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 β-(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.
The heterogeneous complexity of sludges and wastewaters has created gross uncertainty and deviations in predictions of suitable models for their measurement. At the same time, it is becoming ...increasingly obvious that the current paradigms and ideologies are wrought with problems and limitations suggesting the need to move to a more consolidated analytical objective evaluation. Rapid developments in understanding activated sludge processes and wastewater remediation warrants exploitation of different strategies for studying their degradation.
It is time to replace subjective terms like sludge volume index (SVI), zone settling velocity (ZSV), filament index (
F
I), fractal dimension (
D), flocculating ability, surface charge (
ζ), degree of hydrophobicity, chemical oxygen demand (COD) with a mathematical one that can provide an absolute quantitative relationship for the properties of wastewater and/or a sludge floc. There are no current objective values that can be introduced to represent the plethora of biological remediation terminologies such as bioleaching, biosorption, bioaugmentation, biostimulation, biopulping, biodeterioration, biobleaching, bioaccumulation, biotransformation and bioattenuation.
Enzyme technology has been receiving increased attention and this review focuses on the latest developments on the enzymology of biological remediation. It discusses the present pitfalls with current strategies and suggests that sludge–floc parameters, such as internal structure and composition, sludge retention time, microbial ecology, nutrient concentration, dissolved oxygen and type of industrial wastewater, whether from an aerobic or anaerobic system, are replaced by quantitative kinetic terms (
K
m,
V
max,
K
cat) associated with the enzymology of the sludge floc and/or wastewater.
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.
Fluorescence quenching, thermodynamic analysis and FRET have shown that arginine kinase from T. brucei interacted with silver or gold nanoparticles.
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.
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.
The nanoparticles of silver and gold interact with arginine kinase from T. brucei and may prove to have far reaching consequences in clinical trials.
Proposed structure of the binding sites for TbAK showing the interaction of silver/gold nanoparticles through Cys271, interfering with N1 of the arginine substrate. The interatomic distance between the thiolate atom of Cys271 and N1 of the arginine substrate is 3.3Å. The interatomic distance between Trp104 and N1 is 22.2Å while that distance between Trp104 and bound Ag/Au nanoparticles is 21.1Å and 22.6Å respectively. Nitrate not shown. Display omitted
•Interaction of silver and gold nanoparticles with arginine kinase from Trypanosoma brucei by fluorescence quenching•FRET showed that the nanoparticles were bound 2.11nm Ag and 2.26nm Au from a single surface tryptophan.•The nanoparticles bind close to the arginine substrate through a cysteine residue crucial for the enzymes reaction mechanism.•These nanoparticles could have implications against trypanosomiasis in clinical trials.
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.
A transferase was isolated, purified and characterised from
Aspergillus aculeatus. The enzyme exhibited a pH and temperature optima of 6.0 and 60
°C, respectively and under such conditions remained ...stable with no decrease in activity after 5
h. The enzyme was purified 7.1 fold with a yield of 22.3% and specific activity of 486.1
U
mg
−1 after dialysis, concentration with polyethyleneglycol (30%) and DEAE-Sephacel chromatography. It was monomeric with a molecular mass of 85
kDa and K
m and V
max values of 272.3
mM and 166.7
μmol
min
−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 600
mg
ml
−1. Sucrose concentration (400
mg
ml
−1) temperature (60
°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 4
h and GF
4 after 8
h. A prolonged incubation of 16
h resulted in the conversion of GF
4 into GF
2 as a result of self hydrolase activity.
A mixed consortium of sulphate-reducing bacteria was used to investigate the enzymatic mechanism for the total bioreduction of platinum (IV) into platinum (0) nanoparticles. It was established that ...two different hydrogenase enzymes were involved. First the platinum (IV) was reduced to platinum (II) by a two-electron bioreduction using an oxygen-sensitive novel cytoplasmic hydrogenase. Second the platinum (II) ion was reduced to platinum (0) nanoparticle by another two-electron bioreduction involving an oxygen-tolerant/protected periplasmic hydrogenase. The enzyme was identified from its reaction with Cu(II), an active inhibitor of periplasmic hydrogenases. No exogenous electron donors were necessary as endogenous production of hydrogen/electrons, via the oxidation of metabolites, was generated
in situ by the cytoplasmic hydrogenase. The hydrogen then dispersed through the cell to the periplasm where it became available for use by the periplasmic hydrogenase. The endogenous electrons were used, in the absence of sulphate, for the reduction of platinum (II) by the periplasmic hydrogenase. It was found that the Pt(IV) ion must be fully reduced before reduction of the Pt(II) ion would begin. Transmission electron microscopy and energy dispersive X-ray analysis confirmed the deposit of platinum particles into the periplasmic space.
Oligosaccharides of chain length up to degree of polymerisation (DP) 5 have been synthesised using a purified endoinulinase and response surface methodology with central composite experimental design ...for the optimisation of the product yield. The endoinulinase was purified through a three-step procedure in a yield of 1.11% and 3.5-fold and its molecular weight was 68.1
kDa by SDS-PAGE. It had optimum pH and temperature values of 6 and 60
°C, respectively with only 10% loss in activity over 60
min under these conditions. From the kinetic studies
K
m and
V
max were estimated at 3.53
mM and 666.7
μmol
min
−1
ml
−1, respectively. Using the purified endoinulinase preparation isolated from
Aspergillus niger, at a temperature (60
°C), inulin concentration (150
mg
ml
−1), incubation time (48
h), pH (6.0) and enzyme dosage (60
U
ml
−1) produced high yields of inulotrioses (70.3
mM), inulotetraoses (38.8
mM), and inulopentaoses (3.5
mM). Response surface regression predicted similar product levels under the same conditions. A comparative analysis of the yields of individual oligosaccharides produced from both the crude and purified enzyme is also presented.
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.
Abstract Aggregated β-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 Aβ17-21 and glycine zipper peptide Aβ29-33 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 Aβ17-21r ; Aβ29-33r and two peptides with Phe19 , Phe20 and Ile31 , Ile32 substituted with polar glutamic acid Aβ17-21p ; Aβ29-33p . 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 ( KSV ) for Aβ17-21p and Aβ29-33p were 7.2 × 10−3 and 6.1 × 10−3 μM−1 respectively at 298 K some 2–3 fold lower than the corresponding Aβ17-21r ; Aβ29-33r . With temperature increase there was an increase in KSV 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 Aβ17-21r and Aβ17-21p was 10% greater, while for Aβ29-33r and Aβ29-33p 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.
Abstract Five peptide fragments Aβ17–21 ; Aβ25–29 ; Aβ29–33 ; Aβ33–37 ; Aβ25–37 of the toxic Aβ1–40(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 Aβ17–21 to about 8–12 μM for the other peptides. According to the van’t Hoff relationship the thermodynamic parameters, Δ H , Δ S and Δ G 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 Aβ aggregation had to, initially, overcome an activation barrier. Since the Δ G 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 Aβ25–37.
PDF
