Xylan oligosaccharides and cellobiohydrolase I (TrCel7A) interaction and effect on activity

<p>Abstract</p> <p>Background</p> <p>The well-studied cellulase mixture secreted by <it>Trichoderma reesei </it>(anamorph to <it>Hypocrea jecorina</it>) contains two cellobiohydolases (CBHs), cellobiohydrolase I (<it>Tr</it>Cel7A) and cellobiohydrolase II (<it>Tr</it>CeI6A), that are core enzymes for the solubilisation of cellulose. This has attracted significant research interest because of the role of the CBHs in the conversion of biomass to fermentable sugars. However, the CHBs are notoriously slow and susceptible to inhibition, which presents a challenge for the commercial utilisation of biomass. The xylans and xylan fragments that are also present in the biomass have been suggested repeatedly as one cause of the reduced activity of CHBs. Yet, the extent and mechanisms of this inhibition remain poorly elucidated. Therefore, we studied xylan oligosaccharides (XOSs) of variable lengths with respect to their binding and inhibition of both <it>Tr</it>Cel7A and an enzyme variant without the cellulose-binding domain (CBM).</p> <p>Results</p> <p>We studied the binding of XOSs to <it>Tr</it>Cel7A by isothermal titration calorimetry. We found that XOSs bind to <it>Tr</it>Cel7A and that the affinity increases commensurate with XOS length. The CBM, on the other hand, did not affect the affinity significantly, which suggests that XOSs may bind to the active site. Activity assays of <it>Tr</it>Cel7A clearly demonstrated the negative effect of the presence of XOSs on the turnover number.</p> <p>Conclusions</p> <p>On the basis of these binding data and a comparison of XOS inhibition of the activity of the two enzyme variants towards, respectively, soluble and insoluble substrates, we propose a competitive mechanism for XOS inhibition of <it>Tr</it>Cel7A with phosphoric swollen cellulose as a substrate.</p

Association of ethanol with lipid membranes containing cholesterol, sphingomyelin and ganglioside: a titration calorimetry study

AbstractThe association of ethanol at physiologically relevant concentrations with lipid bilayers of different lipid composition has been investigated by use of isothermal titration calorimetry (ITC). The liposomes examined were composed of combinations of lipids commonly found in neural cell membranes: dimyristoyl phosphatidylcholine (DMPC), ganglioside (GM1), sphingomyelin and cholesterol. The calorimetric results show that the interaction of ethanol with fluid lipid bilayers is endothermic and strongly dependent on the lipid composition of the liposomes. The data have been used to estimate partitioning coefficients for ethanol into the fluid lipid bilayer phase and the results are discussed in terms of the thermodynamics of partitioning. The presence of 10 mol% sphingomyelin or ganglioside in DMPC liposomes enhances the partitioning coefficient by a factor of 3. Correspondingly, cholesterol (30 mol%) reduces the partitioning coefficient by a factor of 3. This connection between lipid composition and partitioning coefficient correlates with in vivo observations. Comparison of the data with the molecular structure of the lipid molecules suggests that ethanol partitioning is highly sensitive to changes in the lipid backbone (glycerol or ceramide) while it appears much less sensitive to the nature of the head group

Gradual crossover in molecular organization of stable liquid H2O at moderately high pressure and temperature

Using the literature raw data of the speed of sound and the specific volume, the isothermal compressibility, κT, a second derivative thermodynamic quantity of G, was evaluated for liquid H2O in the pressure range up to 350 MPa and the temperature to 50 ºC. We then obtained its pressure derivative, dκT/dp, a third derivative numerically without using a fitting function to the κT data. On taking yet another p-derivative at a fixed T graphically without resorting to any fitting function, the resulting d2κT/dp2, a fourth derivative, showed a weak but clear step anomaly, with the onset of the step named point X and its end point Y. In analogy with another third and fourth derivative pair in binary aqueous solutions of glycerol, dαp/dxGly and d2αp/dxGly2, at 0.1 MPa (αp is the thermal expansivity and xGly the mole fraction of solute glycerol) in our recent publication [J. Solution Chem. 43, 663-674 (2014); DOI:10.1007/s10953-013-0122-7], we argue that there is a gradual crossover in the molecular organization of pure H2O from a low to a high p-regions starting at point X and ending at Y at a fixed T. The crossover takes place gradually spanning for about 100 MPa at a fixed temperature. The extrapolated temperature to zero p seems to be about 70 – 80 °C for points X and 90 – 110 °C for Y. Furthermore, the mid-points of X and Y seem to extrapolate to the triple point of liquid, ice Ih and ice III. Recalling that the zero xGly extrapolation of point X and Y for binary aqueous glycerol at 0.1 MPa gives about the same T values respectively, we suggest that at zero pressure the region below about 70 °C the hydrogen bond network is bond-percolated, while above about 90 ºC there is no hydrogen bond network. Implication of these findings is discussed