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 °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 μmol 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 °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 °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.
On the enzymatic formation of platinum nanoparticles Govender, Y.; Riddin, T. L.; Gericke, M. ...
Journal of nanoparticle research : an interdisciplinary forum for nanoscale science and technology,
2010/1, Letnik:
12, Številka:
1
Journal Article
A dimeric hydrogenase enzyme (44.5 and 39.4 kDa sub units) was isolated in a 39.5% yield from the fungus
Fusarium oxysporum
and purified 4.64-fold by ion exchange chromatography on Sephacryl S-200. ...Characterisation of the enzyme afforded pH and temperature optima of 7.5 and 38 °C, respectively, a half-life stability of 36 min and a
V
max
and
K
m
of 3.57 nmol min
−1
mL
−1
and 2.25 mM, respectively. This enzyme was inhibited (non-competitively) by hydrogen hexachloroplatinic acid (H
2
PtCl
6
) at 1 or 2 mM with a
K
i
value of 118 μM. Incubation of the platinum salt with the pure enzyme under an atmosphere of hydrogen and optimum enzyme conditions (pH 7.5, 38 °C) afforded <10% bioreduction after 8 h while at conditions suitable for platinum nanoparticle formation (pH 9, 65 °C) over 90% reduction took place after the same length of time. Cell-free extract from the fungal isolates produced nearly 90% bioreduction of the platinum salt under both pH and temperature conditions. The bioreduction of the platinum salt by a hydrogenase enzyme takes place by a passive process and not an active one as previously understood.
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.
•Recombinant His-tagged thiazole kinase isolated, purified from genomic DNA of Plasmodium falciparum.•Monomeric enzyme with mass 34kDa, specific activity 295nmolmg−1 temperature and pH optima of 37°C ...and 7.5.•Km (65μM) and Vmax (36.8μmolml−1min−1) purified by FPLC on Sephacryl S100HR.•Non-competitive inhibition by Ag (79%; Ki=6.45μM).•Nanoparticles interact through Met1, Cys206 on surface of enzyme.
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 34kDa, a specific activity of 295.04nmolmin−1mg−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 (2–6nm); Ki=6.45μM. A mechanism is suggested for the interaction of the silver nanoparticle with PfThzK through two sulphur bearing amino acids (Met1, Cys206) on the surface of each subunit of the enzyme.
Ultrasound tissue characterization (UTC) imaging has been previously used to describe the characteristics of patellar and Achilles tendons. UTC imaging compares and correlates successive ...ultrasonographic transverse tendon images to calculate the distribution of four color-coded echo-types that represent different tendon tissue types. However, UTC has not been used to describe the characteristics of patellar tendons after anterior cruciate ligament reconstruction (ACLR). The aim of this cross-sectional study was to assess the intra and inter-rater reliability of the UTC in unharvested and harvested patellar tendons of patients undergoing ACLR.
Intra and inter-rater reliability of both UTC data collection and analysis were assessed. Ten harvested and twenty unharvested patellar tendons from eighteen participants were scanned twice by the same examiner. Eleven harvested and ten unharvested patellar tendons from sixteen participants were scanned and analyzed twice by two different examiners. Twenty harvested and nineteen unharvested patellar tendons from twenty-three participants were analyzed twice by two examiners.
Quantification of the proportion of echo-types I, II, III and IV in the areas of interest: (1) patella apex, (2) proximal tendon, (3) mid tendon, (4) distal tendon, and overall tendon of harvested and unharvested patellar tendons all displayed excellent intra-rater reliability (ICC
: 0.94 to 0.99), excellent inter-rater reliability for harvested and unharvested patellar tendon scanning and analysis (ICC
: 0.89 to 0.98), and excellent inter-rater reliability for analysis (ICC
: 0.95 to 0.99). Intra-rater reliability for the measure of volume was good (ICC
: 0.69 harvested, 0.67 unharvested), whilst mixed results were observed for the measure of mid tendon thickness (ICC
: 0.88 harvested, 0.57 unharvested). Inter-rater reliability for scanning and analysis was good for volume (ICC
: 0.67) and excellent for thickness (ICC
: 0.97), while the inter-rater reliability for analysis was fair to poor for volume (ICC
: 0.59 harvested, 0.30 unharvested), and excellent to poor for mid tendon thickness (ICC
: 0.85 harvested, 0.24 unharvested).
UTC imaging is a reliable tool to characterize the quality of most aspects of unharvested and harvested patellar tendons in subjects undergoing ACLR.
A novel biological method for the synthesis of platinum nanoparticles using the horse spleen apoferritin (HSAF) is reported. When HSAF was incubated with K₂PtCl₆ at 23°C) for 48 h followed by ...subsequent reduction with NaBH₄ 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.
The incubation of neuronal nitric oxide synthase with the five amyloid peptide fragments Aβ
17–21
; Aβ
25–29
; Aβ
29–33
; Aβ
33–37
; Aβ
25–37
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 Aβ
17–21
, the three glycine zipper peptides Aβ
25–29
; Aβ
29–33
; Aβ
33–37
and Aβ
25–37
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-Aβ incubation. The addition of nNOS and/or Aβ to co-incubated solutions of nanoparticle-Aβ or nanoparticle-nNOS respectively did not prevent fibril formation but reversed it. Three mechanisms for this reversal were proposed: (1) depletion of free Aβ 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 Aβ peptide and nanoparticle (3) disruption of binary adducts between Aβ-peptides and nNOS by nanoparticles.
Not only are β-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 β-amyloid peptides, in the glycine zipper region Aβ29-33 and Aβ25-37 of Aβ1-40 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 Aβ25-37, Aβ29-33 and Aβ1-40, respectively; the number of binding sites on the enzyme for the peptides was 1.
Two endoglucanases and a β-glucosidase have been isolated, purified and characterised from an anaerobic sulphidogenic bioreactor. The enzymes, associated predominantly with the organic particulate ...matter, exhibited a pH optima of 6 and 6.5, respectively and temperature optima of 50
°C. Under such conditions the endoglucanases remained stable and exhibited no decrease in activity after 60
min while only 30% glucosidase remained after the same period. The endoglucanases were purified 13- and 25-fold after sonication, PEG concentration and DEAE chomatography. They were inhibited slightly by increasing concentrations of sulphate but stimulated some 4–6.5-fold by sulphide levels above 400
mg
l
−1. The
K
m value was 4.0
mg
ml
−1 (carboxymethylcellulose) and 5.1
mg
ml
−1 (hydroxyethylcellulose) with
V
max of 0.3 and 0.19
μmol
min
−1
ml
−1, respectively. Divalent ions like Cu, Ni and Zn proved to be inhibitory while Fe, Mg and Ca stimulated the enzyme at concentrations above 400
mg
l
−1. Volatile fatty acids such as acetic, propionic and butyric acid proved slightly inhibitory to endoglucanases with 20–40% inhibition occurring at concentrations of 800
mg
l
−1. β-Glucosidase was purified 5-fold after acetone precipitation, affinity chromatography with Whatman cellulose CC31 and gel exclusion on Sepharose 4B. The
K
m value was 84.2
μM (methyl-umbelliferyl-β-
d-glucopyranoside) and the
V
max 4.4
μmol
min
−1
ml
−1. All of the transition metals inhibited β-glucosidase above 200
mg
l
−1 while the volatile fatty acids afforded similar effects to those of the endoglucanases. Acetic acid enhanced activity at lower concentrations.
The ferroxidase activity of horse spleen apoferritin (HSAF) is increased by nine-fold in the presence of platinum nanoparticles. HSAF was mixed with varying concentrations of K2PtCl4 followed by a ...20-fold concentration of sodium borohydride to afford Pt:HSAF nanoparticle complexes in a ratio of between 1:250 and 1:4000. Typical colour changes, from colourless or pale yellow to brown, occurred that were dependent on the amount of platinum present. These complexes were characterized by UV/vis, inductively coupled plasma optical emission spectroscopy, Fourier transform infrared, transmission electron microscopy and energy dispersive x-ray spectroscopy. Transmission electron microscopy analysis revealed that the size of nanoparticles increased as the molar ratio of platinum to HSAF increased with an average size diameter of 2-6 nm generated with HSAF:platinum molar ratios of 1:250-1:4000. Fourier transform infrared spectroscopy (FTIR) spectra showed no distinct changes in the structure of HSAF but confirmed that the nanoparticles were attached to the protein. The effect of platinum nanoparticles on the ferroxidase activity of HSAF showed a specific activity of 360 ρmol min(-1) mg(-1), (nine-fold increase over the control) at the molar ratio of HSAF:platinum nanoparticles of 1:1000.
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