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.     

Developing a Remediation Strategy for Phosphorus Immobilization: Effect of Co-blending,Al-residual and Ca-Mg Amendments in a Manure-Impacted Spodosol

A remediation strategy called “co-blending” was developed for rapid phosphorus (P) immobilization. Immobilization was achieved through the combination of an Al-based water treatment residual (Al-WTR) with Ca-Mg-based materials (Slag and magnesium oxide) for use in incubation and leaching experiments. Al-WTR was co-blended with Slag and MgO as “Al-WTR+Slag” and “Al-WTR+MgO,” respectively. Sequential extraction was used to delineate P species into operationally defined fractions: soluble or exchangeable, Al-Fe, and Ca-Mg-bound pools. Results from soils used in the incubation experiment showed that Al-amended material tended to sequester P bound (～26%) to the Al-Fe pool. On the other hand, Ca-Mg-based materials tended to sorb (～70%) of P greatly associated to the Ca-Mg pool. Amendments were applied at 2% or 20 g kg^?1 as Al-WTR, MgO, and Slag and at 1%+1% or 10 g kg^?1+10 g kg^?1 as co-blended Al-WTR+MgO and Al-WTR+Slag, respectively, on mass basis. Results from leaching data suggest that treatment effects on pH are significant at (p < 0.0001) and also significant (p < 0.01) with weeks of leaching. A similar significant (p < 0.0001) trend was observed for effects of treatment on redox potential (Eh). However, treatment effects on weeks of leaching were not significant. Cumulative soluble P (mg) of leachate showed linear reduction (96%) from the control (without amendments) using a regression model. Potential co-blended material selected was Al-WTR+Slag (1%+1%) due to less P in leachates, moderate effect on pH, and fewer amounts of Al-WTR and Slag used compared with 2% Al-WTR and Slag, respectively. In addition, the RMSE of Al-WTR+Slag data fitted to a regression model was the least. Results suggested that the metal cations Al, Ca, and Mg tended to bind different forms of total P at any given pH. Through co-blending, less soluble P may be lost to the environment than using sorption materials independently.     

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.     

有机碳对海洋着色菌YL28去除无机三态氮的影响

【目的】在无机三态氮(氨氮、亚硝氮和硝氮)共存的模拟海水水体中,阐明有机碳尤其是海藻寡糖对海洋着色菌(Marichromatium gracile)YL28生长及去除无机三态氮的影响规律。【方法】采用次溴酸钠氧化法、N-(1-萘基)-乙二胺分光光度法和紫外分光光度法分别测定水体中氨氮、亚硝氮和硝氮的含量,菌体生物量采用比浊法测定。【结果】在光照厌氧环境中,小分子有机酸盐(乙酸钠、丙酮酸钠、琥珀酸钠和柠檬酸钠)是YL28生长和去除无机三态氮的良好有机碳,亚硝氮、硝氮和氨氮去除率分别达到97.92%、99.98%、73.23%-87.15%。单糖(葡萄糖和果糖)、双糖(麦芽糖和蔗糖)和寡糖(壳寡糖和海藻寡糖)是YL28可利用的有机碳,亚硝氮和硝氮去除率分别达到99%和87%以上,氨氮去除率在44.82%-54.53%之间。多糖(β-环糊精、淀粉、黄原胶、琼脂粉、海藻酸钠和卡拉胶)不是菌体利用和去除无机三态氮的有机碳。酵母提取物可作为菌体生长、去除硝氮和亚硝氮的良好有机碳,但严重抑制氨氮去除。海藻酸钠、β-环糊精和卡拉胶分别与乙酸钠共存时,YL28生长和对无机三态氮去除能力与乙酸钠为唯一有机碳的水平相当。乙酸钠体系中添加海藻寡糖,YL28生长速率、最大生物量以及氨氮的去除速率和最大去除率均升高,添加酵母提取物时,生长速率和最大生物量升高,但氨氮去除速率和最大去除率降低。黑暗厌氧环境下,以乙酸钠和氨氮为唯一有机碳和氮源时,YL28不生长,但在无机三态氮共存时,则能良好生长并去除无机三态氮。【结论】在无机三态氮共存海水体系和厌氧条件下,无论是光照还是黑暗环境,YL28均能良好地生长和去除无机三态氮,小分子有机酸盐(乙酸钠、丙酮酸钠、琥珀酸钠、柠檬酸钠)是其良好的有机碳,相对而言,乙酸钠和丙酮酸钠更好。海藻寡糖与乙酸钠复合可提高菌体生长和脱氮能力。本研究为研制开发高效脱氮微生物制剂及其合理性应用提供了指导。     

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.