Different temperature sensitivity and kinetics of soil enzymes indicate seasonal shifts in C,N and P nutrient stoichiometry in acid forest soil

Acid forest soils in the Bohemian Forest in Central Europe are biogeochemically imbalanced in organic C, N and P processing. We hypothesized that these imbalances can be due to different temperature sensitivities of soil enzyme activities and their affinities to substrate in litter and organic soil horizons. We measured potential activities of five main soil enzymes (β-glucosidase, cellobiohydrolase, Leu-aminopeptidase, Ala-aminopeptidase, and phosphatase) responsible for organic carbon, nitrogen and phosphorus acquisition. We also modeled potential in situ enzyme activities and nutrient release based on continuous in situ temperature measurements. We determined basic kinetic parameters (K_m, V_max), enzyme efficiencies (k_cat) and temperature sensitivities (E_a and Q₁₀) according to Michaelis–Menten kinetic and modified Arrhenius models. Our results showed significant differences in substrate affinities between the litter and organic soil horizons. Higher aminopeptidase affinity (lower K_m) in the litter soil horizon can lead to leaching of peptidic compounds to lower soil horizons. β-Glucosidase and phosphatase showed high temperature response following the Arrhenius model. However, both aminopeptidases showed no or even decreased activity with increasing temperature. The aminopeptidase temperature insensitivity means that peptidic compounds are degraded at the same or even lower rate in warmer and colder periods of the year in acid forest soils. This imbalance results in different release of available nutrients from plant litter and soil organic matter which may affect bacterial and fungal community composition and nutrient leaching from these ecosystems.     

Enzyme activities of aerobic lignocellulolytic bacteria isolated from wet tropical forest soils

Lignocellulolytic bacteria have promised to be a fruitful source of new enzymes for next-generation lignocellulosic biofuel production. Puerto Rican tropical forest soils were targeted because the resident microbes decompose biomass quickly and to near-completion. Isolates were initially screened based on growth on cellulose or lignin in minimal media. 75 Isolates were further tested for the following lignocellulolytic enzyme activities: phenol oxidase, peroxidase, β-d-glucosidase, cellobiohydrolase, β-xylopyranosidase, chitinase, CMCase, and xylanase. Cellulose-derived isolates possessed elevated β-d-glucosidase, CMCase, and cellobiohydrolase activity but depressed phenol oxidase and peroxidase activity, while the contrary was true of lignin isolates, suggesting that these bacteria are specialized to subsist on cellulose or lignin. Cellobiohydrolase and phenol oxidase activity rates could classify lignin and cellulose isolates with 61% accuracy, which demonstrates the utility of model degradation assays. Based on 16S rRNA gene sequencing, all isolates belonged to phyla dominant in the Puerto Rican soils, Proteobacteria, Firmicutes, and Actinobacteria, suggesting that many dominant taxa are capable of the rapid lignocellulose degradation characteristic of these soils. The isolated genera Aquitalea, Bacillus, Burkholderia, Cupriavidus, Gordonia, and Paenibacillus represent rarely or never before studied lignolytic or cellulolytic species and were undetected by metagenomic analysis of the soils. The study revealed a relationship between phylogeny and lignocellulose-degrading potential, supported by Kruskal–Wallis statistics which showed that enzyme activities of cultivated phyla and genera were different enough to be considered representatives of distinct populations. This can better inform future experiments and enzyme discovery efforts.     

氮添加对湿地松林土壤水解酶和氧化酶活性的影响

通过3个水平野外氮添加控制试验(0、40、120 kg N·hm^-2·a^-1),研究氮添加对亚热带湿地松林土壤水解酶和氧化酶活性的影响.结果表明: 氮添加显著抑制了土壤有机质中碳、氮、磷水解酶和氧化酶的活性,导致β-1,4-葡糖苷酶(BG)、纤维素二糖水解酶(CBH)、β-1,4-乙酰基-葡糖胺糖苷酶(NAG)、过氧化物酶(PER)活性下降16.5%～51.1%,并且高水平氮添加对酶活性抑制效果更明显;氮添加导致α-1,4-葡糖苷酶(aG)、β-1,4-木糖苷酶(BX)、酸性磷酸酶(AP)、多酚氧化酶(PPO)活性降低14.5%～38.6%,不同水平氮添加处理间差异不显著.土壤酶活性存在明显的季节性差异,BG、NAG、BX、CBH、AP、PPO活性表现为3月>6月>10月,aG、PER活性表现为10月>3月>6月.多数土壤水解酶和氧化酶与pH呈显著正相关,与NO₃^--N含量呈显著负相关,表明氮添加导致pH降低和土壤中硝化作用增强,抑制了土壤水解酶和氧化酶活性.氮添加不利于亚热带土壤有机质的矿化和周转,并且随着氮添加量的增加,效果更明显.     

Priming and microbial nutrient limitation in lowland tropical forest soils of contrasting fertility

Priming is an increase in soil organic carbon decomposition following input of labile organic carbon. In temperate soils where biological activity is limited commonly by nitrogen availability, priming is expected to occur through microbial acquisition of nitrogen from organic matter or stimulated activity of recalcitrant-carbon degrading microorganisms. However, these priming mechanisms have not yet been assessed in strongly weathered tropical forest soils where biological activity is often limited by the availability of phosphorus. We examined whether microbial nutrient limitation or community dynamics drive priming in three lowland tropical forest soils of contrasting fertility (‘low’, ‘mid’ and ‘high’) by applying C₄-sucrose (alone or in combination with nutrients; nitrogen, phosphorus and potassium) and measuring (1) the δ¹³C-signatures in respired CO₂ and in phospholipid fatty acid (PLFA) biomarkers, and (2) the activities of enzymes involved in nitrogen (N-acetyl β-glucosaminidase), phosphorus (phosphomonoesterase) and carbon (β-glucosidase, cellobiohydrolase, xylanase, phenol oxidase) acquisition from organic compounds. Priming was constrained in part by nutrient availability, because priming was greater when sucrose was added alone compared to when added with nutrients. However, the greatest priming with sucrose addition alone was detected in the medium fertility soil. Priming occurred in parallel with stimulated activity of phosphomonoesterase and phenol oxidase (but not N-acetyl β-glucosaminidase); when sucrose was added with nutrients there were lower activities of phosphomonoesterase and phenol oxidase. There was no evidence according to PLFA δ¹³C-incorporation that priming was caused by specific groups of recalcitrant-carbon degrading microorganisms. We conclude that priming occurred in the intermediate fertility soil following microbial mineralization of organic nutrients (phosphorus in particular) and suggest that priming was constrained in the high fertility soil by high nutrient availability and in the low fertility soil by the low concentration of soil organic matter amenable to priming. This first study of priming mechanisms in tropical forest soils indicates that input of labile carbon can result in priming by microbial mineralization of organic nutrients, which has important implications for understanding the fate of organic carbon in tropical forest soils.     

Denitrification in Low pH Spodosols and Peats Determined with the Acetylene Inhibition Method

Potential denitrification rates were determined for predominantly acid (pH ≥ 3.6) horizons of forestal, miry, and agricultural soils from 22 locations in southern Finland. The acetylene inhibition method was used with nitrate-amended water-logged soils incubated in an N₂ atmosphere containing 2.5 or 5% C₂H₂. Complete inhibition of the reduction of N₂O to N₂ was observed in 99.3% of the samples. The denitrification rates varied from 0.12 to 53.8 μg of N·cm^-3·day^-1. Correlation between denitrification rate and soil pH was highly significant: r = 0.84 on a volume basis, and r = 0.44 on a weight basis. Vegetation type and amount of soil organic matter had a minor or no effect, respectively. In spodosolized soils the rates were significantly higher for B horizons than for A horizons. These results show that denitrification can occur in acid soils.     

Effects of dissolved organic matter from different plant sources on soil enzyme activities in subtropical forests

探究不同植物来源可溶性有机质(DOM)进入土壤后对酶活性的影响, 可以为降水淋溶下亚热带地区不同森林生态系统土壤碳循环提供科学依据。该研究提取杉木(Cunninghamia lanceolata)、木荷(Schima superba)和楠木(Phoebe zherman) 3种植物鲜叶中的DOM分别输入杉木人工林土壤中, 以等量的去离子水添加为对照, 进行25天的室内培养。培养结束后测定土壤理化性质、微生物生物量和酶活性等指标。结果表明: 与对照处理(CT)相比, 添加3种叶片DOM后, 土壤总有机碳(SOC)、总氮(TN)含量和碳氮比均无显著变化。杉木叶片DOM添加处理(CL)的TN含量显著低于木荷叶片DOM添加处理(SL)和楠木叶片DOM添加处理(PL), 碳氮比显著高于SL和PL。3种叶片DOM输入整体上提高了土壤溶解有机碳(DOC)和溶解有机氮(DON)的含量。叶片DOM输入后土壤微生物生物量碳(MBC)含量无显著变化, 然而CL和SL的土壤微生物生物量氮(MBN)含量分别比CT降低了50.9%和51.1%, PL的MBN含量比CT提高了54.0%。与CT相比, 不同植物来源DOM输入后, β-葡萄糖苷酶(βG)、纤维素水解酶(CBH)和过氧化物酶(PEO) 3种酶活性均显著上升, 而多酚氧化酶(PPO)活性则显著下降; 此外, βG和CBH活性均表现出CL > SL > PL的特征。相关性分析的结果表明, 添加叶片DOM 3种处理的SOC、TN、MBN含量和βG、CBH活性都与所输入DOM的DOC含量和腐殖化指数(HIX)显著相关, 此外, 土壤MBN含量和PPO活性与输入叶片DOM的pH呈正相关关系。冗余分析(RDA)结果表明, 叶片DOM输入后引起土壤酶活性变化的关键因子是DON和DOC含量。总体来说, 不同植物来源DOM性质的差异会影响土壤碳循环水解酶的活性, 而叶片DOM输入后增加了土壤碳和氮的有效性, 引起4种碳循环酶的不同响应。     

不同植物来源可溶性有机质对亚热带森林土壤酶活性的影响

探究不同植物来源可溶性有机质(DOM)进入土壤后对酶活性的影响, 可以为降水淋溶下亚热带地区不同森林生态系统土壤碳循环提供科学依据。该研究提取杉木(Cunninghamia lanceolata)、木荷(Schima superba)和楠木(Phoebe zherman) 3种植物鲜叶中的DOM分别输入杉木人工林土壤中, 以等量的去离子水添加为对照, 进行25天的室内培养。培养结束后测定土壤理化性质、微生物生物量和酶活性等指标。结果表明: 与对照处理(CT)相比, 添加3种叶片DOM后, 土壤总有机碳(SOC)、总氮(TN)含量和碳氮比均无显著变化。杉木叶片DOM添加处理(CL)的TN含量显著低于木荷叶片DOM添加处理(SL)和楠木叶片DOM添加处理(PL), 碳氮比显著高于SL和PL。3种叶片DOM输入整体上提高了土壤溶解有机碳(DOC)和溶解有机氮(DON)的含量。叶片DOM输入后土壤微生物生物量碳(MBC)含量无显著变化, 然而CL和SL的土壤微生物生物量氮(MBN)含量分别比CT降低了50.9%和51.1%, PL的MBN含量比CT提高了54.0%。与CT相比, 不同植物来源DOM输入后, β-葡萄糖苷酶(βG)、纤维素水解酶(CBH)和过氧化物酶(PEO) 3种酶活性均显著上升, 而多酚氧化酶(PPO)活性则显著下降; 此外, βG和CBH活性均表现出CL > SL > PL的特征。相关性分析的结果表明, 添加叶片DOM 3种处理的SOC、TN、MBN含量和βG、CBH活性都与所输入DOM的DOC含量和腐殖化指数(HIX)显著相关, 此外, 土壤MBN含量和PPO活性与输入叶片DOM的pH呈正相关关系。冗余分析(RDA)结果表明, 叶片DOM输入后引起土壤酶活性变化的关键因子是DON和DOC含量。总体来说, 不同植物来源DOM性质的差异会影响土壤碳循环水解酶的活性, 而叶片DOM输入后增加了土壤碳和氮的有效性, 引起4种碳循环酶的不同响应。     

Synergistic Saccharification, and Direct Fermentation to Ethanol, of Amorphous Cellulose by Use of an Engineered Yeast Strain Codisplaying Three Types of Cellulolytic Enzyme

A whole-cell biocatalyst with the ability to induce synergistic and sequential cellulose-degradation reaction was constructed through codisplay of three types of cellulolytic enzyme on the cell surface of the yeast Saccharomyces cerevisiae. When a cell surface display system based on α-agglutinin was used, Trichoderma reesei endoglucanase II and cellobiohydrolase II and Aspergillus aculeatus β-glucosidase 1 were simultaneously codisplayed as individual fusion proteins with the C-terminal-half region of α-agglutinin. Codisplay of the three enzymes on the cell surface was confirmed by observation of immunofluorescence-labeled cells with a fluorescence microscope. A yeast strain codisplaying endoglucanase II and cellobiohydrolase II showed significantly higher hydrolytic activity with amorphous cellulose (phosphoric acid-swollen cellulose) than one displaying only endoglucanase II, and its main product was cellobiose; codisplay of β-glucosidase 1, endoglucanase II, and cellobiohydrolase II enabled the yeast strain to directly produce ethanol from the amorphous cellulose (which a yeast strain codisplaying β-glucosidase 1 and endoglucanase II could not), with a yield of approximately 3 g per liter from 10 g per liter within 40 h. The yield (in grams of ethanol produced per gram of carbohydrate consumed) was 0.45 g/g, which corresponds to 88.5% of the theoretical yield. This indicates that simultaneous and synergistic saccharification and fermentation of amorphous cellulose to ethanol can be efficiently accomplished using a yeast strain codisplaying the three cellulolytic enzymes.     

Interactive effects of wildfire and permafrost on microbial communities and soil processes in an Alaskan black spruce forest

Boreal forests contain significant quantities of soil carbon that may be oxidized to CO₂ given future increases in climate warming and wildfire behavior. At the ecosystem scale, decomposition and heterotrophic respiration are strongly controlled by temperature and moisture, but we questioned whether changes in microbial biomass, activity, or community structure induced by fire might also affect these processes. We particularly wanted to understand whether postfire reductions in microbial biomass could affect rates of decomposition. Additionally, we compared the short‐term effects of wildfire to the long‐term effects of climate warming and permafrost decline. We compared soil microbial communities between control and recently burned soils that were located in areas with and without permafrost near Delta Junction, AK. In addition to soil physical variables, we quantified changes in microbial biomass, fungal biomass, fungal community composition, and C cycling processes (phenol oxidase enzyme activity, lignin decomposition, and microbial respiration). Five years following fire, organic surface horizons had lower microbial biomass, fungal biomass, and dissolved organic carbon (DOC) concentrations compared with control soils. Reductions in soil fungi were associated with reductions in phenol oxidase activity and lignin decomposition. Effects of wildfire on microbial biomass and activity in the mineral soil were minor. Microbial community composition was affected by wildfire, but the effect was greater in nonpermafrost soils. Although the presence of permafrost increased soil moisture contents, effects on microbial biomass and activity were limited to mineral soils that showed lower fungal biomass but higher activity compared with soils without permafrost. Fungal abundance and moisture were strong predictors of phenol oxidase enzyme activity in soil. Phenol oxidase enzyme activity, in turn, was linearly related to both ¹³C lignin decomposition and microbial respiration in incubation studies. Taken together, these results indicate that reductions in fungal biomass in postfire soils and lower soil moisture in nonpermafrost soils reduced the potential of soil heterotrophs to decompose soil carbon. Although in the field increased rates of microbial respiration can be observed in postfire soils due to warmer soil conditions, reductions in fungal biomass and activity may limit rates of decomposition.     

Stoichiometry of soil enzyme activity at global scale

Extracellular enzymes are the proximate agents of organic matter decomposition and measures of these activities can be used as indicators of microbial nutrient demand. We conducted a global-scale meta-analysis of the seven-most widely measured soil enzyme activities, using data from 40 ecosystems. The activities of beta-1,4-glucosidase, cellobiohydrolase, beta-1,4-N-acetylglucosaminidase and phosphatase g(-1) soil increased with organic matter concentration; leucine aminopeptidase, phenol oxidase and peroxidase activities showed no relationship. All activities were significantly related to soil pH. Specific activities, i.e. activity g(-1) soil organic matter, also varied in relation to soil pH for all enzymes. Relationships with mean annual temperature (MAT) and precipitation (MAP) were generally weak. For hydrolases, ratios of specific C, N and P acquisition activities converged on 1 : 1 : 1 but across ecosystems, the ratio of C : P acquisition was inversely related to MAP and MAT while the ratio of C : N acquisition increased with MAP. Oxidative activities were more variable than hydrolytic activities and increased with soil pH. Our analyses indicate that the enzymatic potential for hydrolyzing the labile components of soil organic matter is tied to substrate availability, soil pH and the stoichiometry of microbial nutrient demand. The enzymatic potential for oxidizing the recalcitrant fractions of soil organic material, which is a proximate control on soil organic matter accumulation, is most strongly related to soil pH. These trends provide insight into the biogeochemical processes that create global patterns in ecological stoichiometry and organic matter storage.     

Ferrous Iron Stimulates Phenol Oxidase Activity and Organic Matter Decomposition in Waterlogged Wetlands

Soil organic matter decomposition is limited at waterlogged conditions by the low activity of extracellular enzymes like phenol oxidases. In this paper, we show that ferrous iron (Fe²⁺), which is abundant in waterlogged soils, significantly stimulates phenol oxidase activity both in pure enzyme assays and in waterlogged soil slurries from nutrient-poor dune slacks. However, the effects in soil slurries were less strong than in enzyme assays. Both the addition of Fe²⁺ and the initial presence of Fe²⁺ stimulated phenol oxidase activity at the microaerophilic conditions tested. This stimulation is attributed to the catalysis of additional OH radical production, promoting the oxidation of phenolics. Subsequently, the presence of Fe²⁺ strongly increased total decomposition rates of soil organic matter, measured as CO₂ production and Cotton strip Tensile Strength Loss. There is circumstantial evidence that this stimulation by Fe²⁺ could be important for decomposition in wetlands at field conditions, but its relevance compared to the effects of other compounds still needs to be elucidated. These results emphasise the crucial role of water quality in determining extracellular enzyme activity and decomposition in waterlogged wetlands.     

Using hydrogel and clay to improve the water status of seedlings for dryland restoration

The decomposition of soil organic matter is mediated by extracellular enzymes. The aim of this work was to identify the factors determining the activity and size of the mobile fraction of extracellular enzymes (laccase, Mn-peroxidase, endocellulase, cellobiohydrolase, ??-glucosidase, endoxylanase, ??-xylosidase, ??-glucosidase, chitinase, arylsulfatase, phosphatase, phosphodiesterase, alanine and leucine aminopeptidase) using a set of soils covering a wide range of physico-chemical properties. Organic matter content had a major effect on enzyme activity both in forest and grassland soils, while the effects of pH and humic compounds content were only important in forest soils, and the molecular mass of humic compounds and Ca content were only important in grasslands. Specific enzyme activity was either comparable between the soil types or higher in grasslands. With the exception of Mn-peroxidase and ??-glucosidase, the specific activities of all enzymes in arable fields under tillage were similar to those in grasslands. Mobility differed among the enzymes and ranged from <1% for arylsulfatase and phosphodiesterase up to 20?C40% for ??-glucosidase and aminopeptidases, with pH being the most important variable. These results demonstrate that the factors regulating enzyme activity are likely to be different in forest soils and grasslands and that enzyme mobility is a characteristic feature of each individual enzyme.     

Glomalin, an arbuscular-mycorrhizal fungal soil protein, responds to land-use change

Glomalin is a soil proteinaceous substance produced by arbuscular mycorrhizal fungi. Most of the information available concerning this protein has been collected in relation to its role in soil aggregation. In this study, we explored the distribution of glomalin across soil horizons, decomposition of glomalin, and relationship with soil C and N in an agricultural field, a native forest, and an afforested system. Glomalin was present in A, B, and C horizons in decreasing concentrations. Land-use type significantly affected glomalin concentrations (mg cm^–3), with native forest soils having the highest concentrations of the three land-use types in both A and B horizons. In terms of glomalin stocks (Mg ha^–1), calculated based on corrected horizon weights, the agricultural area was significantly lower than both afforested and native forest areas. As measured after a 413 day laboratory soil incubation, glomalin was least persistent in the A horizon of the afforested area.. In agricultural soils and native soils, ca. 50% of glomalin was still remaining after this incubation, indicating that some glomalin may be in the slow or recalcitrant soil C fraction. Comparison of glomalin decomposition with CO₂-C respired during incubation indicates that glomalin makes a large contribution to active soil organic C pools. Soil C and N were highly correlated with glomalin across all soils and within each land-use type, indicating that glomalin may be under similar controls as soil C. Our results show that glomalin may be useful as an indicator of land-use change effects on deciduous forest soils.     

Production and Distribution of Endoglucanase, Cellobiohydrolase, and β-Glucosidase Components of the Cellulolytic System of Volvariella volvacea, the Edible Straw Mushroom

The edible straw mushroom, Volvariella volvacea, produces a multicomponent enzyme system consisting of endo-1,4-β-glucanase, cellobiohydrolase, and β-glucosidase for the conversion of cellulose to glucose. The highest levels of endoglucanase and cellobiohydrolase were recorded in cultures containing microcrystalline cellulose (Avicel) or filter paper, while lower but detectable levels of activity were also produced on carboxymethyl cellulose, cotton wool, xylitol, or salicin. Biochemical analyses of different culture fractions in cultures exhibiting peak enzyme production revealed that most of the endoglucase was present either in the culture filtrate (45.8% of the total) or associated with the insoluble pellet fraction remaining after centrifugation of homogenized mycelia (32.6%). Cellobiohydrolase exhibited a similar distribution pattern, with 58.9% of the total enzyme present in culture filtrates and 31.0% associated with the pellet fraction. Conversely, most β-glucosidase activity (63.9% of the total) was present in extracts of fungal mycelia whereas only 9.4% was detected in culture filtrates. The endoglucanase and β-glucosidase distribution patterns were confirmed by confocal laser scanning microscopy combined with immunolabelling. Endoglucanase was shown to be largely cell wall associated or located extracellularly, with the highest concentrations being present in a region 1 to 2 μm wide immediately adjacent to the outer surface of (and possibly including) the hyphal wall and extending 60 to 70 μm from the hyphal tip. Immunofluorescence patterns indicated little if any intracellular endoglucanase. Most β-glucosidase was located intracellularly in the apical area extending 60 to 70 μm below the hyphal tip, although enzyme was also evident in the extracellular region extending approximately 15 μm all around the hyphal tip and trailing back along the length of the hypha. The regions of the hypha located some distance from the apical region appeared to be devoid of intracellular β-glucosidase, and the enzyme appears to be associated almost exclusively with, or located on the outside surface of, the hyphal wall.     

A digestive beta-glucosidase from the silkworm, Bombyx mori: cDNA cloning, expression and enzymatic characterization

A digestive β-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori β-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori β-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori β-glucosidase to be a single gene. Northern blot analysis of B. mori β-glucosidase gene confirmed larval midgut-specific expression. The B. mori β-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori β-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant β-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori β-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant β-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per μg of recombinant B. mori β-glucosidase. The purified recombinant B. mori β-glucosidase showed the highest activity at 35 °C and pH 6.0, and were stable at 50 °C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori β-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.     

Washingtonia filifera seed extracts inhibit the islet amyloid polypeptide fibrils formations and α-amylase and α-glucosidase activity

Washingtonia filifera seeds have revealed to possess antioxidant properties, butyrylcholinesterase and xanthine oxidase inhibition activities. The literature has indicated a relationship between Alzheimer’s disease (AD) and type-2 diabetes (T2D). Keeping this in mind, we have now evaluated the inhibitory properties of W. filifera seed extracts on α-amylase, α-glucosidase enzyme activity and the Islet Amyloid Polypeptide (IAPP) fibrils formation.Three extracts from seeds of W. filifera were evaluated for their enzyme inhibitory effect and IC₅₀ values were calculated for all the extracts. The inhibition mode was investigated by Lineweaver-Burk plot analysis and the inhibition of IAPP aggregate formation was monitored.W. filifera methanol seed extract appears as the most potent inhibitor of α-amylase, α-glucosidase, and for the IAPP fibril formation.Current findings indicate new potential of this extract that could be used for the identification or development of novel potential agents for T2D and AD.     

The purification and some properties of the polyphenol oxidase from tea (Camellia sinensis L.)

1. Polyphenol oxidase (EC 1. 10. 3.–) from the shoots of the tea plant was purified about 5000-fold on a dry-weight basis. 2. At an intermediate stage of purification four soluble yellow fractions were obtained. They are believed to represent complexes of a basic enzyme protein with acidic phenolic oxidation products and nucleic acids. After removal of the complex-forming materials the fractions were blue and similar to each other. About 40% of the activity could not be extracted from the acetone-dried powder. 3. Each of the four blue fractions was resolved further into two species, A and B. The following results refer to species A. 4. The enzyme showed absorption maxima at 279mμ (E^1%_1cm., 13·5) and 611mμ (E^1%_1cm., 0·84) with a shoulder at 330mμ. The enzyme was bleached by substrate under anaerobic conditions and the colour was restored by oxygen. 5. The molecular weight measured by sedimentation and diffusion was 144000±16000. The copper content was 0·32% (w/w). 6. Kinetic constants are given for a number of substrates and inhibitors, including the natural substrates of the tea leaf. The specific activity towards pyrogallol was 373 units/mg. at 30°. 7. The best substrates were o-dihydric phenols. Quinol and p-phenylenediamine were slowly oxidized. Monohydric phenols and ascorbic acid were not oxidized. 8. The kinetics of oxidation of most substrates are consistent with a mechanism in which oxidized and reduced forms of the enzyme form binary complexes with phenol and oxygen respectively. A modified mechanism is postulated for the oxidation of chlorogenic acid. 9. The relation of the results to the mechanism of tea fermentation is discussed.     

Microbial Community Structure and Oxidative Enzyme Activity in Nitrogen-amended North Temperate Forest Soils

Large regions of temperate forest are subject to elevated atmospheric nitrogen (N) deposition which can affect soil organic matter dynamics by altering mass loss rates, soil respiration, and dissolved organic matter production. At present there is no general model that links these responses to changes in the organization and operation of microbial decomposer communities. Toward that end, we studied the response of litter and soil microbial communities to high levels of N amendment (30 and 80 kg ha^–1 yr^–1) in three types of northern temperate forest: sugar maple/basswood (SMBW), sugar maple/red oak (SMRO), and white oak/black oak (WOBO). We measured the activity of extracellular enzymes (EEA) involved directly in the oxidation of lignin and humus (phenol oxidase, peroxidase), and indirectly, through the production of hydrogen peroxide (glucose oxidase, glyoxal oxidase). Community composition was analyzed by extracting and quantifying phospholipid fatty acids (PLFA) from soils. Litter EEA responses at SMBW sites diverged from those at oak-bearing sites (SMRO, BOWO), but the changes were not statistically significant. For soil, EEA responses were consistent across forests types: phenol oxidase and peroxidase activities declined as a function of N dose (33–73% and 5–41%, respectively, depending on forest type); glucose oxidase and glyoxal oxidase activities increased (200–400% and 150–300%, respectively, depending on forest type). Principal component analysis (PCA) ordinated forest types and treatment responses along two axes; factor 1 (44% of variance) was associated with phenol oxidase and peroxidase activities, factor 2 (31%) with glucose oxidase. Microbial biomass did not respond to N treatment, but nine of the 23 PLFA that formed >1 mol% of total biomass showed statistically significant treatment responses. PCA ordinated forest types and treatment responses along three axes (36%, 26%, 12% of variance). EEA factors 1 and 2 correlated negatively with PLFA factor 1 (r = –0.20 and –0.35, respectively, n = 108) and positively with PLFA factor 3 (r = +0.36 and +0.20, respectively, n = 108). In general, EEA responses were more strongly tied to changes in bacterial PLFA than to changes in fungal PLFA. Collectively, our data suggests that N inhibition of oxidative activity involves more than the repression of ligninase expression by white-rot basidiomycetes.This revised version was published online in November 2004 with corrections to Volume 48.     

Some kinetic properties of polyphenol oxidase obtained from dill (Anethum graveolens)

Polyphenol oxidase (PPO) was partially purified from dill by (NH₄)₂SO₄ precipitation followed by dialysis and gel filtration chromatography. Polyphenol oxidase activity was measured spectrophotometrically at 420 nm using catechol, dopamine and chlorogenic acid as substrates. Optimum pH, temperature, and ionic strength were determined with three substrates. The best substrate of dill PPO was found to be chlorogenic acid. Some kinetic properties of the enzyme such as V_max, K_M and V_max/K_M were determined for all three substrates. The effects of various inhibitors on the reaction catalysed by the enzyme were tested and I₅₀ values calculated. The most effective inhibitor was l-cysteine. Activation energies, E_a, were determined from the Arrhenius equation. In addition, activation enthalpy, ΔH_a, and Q₁₀ values of the enzyme were also calculated.     

Vertical distribution of biological and geochemical phosphorus subcycles in two southern Appalachian forest soils

We measured Al, Fe, and P fractions by horizon in two southern Appalachian forest soil profiles, and compared solution PO₄ ^–1 removal in chloroform-sterilized and non-sterilized soils, to determine whether biological and geochemical P subcycles were vertically stratified in these soils. Because organic matter can inhibit Al and Fe oxide crystallization, we hypothesized that concentrations of non-crystalline (oxalate-extractable) Al (Al₀) and Fe (Fe₀), and concomitantly P sorption, would be greatest in near-surface mineral (A) horizons of these soils.Al₀ and Fe₀ reached maximum concentrations in forest floor and near-surface mineral horizons, declined significantly with depth in the mineral soil, and were highly correlated with P sorption capacity. Small pools of readily acid-soluble (AF-extractable) and readily-desorbable P suggested that PO₄ ^3– was tightly bound to Al and Fe hydroxide surfaces. P sorption in CHCl₃-sterilized mineral soils did not differ significantly from P sorption in non-sterilized soils, but CHCl₃ sterilization reduced P sorption 40–80% in the forest floor. CHCl₃ labile (microbial) P also reached maximum concentrations in forest floor and near-surface mineral horizons, comprising 31–35% of forest floor organic P. Combined with previous estimates of plant root distributions, data suggest that biological and geochemical P subcycles are not distinctly vertically stratified in these soils. Plant roots, soil microorganisms, and P sorbing minerals all reach maximum relative concentrations in near-surface mineral horizons, where they are likely to compete strongly for PO₄ ^3– available in solution.