Production and characterization of laccase and manganese peroxidase from the ligninolytic fungus Fomes sclerodermeus

Fomes sclerodermeus was grown on semi‐defined media based on yeast extract, peptone and glucose (YPG). The fungus produced a minimum basal level of laccase activity irrespective of culture medium. The highest laccase production (20 U cm^?3) was obtained in cultures supplemented with CuSO₄. Manganese peroxidase (MnP) could only be detected when MnSO₄ was added to the medium. None of the aromatic compounds tested stimulated further laccase or MnP production. Laccase and MnP stimulated by Cu²⁺ or Mn²⁺ respectively were purified. Two different laccase isoenzymes with the same molecular mass (67 kDa) and N‐linked carbohydrate content (3%) and a slight difference in their pI values (3.41 and 3.48) were characterized. In addition, two different MnP isoenzymes with the same molecular mass (47 kDa) and N‐linked carbohydrate content (4%) and different pI values (3.35 and 3.45) were characterized. Both enzymes showed good stability at 25 °C and over a wide range of pH. Both laccases oxidize ABTS (2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) more efficiently than 2,6‐dimethoxyphenol (DMP) with similar efficiency values (K_cat/K_m) while the MnP I, the major peroxidase isoenzyme in the studied conditions, oxidizes the Mn²⁺ and Mn‐mediated activity on DMP more efficiently than MnP II. Copyright © 2006 Society of Chemical Industry     

Browning in processed yams: peroxidase or polyphenol oxidase?

Polyphenol oxidase and peroxidase were purified from white yam (Dioscorea rotundata) using DEAE‐cellulose ionexchange chromatography. Thermoinactivation curves for polyphenol oxidase showed monophasic kinetics, while those for peroxidase were biphasic. Urea partially stabilised peroxidase against irreversible thermoinactivation, but did not do so in the case of polyphenol oxidase. Only peroxidase was capable of regenerating activity after thermoinactivation. The results showed that thermoinactivation of peroxidase was mainly due to conformational changes, while that of polyphenol oxidase was probably due to covalent damage. Peroxidase reactivation might play an important role in the browning of processed yam. © 2002 Society of Chemical Industry     

Removal of aqueous phenolic compounds from a model system by oxidative polymerization with turnip (Brassica napus L var purple top white globe) peroxidase

Turnip roots, which are readily available in Mexico, are a good source of peroxidase, and because of their kinetic and biochemical properties have a high potential as an economic alternative to horseradish peroxidase (HRP). The efficiency of using turnip peroxidase (TP) to remove several different phenolic compounds as water‐insoluble polymers from synthetic wastewater was investigated. The phenol derivatives studied included phenol, 2‐chlorophenol, 3‐chlorophenol, o‐cresol, m‐cresol, 2,4‐dichlorophenol and bisphenol‐A. The effect of pH, substrate concentration, amount of enzyme activity, reaction time and added polyethylene glycol (PEG) was investigated in order to optimize reaction conditions. A removal efficiency ≥85% was achieved for 0.5 mmol dm^?3 phenol derivatives at pH values between 4 and 8, after a contact time of 3 h at 25 °C with 1.28 U dm^?3 of TP and 0.8 mmol dm^?3 H₂O₂. Addition of PEG (100–200 mg dm^?3) significantly reduced the reaction time required (to 10 min) to obtain >95% removal efficiency and up to 230% increase in remaining TP activity. A relatively low enzyme activity (0.228 U dm^?3) was required to remove >95% of three phenolic solutions in the presence of 100–200 mg dm^?3 PEG. TP showed efficient and fast removal of aromatic compounds from synthetic wastewaters in the presence of hydrogen peroxide and PEG. These results demonstrate that TP has good potential for the treatment of phenolic‐contaminated solutions. © 2002 Society of Chemical Industry     

Effect of some thermal and chemical pretreatments on smoked Oyster mushroom quality

The effect of different thermal and chemical pretreatments on quality and enzyme activities of smoked mushroom was investigated. Mushrooms were blanched (water and steam) and dipped in different concentrations of sulphites (SO₂), hydrogen peroxide (H₂O₂), ethylene‐di‐amine tetra‐acetic acid (EDTA) and citric acid for 10 min before smoking. Enzyme activities, colour characteristics, microbiological and sensory examinations were carried out every 2 weeks up to 8 weeks of storage in refrigerator at 4 °C. Results could be concluded that smoked mushroom pretreated with SO₂, H₂O₂ and steam blanching had the best colour values, the better score for all sensory characteristics and lower non‐enzymatic browning compared with other pretreatments. The most effective pretreatment against total aerobic bacteria and yeast & moulds were citric acid, EDTA and steam, and then smoking of mushroom can be attributed to the reduction of microbial counts. The most effective pretreatments on quality and safety of smoked mushrooms were H₂O₂ and steam. It can be concluded that thermal and chemical treatments, rather than smoking of mushroom, reduce enzyme activities and are suitable to preserve mushrooms.     

Mn‐peroxidase production by Panus tigrinus CBS 577.79: response surface optimisation and bioreactor comparison

This study reports the statistical optimisation through response surface methodology of the growth medium for Panus tigrinus manganese‐dependent peroxidase (MnP) production in shaken culture. Three crucial variables, including carbon source, malonic acid and Mn²⁺, were optimised in a nitrogen‐limited medium. Sucrose was the best carbon source for MnP production. Mn²⁺ ions and malonic acid significantly stimulated MnP production at an optimal concentration of 53 mg dm^?3 and 8.2 mmol dm^?3, respectively, resulting in 0.83 U cm^?3. Further experiments were performed in lab‐scale stirred tank (STR) and bubble‐column (BCR) reactors using the previously optimised liquid medium. BCR proved to be more adequate than STR in supporting MnP production, leading to 3700 U dm^?3 after 144 h with a productivity of 25.7 U dm^?3 h^?1. On a comparative basis with other production data in lab‐scale reactors, these results appear to be compatible with scale transfer. Copyright © 2006 Society of Chemical Industry     

Immobilization of horseradish peroxidase on self-assembled (3-mercaptopropyl)trimethoxysilane film: Characterization, direct electrochemistry, redox thermodynamics and biosensing

Highly organized (3-mercaptopropyl)trimethoxysilane (3-MPT) films have been prepared via self-assembled coupled with sol–gel linking technology. Horseradish peroxidase (HRP) is successfully immobilized onto the densely packed three-dimensional (3D) 3-MPT network and the direct electrochemistry of HRP is achieved without any electron mediators or promoters. Redox thermodynamics of HRP on the 3-MPT films, which is obtained from the temperature dependence of the reduction potential, suggests that the positive shift of redox potentials of HRP at the interface of 3-MPT originates from the solvent reorganization effects and conformational change of the polypeptide chain of HRP. Based on the direct electrochemistry and electrocatalytic ability of HRP, a sensitive third-generation amperometric H₂O₂ biosensor is developed with two linear dependence ranges of 5.0 × 10⁻⁷ to 1.0 × 10⁻⁴ and 1.0 × 10⁻⁴ to 2.0 × 10⁻² mol L⁻¹.     

The use of specimen tilt in transmission electron microscopy of the central nervous system

Thin sections of nervous tissue were viewed at different tilt angles using a transmission electron microscope equipped with a eucentric goniometer stage. In a comparison study of various degrees of tilt, one can observe additional morphological features within synaptic profiles, define subsynaptic structures such as Taxi-bodies, and clearly see the crystalline formation of cytochemical tracers. This study demonstrates the value of tilting thin-sections in the analysis of synapses and other biological material at the ultrastructural level.     

Is there a future for antioxidants in atherogenesis?

Antioxidants, preferentially those of dietary origin, have for a long time been considered to help against diseases that are presumably aggravated by oxidative stress, such as cardiovascular diseases, cancer, and neurodegenerative disorders. The outcome of clinical trials undertaken to corroborate this hypothesis, however, remained largely inconclusive. Evidence is now emerging that some dietary "antioxidants" influence signaling pathways and the expression of genes relevant in atherosclerosis by mechanisms other than antioxidative ones. By concrete examples we show that (1) vitamin E has gene regulatory functions which might be more important than acting as an antioxidant in vivo, (2) selenium itself is not an antioxidant at all, and even not in general when incorporated into glutathione peroxidases, and (3) a moderate oxidative stress is beneficial rather than detrimental since it can induce defense mechanisms counteracting xenobiotic and oxidative stress. Thus, there is only a future for antioxidants in the prevention of any disease if their real mechanism of action is considered and suitable read-outs and biomarkers are established.     

Drip loss,peroxidase and sensory changes in kiwi fruit slices during frozen storage

The biochemical, sensory and drip loss changes that occur during processing and prolonged frozen storage of kiwi fruit slices (cvs Abbott and Hayward) were studied. Fruit slices were frozen at ?40°C, packed in polyethylene bags and stored at ?18°C for 11 months. Maturity characteristics (pH, acidity, dry matter, soluble solids) were determined on raw fruit. Objective (proteins, peroxidase (EC 1.11.1.7) activity, drip loss) and subjective (sensory tests) analyses were carried out during processing and storage, and indicated a good quality of the frozen kiwi fruit slices after 11 months of storage. There were significant differences (P < 0.05) between the studied varieties with respect to drip loss during frozen storage and colour after 11 months. Best results were obtained with cv Hayward. This variety showed less drip loss after thawing and after 11 months storage presented the same green colour as after freezing, while cv Abbott became yellowish-green.     

基于纳米金类过氧化物酶活性构建RGB可视化检测体系的研究

目的 利用金纳米粒子（gold nanoparticles,AuNPs）的类过氧化物酶催化活性,结合智能手机RGB识别进行可视化信号采集,构建颜色-RGB数值-模拟曲线响应模型。方法 采用柠檬酸钠还原法合成粒径均一的AuNPs分散液。利用一级动力学模型对AuNPs催化活性进行测定并探究不同反应条件对催化活性的影响。借助智能手机软件Color Meter获得反应后图像的RGB参数信息,选择最优定量公式拟合实验数据。结果 AuNPs表观催化活性为3.5×10-3 s-1/mL。反应条件在40~50 ℃、pH 3.5~4.0、醋酸钠缓冲液浓度大于1 mmol/L、反应时间大于15 min时AuNPs催化活性最高,AuNPs的量与反应后溶液显色程度在一定范围内呈现良好的线性关系。结论 AuNPs可通过控制反应参数呈现不同的类过氧化物酶催化活性,利用智能手机RGB识别技术可快速、直观地展现不同量AuNPs催化显色反应的结果,完成定量分析。该方法具有良好的准确度和可靠性,可作为一种可视化检测手段,为利用AuNPs构建的生物传感检测体系提供参考。