Limits of Non-invasive Enzymatic Activation by Local Temperature Control.

Enzymatic activity depends on and can therefore be regulated by temperature. Selective modulation of the activity of different enzymes in one reaction pot would require temperature control local to each type of enzyme. It has been suggested previously that immobilization of enzyme on magnetic nanoparticles and exposing them to alternating magnetic field can enhance the reaction rate. This enhancement has been explained as being mediated by temperature increase caused by dissipation of the absorbed field energy in the form of heat. However, the possibility of spatially limiting this temperature increase on the microscale has been questioned. Here, it is investigated whether an activity enhancement of the enzyme sucrose phosphorylase immobilized on magnetic beads can be achieved, how this effect is related to the increase in temperature, and whether temperature differences within one reaction pot could be generated in this way. It is found that alternating magnetic field stimulation leads to increased enzymatic activity fully attributable to the increase of bulk temperature. Both theoretical analysis and experimental data indicate that no local heating near the particle surface takes place. It is further concluded that relevant increase of surface temperature can be obtained only with macroscopic, millimeter-sized, magnetic particles.

Energetics of the Glycosyl Transfer Reactions of Sucrose Phosphorylase.

From its structure and mechanism, sucrose phosphorylase is a specialized glycoside hydrolase that uses phosphate ions instead of water as the nucleophile of the reaction. Unlike the hydrolysis reaction, the phosphate reaction is readily reversible and, here, this has enabled the study of temperature effects on kinetic parameters to map the energetic profile of the complete catalytic process via a covalent glycosyl enzyme intermediate. Enzyme glycosylation from sucrose and α-glucose 1-phosphate (Glc1P) is rate-limiting in the forward (kcat = 84 s-1) and reverse direction (kcat = 22 s-1) of reaction at 30 °C. Enzyme-substrate association is driven by entropy (TΔSb ≥ +23 kJ/mol), likely arising from enzyme desolvation at the binding site for the leaving group. Approach from the ES complex to the transition state involves uptake of heat (ΔH⧧ = 72 ± 5.2 kJ/mol) with little further change in entropy. The free energy barrier for the enzyme-catalyzed glycoside bond cleavage in the substrate is much lower than that for the non-enzymatic reaction (knon), ΔΔG⧧ = ΔGnon⧧ - ΔGenzyme⧧ = +72 kJ/mol; sucrose. This ΔΔG⧧, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (∼1014 M-1), is almost entirely enthalpic in origin. The enzymatic rate acceleration (kcat/knon) is ∼1012-fold and similar for reactions of sucrose and Glc1P. The 103-fold lower reactivity (kcat/Km) of glycerol than fructose in enzyme deglycosylation reflects major losses in the activation entropy, suggesting a role of nucleophile/leaving group recognition by the enzyme in inducing the active-site preorganization required for optimum transition state stabilization by enthalpic forces.

Novel Two-Step Process in Cellulose Depolymerization: Hematite-Mediated Photocatalysis by Lytic Polysaccharide Monooxygenase and Fenton Reaction.

To transform cellulose from biomass into fermentable sugars for biofuel production requires efficient enzymatic degradation of cellulosic feedstocks. The recently discovered family of oxidative enzymes, lytic polysaccharide monooxygenase (LPMO), has a high potential for industrial biorefinery, but its energy efficiency and scalability still have room for improvement. Hematite (α-Fe2O3) can act as a photocatalyst by providing electrons to LPMO-catalyzed reactions, is low cost, and is found abundantly on the Earth's surface. Here, we designed a composite enzymatic photocatalysis-Fenton reaction system based on nano-α-Fe2O3. The feasibility of using α-Fe2O3 nanoparticles as a composite catalyst to facilitate LPMO-catalyzed cellulose oxidative degradation in water was tested. Furthermore, a light-induced Fenton reaction was integrated to increase the liquefaction yield of cellulose. The innovative approach finalized the cellulose degradation process with a total liquefaction yield of 93%. Nevertheless, the complex chemical reactions and products involved in this system require further investigation.

A comparative study on the chemo-enzymatic upgrading of renewable biomass to 5-Hydroxymethylfurfural.

5-hydroxymethylfurfural (HMF) obtained from renewable biomass-derived carbohydrates is a potential sustainable substitute to petroleum-based building blocks. In the present work, we constituted a comparative study on the production of HMF from two widely available real biomasses in India- Agave americana and Casuarina equisetifolia. In the initial hydrolysis studies for the production of reducing sugars, 649.5 mg/g of fructose was obtained from the hydrolysis of 5% (w/v) A. americana biomass by the enzyme inulinase in 3 h at 50°C. Similarly, upon hydrolysis of 15% (w/v) C. equisetifolia biomass by the lignocellulolytic enzymes (laccase, cellulase and xylanase) from Trichoderma atroviride, 456.65 mg/g of reducing sugars was released in 24 h at 30°C. Subsequently, the dehydration of the obtained reducing sugars to HMF was achieved with titanium dioxide as the catalyst. The dehydration of A. americana-derived fructose at 140°C led to a maximum HMF yield of 92.6% in 15 min with 10% catalyst load. Contrarily, upon optimizing the process parameters for dehydration of C. equisetifolia derived reducing sugars, the maximum HMF yield of 85.7% was obtained at 110°C in 25 min with a TiO2 concentration of 10%. This study reports for the first time the utilization of C. equisetifolia biomass for HMF production and thus, by utilizing these inexpensive, abundantly available and highly functionalized polysaccharides, a strategical approach can be developed for the production of fine chemicals, eliminating the need of fossil-based chemicals. Implications: The catalytic upgrading of lignocellulosic biomass into high-valued platform chemicals like 5-Hydroxymethylfurfural (HMF) implies an extremely significant challenge to the attempts of establishing a green economy. Casuarina equisetifolia and Agave americana represents a sustainable feedstock for the production of HMF through catalytic integration. The present work describes a two-step reaction process where the initial depolymerization step comprises of an enzymatic hydrolysis followed by a chemical-catalyst mediated dehydration process. The utilization of a biocatalytic approach followed by mild chemical catalyst eliminates the need of hazardous chemical conversion processes. Thus, the HMF produced via sustainable can bridge the gap between carbohydrate chemistry and petroleum-based industrial chemistry because of the wide range of chemical intermediates and end-products that can be derived from this compound.

Preparation of 4-Deoxy-L-erythro-5-hexoseulose Uronic Acid (DEH) and Guluronic Acid Rich Alginate Using a Unique exo-Alginate Lyase from Thalassotalea crassostreae.

Marine multicellular algae are considered promising crops for the production of sustainable biofuels and commodity chemicals. However, their commercial exploitation is currently limited by a lack of appropriate and efficient enzymes for converting alginate into metabolizable building blocks, such as 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Herein, we report the discovery and characterization of a unique exo-alginate lyase from the marine bacterium Thalassotalea crassostreae that possesses excellent catalytic efficiency against poly-ß-D-mannuronate (poly M) alginate, with a kcat of 135.8 s-1, and a 5-fold lower kcat of 25 s-1 against poly-α-L-guluronate (poly G alginate). We propose that this preference for poly M is due to a structural feature of the protein's active site. The mode of action and specificity of this enzyme has made it possible to design an effective and environmentally friendly process for the production of DEH and low molecular weight guluronate-enriched alginate.