Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes.

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo- and exo-lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo-acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo-exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo-exo synergy was caused by an auxiliary endo-lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo-lytic activity of both Cel6A and Cel7A were 103 -104 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo-lytic activity of Cel7A was 2-3-fold higher than for Cel6A, and we suggest that endo-like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo-exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639-1647. © 2017 Wiley Periodicals, Inc.

A Reinvestigation of the Ionic Liquid Diisopropylethylammonium Formate by NMR and DFT Methods.

The complex between diisopropylethylamine (DIPEA) and formic acid has been reinvestigated. Mixing the compounds in the ratio 1:1 leads to a phase separation in which the upper phase is DIPEA and the lower phase is the "ionic liquid" named DIPEF. A combined NMR and DFT study shows that the lower phase primarily is formic acid:formate and diisopropylammonium ions in the ratio 2:1 (acid:base) plus the formic acid dimer. Addition of more acid leads to more and more of the acid dimer. The proton transfer in the system is 65-80%. The structural picture presented in this paper is very different from that presented elsewhere. However, the present picture should be considered using acids and bases with a pKa difference less than 8. The formic acid content in the DIPEF ionic liquid causes desorption of the dye-sensitized solar cell (DSC) dye N719 from the photo anode, and DIPEF is therefore not a suitable electrolyte for DSCs.

Role of Electrostatic Interactions on the Transport of Druglike Molecules in Hydrogel-Based Articular Cartilage Mimics: Implications for Drug Delivery.

In the field of drug delivery to the articular cartilage, it is advantageous to apply artificial tissue models as surrogates of cartilage for investigating drug transport and release properties. In this study, artificial cartilage models consisting of 0.5% (w/v) agarose gel containing 0.5% (w/v) chondroitin sulfate or 0.5% (w/v) hyaluronic acid were developed, and their rheological and morphological properties were characterized. UV imaging was utilized to quantify the transport properties of the following four model compounds in the agarose gel and in the developed artificial cartilage models: H-Ala-ß-naphthylamide, H-Lys-Lys-ß-naphthylamide, lysozyme, and α-lactalbumin. The obtained results showed that the incorporation of the polyelectrolytes chondroitin sulfate or hyaluronic acid into agarose gel induced a significant reduction in the apparent diffusivities of the cationic model compounds as compared to the pure agarose gel. The decrease in apparent diffusivity of the cationic compounds was not caused by a change in the gel structure since a similar reduction in apparent diffusivity was not observed for the net negatively charged protein α-lactalbumin. The apparent diffusivity of the cationic compounds in the negatively charged hydrogels was highly dependent on the ionic strength, pointing out the importance of electrostatic interactions between the diffusant and the polyelectrolytes. Solution based affinity studies between the model compounds and the two investigated polyelectrolytes further confirmed the electrostatic nature of their interactions. The results obtained from the UV imaging diffusion studies are important for understanding the effect of drug physicochemical properties on the transport in articular cartilage. The extracted information may be useful in the development of hydrogels for in vitro release testing having features resembling the articular cartilage.

The molar hydrodynamic volume changes of factor VIIa due to GlycoPEGylation.

The effects of GlycoPEGylation on the molar hydrodynamic volume of recombinant human rFVIIa were investigated using rFVIIa and two GlycoPEGylated recombinant human FVIIa derivatives, a linear 10kDa PEG and a branched 40kDa PEG, respectively. Molar hydrodynamic volumes were determined by capillary viscometry and mass spectrometry. The intrinsic viscosities of rFVIIa, its two GlycoPEGylated compounds, and of linear 8kDa, 10kDa, 20kDa and branched 40kDa PEG polymers were determined. The measured intrinsic viscosity of rFVIIa is 6.0mL/g, while the intrinsic viscosities of 10kDa PEG-rFVIIa and 40kDa PEG-rFVIIa are 29.5mL/g and 79.0mL/g, respectively. The intrinsic viscosities of the linear PEG polymers are 20, 22.6 and 41.4mL/g for 8, 10, and 20kDa, respectively, and 61.1mL/g for the branched 40kDa PEG. From the results of the intrinsic viscosity and MALDI-TOF measurements it is evident, that the molar hydrodynamic volume of the conjugated protein is not just an addition of the molar hydrodynamic volume of the PEG and the protein. The molar hydrodynamic volume of the GlycoPEGylated protein is larger than the volume of its composites. These results suggest that both the linear and the branched PEG are not wrapped around the surface of rFVIIa but are chains that are significantly stretched out when attached to the protein.

Thermodynamic investigations of methyl tert-butyl ether and water mixtures.

Interactions between methyl tert-butyl ether (MTBE) and water have been investigated by scanning calorimetry, isothermal titration calorimetry, densitometry, IR-spectroscopy, and gas chromatography. The solubilization of MTBE in water at 25 °C at infinite dilution has ΔH° = -17.0 ± 0.6 kJ mol(-1); ΔS° = -80 ± 2 J mol(-1) K(-1); ΔC(p) = +332 ± 15 J mol(-1) K(-1); ΔV° = -18 ± 2 cm(3) mol(-1). The signs of these thermodynamic functions are consistent with hydrophobic interactions. The occurrence of hydrophobic interaction is further substantiated as IR absorption spectra of MTBE-water mixtures show that MTBE strengthens the hydrogen bond network of water. Solubilization of MTBE in water is exothermic whereas solubilization of water in MTBE is endothermic with ΔH° = +5.3 ± 0.6 kJ mol(-1). The negative mixing volume is explained by a large negative contribution due to size differences between water and MTBE and by a positive contribution due to changes in the water structure around MTBE. Henry's law constants, K(H), were determined from vapor pressure measurements of mixtures equilibrated at different temperatures. A van't Hoff analysis of K(H) gave ΔH(H)° = 50 ± 1 kJ mol(-1) and ΔS(H)° = 166 ± 5 J mol(-1) K(-1) for the solution to gas transfer. MTBE is excluded from the ice phase water upon freezing MTBE-water mixtures.

Basic rheology for biologists.

Many cellular processes lead to changes in elastic and viscous properties of cells. Rheology is the science that deals with deformation and flow of materials. Fundamental rheologic concepts are explained, and some of the main techniques are discussed. Nonperturbing oscillatory techniques are especially useful for monitoring structure formation including gelation, whereas other techniques such as steady shear flow and creep are useful for determining flow properties. Sample preparation is often a major obstacle, and advantages of different deformation geometries are discussed. Simple biological samples such as purified biopolymers can be investigated with a range of rheologic techniques, and factors affecting gelation of, for example, blood or cytoskeletal proteins can be studied in detail. More complex biological systems such as intact tissues can often only be studied with more qualitative techniques and results. With proper choice of experimental setup, rheologic techniques can give valuable information about cellular systems and dynamics on a timescale that is closely related to biological functions.

Elastic contributions dominate the viscoelastic properties of sputum from cystic fibrosis patients.

Sputum samples from cystic fibrosis (CF) patients were investigated by oscillatory, creep and steady shear rheological techniques over a range of time scales from 10(-3) to 10(6) s. The viscoelastic changes obtained by mixing sputa with the actin-filament-severing protein gelsolin and with the thiol-reducing agent dithiothreitol (DTT) were also investigated. At small strains sputum behaves like a viscoelastic solid rather than a liquid. A nearly constant steady shear viscosity at low shear rates is only observed after long shearing times which cause irreversible changes in the samples. Creep-recovery tests confirm that sputa exhibit viscoelastic properties, with a significant elastic recovery. The results suggest that measurements of elastic moduli, rather than viscosities are more closely related to the mechanical properties of sputum in situ. Severing of actin filaments lowers the elastic modulus by 30-40%, but maintains viscoelastic integrity, while reduction of thiols in the glycoproteins nearly completely fluidizes the samples.

Micellization of block copolymer P94 in aqueous solution.

P94 is a triblock copolymer of poly(oxyethylene) and poly(oxypropylene), type EPE. Eluent gel permeation chromatography has been used to demonstrate the formation of large micelles in solutions of the copolymer at temperatures immediately above the critical micelle temperature. Values of the critical micelle temperature (cmt) obtained in this way are in excellent agreement with results from differential scanning calorimetry and surface tensiometry. A van't Hoff plot was used to define the concentration dependence of the cmt for dilute solutions (c < or =2 wt%) and to obtain an apparent value of the standard enthalpy of micellization. Using published data, comparison is made with the thermodynamics of micellization in concentrated solution (c > or =20 wt%).

Effects of poloxamer polydispersity on micellization in water.

The poloxamer 284 (Pluronic P94) is a triblock copolymer of poly(ethylene oxide) and poly(propylene oxide). P94 and fractions of P94 obtained after centrifugation at temperatures where solutions contain both dissolved unimers and micelles have been investigated by differential scanning calorimetry, mass spectrometry, and NMR. The results show that the P94 sample is heterogeneous with respect to both chemical composition and molar mass. The first micelles formed, when the temperature is increased, contain poloxamers with a significantly higher propylene oxide content and a higher molar mass in agreement with theoretical predictions. The characteristic temperatures of micellization, sphere-to-rod, and phase separation transitions observed in thermograms are influenced by polydispersity, which results in broader transitions.