Lime pretreatment of sugar beet pulp and evaluation of synergy between ArfA, ManA and XynA from Clostridium cellulovorans on the pretreated substrate

Sugar beet pulp (SBP) is a waste product from the sugar beet industry and could be used as a potential biomass feedstock for second generation biofuel technology. Pretreatment of SBP with ‘slake lime’ (calcium hydroxide) was investigated using a 23 factorial design and the factors examined included lime loading, temperature and time. The pretreatment was evaluated for its ability to enhance enzymatic degradation using a combination of three hemicellulases, namely ArfA (an arabinofuranosidase), ManA (an endo-mannanase) and XynA (an endo-xylanase) from C. cellulovorans to determine the conditions under which optimal activity was facilitated. Optimal pretreatment conditions were found to be 0.4 g lime/g SBP, with 36 h digestion at 40 °C. The synergistic interactions between ArfA, ManA and XynA from C. cellulovorans were subsequently investigated on the pretreated SBP. The highest degree of synergy was observed at a protein ratio of 75% ArfA to 25% ManA, with a specific activity of 2.9 U/g protein. However, the highest activity was observed at 4.2 U/g protein at 100% ArfA. This study demonstrated that lime treatment enhanced enzymatic hydrolysis of SBP. The ArfA was the most effective hemicellulase for release of sugars from pretreated SBP, but the synergy with the ManA indicated that low levels of mannan in SBP were probably masking the access of the ArfA to its substrate. XynA displayed no synergy with the other two hemicellulases, indicating that the xylan in the SBP was not hampering the access of ArfA or ManA to their substrates and was not closely associated with the mannan and arabinan in the SBP

Both helix topology and counterion distribution contribute to the more effective charge screening in dsRNA compared with dsDNA

The recent discovery of the RNA interference mechanism emphasizes the biological importance of short, isolated, double-stranded (ds) RNA helices and calls for a complete understanding of the biophysical properties of dsRNA. However, most previous studies of the electrostatics of nucleic acid duplexes have focused on DNA. Here, we present a comparative investigation of electrostatic effects in RNA and DNA. Using resonant (anomalous) and non-resonant small-angle X-ray scattering, we characterized the charge screening efficiency and counterion distribution around short (25 bp) dsDNA and RNA molecules of comparable sequence. Consistent with theoretical predictions, we find counterion mediated screening to be more efficient for dsRNA than dsDNA. Furthermore, the topology of the RNA A-form helix alters the spatial distribution of counterions relative to B-form DNA. The experimental results reported here agree well with ion-size-corrected non-linear Poisson–Boltzmann calculations. We propose that differences in electrostatic properties aid in selective recognition of different types of short nucleic acid helices by target binding partners

Nanomaterial-Assisted Signal Enhancement of Hybridization for DNA Biosensors: A Review

Detection of DNA sequences has received broad attention due to its potential applications in a variety of fields. As sensitivity of DNA biosensors is determined by signal variation of hybridization events, the signal enhancement is of great significance for improving the sensitivity in DNA detection, which still remains a great challenge. Nanomaterials, which possess some unique chemical and physical properties caused by nanoscale effects, provide a new opportunity for developing novel nanomaterial-based signal-enhancers for DNA biosensors. In this review, recent progress concerning this field, including some newly-developed signal enhancement approaches using quantum-dots, carbon nanotubes and their composites reported by our group and other researchers are comprehensively summarized. Reports on signal enhancement of DNA biosensors by non-nanomaterials, such as enzymes and polymer reagents, are also reviewed for comparison. Furthermore, the prospects for developing DNA biosensors using nanomaterials as signal-enhancers in future are also indicated

Starch digestion in tropical fishes : isolation, structural studies and inhibition kinetics of alpha-amylases from two tilapias Oreochromis niloticus and Sarotherodon melanotheron

Alpha-amylases from the intestinal cavity of two tilapia species, #Oreochromis niloticus (ONI-AMY) and #Sarotherodon melanotheron (SME-AMY), were purified using ammonium sulfate precipitation, affinity chromatography and chromatofocusing procedures. The purification was approximately 100-fold. The amylolytic activity, specific activity, product distribution, pH and temperature profile of ONI-AMY and SME-AMY are quite similar. The molecular mass differs slightly : 56 600 (ONI-AMY) vs. 55 500 (SME-AMY). As shown by isoelectric focusing analysis, both amylases contain two isoforms A and B with distinct pI : 7.2 (A) and 7.8 (B), vs. 8.3 (A) and 8.8 (B), respectively. It was not possible to isolate B, since B converts into A with time. The kinetics of the inhibition of ONI-AMY and SME-AMY activity by alpha, Beta, and gamma-cyclodextrin (alpha-, Beta- and gamma-CD) were investigated using amylose as the substrate. Statistical analysis of the kinetic data expressed using a general velocity equation and assuming rapid equilibrium showed that the inhibition is of the mixed noncompetitive type. Similar results were obtained with ONI-AMY and SME-AMY. Beta- and gamma-CD are stronger inhibitors than alpha-CD. ONI-AMY and SME-AMY are then closely related and show the general features common to the members of the alpha-amylase class (family 13). They enable ONI and SME tilapias to digest starch in food. (Résumé d'auteur

Cysteine Alkylation Blocks siRNA Binding to a p19 Viral Suppressor of RNA Silencing

NRC publication: Ye

Studies of a viral suppressor of RNA silencing p19-CFP fusion protein: A FRET-based probe for sensing double stranded fluorophore tagged small RNAs

Eukaryotes have evolved complex cellular responses to double-stranded RNA. One response that is highly conserved across many species is the RNA silencing pathway. Tombusviruses have evolved a mechanism to evade the RNA silencing pathway that involves a small protein, p19, that acts as a suppressor of RNA silencing. This protein binds specifically to small-interfering RNAs (siRNAs) with nanomolar affinity in a sequence-independent manner and with size selectivity.Peer reviewed: YesNRC publication: N