Enzyme activity in soil: Location and a possible role in microbial ecology

The activity of any particular enzyme in soil is a composite of activities associated with various biotic and abiotic components, e.g. proliferating cells, latent cells, cell debris, clay minerals, humic colloids and the soil aqueous phase. The location of the enzyme is at least partially determined by such factors as the size and solubility of its substrate, the species of microorganism, and the physical and chemical nature of the soil colloids. However, enzymes may change location with time, for example, many hydrolases are intracellular sensu stricto but are also found associated with cell debris and clay and organic colloids. There are difficulties in quantifying the various activities, but this may be possible by employing different types of assays, the prudent use of controls and the study of crude enzyme extracts from soil.Enzymes bound to clay and humic colloids (the immobilized or accumulated enzyme fraction) have a residual activity not found in enzymes free in the soil aqueous phase. However, the mere adsorption of enzymes to soil surfaces does not guarantee subsequent activity, and it appears that some mechanism of association with the humic polymer offers the best form of protection, yet permits the retention of enzyme activity.The catalytic activity of extracellular enzymes is discussed and a possible relationship between soil microorganisms, exogenous substrates and immobilized enzymes is suggested.     

Activity and stability of urease-hydroxyapatite and urease-hydroxyapatite-humic acid complexes

In agricultural calcareous soils, hydroxyapatite (APA) may well represent an important support for urease immobilisation and could be present in both mineral and organo-mineral complexes. In this paper we studied the formation of APA-urease-humic acid (HA) complexes after the addition of urease either before or after HAs. We then proceeded to evaluate the role of HAs on the activity and stability of the complexes as compared to the APA-urease complexes and free urease. When increasing amounts of HAs were added after urease, they did not affect the activity of the enzymes that had already adsorbed onto the complexes. On the contrary, adding the same amount of HA before the enzyme caused a significant reduction in the amount of enzyme adsorbed. However, when urease adsorption onto the APA-HA complexes was carried out in the presence of NaCl, the enzyme activity of the complexes increased sharply to 86% of the initial activity. The immobilisation of the enzyme on the support increased urease stability against pronase treatment as well as directly in soil over time. The inhibition of urease activity by Cu ²⁺was reduced by urease immobilisation. However, the presence of HA did not alter the stabilisation capability of APA when alone.     

Inhibitory effects of plant phenols on the activity of selected enzymes

Selected enzymes (alpha-amylase, trypsin, and lysozyme) were allowed to react with some simple phenolic and related compounds (caffeic acid, chlorogenic acid, ferulic acid, gallic acid, m-, o-, and p-dihydroxybenzenes, quinic acid, and p-benzoquinone). The derivatized enzymes obtained were characterized in terms of their activity. In vitro experiments showed that the enzymatic activity of the derivatives was adversely affected. This enzyme inhibition depended on the reactivity of the phenolic and related substances tested as well as on the kind of substrate applied. The decrease in the activity was accompanied by a reduction in the amount of free amino and thiol groups, as well as tryptophan residues, which resulted from the covalent attachment of the phenolic and related compounds to these reactive nucleophilic sites in the enzymes. The enzyme inhibition correlates well with the blocking of the mentioned amino acid side chains.     

A theoretical model of C- and N-acquiring exoenzyme activities,which balances microbial demands during decomposition

We developed an Extracellular EnZYme model (EEZY) of decomposition that produces two separate pools of C- and N-acquiring enzymes, that in turn hydrolyze two qualitatively different substrates, one containing only C (e.g., cellulose) and the other containing both C and N (e.g., chitin or protein). Hence, this model approximates the actions of commonly measured indicator enzymes ß-1,4-glucosidase and ß-1,4-N-acetylglucosaminidase (or leucine aminopeptidase) as they hydrolyze cellulose and chitin (or protein), respectively. EEZY provides an analytical solution to the allocation of these two enzymes, which in turn release C and N from the two substrates to maximize microbial growth. Model behaviors were both qualitatively and quantitatively consistent with patterns of litter decay generated by other decomposition models. However, EEZY demonstrated greater sensitivity to the C:N of individual substrate pools in addition to responding to factors directly affecting enzyme activity. Output approximated field observations of extracellular enzyme activities from studies of terrestrial soils, aquatic sediments, freshwater biofilm and plankton communities. Although EEZY is largely a theoretical model, simulated C- and N-acquiring enzyme activities approximated a 1:1 ratio, consistent with the bulk of these field observations, only when the N-containing substrate had a C:N ratio similar to commonly occurring substrates (e.g., proteins or chitin). This result supported the emerging view of the stoichiometry of extracellular enzyme activities from an environmental context, which suggests that a relatively narrow range of microbial C:N, carbon use efficiency and soil/sediment organic matter C:N across ecosystems explains the tendency towards this 1:1 ratio of enzyme activities associated with C- and N-acquisition. Sensitivity analyses indicated that simulated extracellular enzyme activity was most responsive to variations in carbon use efficiency of microorganisms, although kinetic characteristics of enzymes also had significant impacts. Thus EEZY provides a quantitative framework in which to interpret mechanisms underlying empirical patterns of extracellular enzyme activity.     

A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R. Br.: purification and biochemical properties

Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.     

Effects of wood char and activated carbon on the hydrolysis of cellobiose by β-glucosidase from Aspergillus niger

Biochar amendments to soils have been suggested as a strategy to sequester carbon and therefore mitigate global climate change. The enrichment of soils with charred materials also increases their fertility. This fertilising effect of biochar may be caused by various mechanisms; an acceleration of nutrient cycling has been suggested as one such mechanism. The rate-limiting step in nutrient cycling is thought to be the extracellular enzymatic attack on biological macromolecules. In this study, therefore, the effects of chestnut wood char (specific surface area 2.0 m² g⁻¹) and of activated carbon (specific surface area approximately 900 m² g⁻¹) on an extracellular enzymatic reaction involved in the degradation of cellulose (i.e., hydrolysis of cellobiose by β-glucosidase from Aspergillus niger) were investigated. Cellobiose was not adsorbed by chestnut wood char, whereas activated carbon absorbed more than 97% of it. Both charred materials adsorbed more than 99% of β-glucosidase. For chestnut wood char, adsorption of the enzyme caused a decrease of approximately 30% in the reaction rate, whereas for activated carbon, the nearly complete absorption of both substrate and enzyme entirely inhibited the reaction. These results show that β-glucosidase from A. niger retains most of its activity when adsorbed to chestnut wood char and that the reaction it catalyses in nature is only slightly affected by this charred material. On the other hand, a material characterised by a high specific surface area and high porosity, such as activated carbon, can make even a highly soluble substrate unavailable for soil enzymes and therefore completely inhibit the reaction. Thus, charred materials may affect nutrient cycling mainly by regulating the availability of substrates: the degradation of highly soluble substrates may be accelerated by materials with low specific surface area, which maintain an active and protected enzyme pool, whereas materials with high specific surface and high porosity may slow down the degradation by making substrates unavailable.     

Kinetic properties and stability of potato acid phosphatase immobilized on Ca-polygalacturonate

 Acid phosphatase from potato was adsorbed and immobilized on a pre-formed network of Ca-polygalacturonate, a substrate which has a composition and morphology similar to the mucigel present at the root-soil interface. The influence of different types of organic buffers on enzyme adsorption and activity was investigated. The highest enzyme activity, for free and adsorbed enzyme, was obtained with Na-maleate buffer at pH 6.0, which was used for all subsequent experiments. The Michaelis-Menten kinetic parameters, V_max and K_m, were determined for free and adsorbed phosphatase. V_max showed a 60% decrease upon adsorption (2.09±0.30 U/mg, for the soluble form and 0.84±0.15 U/mg, for the adsorbed enzyme), whereas K_m increased from 0.49±0.15 mM for the free enzyme to 0.99±0.20 mM for adsorbed phosphatase. Phosphatase adsorption decreased as the concentration of NaCl increased, indicating that the enzyme is bound to the carrier gel through coulombic interactions. Adsorption increased stability of the enzyme as compared with the free enzyme (t _1/2 of the activity was 9.4 days and 5.8 days, respectively), but increased thermal and proteolytic inactivation. The pH/activity profile revealed no change in terms of shape or optimum pH (4.5) upon adsorption of the enzyme. These results indicate that adsorption of acid phosphatase on Ca-polygalacturonate induces changes in the kinetic properties and stability of the enzyme, and the same type of response can be extrapolated from these results for acid phosphatases of the rhizosphere. Received: 1 July 1997     

Influence of polysaccharide structure on dextran adsorption by montmorillonite

Two dextrans of similar molecular weight (?2 × 10⁶) but containing different structural linkages (B-215F: 95% α-1→ 6 and 5% α-1→ 3 and Polytran: 75% β-1→ 3 and 25% β-1→ 6) were adsorbed on Na-montmorillonite. Adsorption isotherms showed strong, H-2-type (high-affinity. Langmuir mono-layer adsorption) clay-dextran interactions for both polymers. Maximum adsorption of the Polytran dextran (60 $\text{mg}\text{100mg}$ clay) was 33 per cent greater than that of the B-512F dextran (44.5 $\text{mg}\text{100mg}$ clay) for comparative equilibrium systems. Stable clay-Polytran complexes containing up to 47% dextran were prepared. Excess Na₂SO₄ (0.1 m) did not affect the quantity of dextran adsorption by montmorillonite at the 15% dextran concentration. Acid hydrolysis and modified Pregl method-C analyses did not quantitatively recover adsorbed dextran from complexes containing more than 13 mg of dextran adsorbed per 100 mg of clay. Loss on ignition determinations were in good agreement with the difference measurements of dextran adsorption, suggesting that part of the C was expelled as something other than CO₂ in the ignition determinations. The maximum adsorption segment for Polytran was much smaller than the individual molecule. In contrast, the maximum adsorption segment for the B-512F dextran appeared to be the same magnitude as the individual polymer molecule. Adsorption segment length was regarded as a manifestation of the relative proportions of primary and secondary alcohol groups of the molecules.     

Purification and characterization of a stable cysteine protease ervatamin B, with two disulfide bridges, from the latex of Ervatamia coronaria

Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.     

Reconciling apparent variability in effects of biochar amendment on soil enzyme activities by assay optimization

We studied the effects of a biochar made from fast pyrolysis of switchgrass on four soil enzymes (β-glucosidase, β-N-acetylglucosaminidase, lipase, and leucine aminopeptidase) to determine if biochar would consistently modify soil biological activities. Thus, we conducted a series of enzyme assays on biochar-amended soils. Inconsistent results from enzyme assays of char-amended soils suggested that biochar had variable effects on soil enzyme activities, thus we conducted a second experiment to determine if biochar reacts predictably with either enzyme or substrate in in vitro reactions. Both colorimetric and fluorescent assays were used for β-glucosidase and β-N-acetylglucosaminidase. Seven days after biochar was added to microcosms of 3 different soils, fluorescence-based assays revealed some increased enzyme activities (up to 7-fold for one measure of β-glucosidase in a shrub-steppe soil) and some decreased activities (one-fifth of the unamended control for lipase measured in the same shrub-steppe soil), compared to non-amended soil. In an effort understand the varied effects, purified enzymes or substrates were briefly exposed to biochar and then assayed. In contrast to the soil assays, except for β-N-acetylglucosaminidase, the exposure of substrate to biochar reduced the apparent activity of the enzymes, suggesting that sorption reactions between substrate and biochar impeded enzyme function. Our findings indicate that fluorometric assays are more robust to, or account for, this sorption better than the colorimetric assays used herein. The activity of purified β-N-acetylglucosaminidase increased 50-75% following biochar exposure, suggesting a chemical enhancement of enzyme function. In some cases, biochar stimulates soil enzyme activities, to a much greater degree than soil assays would indicate, given that substrate reactivity can be impeded by biochar exposure. We conclude that the effects of biochar on enzyme activities in soils are highly variable; these effects are likely associated with reactions between biochar and the target substrate.     

Browning of pyrogallol as affected by clay minerals

One of the authors, Kumada(1), has presented the idea that humification must be regarded as browning phenomena of organic matter in soils. The browning reaction can be accelerated non-enzymatically as well as enzymatically, and it is considered that humification would be conducted under the influence of some catalytic actions of clay fraction, composed of various kinds of clay minerals, free oxides and electrolytes, as well as soil enzymes, under the prevailing hydrothermal conditions. This was well illustrated by Kyuma and Kawaguchi(2), who definitely demonstrated the catalytic effect of allophane on the oxidative polymerization of polyphenols.     

Microparticle dispensers for the controlled release of insect pheromones

The potential utility of micrometer-sized particles as controlled-release devices for the volatilization of insect pheromones for mating disruption applications is evaluated in this study for two pheromone/model compound systems (codlemone/1-dodecanol and disparlure/1,2-epoxyoctadecane). To expedite the measurement of release rates from these particle devices, two techniques based on thermogravimetric analysis (TGA) have been exploited: isothermal TGA (I-TGA) at elevated temperatures (40-80 degrees C) with N(2) convection and volatilization temperature (VT) by dynamic TGA. A correlation between these two methods has been established. Samples that exhibit a higher VT provide a lower release rate from a particle substrate. Using these techniques, it has been demonstrated that chemical interactions between adsorbed liquids and particle surfaces may play a small role in defining release characteristics under conditions of low surface area, whereas parameters associated with total surface area and micropore structure appear to be much more significant in retarding evaporation for uncoated particles containing an adsorbed liquid. Additional regulation of release rates was achieved by coating the particle systems with water-soluble or water-dispersible polymers. By careful selection of particle porosity and coating composition, it is envisioned that the evaporation rate of pheromones can be tailored to specific insect control applications.     

Einfluß von Zwei- und Dreischichttonmineralen auf die Dehydrogenase-, saure Phosphatase- und Urease-Aktivität in Modellversuchen

Influence of two- and three-layered clay minerals on the Dehydrogenase-, acid Phosphatase and Urease Activity in model experiments The influence of different Ca-homo-ionic clay minerals as well as humic acid (Roth) on the dehydrogenase activity (DHA) as well as on urease and acid phosphatase were examined in model experiments. The following results were obtained: 1. The DHA of a fertile soil and the activity of urease and acid-phosphatase decreased remarkably and specifically with increasing amounts of sorbents. Generally speaking, inactivation was complete at approximately 0.6 g of each additive. Both urease- and acid phosphatase activity were inactivated specifically. The inactivation of urease decreased in the sequence humic acid < montmorillonite < illite < pyrophyllite < halloysite < kaolinite < bentonite, whereas the following order of succession pyrophyllite < montmorillonite < bentonite < halloysite < kaolinite < illite was recorded with acid phosphatase. The yellow colour of humic acid excluded it from experiments with acid phosphatase. 2. No relationship could be found between the degree of inactivation and the cation exchange capacity (CEC) or the total surface area of the clays. However, nearly the same sequence of inactivation with the three enzyme systems was revealed, if their activity was related to the specific surface charge of the clay minerals. Apparently, both the total surface area as well as CEC are involved in the specific sorption of cells and enzymes. The possible mechanisms of sorption on clay surfaces are discussed.     

Redox dependence of papain enzyme activity on charged kaolinite surfaces and in solution

The relative activity of an SH dependent enzyme, papain, is decreased by increasing the ratio of oxidizing disulfide to reducing thiol in solution. The same applies to papain adsorbed on negatively charged clay particles, but the effect is more pronounced if positively charged disulfides can be formed in the thiol-disulfide exchange equilibrium. Disulfides with a double positive charge are apparently more strongly attracted to clay than are singly charged, neutral, or negatively charged products of exchange. Preferential attraction of oxidizing disulfides to the particulate surfaces increases local, microenvironmental oxidizing potentials.     

Eggplant lipoxygenase (Solanum melongena): product characterization and effect of physicochemical properties of linoleic acid on the enzymatic activity

Lipoxygenase (LOX) from eggplant (Solanum melongena L. cv. Belleza negra) was partially purified, and the products and kinetics of the enzyme were studied. Linoleic acid (LA) was the best substrate for this enzyme. Product analysis by HPLC and GC/MS revealed that, at its pH optimum (pH 7.0), the enzyme converted LA almost totally into the 9-hydroperoxy isomer, whereas the 13-hydroperoxy isomer was only a minor product. At this pH, the enzyme had K(m) and V(max) values for LA of 1.4 microM and 2.2 micromol min(-1) (mg of protein)(-1), respectively, when the monomeric form of LA was used as substrate. The dependence of eggplant LOX activity on the physicochemical properties of LA was also studied. Experiments revealed that LA aggregates were used more efficiently than monomeric LA as substrate. The apparent substrate cooperativity observed may be due to the different activities exhibited toward monomers and aggregates. This result can be interpreted as a substrate-aggregation dependent activity.     

Interaction of phytases with minerals and availability of substrate affect the hydrolysis of inositol phosphates

The stability and activity of phytases in the soil environment may be affected by their sorption on soil particle surfaces and by substrate availability with important consequences for P cycling and nutrient bioavailability. This work evaluated the interaction of phytases with goethite, haematite, kaolinite, montmorillonite and two oxisol clays and investigated how this interaction is affected when myo-inositol hexakisphosphate (InsP₆) was sorbed on the mineral surfaces. phyA histidine acid phosphatases of fungal origin were used and their ability to release orthophosphate from the InsP₆-saturated minerals was evaluated.The phytases showed a high affinity for the mineral surfaces, with a loss of enzyme activity generally being observed over 24 h (up to 95% of the initially added activity). The loss of phytase activity was dependent on the type of mineral, with kaolinite and montmorillonite showing the greatest effect. Retention of enzyme activity was higher with the two oxisol clays, suggesting that the heterogeneous nature of clay surfaces and the presence of endogenous organic matter may limit the inhibition caused by interaction with minerals.In the presence of mineral surfaces saturated with InsP₆, the partitioning of enzyme activity between the solution and the solid phase was shifted more towards the solution phase, presumably due to the mineral surfaces being occupied by the substrate. However, phytases were not able to release any orthophosphate directly from InsP₆-saturated goethite and haematite, and hydrolysed InsP₆ that was desorbed from haematite. Conversely, in the case of kaolinite and of the oxisol clays, where desorption was limited, phytases appeared to be able to hydrolyse a small fraction of the InsP₆ adsorbed on the surfaces. These findings suggest that the bioavailability of P from inositol phosphates is governed to a large extent by the mineral composition of soil and by competitive effects for sorption on reactive surfaces among inositol phosphates and phytases.     

Enzymatic transformation of humic substances by NDO

Enzymatic transformation of humic acids (HA), fulvic acids (FA) and indole was examined using naphthalene 1,2-dioxygenase (NDO). NDO was used as a model for dioxygenase enzymes found in various microbial species. Indole was used as a model substrate for NDO-catalyzed reactions resulting in condensation products. Although NDO is not classified as a soil enzyme, all HA and FA tested were susceptible to NDO-induced transformation. The extent of NDO-specific NADH oxidation in solutions containing HA and FA paralleled the percent aromaticity of the HA and FA. Furthermore, the UV–Vis absorptive properties of NDO-treated HA and FA were altered in a manner suggesting condensation reactions similar to the formation of indigo from indole. Condensation reactions were enhanced in NDO-treated mixtures containing indole and an FA. NDO retained activity for 2 weeks under ambient conditions, and retained some enzymatic activity for 9 days based on detection of specific metabolites by HPLC, suggesting prolonged extracellular activity. Humic substances have not previously been known to be substrates for dioxygenases; even more significant was that dioxygenase enzymes can facilitate condensation reactions between indole-like functional groups well-known to be present in HA and FA. These results illustrate how dioxygenases can be potential humic-modifying enzymes when released into the environment upon microbial death and concurrent cell lysis which could alter the bioavailability of organic contaminants associated with dissolved organic matter through specific modulation of enzyme activity involving substrate competition.     

Einfluß pedogener Tonminerale auf die Kinetik (km und Vmax) von alkalischer und saurer Phosphatase

Effect of pedogenic clay minerals on the kinetics (Km and Vmax) of alkaline and acid phosphatase The influence of various pedogenic clay minerals, derived from 5 different Pelosols (=Vertisols), on the activity and kinetics (Km and Vmax) of alkaline and acid phosphatase (AP and SP, respectively) was studied in model experiments. Generally speaking, Vmax of both enzymes decreased with increasing immobilization. Km remained constant or increased considerably depending on the composition of the clay minerals. The changes in Vmax and Km were both clay- and enzyme specific. Immobilization of AP by most clays was reflected by a mixed type of inhibition, while that of SP resulted in a competitive type of interaction. Non-competitive inhibition was recorded with AP after sorption to mixed and interlayered clays of the illite-montmorillonite-chlorite type (profile V) and with SP in the presence of montmorillonite clay (profile I). The types of inhibition were compared with those observed with two- and three-layered clays of sedimentological origin.     

Soil enzymes in a changing environment: Current knowledge and future directions

This review focuses on some important and challenging aspects of soil extracellular enzyme research. We report on recent discoveries, identify key research needs and highlight the many opportunities offered by interactions with other microbial enzymologists. The biggest challenges are to understand how the chemical, physical and biological properties of soil affect enzyme production, diffusion, substrate turnover and the proportion of the product that is made available to the producer cells. Thus, the factors that regulate the synthesis and secretion of extracellular enzymes and their distribution after they are externalized are important topics, not only for soil enzymologists, but also in the broader context of microbial ecology. In addition, there are many uncertainties about the ways in which microbes and their extracellular enzymes overcome the generally destructive, inhibitory and competitive properties of the soil matrix, and the various strategies they adopt for effective substrate detection and utilization. The complexity of extracellular enzyme activities in depolymerising macromolecular organics is exemplified by lignocellulose degradation and how the many enzymes involved respond to structural diversity and changing nutrient availabilities. The impacts of climate change on microbes and their extracellular enzymes, although of profound importance, are not well understood but we suggest how they may be predicted, assessed and managed. We describe recent advances that allow for the manipulation of extracellular enzyme activities to facilitate bioremediation, carbon sequestration and plant growth promotion. We also contribute to the ongoing debate as to how to assay enzyme activities in soil and what the measurements tell us, in the context of both traditional methods and the newer techniques that are being developed and adopted. Finally, we offer our collective vision of the future of extracellular enzyme research: one that will depend on imaginative thinking as well as technological advances, and be built upon synergies between diverse disciplines.     

Response of the bacterial diversity and soil enzyme activity in particle-size fractions of Mollisol after different fertilization in a long-term experiment

Particle-size soils were fractionated for evaluating changes in the composition of bacterial community and enzyme activity in response to 13 years of fertilization. This study focused on Mollisol and its particle-size fractions of 200–2,000 μm (coarse sand sized), 63 to 200 μm (fine sand sized), 2 to 63 μm (silt sized), and 0.1 to 2 to μm (clay-sized). Long-term chemical fertilization lowered the pH of all particle fractions, whereas organic fertilizer application mitigated soil acidification. Nutrient concentrations depended on both fertilizer treatment and particle fractions and enzymes were unevenly active throughout the soil. Generally, the highest enzyme activities were observed in the silt and clay fractions of control soil and the soil treated with chemical fertilizer (N, P, and K (NPK)) and in the sand-sized fraction of soil treated with manure and chemical fertilizer (MNPK). Except for acid phosphomonoesterase, the other tested enzyme activities in coarse-sized fractions of MNPK soil were significantly higher than those of the control and NPK soils. Fertilization and soil fraction interactively (p?<?0.05) affected the enzyme activity. Denaturing gradient gel electrophoresis analysis showed that the bacterial community structure significantly differed in different particle sizes with a higher bacterial diversity in small-sized than in coarse-sized fractions. Dominant bands were excised and sequenced. We have found the following bacterial groups: Actinobacteria, γ-proteobacteria, and Acidobacteria. In addition, enrichment of organic matter in coarser fractions was related to greater bacterial diversity than any other treatment. Principal component analysis showed a smaller variability among fractions of the organic amended treatment. Redundancy analysis showed that the tested properties significantly affected the composition of bacterial community with the exception of C/N and available P. No significant correlation between enzyme activity and bacterial community composition was detected, whereas positive correlations between other soil properties and enzyme activities were observed to various extents. Probably, enzyme activities might be affected by specific functional bacterial communities rather than by the overall bacterial community. We concluded that the long-term application of organic manures contributed to the increase of soil organic matter content of particles higher than 200 mm, with higher bacterial diversity and increases in most of the enzyme activities.