Increased mixing intensity is not necessary for more efficient cellulose hydrolysis at high solid loading.

To enhance the cellulose hydrolysis at high solid loadings, an increased mixing intensity is generally required for the high solid loading hydrolysis, while it leads to higher energy consumption. In this study, the impact of mixing intensity on cellulose conversion during hydrolysis at different solid loadings were systematically studied. It was found that the increased mixing intensity is not necessary for more efficient cellulose hydrolysis. For cellulose hydrolysis at higher solid loadings, a lower mixing intensity is needed for a higher cellulose conversion. Although the increased mixing intensity promoted enzyme adsorption, it strengthened product inhibition and caused severer enzyme deactivation. Besides, mixing at the initial stage of cellulose hydrolysis was more crucial, while continuous mixing throughout the hydrolysis was not required for more efficient cellulose hydrolysis. Based on the mechanism study, a combined mixing strategy was developed to achieve efficient cellulose hydrolysis with about two-third reduction in energy consumption.

Effect of cold and hot enzyme deactivation on the structural and functional properties of rice dreg protein hydrolysates.

This study explored the effect of three different enzyme deactivation treatments: 4 °C slow cold deactivation (RDPH-(4 °C)), -18 °C rapid cold deactivation (RDPH-(-18 °C)) and 100 °C water bath (RDPH-(100 °C)), compared to that without enzyme deactivation (RDPH-(control)) on the structural and functional properties of rice dreg protein hydrolysates (RDPHs). The RDPHs from the different enzyme deactivation methods led to significant differences in the degree of hydrolysis, surface hydrophobicity, average particle size, intrinsic fluorescence and emulsion stability. FTIR analysis revealed that the strength of RDPH-(100 °C) spectrum peaks decreased significantly. All samples showed high solubility (>85%) and potent antioxidant capacity: DPPH (~90%), ABTS (~99%), and reducing power (0.86-1.03). Among the hydrolysates evaluated, the RDPH-(100 °C) led to the lowest reducing power and hydroxyl radical scavenging activity. Results reported here will be instrumental for the development of rice protein-based products and in the optimization and scale up of manufacturing process.

Study on the effects of several operational variables on the enzymatic batch saccharification of orange solid waste.

In this work, batch enzyme-aided extraction and enzymatic saccharification of blade-milled orange waste was studied. The operation variables for this process were thoroughly analysed. It was determined that batch runs with initial pH values of 5.0 and 5.2 controlled during the first hour, 50°C and 300-500r.p.m. agitation resulted in the best yields, with a limited total and partial first-order enzyme deactivation (for cellulases and polygalacturonidase, respectively). Orange peel waste (OPW) at 6.7% w/w dry solid, 0.22 filter paper units (FPU)/g DS and proportional activities of other enzymes led to over 40g/L free monosaccharides and global yields to glucose over 80%. When using 10.1% w/w DS in these conditions, glucose yield was 63%, with total monosaccharide concentration on top of 50g/L. Similar concentrations were obtained by additional partial drying of OPW to 60% humidity at DS/L ratios near 7.5% (glucose yield >80%).

The promotional effect of water-soluble extractives on the enzymatic cellulose hydrolysis of pretreated wheat straw.

Enzymatic cellulose hydrolysis of pretreated wheat straw pulp to glucose is enhanced when the hydrolysis is performed in the presence of an aqueous extract of the wheat straw. A relative digestibility increase of about 10% has been observed for organosolv, alkaline and dilute acid pretreated wheat straw pulp (enzyme dose 2.5FPU/g pulp). At lower enzyme doses, the extract effect increases leading to an enzyme dose reduction of 40% to obtain a glucose yield of 75% within 48h using organosolv wheat straw pulp. Possibly, cellulase deactivation by irreversible binding to pulp lignin is reduced by competition with proteins in the extract. However, since the extract effect has also been demonstrated for lignin-lean substrates, other effects like improved accessibility of the pulp cellulose (amorphogenesis) cannot be excluded. Overall, this contribution demonstrates the positive effect of biomass extractives on enzymatic cellulose digestibility, thereby reducing costs for 2G biofuels and bio-based chemicals.

A new method to determine optimum temperature and activation energies for enzymatic reactions.

A new method for determination of the optimum temperature and activation energies based on an idea of the average rate of enzymatic reaction has been developed. A mathematical model describing the effect of temperature on a dimensionless activity for enzyme deactivation following the first-order kinetics has been derived. The necessary condition for existence of the function extreme of the optimal temperature has been applied in the model. The developed method has been verified using the experimental data for inulinase from Kluyveromyces marxianus.

Utilization of parameters developed in layer-by-layer fabrication of protein-containing films for enzyme immobilization.

The layer-by-layer (LbL) self-assembly method has found a broad range of applications in biologically important materials. Developed upon experience based on polyelectrolyte systems, various types of biomolecules have been successfully incorporated into ultrathin films with thickness in the nanometer range. We employed orthogonal experimental design to analyze the factors influencing the buildup of protein-containing LbL ultrathin films, first selecting bovine serum albumin as the exemplary protein. Among the factors, we found that the protein concentration was the most influential factor, followed by protein solution pH. In comparison, the counter polyelectrolyte concentration and solution pH play smaller roles in affecting the film structure. In a preliminary attempt, we employed horseradish peroxidase (HRP) to fabricate ultrathin films and tested the enzymatic activity of these films. We found that the total enzymatic activity increased with more HRP incorporated until reaching four bilayers. After that, the total enzymatic activity became retarded, probably due to amplified diffusion resistance by the added assembly components. The immobilized HRP demonstrated diminished enzymatic activity while recycling. The suspected cause was assigned to the enzyme deactivation by relatively high H2O2 concentrations, employed for lower substrate detection limits. When a low H2O2 concentration was applied during the enzymatic activity measurements, the HRP LbL film maintained the activity level even after nine runs.

Biocatalytic carboxylation of phenol derivatives: kinetics and thermodynamics of the biological Kolbe-Schmitt synthesis.

Microbial decarboxylases, which catalyse the reversible regioselective ortho-carboxylation of phenolic derivatives in anaerobic detoxification pathways, have been studied for their reverse carboxylation activities on electron-rich aromatic substrates. Ortho-hydroxybenzoic acids are important building blocks in the chemical and pharmaceutical industries and are currently produced via the Kolbe-Schmitt process, which requires elevated pressures and temperatures (≥ 5 bar, ≥ 100 °C) and often shows incomplete regioselectivities. In order to resolve bottlenecks in view of preparative-scale applications, we studied the kinetic parameters for 2,6-dihydroxybenzoic acid decarboxylase from Rhizobium sp. in the carboxylation- and decarboxylation-direction using 1,2-dihydroxybenzene (catechol) as starting material. The catalytic properties (K(m), V(max)) are correlated with the overall thermodynamic equilibrium via the Haldane equation, according to a reversible random bi-uni mechanism. The model was subsequently verified by comparing experimental results with simulations. This study provides insights into the catalytic behaviour of a nonoxidative aromatic decarboxylase and reveals key limitations (e.g. substrate oxidation, CO2 pressure, enzyme deactivation, low turnover frequency) in view of the employment of this system as a 'green' alternative to the Kolbe-Schmitt processes.

Thermodynamics of a Ca(2+)-dependent highly thermostable alkaline protease from a haloalkliphilic actinomycete.

An alkaline protease from salt-tolerant alkaliphilic actinomycetes, Nocardiopsis alba OK-5 was purified by a single-step hydrophobic interaction chromatography and characterized. The purified protease with an estimated molecular mass of 20 kDa was optimally active at 70 °C in 0-3 M NaCl and 0-100 mM Ca(2+) displaying significant stability at 50-80 °C. The enzyme was stable at 80 °C in 100 mM Ca(2+) with Kd of 17 × 10(-3) and t1/2 of 32 min. The activation energy (Ea), enthalpy (ΔH*), and entropy (ΔS*) for the protease deactivation calculated in the presence of 200 mM Ca(2+) were 38.15 kJ/mol, 35.49 kJ/mol and 183.48 J/mol, respectively. The change in free energy (ΔG*) for protease deactivation at 60 °C in 200 mM Ca(2+) was 95.88 kJ/mol. Decrease in ΔH* reflected reduced cooperativity of deactivation and unfolding. The enzyme was intrinsically stable that counteracted heat denaturation by a weak cooperativity during the unfolding. Further, the enzyme was highly stable in the presence of various cations, surfactants, H2O2, ß-mercaptoethanol, and commercial detergents. The compatibility of the enzyme with various cations, surfactants, and detergent matrices suggests its suitability as an additive in the detergents and peptide synthesis.

Microwave-driven enzyme deactivation using imidazolium salt-presenting silica nanoparticles.

Thermal enzyme deactivation by the imidazolium-presenting silica nanoparticles with the microwave irradiation is presented in this manuscript. The modified nanoparticles were synthesized, and it was observed that the modified nanoparticles can be a heat source in the buffer under the weak-power microwave irradiation. Finally, based on the heat-generating ability of these nanoparticles, deactivation of glutathione reductase and alkaline phosphatase with the modified nanoparticles were demonstrated.

Inactivation and reactivation of ribonuclease A studied by computer simulation.

The year 2011 marked the half-centenary of the publication of what came to be known as the Anfinsen postulate, that the tertiary structure of a folded protein is prescribed fully by the sequence of its constituent amino acid residues. This postulate has become established as a credo, and, indeed, no contradictions seem to have been found to date. However, the experiments that led to this postulate were conducted on only a single protein, bovine ribonuclease A (RNAse). We conduct molecular dynamics (MD) simulations on this protein with the aim of mimicking this experiment as well as making the methodology available for use with basically any protein. There have been many attempts to model denaturation and refolding processes of globular proteins in silico using MD, but only a few examples where disulphide-bond containing proteins were studied. We took the view that if the reductive deactivation and oxidative reactivation processes of RNAse could be modelled in silico, this would provide valuable insights into the workings of the classical Anfinsen experiment.