In Vivo; In Vitro Interaction of Silver Nanoparticles with Leucine Aminopeptidase from Human and Plasmodium falciparum.

There is increasing requirement for the development of new drug protocols against malaria, a fatal disease caused by the lethal parasite Plasmodium falciparum. Leucine aminopeptidase (Pf LAP) of Plasmodium falciparum, is being pursued as a promising target for the discovery of novel antimalarials. The effects of silver nanoparticles (AgNPs) against P. falciparum leucine amino-peptidase (Pf LAP) and the human homolog (HsLAP) were compared. Pf LAP and HsLAP were expressed in Escherichia coli, and AgNPs (3-10 nm) characterized by ultra-violet spectroscopy and transmission electron microscopy. Pf LAP indicated a Km of 694 µM towards leucine-p-nitroanilide and a Vmax of 57.9 µmol.ml-1 · min-1 while HsLAP had a Km of 1.6 mM and Vmax of 119.6 µmol · ml-1 · min-1. On interaction with AgNPs (670 nM) Pf LAP was selectively inhibited (57.1%; Ki = 610 nM) relative to HsLAP (10.8%; Ki = 5.22 µM). Structural differences between the enzyme variants, particularly the orientation and distance of surface Met349 in Pf LAP and Met306 in HsLAP to the zinc binding sites were significant and may allow for selective targeting of Pf LAP by AgNPs. The viability of P. falciparum parasites was decreased when exposed to silver nanoparticles, with an IC50 value of 6.96 µM, compared to an IC50 value of 647.7 µM for human HeLa cells.

Interaction of Silver Nanoparticles with Triosephosphate Isomerase from Human and Malarial Parasite (Plasmodium falciparum): A Comparative Study.

Recombinant triosephosphate isomerase from Plasmodium falciparum (PfTIM) and humans (hTIM) were expressed, purified and characterised. High specific activity (1207 U x mg(-1)) with a fold purification of -1.8 and a yield of 48% were obtained for hTIM after gel filtration while, in contrast PfTIM afforded a specific activity of 1387 U x mg(-1) with a fold purification of -6.8 and a yield of 57% after gel filtration and prior to dialysis. PfTIM had an optimal pH and temperature, K(m) and V(max) of 5.25, 25 degrees C, 12.8 mM and 1.13 µmol x mL(-1) min(-1) respectively while for hTIM the pH and temperature optima, K(m) and V(max) were 6.75, 30 degrees C; 8.2 mM and 1.35 µmol x ml(-1) min(-1). Polyvinylpyrrolidone stabilised silver nanoparticles (60 nM; 2-6 nm diameter) selectively inhibited PfTIM with a 7-fold decrease in enzyme catalytic efficiency (K(cat)/K(m)) over hTIM. Respective K(i) values were 283 nM [hTIM] and 85.7 nM [PfTIM]. Key structural differences between the two enzyme variants, especially with Cys13 at the dimer interface of PfTIM, were significant enough to suggest unique characteristics allowing for selective targeting of PfTIM by AgNPs.

Isolation, characterization, interaction of a thiazolekinase (Plasmodium falciparum) with silver nanoparticles.

Malaria, a mosquito-borne infectious disease, is caused by the Plasmodium genus, and remains one of the greatest health challenges worldwide. The malarial parasite possess a biosynthetic pathway for the B-group vitamin incorporating the thiamine metabolizing enzymes; humans on the other hand cannot synthesize the vitamin and require it from within their diet. The vitamin B1 biosynthetic enzyme 5-(2-hydroxyethyl)-4-methylthioazolekinase [EC. 2.7.1.50] from Plasmodium (PfThzK) is particularly attractive as a biomedical target since any inhibition of this enzyme may lead to an effective treatment for malaria. In the present study, PfThzK was recombinantly produced as a 6× His fusion protein in Escherichia coli BL21(DE3) and purified using nickel affinity and size exclusion chromatography. The enzyme was monomeric with a molecular mass of 34 kDa, a specific activity of 295.04 nmol min(-1) mg(-1) and showed an optimum temperature and pH of 37 °C and 7.5, respectively. The purified PfThzK was non-competitively inhibited (79%) by silver nanoparticles (2-6 nm); Ki=6.45 µM. A mechanism is suggested for the interaction of the silver nanoparticle with PfThzK through two sulphur bearing amino acids (Met(1), Cys(206)) on the surface of each subunit of the enzyme.

Microbial enzymatic production and applications of short-chain fructooligosaccharides and inulooligosaccharides: recent advances and current perspectives.

The industrial production of short-chain fructooligosaccharides (FOS) and inulooligosaccharides is expanding rapidly due to the pharmaceutical importance of these compounds. These compounds, concisely termed prebiotics, have biofunctional properties and hence health benefits if consumed in recommended dosages. Prebiotics can be produced enzymatically from sucrose elongation or via enzymatic hydrolysis of inulin by exoinulinases and endoinulinases acting alone or synergistically. Exoinulinases cleave the non-reducing ß-(2, 1) end of inulin-releasing fructose while endoinulinases act on the internal linkages randomly to release inulotrioses (F3), inulotetraoses (F4) and inulopentaoses (F5) as major products. Fructosyltransferases act by cleaving a sucrose molecule and then transferring the liberated fructose molecule to an acceptor molecule such as sucrose or another oligosaccharide to elongate the short-chain fructooligosaccharide. The FOS produced by the action of fructosyltransferases are 1-kestose (GF2), nystose (GF3) and fructofuranosyl nystose (GF4). The production of high yields of oligosaccharides of specific chain length from simple raw materials such as inulin and sucrose is a technical challenge. This paper critically explores recent research trends in the production and application of short-chain oligosaccharides. Inulin and enzyme sources for the production of prebiotics are discussed. The mechanism of FOS chain elongation and also the health benefits associated with prebiotics consumption are discussed in detail.

Interaction of metal nanoparticles with recombinant arginine kinase from Trypanosoma brucei: thermodynamic and spectrofluorimetric evaluation.

BACKGROUND: Trypanosoma brucei, responsible for African sleeping sickness, is a lethal parasite against which there is need for new drug protocols. It is therefore relevant to attack possible biomedical targets with specific preparations and since arginine kinase does not occur in humans but is present in the parasite it becomes a suitable target. METHODS: Fluorescence quenching, thermodynamic analysis and FRET have shown that arginine kinase from T. brucei interacted with silver or gold nanoparticles. RESULTS: The enzyme only had one binding site. At 25°C the dissociation (Kd) and Stern-Volmer constants (KSV) were 15.2nM, 0.058nM(-1) [Ag]; and 43.5nM, 0.052nM(-1) [Au] and these decreased to 11.2nM, 0.041nM(-1) [Ag]; and 24.2nM, 0.039nM(-1) [Au] at 30°C illustrating static quenching and the formation of a non-fluorescent fluorophore-nanoparticle complex. Silver nanoparticles bound to arginine kinase with greater affinity, enhanced fluorescence quenching and easier access to tryptophan molecules than gold. Negative ΔH and ΔG values implied that the interaction of both Ag and Au nanoparticles with arginine kinase was spontaneous with electrostatic forces. FRET confirmed that the nanoparticles were bound 2.11nm [Ag] and 2.26nm [Au] from a single surface tryptophan residue. CONCLUSIONS: The nanoparticles bind close to the arginine substrate through a cysteine residue that controls the electrophilic and nucleophilic characters of the substrate arginine-guanidinium group crucial for enzymatic phosphoryl transfer between ADP and ATP. GENERAL SIGNIFICANCE: The nanoparticles of silver and gold interact with arginine kinase from T. brucei and may prove to have far reaching consequences in clinical trials.

Nanomedicine: action of metal nanoparticles on neuronal nitric oxide synthase-fluorimetric analysis on the mechanism for fibrillogenesis.

The incubation of neuronal nitric oxide synthase with the five amyloid peptide fragments [Aß17-21; Aß25-29; Aß29-33; Aß33-37; Aß25-37] catalyzed the formation of fibrils. The role of neuronal isomer (nNOS) involved the entrapment of free monomers and seed aggregates to initiate the events of nucleation and elongation, critical for the formation of fibrils. It was evident that the hydrophobic nature of Aß17-21, the three glycine zipper peptides [Aß25-29; Aß29-33; Aß33-37] and Aß25-37 was a trigger in the formation of fibrils and was a force critical in the association of the peptides with the enzyme. Gold and silver nanoparticles (average 4.0 nm) inhibited fibril formation when added to the induced fibrils from nNOS-Aß incubation. The addition of nNOS and/or Aß to co-incubated solutions of nanoparticle-Aß or nanoparticle-nNOS respectively did not prevent fibril formation but reversed it. Three mechanisms for this reversal were proposed: (1) depletion of free Aß monomer in solution and blocking potential aggregation sites on the nNOS molecule due to large surface area of the nanoparticle (2) hydrophobic interaction between the Aß peptide and nanoparticle (3) disruption of binary adducts between Aß-peptides and nNOS by nanoparticles.

Etiology of Alzheimer's disease: kinetic, thermodynamic and fluorimetric analyses of interactions of pseudo Aß-peptides with neuronal nitric oxide synthase.

Aggregated ß-amyloid deposit is a hallmark in the neuropathology of Alzheimer's disease but their mechanism of formation still remains unresolved. Previously we reported that a normal pentapeptide Aß(17-21) and glycine zipper peptide Aß(29-33) strongly inhibited nitric oxide synthase and rapidly initiated fibrillogenesis. Critical amino acids within these fragments were not identified. We now report on the interaction of four pseudo-peptides with nNOS - two peptides with a reversed amino acid sequence [Aß(17-21r); Aß(29-33r)] and two peptides with Phe19, Phe20 and Ile31, Ile32 substituted with polar glutamic acid [Aß(17-21p); Aß(29-33p)]. It was shown that while the inhibitor constants (Ki) increased 2-3 fold for each of the pseudo-peptides when compared with the normal peptides the dissociation constant Kd increased between 20 and 50 fold. Stern-Volmer fluorescence quenching constants (K(SV)) for Aß(17-21p) and Aß(29-33p) were 7.2×10(-3) and 6.1×10(-3) µM(-1) respectively at 298 K some 2-3 fold lower than the corresponding Aß(17-21r); Aß(29-33r). With temperature increase there was an increase in K(SV) and Kd, suggesting a dynamic quenching mechanism. Thermodynamic parameters, ΔH, ΔS and ΔG were all positive indicating endothermic, non-spontaneous, hydrophobic-hydrophobic associations of the pseudo-peptides with the enzyme. By FRET analysis the efficiency of fluorescence transfer between enzyme tryptophans and the pseudo-peptides was 90% (compared to 97% for the natural substrate). The distance the tryptophans moved after interaction with Aß(17-21r) and Aß(17-21p) was 10% greater, while for Aß(29-33r) and Aß(29-33p) it was 20-25% greater, than with the normal peptides; the fluorescence intensity was 20-75% higher. This increase in distance, fluorescent intensity and transfer efficiency illustrate an increase in interaction energy for the pseudo-peptides with nNOS lending support for the strategic position of the Phe19, Phe20, Ile31 and Ile32 in the original peptides not only for inhibition of the nNOS but for initiation of fibrillogenesis.

Interaction of glycine zipper fragments of Aß-peptides with neuronal nitric oxide synthase: kinetic, thermodynamic and spectrofluorimetric analysis.

Five peptide fragments [Aß(17-21); Aß(25-29); Aß(29-33); Aß(33-37); Aß(25-37)] of the toxic Aß(1-40(42)) amyloid peptide were shown to bind with neuronal nitric oxide synthase by means of hydrophobic-hydrophobic forces. The enzyme has a single site for the amyloid peptide binding, which resulted in a quenching of the intrinsic fluorescence of the enzyme. Binding constants determined from Stern-Volmer analysis were between 9×10(-3) and 1.8×10(-2) µM(-1). As temperature increased these binding constants increased reflecting that the interaction of the amyloid peptides with nNOS was endothermic and the quenching was dynamic. Kinetic analysis revealed a non-competitive interaction of the amyloid peptides to the enzyme with inhibitor constants of 5.1 µM for Aß(17-21) to about 8-12 µM for the other peptides. According to the van't Hoff relationship the thermodynamic parameters, ΔH, ΔS and ΔG for the interaction of the amyloid peptides were all positive and between 41.28 and 77.86 kJ mol(-1)K(-1), 104.92 and 220.82 J mol(-1)K(-1) and 9.92 and 13.13 kJ mol(-1)K(-1), respectively. This suggested that the transition state, created by the amyloid peptide-nNOS complex and generated during the initial stages of Aß aggregation had to, initially, overcome an activation barrier. Since the ΔG values decreased as temperature increased it not only implied a non-spontaneous interaction but that hydrophobic forces were operative during the binding. By FRET analysis the distance between the donor enzyme and the acceptor amyloid peptide was between 2.7 and 2.8 nm. As the temperature increased from 298 K through 313 K (and higher) the fraction of these tryptophan residues that became exposed increased, to approach a value of 1. There was strong support for the initial interaction being through the glycine zipper regions of Aß(25-37).

Interaction of superoxide dismutase with the glycine zipper regions of ß-amyloid peptides: is there an implication towards Alzheimer's disease and oxidative stress?

Not only are ß-amyloid peptides and senile plaque deposits characteristics in Alzheimer's disease but there is growing evidence to suggest that oxidative stress also plays a role with a decrease in levels of brain superoxide dismutase (SOD), an enzyme that catalyses the dismutation of superoxide radicals into molecular oxygen and hydrogen peroxide. We show through kinetic and fluorescence analysis that ß-amyloid peptides, in the glycine zipper region [Aß29₋33 and Aß25₋37] of Aß1₋40 interact with, and inhibit, SOD directly. The enzyme was purified 15.7-fold from bovine brain by DEAE-Sepharose ion exchange chromatography in a yield of 68.8% and specific activity of 3.66 U.mg(-1). The subunit structure of the enzyme was monomeric with a molecular mass of 13 kDa, as estimated by SDS-PAGE. Inhibitor constants (Ki) and dissociation constants (Kd) were calculated as 14.44, 13.16 and 11.72 µM and 9.38, 15.7 and 12.13 for Aß25₋37, Aß29₋33 and Aß1₋40, respectively; the number of binding sites on the enzyme for the peptides was 1.

Biological synthesis of platinum nanoparticles: Effect of initial metal concentration.

The unusual and novel properties of metal nanoparticles are highly sought after in a number of new and existing industries. Current chemical methods of nanoparticle synthesis have shown limited success and it is expected that the use of a biological approach may overcome many of these obstacles. The exploitation of microorganisms for the biosynthesis of metal nanoparticles is an area of research that has received increasing interest over the last decade. The use of living microbes as a tool for nanoparticle biosynthesis has been researched extensively, however the use of the cellular extract within the cells, excluding the living organism as a whole, has not received much attention. In this investigation, the cell-free, cell-soluble protein extract from a consortium of sulfate-reducing bacteria was used successfully in the biosynthesis of geometric Pt(0) nanoparticles, where previously, whole cells from the same culture had only resulted in amorphous Pt(0) deposits. It appears that by removing the spatial restrictions imposed by the cell itself, nanoparticles could form. It was also found that by altering the ratio of Pt(IV) to protein concentration in solution, a variety of particle morphologies resulted.

Biological synthesis of platinum nanoparticles with apoferritin.

A novel biological method for the synthesis of platinum nanoparticles using the horse spleen apoferritin (HSAF) is reported. When HSAF was incubated with K(2)PtCl(6) at 23 degrees C) for 48 h followed by subsequent reduction with NaBH(4) it resulted in the formation of spherical platinum nanoparticles, size 4.7 +/- 0.9 nm, with narrow particle size distribution confirmed by transmission electron microscopy and energy dispersive X-ray analysis. As the initial platinum concentration increased through 0.155, 0.31, 0.465 to 0.62 mM the efficiency of its removal from solution by the apoferritin was 99, 99, 84 and 71% respectively. The maximum uptake of platinum salt per mM apoferritin was estimated at 12.7 mmol l(-1) h(-1). These results clearly indicate that the HSAF protein cage can successfully serve as a suitable size-constrained support matrix for the biological synthesis of platinum nanoparticles.

Controlled production of fructose by an exoinulinase from Aspergillus ficuum.

An exoinulinase has been isolated, purified and characterised from a commercially available broth of Aspergillus ficuum. The enzyme was purified 4.2-fold in a 21% yield with a specific activity of 12,300 U mg(-1)(protein) after dialysis, ammonium sulphate fractionation and Sephacryl S-200 size exclusion and ion exchange chromatography. The molecular weight of this enzyme was estimated to be 63 kDa by SDS-PAGE. It exhibited a pH and temperature optima of 5.4 and 50 degrees C respectively and under such conditions the enzyme remained stable with 96% and 63.8% residual activity after incubation for 12 h and 72 h respectively. The respective K (m) and V (max) values were 4.75 mM and 833.3 micromol min(-1) ml(-1), respectively. Response surface methodological statistical analysis was evaluated for the maximal production of fructose from the hydrolysis of pure commercial chicory inulin. Incubation of the dialyzed crude exoinulinase (100 U/ml, 48 h, 50 degrees C, 150% inulin, pH 5.0) produced the highest amount of fructose (106.4 mg/ml) under static batch conditions. The purified exoinulinase was evaluated for fructose production and the highest amount (98 mg/ml) was produced after 12 h incubation at 50 degrees C, 150% inulin pH 5.0. The use of a crude exoinulinase preparation is economically desirable and the industrial production of fructose from inulin hydrolysis is biotechnologically feasible.

Response surface methodology: Synthesis of short chain fructooligosaccharides with a fructosyltransferase from Aspergillus aculeatus.

A transferase was isolated, purified and characterised from Aspergillus aculeatus. The enzyme exhibited a pH and temperature optima of 6.0 and 60 degrees C, respectively and under such conditions remained stable with no decrease in activity after 5h. The enzyme was purified 7.1 fold with a yield of 22.3% and specific activity of 486.1Umg(-1) after dialysis, concentration with polyethyleneglycol (30%) and DEAE-Sephacel chromatography. It was monomeric with a molecular mass of 85kDa and K(m) and V(max) values of 272.3mM and 166.7micromolmin(-1)ml(-1). The influence of pH, temperature, reaction time, and enzyme and sucrose concentration on the formation of short-chain fructooligosaccharides (FOS) was examined by statistical response surface methodology (RSM). The enzyme showed both transfructosylation and hydrolytic activity with the transfructosylation ratio increasing to 88% at a sucrose concentration of 600mgml(-1). Sucrose concentration (400mgml(-1)) temperature (60 degrees C), and pH (5.6) favoured the synthesis of high levels of GF(3) and GF(4). Incubation time had a critical effect on the yield of FOS as the major products were GF(2) after 4h and GF(4) after 8h. A prolonged incubation of 16h resulted in the conversion of GF(4) into GF(2) as a result of self hydrolase activity.

Decolorization and degradation of textile dyes with biosulfidogenic hydrogenases.

Successful decolorization of azo dyes (Orange II, Amido Black 10, Reactive Black 5, and Reactive Red 120) and industrial textile dye influents and effluents with sulfate-reducing bacteria from within a biosulfidogenic reactor was achieved with decolorizations ranging from 96% to 49% over 144 h. Concomitant with the decrease in absorbance of the dye in the visible region (480-620 nm) was an increase in the absorbance at 280 nm, over 48 h, suggesting an increase in concentration of single aromatic amines. With an extended period of time there was a subsequent decrease in the absorbance at 280 nm indicating that the aromatic amines had been degraded. The anthraquinone dye, Reactive Blue 2, remained unchanged after 144 h of incubation in the biosulfidogenic reactor and was only rapidly decolored at 192 h, implying that certain factors are induced in the reactor to break down this non-azo dye. The fastest decolorization/degradation rates and highest hydrogenase enzyme production were observed with Orange II, while the slowest decolorization/degradation rate and least enzyme production were with Reactive Blue 2, suggesting that these processes are controlled, to a certain degree, by an enzymatic mechanism. With sulfate-reducing bacteria that had been cultured on a lactate medium, there was complete decolorization of both authentic dyes and industrial influents and effluents as monitored by the decrease of absorbance in the visible region (480-620 nm). There was, however, very little breakdown of the single aromatic compounds as the absorbance at 280 nm remained fairly significant. This supports the suggestion that, within the biosulfidogenic reactor, there are factors other than the identified hydrogenases that are responsible for degradation of the aromatic compounds.

Bioreduction of Pt (IV) from aqueous solution using sulphate-reducing bacteria.

The aims of this study were to investigate the role of sulphate-reducing bacteria in facilitating Pt removal from aqueous solutions and to investigate the role of a hydrogenase enzyme in Pt reduction in vitro. To avoid precipitation of Pt as Pt sulphide, a resting (non-growing) mixed culture was used. A pH-dependent rate of Pt removal from aqueous solution was observed, indicating that metal speciation was the main factor for its removal from solution. The maximum initial concentration of Pt(IV) that the cells can effectively remove from solution was 50 mg/l, while the maximum capacity was only 4 mg of Pt per gram of resting biomass. Transmission electron microscopy and energy dispersive X-ray analyses indicated that Pt was being precipitated in the periplasm, a major area of hydrogenase activity in the cells. In vitro investigation of Pt reduction with hydrogen as the electron donor showed that 49% was removed within 1 h when a relatively pure hydrogenase extract was used, 31% was removed with a cell-free soluble extract and 70% removed by live cells.

Analysis of the inter- and extracellular formation of platinum nanoparticles by Fusarium oxysporum f. sp. lycopersici using response surface methodology.

Fusarium oxysporum fungal strain was screened and found to be successful for the inter- and extracellular production of platinum nanoparticles. Nanoparticle formation was visually observed, over time, by the colour of the extracellular solution and/or the fungal biomass turning from yellow to dark brown, and their concentration was determined from the amount of residual hexachloroplatinic acid measured from a standard curve at 456 nm. The extracellular nanoparticles were characterized by transmission electron microscopy. Nanoparticles of varying size (10-100 nm) and shape (hexagons, pentagons, circles, squares, rectangles) were produced at both extracellular and intercellular levels by the Fusarium oxysporum. The particles precipitate out of solution and bioaccumulate by nucleation either intercellularly, on the cell wall/membrane, or extracellularly in the surrounding medium. The importance of pH, temperature and hexachloroplatinic acid (H(2)PtCl(6)) concentration in nanoparticle formation was examined through the use of a statistical response surface methodology. Only the extracellular production of nanoparticles proved to be statistically significant, with a concentration yield of 4.85 mg l(-1) estimated by a first-order regression model. From a second-order polynomial regression, the predicted yield of nanoparticles increased to 5.66 mg l(-1) and, after a backward step, regression gave a final model with a yield of 6.59 mg l(-1).

The enzymology of sludge solubilisation utilising sulphate reducing systems: the role of lipases.

The first stage in the degradation and recycling of particulate organic matter is the solubilisation and enhanced hydrolysis of complex polymeric organic carbon structures associated with the sulphidogenic environment. An investigation into the enzymology of these processes has shown that lipase enzyme activities were found predominantly associated with the organic particulate matter of the sewage sludge. Sonication of the sludge gave an increase in enzyme activity as the enzymes were released into the supernatant. pH and temperature optimisation studies showed optima at between 6.5 and 8 and 50-60 degrees C, respectively. All the lipase enzymes from the methanogenic bioreactors indicated extensive stability for at least an hour at their respective optimum temperatures and pH; sulphidogenic lipases reflected limited stability at these temperatures and pH during this time period. Though sulphate showed inhibitory properties towards lipases both sulphide and sulphite appeared to enhance the activity of the enzymes. It is argued that these sulphur species, liberated at different times during the sulphate reduction process, disrupt the integrity of the organic particulate floc by neutralising acidic components on the surface. The release of further entrapped enzymes from the organic particulate matter results in a subsequent enhancement of hydrolysis of polymeric material.

Co-digestion of primary sewage sludge and industrial wastewater under anaerobic sulphate reducing conditions: enzymatic profiles in a recycling sludge bed reactor.

The first stage in the degradation and recycling of primary sewage sludge and particulate organic matter is the solubilisation and enhanced hydrolysis of complex polymeric organic carbon structures associated with the anaerobic sulphidogenic environment. Solubilization of complex carbon substrates provides the primary reaction in the BioSURE Process, and is effected in the recycling sludge bed reactor (RSBR). During the process of anaerobic digestion, macromolecules are broken down into simpler low molecular weight compounds in the presence of extracellular enzymes. Though hydrolysis of the complex carbon sources was enhanced under biosulphidogenic conditions, no studies have examined the role of enzymes and the enzymatic profiles in the RSBR. To investigate the overall enzymology in the RSBR variations in COD, pH, sulphate, sulphite and sulphide concentrations, carbohydrates, protein and activities of glucosidases, proteases and lipases were studied over 50 days in the reactor at three different depths: 0-16 cm; 16-32 cm; 32-50 cm. While the pH profile remained fairly constant between 6.9 and 7.3 the sulphate and sulphide levels, as expected, changed dramatically as sulphate reduction took place. Proteases, lipases and glucosidases all showed enhanced activity with depth in the bioreactor. It is suggested that the increased sulphide concentration generated during the sulphate reduction process stimulates the enzymes, leading to enhanced solubilisation of primary sewage sludge.