Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6–12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d /l -, α/β-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.     

Bifunctional Dendrimers: From Robust Synthesis and Accelerated One‐Pot Postfunctionalization Strategy to Potential Applications

A fourth wheel : Two sets of bifunctional AB₂C dendrimers having internal acetylene/azides and external hydroxy groups were constructed utilizing benign synthetic protocols. An in situ postfunctionalization strategy was successfully carried out to illustrate the chemoselective nature of these dendrimers. The dendrimers were also transformed into dendritic nanoparticles or utilized as dendritic crosslinkers for the fabrication hydrogels.

     

An on-line method for pressurized hot water extraction and enzymatic hydrolysis of quercetin glucosides from onions

A novel environmentally sound continuous-flow hot water extraction and enzymatic hydrolysis method for determination of quercetin in onion raw materials was successfully constructed using a stepwise optimization approach. In the first step, enzymatic hydrolysis of quercetin-3,4′-diglucoside to quercetin was optimized using a three level central composite design considering temperature (75–95 °C), pH (3–6) and volume concentration of ethanol (5–15%). The enzyme used was a thermostable β-glucosidase variant (termed TnBgl1A_N221S/P342L) covalently immobilized on either of two acrylic support-materials (Eupergit^® C 250L or monolithic cryogel). Optimal reaction conditions were irrespective of support 84 °C, 5% ethanol and pH 5.5, and at these conditions, no significant loss of enzyme activity was observed during 72 h of use. In a second step, hot water extractions from chopped yellow onions, run at the optimal temperature for hydrolysis, were optimized in a two level design with respect to pH (2.6 and 5.5), ethanol concentration (0 and 5%) and flow rate (1 and 3 mL min⁻¹) Obtained results showed that the total quercetin extraction yield was 1.7 times higher using a flow rate of 3 mL min⁻¹ (extraction time 90 min), compared to a flow rate of 1 mL min⁻¹ (extraction time 240 min). Presence of 5% ethanol was favorable for the extraction yield, while a further decrease in pH was not, not even for the extraction step alone. Finally, the complete continuous flow method (84 °C, 5% ethanol, pH 5.5, 3 mL min⁻¹) was used to extract quercetin from yellow, red and shallot onions and resulted in higher or similar yield (e.g. 8.4 ± 0.7 μmol g⁻¹ fresh weight yellow onion) compared to a conventional batch extraction method using methanol as extraction solvent.     

Improved measurement of /V(cb)/ using B-->D*l nu decays

We determine the weak coupling /V(cb)/ between the b and c quarks using a sample of 3 x 10(6) BB; events in the CLEO detector at the Cornell Electron Storage Ring. We determine the yield of reconstructed B-->D*l nu; decays as a function of w, the boost of the D* in the B rest frame, and from this we obtain the differential decay rate d Gamma/dw. By extrapolating d Gamma/dw to w=1, the kinematic end point at which the D* is at rest relative to the B, we extract the product /V(cb)/F(1), where F(1) is the form factor at w=1. Combined with theoretical results for F(1) we determine /V(cb)/=0.0469+/-0.0014(stat)+/-0.0020(syst)+/-0.0018(theor).