Immobilization of chloroperoxidase on silica-based materials for 4,6-dimethyl dibenzothiophene oxidation

Silica-based materials have been used as effective supports for the immobilization of enzymes. Moreover, the understanding on the oxidation of sulfur compounds by immobilized chloroperoxidase represents a step further in the development of a biocatalytic desulfurization process of fossil fuels. Here, chloroperoxidase from Caldariomyces fumago was immobilized on amorphous and structured silica-based materials either physically or covalently using an organosilane derivative for the oxidation of a recalcitrant organosulfur compound currently found in gas oil and diesel, such as 4,6-dimethyldibenzothiophene (4,6-DMDBT). Such materials were characterized by FTIR, N₂-adsorption, XRD, SEM and TEM. We have found that the chemical differences on the silanol/siloxane groups of SG/67 and SBA15 mesoporous materials deeply modify the enzymatic load, activity, thermal stability and reusability. The physical immobilization of CPO was characterized by a high adsorption capacity (q_m) and affinity constants (K_m) when compared to the covalent approach, but it resulted more sensitive to temperature than free, the silanized and covalently immobilized enzyme. The thermal residual activity as well as reusability of CPO were first improved by silanization, then by covalent immobilization in a support with a large pore size and high silanol/siloxane ratio.     

Chloroperoxidase-catalyzed oxidation of 4,6-dimethyldibenzothiophene as dimer complexes: evidence for kinetic cooperativity

A sigmoidal kinetic behavior of chloroperoxidase for the oxidation of 4,6-dimethyldibenzothiophene (4,6-DMDBT) in water-miscible organic solvent is for the first time reported. Kinetics of 4,6-DMDBT oxidation showed a cooperative profile probably due to the capacity of chloroperoxidase to recognize a substrate dimer (pi-pi dimer) in its active site. Experimental evidence is given for dimer formation and its presence in the active site of chloroperoxidase. The kinetic data were adjusted for a binding site able to interact with either monomer or dimer substrates, producing a cooperative model describing a one-site binding of two related species. Determination of kinetics constants by iterative calculations of possible oxidation paths of 4,6-DMDBT suggests that kinetics oxidation of dimer substrate is preferred when compared to monomer oxidation. Steady-state fluorometry of substrate in the absence and presence of chloroperoxidase, described by the spectral center of mass, supports this last conclusion.     

Amperometric biosensor based on enzymes immobilized in hybrid mesoporous membranes for the determination of acetylcholine

Acetylcholine sensor is successfully prepared by using immobilized enzymes, i.e., acetylcholinesterase and choline oxidase within separate hybrid mesoporous silica membranes with 12 nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide produced by two sequential enzyme reactions. The determination range and the response time are 6.0–800 μM and within approximately 3 min, respectively. The sensor is very stable compared to free enzymes and 80% of the initial response was maintained even after storage for 80 days. These results show that two enzymes are successfully immobilized and well stabilized, and at the same time, two sequential enzyme reactions efficiently proceed within the separate hybrid mesoporous membranes. Further, we studied the possible detection of organophosphorus pesticides in terms of the inhibition of acetylcholinesterase activity, i.e., the decrease of current response, and demonstrated that the nanomolar concentrations of pesticide (DZN-oxon) can be detected with our sensor.     

Methane uptake rates in Japanese forest soils depend on the oxidation ability of topsoil, with a new estimate for global methane uptake in temperate forest

To clarify the reason for the higher CH₄ uptake rate in Japanese forest soils, twenty-seven sites were established for CH₄ flux measurement. The first order rate constant for CH₄ uptake was also determined using soil core incubation at 14 sites. The CH₄ uptake rate had a seasonal fluctuation, high in summer and low in winter, and the rate correlated with soil temperature at 17 sites. The annual CH₄ uptake rates ranged from 2.7 to 24.8 kg CH₄ ha⁻¹ y⁻¹ (the average of these rates was 9.7 or 10.9 kg CH₄ ha⁻¹ y⁻¹, depending on method of calculation), which is somewhat higher than the uptake rates reported in previous literature. The averaged CH₄ uptake rate correlated closely with the CH₄ oxidation rate of the topsoil (0–5 cm) in the study sites. The CH₄ oxidation constant of the topsoil was explained by a multiple regression model using total pore volume of the soil, nitrate content, and C/N ratio (p < 0.05, R ² = 0.684). This result and comparison with literature data suggest that the high CH₄ uptake rate in Japanese forest soils depends on the high porosity probably due to volcanic ash parent materials. According to our review of the literature, the CH₄ uptake rate in temperate forests in Europe is significantly different from that in Asia and North America. A new global CH₄ uptake rate in temperate forests was estimated to be 5.4 Tg y⁻¹ (1 SE is 1.1 Tg y⁻¹) on a continental basis.