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

The location of extracellular enzymes within the soil architecture and their association with the various soil components affects their catalytic potential. A soil fractionation study was carried out to investigate: (a) the distribution of a range of hydrolytic enzymes involved in C, N and P transformations, (b) the effect of the location on their respective kinetics, (c) the effect of long-term N fertilizer management on enzyme distribution and kinetic parameters. Soil (silty clay loam) from grassland which had received 0 or 200 kg N ha(-1) yr(-1) was fractionated, and four particle-size fractions (> 200, 200-63, 63-2 and 0. 1-2 mum) were obtained by a combination of wet-sieving and centrifugation, after low-energy ultrasonication. All fractions were assayed for four carbohydrases (beta-cellobiohydrolase, N-acetyl-beta-glucosammidase, beta-glucosidase and beta-xylosidase), acid phosphatase and leucine-aminopeptidase using a microplate fluorimetric assay based on MUB-substrates. Enzyme kinetics (V-max and K-m) were estimated in three particle-size fractions and the unfractionated soil. The results showed that not all particle-size fractions were equally enzymatically active and that the distribution of enzymes between fractions depended on the enzyme. Carbohydrases predominated in the coarser fractions while phosphatase and leucine-aminopeptidase were predominant in the clay-size fraction. The Michaelis constant (K.) varied among fractions, indicating that the association of the same enzyme with different particle-size fractions affected its substrate affinity. The same values of Km were found in the same fractions from the soil under two contrasting fertilizer management regimes, indicating that the Michaelis constant was unaffected by soil changes caused by N fertilizer management. (C) 2004 Elsevier Ltd. All rights reserved.

Anthraquinone catalysis in the glucose-driven reduction of indigo to leuco-indigo

Anthraquinone immobilised onto the surface of indigo microcrystals enhances the reductive dissolution of indigo to leuco-indigo. Indigo reduction is driven by glucose in aqueous NaOH and a vibrating gold disc electrode is employed to monitor the increasing leuco-indigo concentration with time. Anthraquinone introduces a strong catalytic effect which is explained by invoking a molecular "wedge effect'' during co-intercalation of Na+ and anthraquinone into the layered indigo crystal structure. The glucose-driven indigo reduction, which is in effective in 0.1 M NaOH at 65 degrees C, becomes facile and goes to completion in the presence of anthraquinone catalyst. Electron microscopy of indigo crystals before and after reductive dissolution confirms a delamination mechanism initiated at the edges of the plate-like indigo crystals. Catalysis occurs when the anthraquinone-indigo mixture reaches a molar ratio of 1:400 (at 65 degrees C; corresponding to 3 mu M anthraquinone) with excess of anthraquinone having virtually no effect. A strong temperature effect ( with a composite E-A approximate to 120 kJ mol(-1)) is observed for the reductive dissolution in the presence of anthraquinone. The molar ratio and temperature effects are both consistent with the heterogeneous nature of the anthraquinone catalysis in the aqueous reaction mixture.

Experimental and theoretical evidence for homogeneous catalysis in the gas-phase reaction of SiH2 with H2O (and D2O): a combined kinetic and quantum chemical study

Time-resolved kinetic studies of the reaction of silylene, SiH2, with H2O and with D2O have been carried out in the gas phase at 297 K and at 345 K, using laser flash photolysis to generate and monitor SiH2. The reaction was studied independently as a function of H2O (or D2O) and SF6 (bath gas) pressures. At a fixed pressure of SF6 (5 Torr), [SiH2] decay constants, k(obs), showed a quadratic dependence on [H2O] or [D2O]. At a fixed pressure of H2O or D2O, k(obs) Values were strongly dependent on [SF6]. The combined rate expression is consistent with a mechanism involving the reversible formation of a vibrationally excited zwitterionic donor-acceptor complex, H2Si...OH2 (or H2Si...OD2). This complex can then either be stabilized by SF6 or it reacts with a further molecule of H2O (or D2O) in the rate-determining step. Isotope effects are in the range 1.0-1.5 and are broadly consistent with this mechanism. The mechanism is further supported by RRKM theory, which shows the association reaction to be close to its third-order region of pressure (SF6) dependence. Ab initio quantum calculations, carried out at the G3 level, support the existence of a hydrated zwitterion H2Si...(OH2)(2), which can rearrange to hydrated silanol, with an energy barrier below the reaction energy threshold. This is the first example of a gas-phase-catalyzed silylene reaction.

Bidentates versus monodentates in asymmetric hydrogenation catalysis: synergic effects on rate and allosteric effects on enantioselectivity

C-1-Symmetric phosphino/phosphonite ligands are prepared by the reactions of Ph2P(CH2)(2)P(NMe2)(2) with (S)-1,11'-bi-2-naphthol (to give L-A) or (S)-10,10'-bi-9-phenanthrol (to give L-B). Racemic 10,10'-bi-9-phenanthrol is synthesized in three steps from phenanthrene in 44% overall yield. The complexes [PdCl2(L-A,L-B)] (1a,b), [PtCl2(L-A,L-B)] (2a,b), [Rh(cod)(L-A,L-B)]BF4 (3a,b) and [Rh(L-A,L-B)(2)]BF4 (4a,b) are reported and the crystal structure of la has been determined. A P-31 NMR study shows that M, a 1:1 mixture of the monodentates, PMePh2 and methyl monophosphonite L-1a (based on (S)-1,11'-bi-2-naphthol), reacts with 1 equiv of [Rh(cod)(2)]BF4 to give the heteroligand complex [Rh(cod)(PMePh2)(L-1a)]BF4 (5) and homoligand complexes [Rh(cod)(PMePh2)(2)]BF4 (6) and [Rh(cod)(L-1a)(2)]BF4 (7) in the ratio 2:1:1. The same mixture of 5-7 is obtained upon mixing the isolated homoligand complexes 6 and 7 although the equilibrium is only established rapidly in the presence of an excess of PMePh2. The predominant species 5 is a monodentate ligand complex analogue of the chelate 3a. When the mixture of 5-7 is exposed to 5 atm H-2 for 1 h (the conditions used for catalyst preactivation in the asymmetric hydrogenation studies), the products are identified as the solvento species [Rh(PMePh2)(L-1a)(S)(2)]BF4 (5'), [Rh(S)(2)(PMePh2)(2)]BF4 (6') and [Rh(S)(2)(L-1a)(2)]BF4 (7') and are formed in the same 2:1:1 ratio. The reaction of M with 0.5 equiv of [Rh(cod)(2)]BF4 gives exclusively the heteroligand complex cis-[Rh(PMePh2)(2)(L-1a)(2)]BF4 (8), an analogue of 4a. The asymmetric hydrogenation of dehydroamino acid derivatives catalyzed by 3a,b is reported, and the enantioselectivities are compared with those obtained with (a) chelate catalysts derived from analogous diphosphonite ligands L-2a and L-2b, (b) catalysts based on methyl monophosphonites L-1a and L-1b, and (c) catalysts derived from mixture M. For the cinnamate and acrylate substrates studied, the catalysts derived from the phosphino/phosphonite bidentates L-A,L-B generally give superior enantioselectivities to the analogous diphosphonites L-2a and L-2b; these results are rationalized in terms of delta/lambda-chelate conformations and allosteric effects of the substrates. The rate of hydrogenation of acrylate substrate A with heterochelate 3a is significantly faster than with the homochelate analogues [Rh(L-2a)(cod)]BF4 and [Rh(dppe)(cod)]BF4. A synergic effect on the rate is also observed with the monodentate analogues: the rate of hydrogenation with the mixture containing predominantly heteroligand complex 5 is faster than with the monophosphine complex 6 or monophosphonite complex 7. Thus the hydrogenation catalysis carried out with M and [Rh(cod)(2)]BF4 is controlled by the dominant and most efficient heteroligand complex 5. In this study, the heterodiphos chelate 3a is shown to be more efficient and gives the opposite sense of optical induction t the heteromonophos analogue

Engineering Pt in ceria for a maximum metal-support interaction in catalysis

Conventional supported metal catalysts are metal nanoparticles deposited on high surface area oxide supports with a poorly defined metal−support interface. Typically, the traditionally prepared Pt/ceria catalyzes both methanation (H2/CO to CH4) and water−gas shift (CO/H2O to CO2/H2) reactions. By using simple nanochemistry techniques, we show for the first time that Pt or PtAu metal can be created inside each CeO2 particle with tailored dimensions. The encapsulated metal is shown to interact with the thin CeO2 overlayer in each single particle in an optimum geometry to create a unique interface, giving high activity and excellent selectivity for the water−gas shift reaction, but is totally inert for methanation. Thus, this work clearly demonstrates the significance of nanoengineering of a single catalyst particle by a bottom-up construction approach in modern catalyst design which could enable exploitation of catalyst site differentiation, leading to new catalytic properties.

Micellar catalysis for partial oxidation of toluene to benzoic acid in supercritical CO2: effects of fluorinated surfactants

One of the key hindrances on development of solid catalysts containing cobalt species for partial oxidation of organic molecules at mild conditions in conventional liquid phase is the severe metal leaching. The leached soluble Co species with a higher degree of freedom always out-performs those of solid supported Co species in oxidation catalysis. However, the homogeneous Co species concomitantly introduces separation problems. We have recently reponed for the first time, a new oxidation catalyst system for the oxidation of organic molecules in supercritical CO2 using the principle of micellar catalysis. [CF3(CF2)(8)COO](2)Co.xH(2)O (the fluorinated anionic moiety forms aqueous reverse micelles carrying water-soluble Co2+ cations in scCO(2)) was previously shown to be extremely active for the oxidation of toluene in the presence of sodium bromide in water-CO2 mixture, giving 98% conversion and 99% selectivity to benzoic acid at 120 degreesC. In this study, we show that the effects of varying the type of surfactant counterions and the length of the surfactant chains on catalysis. It is found that the use of [CF3(CF2)(8)COO](2)Mg.yH(2)O/Co(II) acetate is as effective as the [CF3(CF2)(8)COO](2)Co.xH(2)O and the fluorinated chain length used has a subtle effect on the catalytic rate measured. It is also demonstrated that this new type of micellar catalyst in scCO(2) can be easily separated via CO2 depressurisation and be reused without noticeable deactivation. (C) 2003 Elsevier B.V. All rights reserved.

Potential prebiotic activity of oligosaccharides obtained by enzymatic conversion of durum wheat insoluble dietary fibre into soluble dietary fibre

Background and aims: Epidemiological evidence indicates that cereal dietary fibre (DF) may have several cardiovascular health benefits. The underlying mechanisms have not yet been elucidated. Here, the potential nutritional effects of physico-chemical. properties modifications of durum wheat dietary fibre (DWF) induced by enzyme treatment have been investigated. Methods and results: The conversion of the highly polymerised insoluble dietary fibre into soluble feruloyl oligosaccharides of DWF was achieved by a tailored enzymatic treatment. The in vitro fermentation and release of ferulic acid by intestinal microbiota from DWF before and after the enzymatic treatment were assessed using a gut model validated to mimic the human colonic microbial environment. Results demonstrated that, compared to DWF, the enzyme-treated DWF (ETD-WF) stimulated the growth of bifidobacteria and lactobacilli. Concurrently, the release of free ferulic acid by ET-DWF was almost three times higher respect to the control. No effect on the formation of short chain fatty acids was observed. Conclusions: The conversion of insoluble dietary fibre from cereals into soluble dietary fibre generated a gut microbial fermentation that supported bifidobacteria and lactobacilli. The concurrent increase in free ferulic acid from the enzyme-treated DWF might result in a higher plasma ferulic acid concentration which could be one of the reasons for the health benefits reported for dietary fibre in cardiovascular diseases. (c) 2008 Elsevier B.V. All rights reserved.

Enzyme glycation influences product yields during oligosaccharide synthesis by reverse hydrolysis

Possible evidence is presented for Maillard glycation of enzymes during oligosaccharide synthesis by reverse hydrolysis. In 70% (w/v) mannose solutions, 1,2-alpha-mannosidase from Penicillium citrinum lost 40% and alpha-mannosidase from almonds lost 60% activity at 55 degreesC over 2 weeks. Oligosaccharide yields were 15 and 45% respectively. Higher molecular weight glycation adducts were formed in a time-dependent manner as seen by MALDI-TOF. Inhibitors of the Maillard. reaction were able to partially alleviate these effects resulting in reduced loss of enzyme activity and oligosaccharide yield increases of 27-53% relative to the control. (C) 2004 Elsevier B.V. All rights reserved.

Prebiotic evaluation of a novel galactooligosaccharide mixture produced by the enzymatic activity of Bifidobacterium bifidum NCIMB 41171, in healthy humans: a randomized, double-blind, crossover, placebo-controlled intervention study

Background: Galactooligosaccharides are selectively fermented by the beneficial member of the colonic microflora contributing to the health of the host. Objective: We assessed the prebiotic potential of a novel galactooligosaccharide produced through the action of beta-galactosidases, originating from a probiotic Bifidobacterium bifidum strain, against a galactooligosaccharide produced through the action of an industrial P-galactosidase and a placebo. Design: Fifty-nine healthy human volunteers participated in this study. Initially, the effect of the matrix on the prebiotic properties of a commercially available galactooligosaccharide (7 g/d) was assessed during 7-d treatment periods with a 7-d washout period in between. During the second phase, 30 volunteers were assigned to a sequence of treatments (7 d) differing in the amount of the novel galactooligosaccharide (0, 3.6, or 7 g/d). Stools were recovered before and after each intervention, and bacteria numbers were determined by fluorescent in situ hybridization. Results: Addition of the novel galactooligosaccharide mixture significantly increased the bifidobacterial population ratio compared with the placebo (P < 0.05), whereas 7 g/d of the novel galactooligosaccharide significantly increased the bifidobacterial ratio compared with the commercial galactooligosaccharide (P < 0.05). Moreover, a significant relation (P < 0.001) between the bifidobacteria proportion and the novel galactooligosaccharide dose (0, 3.6, and 7 g/d) was observed. This relation was similar to the effect of the novel galactooligosaccharide on the prebiotic index of each dose. Conclusions: This study showed that galactooligosaccharide mixtures produced with different beta-galactosidases show different prebiotic properties and that, by using enzymes originating from bifidobacterial species, an increase in the bifidogenic properties of the prebiotic product is achievable.

Regioselective synthesis of mannobiose and mannotriose by reverse hydrolysis using a novel 1,6-alpha-D-mannosidase from Aspergillus phoenicis

A novel 1,6-alpha-D-mannosidase was produced by Aspergillus phoenicis grown on a commercial manno-oligosaccharide preparation in liquid culture. The enzyme hydrolysed only alpha-D-Manp-(1 --> 6)-D-Manp and did not act on alpha-D-Manp-(1 --> 2)-D-Manp, or alpha-D-Manp-(1 --> 3)-D-Manp. The 1,6-alpha-D-mannosidase was used for synthesis of manno-oligosaccharides by reverse hydrolysis reaction. The highest yields, expressed as percentages (w/w) of total sugar, were similar to21% mannobiose and similar to5% mannotriose, and they were obtained with 45% (w/w) initial mannose concentration at pH 4.5 after 12 days incubation at 55 degreesC. The disaccharide and trisaccharide products were separated and their structures determined by methylation analysis. Only 1-6 linkages were found in both of them. (C) 2003 Elsevier B.V. All rights reserved.