Multienzymatic immobilization of laccases on polymeric microspheres:A strategy to expand the maximum catalytic efficiency

Laccase enzymes of were covalently coimmobilized on poly(glycidyl methacrylate) microspheres. The objective of this work was to create a biocatalyst that works efficiently in a wide range of pH. The coimmobilization was performed using two different strategies to compare the most efficient. The results showed that by correctly selecting the enzymes and concentrations involved in the commobilization, it is possible to obtain a biocatalyst that works efficiently at a wide pH range (2.0-7.0). The maximum activity values reached per gram of support for the obtained biocatalyst were 41.90 U (pH 3.0), 40.89 U (pH 4.0), and 39.54 U (pH 6.0). Moreover, the thermal, storage, and mechanical stabilities were improved compared to the free and single-immobilized laccases. It was concluded that enzymatic coimmobilization is an excellent alternative to obtain a robust biocatalyst that works in a wide pH range, with potential environmental and industrial applications

Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from <i>Bacillus clausii</i>

The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ~0.5-2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (K(M)) but to pH dependence of catalytic turnover: The k(cat) of B. clausii cotA was 1 s⁻¹ at pH 6 and 5 s⁻¹ at pH 8 in contrast to 6 s⁻¹ at pH 6 and 2 s⁻¹ at pH 8 for of B. subtilis cotA. Overall, k(cat)/K(M) was 10-fold higher for B. subtilis cotA at pH(opt). While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500-700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ~20 minutes half-life at 80°C, less than the ~50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH~8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization

Stability mechanisms of a thermophilic laccase probed by molecular dynamics.

Laccases are highly stable, industrially important enzymes capable of oxidizing a large range of substrates. Causes for their stability are, as for other proteins, poorly understood. In this work, multiple-seed molecular dynamics (MD) was applied to a Trametes versicolor laccase in response to variable ionic strengths, temperatures, and glycosylation status. Near-physiological conditions provided excellent agreement with the crystal structure (average RMSD ∼0.92 Å) and residual agreement with experimental B-factors. The persistence of backbone hydrogen bonds was identified as a key descriptor of structural response to environment, whereas solvent-accessibility, radius of gyration, and fluctuations were only locally relevant. Backbone hydrogen bonds decreased systematically with temperature in all simulations (∼9 per 50 K), probing structural changes associated with enthalpy-entropy compensation. Approaching T opt (∼350 K) from 300 K, this change correlated with a beginning "unzipping" of critical β-sheets. 0 M ionic strength triggered partial denucleation of the C-terminal (known experimentally to be sensitive) at 400 K, suggesting a general salt stabilization effect. In contrast, F(-) (but not Cl(-)) specifically impaired secondary structure by formation of strong hydrogen bonds with backbone NH, providing a mechanism for experimentally observed small anion destabilization, potentially remedied by site-directed mutagenesis at critical intrusion sites. N-glycosylation was found to support structural integrity by increasing persistent backbone hydrogen bonds by ∼4 across simulations, mainly via prevention of F(-) intrusion. Hydrogen-bond loss in distinct loop regions and ends of critical β-sheets suggest potential strategies for laboratory optimization of these industrially important enzymes

Pre-treatment of Malaysian agricultural wastes toward biofuel production

Various renewable energy technologies are under considerable interest due to the projected depletion of our primary sources of energy and global warming associated with their utilizations. One of the alternatives under focus is renewable fuels produced from agricultural wastes. Malaysia, being one of the largest producers of palm oil, generates abundant agricultural wastes such as fibers, shells, fronds, and trunks with the potential to be converted to biofuels. However, prior to conversion of these materials to useful products, pre-treatment of biomass is essential as it influences the energy utilization in the conversion process and feedstock quality. This chapter focuses on pre-treatment technology of palm-based agriculture waste prior to conversion to solid, liquid, and gas fuel. Pre-treatment methods can be classified into physical, thermal, biological, and chemicals or any combination of these methods. Selecting the most suitable pre-treatment method could be very challenging due to complexities of biomass properties. Physical treatment involves grinding and sieving of biomass into various particle sizes whereas thermal treatment consists of pyrolysis and torrefaction processes. Additionally biological and chemical treatment using enzymes and chemicals to derive lignin from biomass are also discussed