Extraction and properties of phosphodiesterase from a forest soil

Phosphodiesterase together with brown-coloured compounds was extracted from a forest soil using 0.1 M phosphate buffer (pH 7). Use of KCl and EDTA with the buffer facilitated phosphodiesterase extraction. Distilled water extracted little enzyme activity. A curvilinear relationship such as the Langmuir type was found between solution volume and phosphodiesterase activity of the extract. The results implied that the phosphodiesterase extracted was extracellular and was adsorbed on the surface of soil particles by ionic bonding. Brown-coloured compounds in the extract were removed by precipitation with protamine sulfate. The phosphodiesterase activity of the extract treated with protamine sulfate was lost on keeping at 80°C for 10 min and was optimal at pH 5.2–6.0. The extract hydrolyzed either the 3'- or the 5'-phosphodiester bond of deoxythymidine p-nitrophenyl phosphate.     

Extraction of β-glucosidase activity from PEA field soil

Brown compounds with β-glucosidase activity were extracted from a cultivated soil in 0.1 m phosphate buffer (pH 7), 0.3 m KCl and 10 mm EDTA. A curvilinear relationship of the Langmuir type was observed between the solution volume and β-glucosidase activity of the extract. The results indicated the β-glucosidase was extracellular and was adsorbed on the surfaces of soil particles by ionic bonds. The preparation was treated with protamine sulfate and its enzymatic properties were investigated. The activity of the preparation was optimal at pH 5.4 and was lost after 10 min at 80°C. The extract hydrolyzed p-nitrophenyl β-glucoside(100), phenyl β-glucoside(19), salicin(8), amygdalin(33), cellobiose(52) and gentiobiose(55) (relative activities shown in parentheses). But, the extract had no effect on methyl β-glucoside and phlorizin. The substrate specificity and optimum pH of the enzymatic activity of the soil extract was similar to those of β-glucosidases from various fungi.     

Glucose oxidase activity in soil and its possible interference in assays of cellulase activity

The occurrence of naturally-occurring glucose oxidase activity in a soil collected at a depth of 0–8 cm under tussock has been indicated. This activity was probably responsible for utilization of some of the glucose added to a system of soil, buffer and toluene that was incubated at 30°C; no loss of added glucose was apparent at 50°C.The cellulase activity of this soil was assessed by measuring rates of hydrolysis of added cellulose with Somogyi and anthrone reagents in systems of soil, buffer and toluene at 30 and 50°C. The production of reducing sugars was approximately linear over the first 48 or 72 h of incubation but appeared to decline subsequently at both temperatures. Interference by the activity of the naturally-occurring glucose oxidase at 30°C is suggested. Interference by glucose oxidase appears less likely at 50°C because of the apparent stability of added glucose at this temperature and the instability of added glucose oxidase. An incubation period of 48 h at 30°C appears generally satisfactory for assaying the cellulase activity of this soil.The possible role of glucose oxidase in interfering in assays of other soil enzymes in which the production of glucose is measured is discussed.     

Numerical characterization of forest soils using biological and biochemical properties

Summary To find if surface soils could be grouped by their biological and biochemical properties, soil samples (0–5 cm) were collected at 4-week intervals for 56 weeks from 48 woods in and around the English Lake District, and pH, loss-on-ignition (LOI), moisture content, oxygen uptake, and cellulase and phosphatase activities were measured. Results expressed on a loss-on-ignition basis were more informative than those on an oven-dry basis. In a principal component analysis of each property over the 14 samplings, the first component values represent smoothed between-plot differences; other components identify plots which behave differently from the majority at certain times. Analysis of variance showed very highly significant differences between plots for all the properties. pH and loss-on-ignition showed the smallest, but significant, differences between samplings. On a loss-on-ignition basis, the only significant correlations between first component values, and between plot means, were phosphatase with oxygen uptake and cellulase with pH. It is concluded that no individual physiological property can be used as a measure of soil bioactivity, which supports the conclusions of other authors. None of the principal component analyses of individual or combined properties showed any evidence of the existence of distinct clusters of plots. On a loss-on-ignition basis, a priori groups based on (1) pH < 3.8, (2) pH 3.8 – 5.0 and (3) pH > 5.0, showed no significant difference in moisture content. However, oxygen uptake was significantly lower in (1) than in (2). Cellulase activity was significantly greater in (3) than in (1) and (2). Phosphatase activity was significantly lower in (1) than in (2), and there appeared to be a peak at pH 3.8 – 5.0.     

Formation of [14C]cellulase-humic complexes and their stability in soil

[¹⁴C]cellulase was extracted from the culture medium of Trichoderma viride and an attempt made to complex it with humic acid by adsorption. The results showed that the humic acid extracted from soil does not form a stable complex with [¹⁴C]cellulase. In contrast, the flocculation of humic acid by 24 mM Ca²⁺ in the presence of the cellulase resulted in the formation of stable humic-cellulase complexes. These complexes showed great resistance to proteolysis and storage at high temperatures. DEAE cellulose chromatography of cellulase-humic complexes revealed that cellulase could not be separated from the humic acid. Enzyme activity was only eluted along with humic acid upon increasing gradient concentration from 1.0 to 1.5 m NaCl. Furthermore, in order to test their stability, the enzyme-humic complexes were incorporated into fresh soil for 90 days. The cellulase-humic complexes were then extracted from soil. Fractionation of the extract on DEAE cellulose and G 100 Sephadex revealed that cellulase activiiy could not be separated from humic acid and was again eluted in the form of enzyme-humic complexes. This confirmed the stability of cellulase-humic complexes in soil.     

Oxidative coupling activity in soil extracts

An assay was developed in which 50 mm citrate buffer was used to extract catalysts responsible for oxidative coupling reactions in soil. The assay was based on the formation of the dimerized quinone from the oxidative coupling of 2,6-dimethoxyphenol. Oxidative coupling activity was destroyed by heating the extracts for 15 min at 100°C and was inhibited by H₂O₂ (98% at 10 mm), KCN (96% at 10 mm), dithiothreitol (100% at 0.1 mm) and 2,3-dimercapto-1-propanol (100% at 0.1 mm). In the assay, a pH of 7.0 and a temperature of 55°C were determined as optimal. Freezing the soil extracts provided the best protection against activity loss, whereas storage caused a decline in oxidative coupling activity as a function of increasing temperatures (5–50°C). If soil had been supplemented with sucrose before extraction, activity increased. Soil extracts reacted with syringaldazine, benzidine, o-dianisidine, guaiacol, and p-cresol, substrates previously used to detect phenoloxidase enzymes.     

Partial purification of polyphenol oxidase from Chinese cabbage Brassica rapa L

Polyphenol oxidase (PPO) was purified and characterized from Chinese cabbage by ammonium sulfate precipitation and DEAE-Toyopearl 650M column chromatography. Substrate staining of the crude protein extract showed the presence of three isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be approximately 65 kDa by gel filtration on Toyopearl HW-55F. On SDS-PAGE analysis, this enzyme was composed of a subunit molecular weight of 65 kDa. The optimum pH was 5.0, and this enzyme was stable at pH 6.0 but was unstable below pH 4.0 or above pH 7.0. The optimum temperature was 40 degrees C. Heat inactivation studies showed temperatures >40 degrees C resulted in loss of enzyme activity. PPO showed activity to catechol, pyrogallol, and dopamine (K(m) and V(max) values were 682.5 mM and 67.6 OD/min for catechol, 15.4 mM and 14.1 OD/min for pyrogallol, and 62.0 mM and 14.9 OD/min for dopamine, respectively). The most effective inhibitor was 2-mercaptoethanol, followed in decreasing order by ascorbic acid, glutathione, and L-cysteine. The enzyme activity of the preparation was maintained for 2 days at 4 degrees C but showed a sudden decreased after 3 days.     

土地利用和轮作方式对旱地红壤生化性质的影响

研究不同土地利用和轮作方式对旱地红壤肥力的影响对提高红壤质量具有十分重要的指导意义。本研究以湖南省桃源县的林地、大豆-油菜轮作、玉米-休闲轮作土壤为研究对象,明确了林地、农地土壤及农地不同轮作方式对土壤化学和生物性质的影响。结果表明,林地土壤的pH、有机碳、速效养分、微生物生物量碳及酶活性(纤维素酶、酸性磷酸酶、转化酶、蛋白酶)均显著高于农地土壤;大豆-油菜轮作土壤的pH、养分含量、微生物生物量碳含量及其微生物熵在大多数情况下高于玉米-休闲轮作,但轮作处理对各酶活性的影响并不完全一致。这种不一致性可能与不同酶对由不同利用和轮作方式导致的土壤性质差异的敏感性不同所致。土壤有机碳和pH与各生物指标均呈显著正相关关系,表明提高该地区的土壤有机碳含量对于维持土壤的生化性质具有重要的作用。     

Development of a New Soil Extractant for Simultaneous Phosphorus,Ammonium, and Nitrate Analysis

Abstract

A new soil extractant (H³A) with the ability to extract NH₄, NO₃, and P from soil was developed and tested against 32 soils, which varied greatly in clay content, organic carbon (C), and soil pH. The extractant (H³A) eliminates the need for separate phosphorus (P) extractants for acid and calcareous soils and maintains the extract pH, on average, within one unit of the soil pH. The extractant is composed of organic root exudates, lithium citrate, and two synthetic chelators (DTPA, EDTA). The new soil extractant was tested against Mehlich 3, Olsen, and water for extractable P, and 1 M KCl and water‐extractable NH₄ and NO₂/NO₃. The pH of the extractant after adding soil, shaking, and filtration was measured for each soil sample (5 extractants×2 reps×32 soils=320 samples) and was shown to be highly influential on extractable P but has no effect on extractable NH₄ or NO₂/NO₃. H³A was highly correlated with soil‐extractable inorganic N (NH₄, NO₂/NO₃) from both water (r=0.98) and 1 M KCl (r=0.97), as well as being significantly correlated with water (r=0.71), Mehlich 3 (r=0.83), and Olsen (r=0.84) for extractable P.     

种还分离玉米秸秆还田对土壤微生物量碳及酶活性的影响

在种还分离模式下采用尼龙网袋法对玉米秸秆还田进行田间原位模拟,比较不同秸秆还田量在不同还田深度下对土壤总有机碳、微生物量碳、纤维素酶活性和过氧化氢酶活性的影响。试验设置秸秆还田量0(R0),0.44%(R1),0.88%(R2)和1.32%(R3)4个水平和3个还田深度0—15,15—30,30—45cm交叉处理。结果表明:还田1年时,在0—15cm土层,土壤总有机碳、微生物量碳、纤维素酶活性和过氧化氢酶活性均随秸秆还田量增加而提高,且R3处理提升效果最显著,分别达到30.98%,101.16%,172.72%,5.40%;在15—30cm土层,秸秆还田处理的土壤总有机碳、微生物量碳含量和过氧化氢酶活性高于R0处理,但R1、R2、R3之间无显著性差异,纤维素酶活性随秸秆还田量增加而变大;在30—45cm土层,R2、R3处理的土壤总有机碳、微生物量碳含量和纤维素酶活性显著高于其他处理,各处理的过氧化氢酶活性并无显著差异。还田2年与还田1年相比,各处理的土壤总有机碳、微生物量碳、纤维素酶活性均降低,降低幅度分别为8.59%~35.36%,6.74%~29.16%,6.18%~31.72%,但过氧化氢酶活性呈增加趋势,以R3增幅最大,随土层加深,提升幅度为14.14%,10.14%,12.11%。说明在种还分离模式下可以高量秸秆还田,并可有效改善土壤肥力。     

大孔树脂分离纯化玫瑰果多酚及其抗氧化性

为了研究大孔树脂对玫瑰果多酚的纯化效果,该试验以玫瑰果多酚粗提液为原料,利用大孔树脂吸附法对其进行纯化,并对其体外抗氧化活性进行了研究。结果表明,大孔吸附树脂纯化较佳工艺为:在20℃条件下,用质量浓度为0.80 mg/m L的玫瑰果多酚粗提液(p H值5.8)以1 m L/min的流速上样200 m L;吸附平衡后用少量蒸馏水洗至洗脱液无色,后用70 m L体积分数为70%的乙醇溶液,以1.5 m L/min的流速进行动态洗脱,洗脱峰相对集中、对称,无拖尾。纯化后,总酚质量分数由纯化前的122.90 mg/g提高到399.42 mg/g。体外抗氧化活性的研究表明:清除1,1-二苯基-2-苦基肼自由基的作用效果顺序为维生素C纯化多酚2,6-二叔丁基-4-甲基苯酚(Butylated hydroxytoluene,BHT)粗多酚;清除超氧阴离子自由基的作用效果顺序为维生素CBHT纯化多酚粗多酚;清除羟基自由基的作用效果顺序为维生素C纯化多酚BHT粗多酚。研究结果为玫瑰果多酚的进一步利用提供依据。     

Function of the methanogenic community in mangrove soils as influenced by the chemical properties of the hydrosphere

Coastal ecosystems represent a potential additional source of the greenhouse gas methane (CH₄) that has been insufficiently quantified. Thus, to understand the mechanisms controlling greenhouse gas emissions in these ecosystems, this study investigated CH₄ emissions from and the related microbial properties of mangrove soils. Soil and gas samples were collected from several plots at different distances from the seashore in Soc Trang and Ca Mau in Vietnam, and the Sundarbans in India. Soil samples were incubated under different conditions, i.e., anaerobic or aerobic, and the microbial properties of each soil sample with the addition of different amounts of seawater were analyzed. Relatively high CH₄ fluxes and production were detected during the aerobic incubation of samples from the seashore plots in Soc Trang and Ca Mau. However, CH₄ production was reduced under anaerobic conditions [soil electrical conductivity (EC): 179–289 mS m^?1, pH (H₂O): 7.45–8.10] compared with aerobic conditions [water content: 38.9–109.2%, EC: 187–299 mS m^?1, pH (H₂O): 6.86–7.72], but it increased with increasing sulfate concentration, soil EC and cellulase activity and lowering soil pH under anaerobic conditions. Furthermore, mangrove soil with a relatively high level of total organic carbon (C) exhibited relatively high CH₄ production when diluted 4-fold with seawater under anaerobic conditions [water content: 38.9–109.2%, EC: 533 mS m^?1, pH (H₂O): 6.67]. Nearly all of the DNA bands excised from polymerase chain reaction-denaturing gradient gel electrophoresis contained identical sequences related to archaea from the class Halobacteria. The high potential of the seashore plot for CH₄ emissions could be due to the enhancement of cellulase activity under the intermittent oxygen supply, which promotes polysaccharide depolymerization and subsequently increases anaerobic methanogenic activities during tidal flooding. This study also indicates that the major archaea responsible for CH₄ production require a particular hydrospheric salt concentration and soil pH.     

Soil enzyme activities in dependence on tree litter and season of a social forest,Burdwan, India

The study was done to evaluate enzyme activities (amylase, cellulase and invertase) from the soils of different vegetation sites, with seasonal variation, of social forest, Burdwan, India. Study results showed significant lower enzymatic activities in the subsoil compared to those of the topsoil. The seasonal variations indicated that amylase, cellulase and invertase enzyme activities had reached peaks during the rainy seasons in different soil depths. Amylase activity was highest in Tectona litter containing soil in all seasons in both the soil layers. All the three enzyme activities have shown significant positive correlation with available nitrogen (p < 0.05) and available phosphorous (p < 0.05) during rainy season in both the soil depths. Correlation study revealed that soil organic carbon was positively correlated with cellulase and invertase activities except in the Anacardium vegetation site in the topsoil during rainy season. Irrespective of the seasons and the depths of soil, control site without vegetation showed much lower levels of organic carbon and enzyme activity compared to those of the experimental sites. Therefore, it is concluded that carbon transformation will be higher during rainy season in the vegetation sites of forest soil under such agroclimatic conditions.     

纤维素降解菌CMC-4的分离鉴定、诱变和酶学特性研究

从秸秆还田土壤中初筛获得一株高效纤维素降解菌CMC-4,根据菌株形态、理化性质及16S rDNA序列分析,初步鉴定为地衣芽孢杆菌(Bacillus licheniformis)。以此为出发菌株,经亚硝酸钠诱变获得一株稳定高产纤维素酶突变株CMC-4-3。对CMC-4和CMC-4-3的产酶条件和酶学性质进行比较分析,结果表明:CMC-4-3纤维素酶活力较CMC-4提高67.5%;其最适产酶条件是:37℃、pH 6.0、葡萄糖为碳源、蛋白胨为氮源、接种量2.0%、装液量60 ml/250 ml。该菌株所产纤维素酶的最适反应pH为6.0,且在4.0~7.0酶活力较稳定;最适反应温度为50℃,在20~80℃范围内均保持稳定;金属离子Fe~(2+)、Mg~(2+)、Co~(2+)、K~+对酶有激活作用,其余离子均有不同程度抑制作用,而Cu~(2+)和Ca~(2+)抑制作用最强,酶活力减弱近50%,是该酶的强效抑制因子。诱变前后菌株产酶条件和酶学性质等部分表型发生了变化,而突变菌株显示出了更宽泛的环境适应范围。据此,获得一株高效产纤维素酶、耐受范围广的具纤维素降解能力的地衣芽孢杆菌,而CMC-4-3和CMC-4的表型可作为深入探讨基因型变化的线索。     

Extraction and characterization of soil hydrogenases oxidizing atmospheric hydrogen

Soils are the largest sink of atmospheric hydrogen contributing about 75% to the total budget. Atmospheric H₂ is assumed to be oxidized in soil by abiontic soil hydrogenases. Extraction of a forest soil with a slightly alkaline (pH 8.5) buffer containing polyethylene glycol (PEG), followed by filtration yielded a bacteria-free extract that oxidized H₂ at ambient concentrations (0.2–2.0 ppmv). Hydrogenase activity was assayed by gas chromatographic analysis of H₂ consumption and by conversion of ³H₂ to tritiated water. Only less than 2% of the original activity was recovered in the extract. Kinetic analysis nevertheless resulted in a biphasic kinetics exhibiting two K_m and V_max values that were similar to those detected in the original soil. In addition, activities of both original soil and soil extract showed similar optima at pH 4–6 and at 30 °C, indicating that representative fractions of soil hydrogenases were recovered in the extract. Precipitation with PEG or ultrafiltration allowed further purification of the activity, albeit only about 20% of that in the crude extract could be recovered in the precipitate or the fraction >100 kDa.     

利用改进的生物反应器研究不同通气条件下土壤中菲的降解

利用自行设计的生物反应器进行多环芳烃菲污染土壤的生物修复研究。在控制土壤水分、养分的情况下 ,设 6个通气处理 ,分析测定各处理的土壤菲降解率、微生物量、多酚氧化酶以及土壤酸度的动态变化。为期 6 0天的试验结果表明 ,通气量为 0 .0 8m3 h-1时 ,菲的降解率最高 ,达 72 .6 % ;与对照相比 ,微生物量最多 ,其中细菌、真菌都显著高于对照 ;多酚氧化酶活性也最高。通气量为 0 .0 8m3 h-1处理还可以控制土壤中酸度的变化 ,保持土壤中pH的稳定 ,从而更快地降解污染土壤中的菲。较高的菲降解率与通气改变土壤条件有关。在本实验的条件下 ,反应器中土壤细菌、真菌数量增加 ,多酚氧化酶活性提高 ,土壤保持稳定的pH值是土壤中菲降解率提高的主要原因。因而 ,改进的反应器具有较高的降解效果。本研究还表明 ,利用生物反应器能够快速、高效地消除土壤中的有机污染物 ,实现有机污染土壤的离位生物修复。     

Long-Term Effects of Different Cropping Systems on Selected Enzyme Activities

The historic Sanborn Field located on University of Missouri campus at Columbia, Missouri, was designed to demonstrate the importance of agricultural practices, such as fertilization, tillage, and crop rotation. Knowledge of the effect of long-term agricultural practices on the soil quality is critical for farmers to make better management decisions to efficiently and sustainably produce crops. This study examined long-term fertilization, tillage, and crop rotation effects on the activities of soil enzymes including phenol oxidase, peroxidase, dehydrogenase, cellulose, and β-glucosaminidase in the top soil layer (0–10 cm). The results showed that pH, organic matter, and cation exchange capacity are important factors that are associated with enzyme activities. Compared to conventional tillage, no-tillage treatment significantly increased activities of phenol oxidase, peroxidase, dehydrogenase, and d β-glucosaminidase. Fertilization increased cellulase and β-glucosaminidase activity, but had mixed effects for phenol oxidase, peroxidase, and dehydrogenase depending on the type of fertilizer applied. The amount of nitrogen applied significantly affected soil enzyme activities. The influences of the crop type and rotation are significant but differ for each enzyme. Due to the sensitivity of soil enzymes to environmental factors, soil enzymes such as phenol oxidase, dehydrogenase, cellulose, and β-glucosaminidase are suitable as indicators of soil quality.     

Activity,origins and location of cellulases in a silt loam soil

Summary Cellulase activity in a silt loam soil was assayed and characterised using a microcrystalline cellulose substrate (Avicel). Activity was maximal between pH 5.3 and pH 6.0. A 64% loss in activity was observed on air-drying the soil. However, the residual activity was stable to storage at 40°C for 7 days and was resistant to the action of added protease. The component endoglucanase and -D-glucosidase activities in field-moist and air-dried soil were also assayed. The proportion of the soil microbial population able to utilise cellulose was investigated and the persistence of two free (soluble) cellulase preparations of microbial origin was determined following their addition to soil. A rapid decline in the endoglucanase activity of a Streptomyces sp. cellulase preparation was observed while 30% of the original activity of a Trichoderma viride cellulase preparation could still be detected after 20 days. From the data obtained in this study it appears that the major portion of the -D-glucosidase activity is bound to and protected by the soil colloids. By contrast, the major portion of the exo- and endoglucanase activity appears to be free in the soil solution, attached to the outer surfaces of cellulolytic microorganisms or associated in enzyme substrate complexes. The low residual activity measured in air-dried soil may owe its stability to an association with soil colloids or with recalcitrant cellulosic material present in soil.     

The effect of artificial acid rain on respiration and cellulase activity in Norway spruce needle litter

Treatment of Norway spruce forest with artificial acid rain at extreme pH values decreased the pH of the needle litter. Extractable cellulase activity showed a low correlation with litter pH and none with moisture. The respiration was correlated with a second degree polynomial of the moisture. When cellulase was included in the multiple regression, with respiration as the dependent variable, the correlation was improved. It is indicated that acid precipitation has a small effect on biological activity in the litter measured as respiration and cellulase.     

Microbial Populations and Enzyme Activities in Soil Treated with Pesticides

The effect of soil treatment with brominal (a herbicide) and theinsecticide selecron (the equivalent field rates and five-fold) on population counts of bacteria, actinomycetes and celluloyticfungi in soil was tested throughout 10 weeks incubation at28 °C. Also, tested their effect on four soil enzymes:cellulase, acid phosphatase, alkaline phosphatase andarylsulphatase. Bacterial and actinomycetes populations in soiltreated with the two pesticides were promoted at fieldapplication rates and inhibited at higher levels. The twopesticides significantly decreased the total number ofcellulolytic fungi and most fungal species after most incubationperiods either by one or the two used levels but the effect ofselecron was more pronounced.Cellulase activity in soil treated with brominal and selecronwas inhibited after most incubation periods. The effect of soiltreatment with the two pesticides on acid phosphatase waspromotive at field application rates after some incubationperiods but the enzyme activity was delayed at the higherapplication doses. Alkaline phosphatase activity in treated soilwas accelerated with both pesticides even at the higherapplication rates, suggesting a direct role of alkaline soil pHin increasing resistance of alkaline phosphatase to pesticides.The effect of soil treatment with pesticides on arylsulphataseactivity fluctuated between promotion and inhibition, butinhibition was predominant.