Michaelis constants of soil enzymes

Studies to determine the Michaelis constants (k_m values) for the arylsulfatase and phosphatase activity in Iowa surface soils showed that the value obtained for either activity was different for different soils. When the incubation technique used to determine k_m did not involve shaking of the soil-substrate mixture, the k_m value for arylsulfatase activity in nine soils studied ranged from 1·37 × 10⁻³m to 5·69 × 10⁻³m, and the k_m value for phosphatase activity ranged from 1·26 × 10⁻³m to 4·58 × 10⁻³m. Shaking the soil-substrate mixture during incubation decreased the k_m value obtained for arylsulfatase or phosphatase activity and reduced the variation in k_m among soils. The maximum enzyme reaction velocity (V_max value) for soil arylsulfatase or soil phosphatase activity was markedly different for different soils and usually increased when the soil-substrate mixture was shaken during incubation. The k_m value for soil arylsulfatase or soil phosphatase activity was not significantly correlated with other soil properties studied (pH, cation-exchange capacity, percentage organic carbon, percentage clay, percentage sand).     

The kinetics of soil acid phosphatase

The kinetics of enzyme systems in soil can reasonably be expected to be similar to those of immobilised enzymes and therefore be modified by factors not considered in the formulation of the classical Michaelis-Menten model of enzyme kinetics. Previous studies of the p-nitrophenyl phosphatase activity of soil have assumed that the system follows Michaelis-Menten kinetics. This paper reports experiments on the p-nitrophenyl phosphatase activity of a kraznozem in which the plotting of V₀, the initial rate. against $\text{V}{}_0\text{[S]}\text{where}$ [S] is the substrate concentration showed that the system, for this soil at least, does not follow Michaelis-Menten kinetics. An evaluation of the graphical techniques used in previous studies showed that the conclusion that the p-nitrophenyl phosphatase activity of soil follows Michaelis-Menten kinetics is based on doubtful premises. Experiments in which both the substrate and the product were measured spectrophotometrically at intervals throughout the hydrolysis of p-nitrophenyl phosphate by the kraznozem yielded evidence for a model of the system which is capable of accounting for the deviation from Michaelis-Menten kinetics.     

缓冲液种类对土壤酸性磷酸酶活性的影响

土壤酸性磷酸酶影响土壤有机磷矿化。选择不同的缓冲液种类和不同p H值测定土壤酸性磷酸酶活性,可影响数据的可比性。为明确缓冲液及p H值对不同土壤磷酸酶活性的影响程度,选取p H值4～8的土壤样本,测定了在醋酸盐缓冲液、柠檬酸盐缓冲液和MUB缓冲液3种不同培养条件下酸性磷酸酶活性变化。结果表明,采用MUB缓冲液条件可获得较稳定的土样区分序列,且与缓冲液的p H值的变化无关。在醋酸盐缓冲液和柠檬酸盐缓冲液条件下,不同p H值范围的不同土样之间土壤酸性磷酸酶活性变异系数变化剧烈,而采用MUB缓冲液获得的变异系数较其他缓冲液大,且趋势平稳。综合考虑,进行土壤酸性磷酸酶活性测定,可优先选用MUB缓冲溶液。     

基于不同方法测定土壤酸性磷酸酶活性的比较

土壤酸性磷酸酶与有机磷的矿化及植物的磷素营养关系最为密切。目前国内学者在测定酸性磷酸酶活性时主要参照关松荫《土壤酶及其研究法》中以磷酸苯二钠为基质的测定方法,而国外学者主要参照Dick《Methods of Soil Enzymology》中以对硝基苯磷酸二钠为基质的测定方法(PNPP)。但是,在以磷酸苯二钠为基质测定生成物的过程中,常出现显色程度不明显的问题;另外,采用不同基质测定酸性磷酸酶活性也造成了测定方法选择的困难。为合理选择土壤酸性磷酸酶活性的测定方法,本研究选用酸性、中性和碱性土壤各10个土样,分别采用以磷酸苯二钠为基质,且在显色阶段分别加入pH5.0醋酸盐缓冲液(DPP 1)和pH9.4硼酸盐缓冲液(DPP 2)的方法,以及PNPP方法测定土壤酸性磷酸酶活性。同时也研究了不同pH缓冲液和苯酚浓度对生成物显色反应的影响。结果表明:以磷酸苯二钠为基质、在显色反应阶段加入pH≤6的缓冲液时,苯酚和2,6-二溴苯醌氯亚胺不显色;当加入pH≥8的缓冲液时,两者之间显色且苯酚浓度和吸光值的Pearson相关系数极显著。这说明pH低是导致高苯酚浓度和2,6-二溴苯醌氯亚胺显色效果差的一个主要原因。此外,采用PNPP方法测定时,在酸性、中性和碱性土壤中,10个样本酸性磷酸酶活性的变异系数分别较DPP 2增加了70.04%、42.44%和21.17%;极差分别是DPP 2的27.18倍、26.85倍和39.43倍。总之,如果选用磷酸苯二钠为基质测定土壤酸性磷酸酶活性,应在显色阶段加入碱性硼酸盐缓冲液;选用对硝基苯磷酸二钠为基质,是更为简单和灵敏的方法。     

Kinetics of acid phosphatase in calcium chloride extractable soil organic matter

The aim of this work was to compare the kinetic parameters of acid phosphatase (EC 3.1.3.2.) extracted from two forest soils under oak or pine. Soil was extracted with 4 mM CaCl₂ and the extract was divided into two fractions by filtration: one >0.2 μm containing microbial cells and soil particulates, and the other <0.2 μm containing fine particles and dissolved organic compounds of soil. The >0.2 μm fraction had higher K_m (0.26–0.82 vs. 0.12–0.39) and V_max (0.07–0.79 vs. 0.06–0.16) values than the <0.2 μm fraction, indicating a higher enzyme-substrate affinity and smaller amount of enzyme in fine particles and dissolved organic matter.     

柠檬酸盐和磷酸盐存在下酸性磷素酶在矿物和土壤胶体上的吸附

The aim of this work was to study the influence of phosphate and citrate, which are common inorganic andorganic anions in soils, on the adsorption of acid phosphatase by kaolin, goethite and the colloids separatedfrom yellow-brown soil (YBS) and latosol (LS) in central-south China. The YBS colloid has the major claymineral composition of 1.4 nm mineral, illite and kaolinite while the LS colloid mainly contains kaolinite andoxides. The adsorption isotherm of acid phosphatase on the examined soil colloids and minerals fitted tothe Langmuir model. The amount of enzyme adsorbed in the absence of ligands was in the order of YBScolloid ＞LS colloid＞kaolin≈goethite. In the presence of phosphate or citrate, the amounts of the enzymeadsorbed followed the sequence YBS colloid＞kaolin＞LS colloid＞goethite. The presence of ligands alsodecreased the binding energy between the enzyme and soil colloids or minerals. With the increase of ligandconcentration from 10 mmol L-1 to 400 m mol L-1, different behaviors for the adsorption of enzyme werefound in the colloid and mineral systems studied. A sharp decrease in enzyme adsorption was observed ongoethite while gradual decreases of enzyme adsorption were recorded in the two soil colloid systems. However,no any decrease was found for the amount of enzyme adsorbed on kaolin at higher ligand concentrations.When phosphate or citrate was introduced to the system before the addition of enzyme, the ligands usuallyenhanced the adsorption of enzyme. The results obtained in this study suggested the important role ofkaolinite mineral in the adsorption of enzyme molecules in acidic soils in the presence of various ligands.     

Components of organic phosphorus in soil extracts that are hydrolysed by phytase and acid phosphatase

 Extracts were prepared from soil using water, 50 mM citric acid (pH ∼2.3) or 0.5 M NaHCO₃ (pH 8.5), and were incubated with excess phytase from Aspergillus niger to determine the amounts of labile P. Two A. niger phytase preparations were used: (1) a purified form which exhibited a narrow substrate specificity and high specific activity against phytate; and (2) a commercial preparation (Sigma) with activity against a broad range of P compounds. A comparatively large proportion (up to 79%, or 5.7 μg g^–1 soil) of the organic P (P_o) extracted with citric acid was hydrolysed by the commercial phytase, while between 28% and 40% (up to 3.1 μg g^–1 soil) was hydrolysed using purified phytase. By comparison, only small quantities of the P_o in water and NaHCO₃ soil extracts were enzyme labile. While extractable P_o was increased both with increasing concentrations of citric acid (up to 50 mM) and increasing pH (pH 2.3–6.0), enzyme-labile P increased only with citric acid concentration. The labile component of P_o in citric acid extracts from soils with contrasting fertiliser histories indicated that enzyme-labile P_o is a relatively large soil P pool and is potentially an important source of P for plants. Received: 29 October 1999     

Water-extractable soil organic matter inhibits phosphatase activity

We show that water-extractable soil organic matter may inhibit activity of added phosphatase. The inhibition was greater for the topsoils than the corresponding subsoils. The enzyme kinetics in buffer and in soil solutions followed Michaelis–Menten kinetics. Vmax decreased and Km increased with increasing soil water-extractable organic carbon. Both kinetic parameters followed similar trends with the intensities of both fluorescence peaks of the solutions. The effect was more marked at pH below the optimum pH. Inhibition was complex, particularly for topsoil solutions where Vmax was decreased and Km increased. This effect could lead to underestimation of catalytic activity of enzymes extracted from soil and could result in the overestimation of decreased specific activity in the adsorbed phase of enzymes added to soils.     

4种农药对枇杷园土壤磷酸酶活性及微生物呼吸的影响

应用室内培养方法研究了甲基托布津、代森锰锌、杀灭菊酯、阿维菌素4种农药对枇杷园土壤磷酸酶活性及微生物呼吸的影响,并对4种农药进行了安全性评价。结果表明,经杀菌剂甲基托布津和代森锰锌处理的土壤,磷酸酶活性受到明显抑制,且抑制效果未见恢复。经杀灭菊酯和阿维菌素处理的土壤,磷酸酶活性变化呈现"抑制-恢复-激活"趋势;抑制作用随农药浓度的升高而增强;但7d后磷酸酶活性受到了一定程度的激活,35d达到最高值;高浓度杀灭菊酯和低浓度阿维菌素显示出较强的激活作用。农药对土壤微生物呼吸作用的影响初期表现为轻微激活,5d后出现抑制作用,12d后基本恢复正常,接近对照水平。通过安全性评价可知,供试4种农药对土壤微生物的危害较小,为无毒害或无实际危害的农药。     

Phytase and acid phosphatase activities in plant feedstuffs

A total of 183 samples representing 24 feedstuffs were analyzed for total phosphorus, phytate phosphorus content, phytase (Phy), and acid phosphatase (AcPh) activities with the objective to predict the capacity to hydrolyze phytic acid and to contribute to formulating environmentally adequate diets for monogastric animals. Of the cereals and cereal byproducts analyzed, only rye (5147 U kg(-)(1); 21 955 U g(-)(1)), wheat (1637 U kg(-)(1); 10 252 U g(-)(1)), rye bran (7339 U kg(-)(1); 56 722 U g(-)(1)), and wheat bran (4624 U kg(-)(1); 14 106 U g(-)(1)) were rich in Phy and AcPh activities. Legume seeds and oilseeds contained negligible Phy activity and a moderate amount of AcPh activity, except for kidney bean (33 433 U g(-)(1)) and full-fat linseed meal (13 263 U g(-)(1)). On the other hand, a significant linear regression between phytate phosphorus (y) and total phosphorus (x) was observed in cereal byproducts (R(2) = 0. 95; y = 0.8458x - 0.0367; P < 0.001) and oil seeds (R(2) = 0.95; y = 0.945x - 0.20; P < 0.001). Phy and AcPh were positively correlated with respect to phytate phosphorus in cereals, cereal byproducts, and other byproducts and negatively correlated in legume seeds and oilseeds. Except for cereals, the highest correlation between enzyme activities and phytate phosphorus was found for phytase. It is not possible to predict Phy and AcPh activities from phytate phosphorus content by linear and quadratic regressions. Finally, only highly significant and positive correlation was found between Phy and AcPh activities for cereals, cereal byproducts, and oilseeds.     

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.     

Global patterns of soil phosphatase responses to nitrogen and phosphorus fertilization

Hydrolysis of organic phosphorus(P) by soil phosphatases is an important process of P cycling in terrestrial ecosystems, significantly affected by nitrogen(N) and/or P fertilization. However, how soil acid phosphatase(ACP) and alkaline phosphatase(ALP) activities respond to N and/or P fertilization and how these responses vary with climatic regions, ecosystem types, and fertilization management remain unclear. This knowledge gap hinders our ability to assess P cycling and availability from a glo...     

Effect of hydrodynamic dispersion on phosphatase reactions in a soil column

Columns of artificially-prepared soil crumbs were perfused with solutions of p-nitrophenyl phosphate and rates of hydrolysis of this substrate as catalyzed by soil phosphatase were measured. Hydrolysis proceeded by a zero-order or by a first-order reaction at high and low concentrations of substrate, respectively. Steady-state concentrations of substrate and products from the columns can be described essentially in terms of plug flow kinetics with but minor corrections for hydrodynamic dispersion. Values of the zero and first-order rate constants corrected for dispersion are 3 and 9 per cent greater, respectively, than those assuming plug flow kinetics. The apparent value of the first-order rate constant is about 70 percent higher than the true value because of electrostatic repulsion between the negatively-charged substrate and the soil particles.     

Critical state soil mechanics analysis of the constant cell volume triaxial test

We have developed a critical state, stress-strain analysis that predicts the entire sequence of states from start to the end of a constant cell volume triaxial test in p-q-v-ɛ_z, space, where p is the spherical stress, q is the deviator stress, v is the specific volume and ɛ_z is the axial strain. The analysis requires five soil properties to be specified, these being the critical state properties (M, λ and κ) and two elastic properties (any two of E, η, G and K—all four can be found from any two). In order to test the analysis, properties taken mostly from one series of constant cell volume triaxial tests are used to simulate the behaviour in a second test series. In both series the two soils (a sand loam and a clay loam) were tested at several different water contents. The first (property estimation) series of tests was performed using large samples, whereas the second (verification) series involved small samples. The behaviour is quite different in the two series. The elastic property E was not determined in the first series of tests but was estimated from the second series. Furthermore, for three very wet samples, λ had to be estimated by fitting the analysis to the data. The verification was not therefore fully independent of the input test data, particularly for the three wet samples. The stress strain analysis simulated the behaviour of both series of tests in all four dimensions of the p-q-v-ɛ_z space. The match in p-q-v space was good for all samples. On a q-ɛ_z plane, the value of q was under-estimated for several samples, but for most of the samples the match was good on this plane. The analysis was generally as good as, and sometimes better than, a previous analysis that deals only with the end point of the test in p-q-v space. The previous analysis did not take account of the elastic properties. The stress-strain analysis therefore seems to offer a useful framework for parameter estimation from constant cell volume triaxial tests. This extends the usefulness of the test itself, as the elastic properties may now be accounted for. The success of the analysis also strengthens the record of success of the critical state concept for unsaturated soils.     

Hydrolysis of organic phosphorus in soil water suspensions after addition of phosphatase enzymes

Additions of enzymes involved in organic phosphorus (P) hydrolysis can be used to characterize the hydrolyzability of molybdate-unreactive P (MUP) in soil water extracts. Our aim was to test the feasibility of enzyme additions to soil water suspensions with respect to (1) suitable enzyme preparations and (2) recovery of molybdate-reactive P (MRP). To this end, we compared the substrate specificity of seven commercially available enzyme preparations (acid and alkaline phosphomonoesterase, phytase, and nuclease preparations) and optimized the assay conditions in microplates. We then measured MRP release after the addition of the enzymes to soil water suspensions and filtrates of two Swiss grassland soils (midland and alpine). In some cases, commercial preparations of the same enzyme differed in their specificity, presumably due to contamination with other enzymes, and also in their efficiency in soil suspensions. Addition of EDTA to the buffer was required to decrease sorption of released P in soil suspensions. Enzymatic release of P was consistently equal or higher in soil suspensions than in soil filtrates. However, also more dissolved MUP was present in soil suspensions than in filtrates, since the buffer interacted with the solid phase. Of the total dissolved MUP in soil suspensions, 94 and 61 % were hydrolyzable in midland and alpine soil, respectively. More specifically, 60 and 17 % of MUP were in nucleic acids, 6 and 39 % in simple monoesters, and 28 and 5 % in inositol hexakisphosphate in midland and alpine soil, respectively. Thus, we show that the characterization of hydrolyzable organic P in soil suspensions with hydrolytic enzyme preparations may be useful to better understand the availability of soil organic P to enzymatic hydrolysis, but that it requires soil-specific adaptation for optimum P recovery.     

Microbial response of an acid forest soil to experimental soil warming

 Effects of increased soil temperature on soil microbial biomass and dehydrogenase activity were examined on organic (O) horizon material in a low-elevation spruce-fir ecosystem. Soil temperature was maintained at 5  °C above ambient during the growing season in the experimental plots, and soil temperature, moisture, microbial biomass, and dehydrogenase activity were measured during the experiment. An incubation study was also conducted under three temperature regimes, 5, 15, and 25  °C, and under four moisture regimes of 20, 120, 220, and 320% to further evaluate these environmental factors on dehydrogenase activity and microbial biomass. Soil moisture content and microbial biomass controls were significantly lower (30% and 2 μg g^–1 soil, respectively) in the heated plots during the treatment period, suggesting that moisture content was important in controlling microbial biomass. In the incubation study, temperature appeared more important than moisture in controlling microbial biomass and dehydrogenase activity. Increasing temperature between 5  °C and 25  °C resulted in significant decreases in microbial biomass and dehydrogenase activity. Received: 7 August 1998     

Estimating critical state soil mechanics parameters from constant cell volume triaxial tests

Compaction, tillage, stresses around growing roots and other soil deformation events may be predicted by the critical state model of soil mechanics, but estimating the parameters is time consuming and expensive. We develop a back analysis of the constant cell volume triaxial test, in which the critical state parameters are derived from the results of a single test. This both saves much labour and provides more information than traditional analyses, which require several triaxial compression tests and an isotropic compression test to yield the same information. The method finds, using a minimization algorithm and a quasi-analytical solution to the stress–strain equations, the simulated soil deformation (and hence the properties used in that simulation) that best fits the test data. The minimization is a form of regression analysis. For normally consolidated samples the method provides stable estimates of the slope of the critical state line (M), the slope of the virgin compression line (λ) and elastic modulus (E). The standard errors of the estimates are small in relation to the means of these parameters. The estimates appear to be more reliable than those of more commonly used estimation procedures. The slope of the rebound line (κ) is estimated, but a measure of the accuracy of the estimate cannot be calculated.     

Fertilization management affects the alkaline phosphatase bacterial community in barley rhizosphere soil

There is increasing interest in good agriculture practices that address the issues of sustainability, reduction in inputs such as fertilizers and pesticides while maintaining crop yield and soil fertility. It is important that soil microbial diversity and function are not impaired by altered agricultural practice. In this study, as indicators of soil quality, the bacterial community structure was evaluated from a long-term field trial managed with conventional and low-input fertilization/pesticide regimes. The low-input plots under study received approximately one fifth less N fertilizer than the conventional-input plots, a maximum of half the recommended application rates of fungicides and pesticides and no externally added P source. A non-culturable approach was taken using polymerase chain reaction–denaturing gradient gel electrophoresis analysis of 16S rRNA and alkaline phosphomonoesterase [phosphatase] (ALP) genes in an attempt to relate bacterial community structure to respective field management regimes. To identify the ALP bacteria in these plots, randomly selected ALP clones were sequenced. The results based on Shannon diversity indices and community structure analysis of ALP genes suggest differences in community diversity and structure under conventional and low-input barley sites in most sampling seasons. We conclude that soil fertilization management affects the ALP bacteria in the barley rhizosphere, while the overall changes in bacterial community in these sites are prominently due to seasonal variation compared to crop or input regimes. The randomly selected ALP sequences identified from these sites were mostly from the Alpha and Gamma classes of Proteobacteria.