a wide pH range and for orthophosphate monoesters at pH 11.0, except for inositol hexakisphosphate at pH 6.0. The inorganic P supply was the main factor in the regulation of phosphatase activities. These findings demonstrated that the periphytic biofilm tested had high hydrolysis capacity for organic and condensed P,especially under P-limited conditions.     

Parameter estimation for models of ligninolytic and cellulolytic enzyme kinetics

While soil enzymes have been explicitly included in the soil organic carbon (SOC) decomposition models, there is a serious lack of suitable data for model parameterization. This study provides well-documented enzymatic parameters for application in enzyme-driven SOC decomposition models from a compilation and analysis of published measurements. In particular, we developed appropriate kinetic parameters for five typical ligninolytic and cellulolytic enzymes (β-glucosidase, cellobiohydrolase, endo-glucanase, peroxidase, and phenol oxidase). The kinetic parameters included the maximum specific enzyme activity (V_max) and half-saturation constant (K_m) in the Michaelis–Menten equation. The activation energy (Ea) and the pH optimum and sensitivity (pH_opt and pH_sen) were also analyzed. pH_sen was estimated by fitting an exponential-quadratic function. The V_max values, often presented in different units under various measurement conditions, were converted into the same units at a reference temperature (20 °C) and pH_opt. Major conclusions are: (i) Both V_max and K_m were log-normal distributed, with no significant difference in V_max exhibited between enzymes originating from bacteria or fungi. (ii) No significant difference in V_max was found between cellulases and ligninases; however, there was significant difference in K_m between them. (iii) Ligninases had higher Ea values and lower pH_opt than cellulases; average ratio of pH_sen to pH_opt ranged 0.3–0.4 for the five enzymes, which means that an increase or decrease of 1.1–1.7 pH units from pH_opt would reduce V_max by 50%. (iv) Our analysis indicated that the V_max values from lab measurements with purified enzymes were 1–2 orders of magnitude higher than those for use in SOC decomposition models under field conditions.     

Hydrolases extracted from soil: Kinetic parameters of several enzymes catalysing the same reaction

The kinetic parameters (K_m and V_max) of ureases, phosphatases, casein and benzoylargininae-mide-hydrolysing proteases extracted from two different soils were determined. The Eadie-Scatchard plot, which is the most sensitive graphical technique for detecting deviations from Michaelis-Menten kinetics, was used. In the case of phosphatases extracted from two soils and of the benzoylarginineamide hydrolysing protease extracted from one soil the relationship between V/S and V produced two straight lines. The presence of at least two enzymes (or two forms of the same enzyme) catalysing the same reaction and characterized by markedly different K_m and V_max values, was indicated. A procedure to determine the true constants was reported. The conclusion was that the presence in soil of high and low-affinity enzymes permits an efficient reaction at either low or high substrate concentration.     

Carbon availability regulates soil respiration response to nitrogen and temperature

The response of soil CO₂ fluxes (R_soil) to interactions between carbon (C) and nitrogen (N) availability or C and temperature conditions is not well understood, but may increasingly affect future C storage under the combined anthropogenic impacts of N deposition and climate change. Here we addressed this uncertainty through a series of laboratory incubation experiments using soils from three contrasting ecosystems to investigate how changes in C, N, and temperature regulate R_soil through changes to Michaelis–Menten parameters (i.e. V_max and K_m). Results of this study demonstrate that R_soil response to N enrichment and changes in temperature are dependent on the C availability of soil substrates. N addition influenced R_soil through both the maximum rate (V_max) and the half saturation constant (K_m). The increase in K_m corresponded to a decrease in R_soil when C was limited. Alternatively, when C was abundant, N enrichment increased R_soil, which corresponded to an increase in V_max. Regulation of temperature sensitivity through V_max and K_m was also dependent on C availability. Both V_max and K_m demonstrated positive temperature responses, supporting the hypothesis of a canceling effect at low C concentrations. While temperature sensitivity was influenced by both C quantity and C complexity, our results suggested that C quantity is a stronger predictor. Despite strong differences in climate, vegetation, and management of our soils, C–N and C-temperature interactions were markedly similar between sites, highlighting the importance of C availability in the regulation of R_soil and justifying the use of Michaelis–Menten kinetics in biogeochemical modeling.     

Ermittlung kinetischer Parameter der sauren Phosphatasen intakter Zuckerrübenwurzeln bei variierter Phosphaternährung

Determination of kinetic parameters of acid phosphatases in intact sugar beet roots of variable phosphorus nutrition Organically bound phosphorus has to be hydrolysed before its P can be taken up by plants. Both microbes and plant roots possess phosphatases, which could be of importance especially in soils with low concentrations of inorganic phosphorus in the soil solution. This could be the reason why nutrient uptake models underestimate the P-uptake by plants when P-mobilization by the phosphatases of roots is not taken into consideration. Therefore the activity of acid phosphatases (P_ase) was determined to answer the following questions: 1) To which extent does the root bound acid phosphatase (P_ase) follow Michaelis-Menten kinetics? 2) By which of the four linear transformations of the Michaelis-Menten equation (Lineweaver/Burk, Hanes, Eadie/Hofstee, Eisenthal/Cornish-Bowden) can plausible values of V_max and K_m be determined? 3) Which effect has the P nutrition of the plant on these kinetic parameters? Sugar beet plants were grown in full nutrient solution containing 1 and 100 μM P respectively. The P_ase activity of the intact roots was measured at pH 5 using p-nitrophenylphosphate (25—15000 μM p-NPP). V_max values were calculated per m root length. Acid phosphatase activity principally followed Michaelis-Menten kinetics. Transformations and calculations of V_max and K_m after Eadie/Hofstee and Eisenthal/Cornish-Bowden suggested the existence of at least two enzyme systems (P_{ase 1}, P_{ase 2}). The following kinetic parameters were found: P_{ase 1}: P deficient plants: V_max: 43—45 nmol m^—1 min^—1, K_m: 31—37 μM NPP; P sufficient plants: V_max: 7 nmol m^—1 min^—1, K_m: 47—53 μM NPP. P_{ase 2}: P deficient plants: V_max: 230—293 nmol m^—1 min^—1, K_m: 1579—3845 μM NPP; P sufficient plants: V_max: 123—171 nmol m^—1 min^—1, K_m: 3027—7000 μM NPP. Thus plants with sufficient P nutrition have a lower affinity to P_org and a lower hydrolysis of P_org. For P nutrition of crops P_{ase 1} might be the most important enzyme.     

Effect of different treatments with crop residues on soil phosphatase activity

Summary Phosphomonoesterase (both acid and alkaline) and phosphodiesterase activity was either activated or inhibited in a soil treated with different crop residues. Phosphotriesterase activity remained unaffected. The kinetic parameters (V _max and K _m) of treated soil samples were modified in the same way: Increases or decreases in the V _max values corresponded to increases or decreases in the K _m values. The V _max values, rather than the K _m values, were found to have a predominant effect on phosphatase activity, thus indicating a fundamental role for the enzyme concentration. A positive and generally significant correlation was found between the activity of each phosphatase, which suggests an unspecific source of these enzymes. The values of the determination coefficients (R ² × 100) show that a low percentage of the variability may be ascribed to interactions among phosphatase activities.     

Kinetic parameters of dehydrogenase in the assessment of the response of soil to vermicompost and inorganic fertilisers

 Kinetic parameters (V _max and K _m) of dehydrogenase activity were determined in order to assess the metabolic response of a soil about 1 year after organic and mineral treatments. The soil was planted with maize (Zea mays) and treated with the following fertilisers: organic (vermicompost; VC), mineral (ammonium phosphate and urea), and an organo-mineral mixture. V _max, which represents a measurement of the quantity of enzyme, markedly increased in organic and organo-mineral treatments, indicating that the addition of organic matter caused an increase in dehydrogenase in the active microbial biomass. K _m, representing enzyme-substrate affinity and/or different sources of the enzymes, was similar in VC-treated soil and control soil, while it doubled in organo-mineral and mineral treatments. These results suggest that the use of VC did not alter the enzyme-substrate affinity, while mineral fertiliser reduced this affinity or changed the composition and activity of soil microbiota. A positive correlation was found between V _max, the metabolic index (dehydrogenase/water-soluble carbon ratio), and the soil organic matter content. The kinetic constants of dehydrogenase activity and the metabolic index may be considered valid parameters to monitor the evolution of microbiological activity in soil. Received: 4 February 2000     

Correlations between Kinetic Parameters of Phosphate Uptake and Internal Phosphorus Concentrations in Maize Plants: Making it Possible to Estimate the Status of Phosphate Uptake according to Shoot Phosphorus Concentrations

Abstract

The relationship between internal phosphorus (P) concentration [P] and kinetics of phosphate (Pi) uptake was investigated in maize seedlings grown hydroponically at different Pi concentrations (0.1–1,000 µM) and in the phase of Pi deprivation (0–10 d). The results indicated when the internal [P] was higher than 85 µmol g^?1 dw, apparent K_m, C_min, and V_max were significantly (P<0.01) related to [P]s in shoots and roots; when the internal [P] was lower than 85 µmol g^?1 dw, K_m and C_min were small and only V_max was significantly related (P<0.01) to internal [P]s. Three equations were deduced from the linear regressions of the kinetic parameters and [P]s in shoots. Using these equations, the values of apparent K_m, C_min, and V_max of Pi uptake of seedlings grown in different circumstances were calculated according to [P]s in shoots. In all the circumstances involved, for K_m and C_min, there was a parallel relationship between the values estimated by [P]s in shoots and by the Pi‐depletion technique; for V_max, the values estimated by [P]s in shoots were consistent with those obtained from Pi‐depletion experiments except the period of supplying Pi to the Pi‐starved seedlings over several days. These results indicated it is possible to estimate the Pi‐uptake status according to shoot P concentrations in maize plants under experimental conditions, which might be helpful to estimate in‐season status of Pi uptake of maize plants in the field.     

Soil urease activity and kinetics

The activity and kinetic properties of urease in several Malaysian soils were examined. The values for K_m and V_max of the soils computed according to the Hanes equation were in general agreement with other reports as far as magnitudes were concerned. A significant correlation between K_m and V_max was also obtained. The urease activity of the soils was variable, and it was noted that expression of the activity as the time required to hydrolyze half of the applied urea has limited use in soils of low activity. In all soils studied, inhibition of urease activity was effectively achieved using Ag⁺, while Cu²⁺ was only effective in two soils, and marginally effective in the other two soils. Urease inhibitors have potential applications in reducing volatilization losses of ammonia derived from urea applied to soils.     

Iron and zinc absorption characteristics and copper inhibitions in cucurbitaceae

Iron and Zn absorption, interactions, and Cu inhibitions were characterized in cucumber (Cucumis sativus L.), watermelon (Citrullus lanatus Thunb.), and pumpkin (Cucurbita moschata Poir.) by kinetic parameters V_max and K_m. Influx and V_max values for Fe and Zn absorption decreased in each species as plant age increased. For the Michaelis constant, K_m, Fe values in cucumber and watermelon and Zn values in watermelon and pumpkin were relatively unchanged with increased plant age. K_m values for Zn absorption in cucumber and Fe absorption in pumpkin decreased as plant age increased. Among species, watermelon appeared to have a particularly effective uptake mechanism for Zn at low solution concentrations. Non‐competitive inhibition of Zn absorption by Fe (20, 50 uM) was indicated in each species. Iron uptake in pumpkin was inhibited non‐competitively by Zn (5, 10 uM), however no significant effects of Zn on Fe absorption were evident in either watermelon or cucumber. Copper (0.5, 1, 5 uM) inhibited uptake of Fe non‐competitively and Zn competitively in each species.     

The effect of soil pH and high urea concentrations on urease activity in soil

The rate of hydrolysis of urea in soil over the wide range of concentrations, up to 10 moles N per dm³ soil solution, found in fertilizer practice, was examined in Begbroke sandy loam adjusted to different pH values. On rewetting air-dry soil, urease activity increased rapidly, reached a maximum within the first 24 h and then decreased slowly to level off after about 4 days. Pretreatment of the soil with urea or ammonium had no effect on the urease activity. Urease activity increased with substrate concentration, reached an optimum value and then decreased with rising urea concentration. The results could be explained by substrate inhibition at higher urea concentrations, and the data are well described by a modified Michaelis-Menten equation involving three parameters, V_max, K_m and K_i where K_i is an inhibition constant. K_m decreased linearily with rise in pH whereas K_i increased slightly between pH 4.9 and 7.0 and steeply between 7.0 and 8.4. V_max increased with rise in pH, reached a maximum value at pH 6.0 and then declined at higher pHs. There was a further reaction, reaching a maximum rate at a urea concentration of about 0.2 molar N in the soil solution, that followed Michaelis-Menten kinetics. K_m for this high affinity reaction increased up to pH 7.2 and then decreased at higher pH values; V_max increased up to pH 6.8 and then decreased. The contribution of the high affinity reaction was small except at low concentrations of urea.     

Influence of natural and synthetic humic substances on the activity of urease

The activity of a purified urease, obtained from Bacillus pasteurii, was inhibited by humic and fulvic acids obtained from an agricultural soil. Enzyme kinetic studies showed that the humic substances affected the affinity of the enzyme for its substrate (K_m) and the maximum velocity of the reaction (V_max). The V_max was inhibited to the same extent by both humic (HA) and fulvic (FA) acids, the precise effect depending on the pH and concentration of humic substance. At pH 4.0, HA concentrations of 25 pg cm^?3 and 10 μg cm^?3 inhibited the V_max by 38.5% and 20% respectively. HA and FA had similar effects on the K_m but in this case the lowering of the affinity of the enzyme for its substrate was not concentration dependent in the range 0–25 μg cm^?3 of humic substance. Typically, the affinity was decreased from a K_M of 50 mM in the control to 67 mM in the presence of HA and FA. The effects were not due primarily to the ash or N contents of the humic substances because de-ashed humic acid and synthetic model humic (made from catechol, guaiacol, pyrogallol, resorcinol and protocatechuic acid) and fulvic acid (made from polymaleic acid), containing virtually no ash or N, were equally as effective. The effect was not related to the phenolic monomers which, before polymerization, had no effect on urease activity.     

Exploring the enzymatic landscape: distribution and kinetics of hydrolytic enzymes in soil particle-size fractions

The location of extracellular enzymes within the soil architecture and their association with the various soil components affects their catalytic potential. A soil fractionation study was carried out to investigate: (a) the distribution of a range of hydrolytic enzymes involved in C, N and P transformations, (b) the effect of the location on their respective kinetics, (c) the effect of long-term N fertilizer management on enzyme distribution and kinetic parameters. Soil (silty clay loam) from grassland which had received 0 or 200 kg N ha⁻¹ yr⁻¹ was fractionated, and four particle-size fractions (>200, 200-63, 63-2 and 0.1-2 μm) were obtained by a combination of wet-sieving and centrifugation, after low-energy ultrasonication. All fractions were assayed for four carbohydrases (β-cellobiohydrolase, N-acetyl-β-glucosaminidase, β-glucosidase and β-xylosidase), acid phosphatase and leucine-aminopeptidase using a microplate fluorimetric assay based on MUB-substrates. Enzyme kinetics (V_max and K_m) were estimated in three particle-size fractions and the unfractionated soil. The results showed that not all particle-size fractions were equally enzymatically active and that the distribution of enzymes between fractions depended on the enzyme. Carbohydrases predominated in the coarser fractions while phosphatase and leucine-aminopeptidase were predominant in the clay-size fraction. The Michaelis constant (K_m) varied among fractions, indicating that the association of the same enzyme with different particle-size fractions affected its substrate affinity. The same values of K_m were found in the same fractions from the soil under two contrasting fertilizer management regimes, indicating that the Michaelis constant was unaffected by soil changes caused by N fertilizer management.     

Contparison of the influence of Cu,Zn, and Cd on the activity and kinetics of free and intntobilized acid phosphatase

The influence of Cu, Zn, and Cd on the activity and kinetics of acid phosphatase immobilized by two soil clays, kaolin or goethite was investigated. The ability of Cu to inhibit the enzyme activity was higher than that of Zn in all the enzyme complexes examined. The ability of Cd was negligible. The inhibitory effects of Cu and Zn on the two soil clay- and kaolin-enzyme complexes were much stronger than those on the goethite-enzyme complex. The V _max and K _m values of the enzyme complexes indicated that both Cu and Zn decreased the maximum reaction velocity of the enzymes, but increased the affinity of the enzymes for the substrate. The degree of the decrease and increase was higher in the Cu systems than in the Zn systems.     

Immobilization of Carbonic Anhydrase on Chitosan Stabilized Iron Nanoparticles for the Carbonation Reaction

Carbonic anhydrase (CA) has been immobilized on chitosan stabilized iron nanoparticles (CSIN) for the biomimetic carbonation reaction. CSIN was characterized using scanning electron microscope, energy dispersive X-ray, X-ray diffraction spectroscopy, and Fourier transform infrared analysis. The effect of various parameters such as pH, temperature and storage stability, on immobilized CA was investigated using a p-NPA assay. Kinetic parameters of immobilized and free CA (K _m and V _max values) were also evaluated. The K _m and V _max for immobilized CA was 1.727?mM and 1.189???mol?min^?1?ml^?1, respectively, whereas for free enzyme the K _m and V _max was 1.594?mM and 1.307???mol?min^?1?ml^?1, respectively. It was observed that the immobilized enzyme had longer storage stability and retained 50?% of its initial activity upto 30?days at room temperature. CA immobilized on CSIN has been used for hydration of CO₂, and the results were validated by using a gas chromatographic method. Proof of concept has been established for the biomimetic carbonation reaction. Immobilized CA show reasonably good CO₂ sequestration capacity of 21.55?mg of CaCO₃/mg of CA as compared to CO₂ sequestration capacity of 34.92?mg of CaCO₃/mg of CA for free CA respectively, under a limiting concentration of CO₂ (14.5?mg of CO₂/10?ml).