DNA-directed immobilization of horseradish peroxidase onto porous SiO2optical transducers

ABSTRACT: Multifunctional porous Si nanostructure is designed to optically monitor enzymatic activity of Horseradish Peroxidase. First, an oxidized PSi optical nanostructure, a Fabry-Perot thin film, is synthesized and is used as the optical transducer element. Immobilization of the enzyme onto the nanostructure is performed through DNA-Directed Immobilization. Preliminary studies demonstrate high enzymatic activity levels of the immobilized Horseradish Peroxidase, while maintaining its specificity. The catalytic activity of the enzymes immobilized within the porous nanostructure is monitored in real time by reflective interferometric Fourier transform spectroscopy. We show that we can easily regenerate the surface for consecutive biosensing analysis by mild de-hybridization conditions.     

Long-term activity of covalent grafted biocatalysts during intermittent use of a glucose/O2 biofuel cell

The operational stability of enzymes in a concentric glucose/O₂ biofuel cell has been significantly improved with the synthesis of grafted enzyme electrodes compared to entrapped enzyme electrodes. The concentric device combined glucose electro-oxidation by glucose oxidase at the anode and oxygen electro-reduction by bilirubin oxidase at the cathode. The entrapped enzyme electrodes were prepared from physical immobilization of the enzymes by a polypyrrole polymer onto the electrode surface. The grafted enzyme electrodes were synthesized by grafting the enzymes via alkyl spacer arms to a poly(aminopropylpyrrole) film onto the electrode surface. From spectrophotometric and electrochemical analyses, it was demonstrated that the spacer arms increased the operational stability and enzyme mobility that favoured electron transfer from their active sites to the electrode.The maximum power output of the assembled biofuel cell was 20 μW cm⁻², at 0.20 V with 10 mM glucose in phosphate buffer pH 7.4. The grafted enzyme electrodes presented an unprecedented operational stability as the maximum of power density of the BFC remains constant after intermittent use over a 45-day period. This was a remarkable improvement compared to electrodes with entrapped enzymes, which lost 74% of their initial power density after intermittent use over a 17-day period.     

Theoretical analysis of inhibition effect on steady states of enzymatic membrane reactor with substrate and product retention for reaction producing weak acid

The aim of this study is to formulate a model of enzymatic membrane reactor (EMR), i.e., a continuous, stirred tank bioreactor with full enzyme recycle, for a reaction producing a weak acid, and to explore the effect of substrate and product inhibition of different mechanisms coupled with transport properties of the membrane on the static behaviour of the system. The inhibition of an enzyme by a substrate leads to the non-monotonicity of reaction rate expression with respect to the substrate concentration. If a product of enzymatic reaction, taking place in the EMR, influences the pH of a reaction mixture this is also the factor causing the non-monotonicity of the substrate and product dependent reaction rate. The character of these dependencies affects substantially the structure of the steady states of the reactor. The bifurcation diagrams, shown in the work, are of different characters depending on the bifurcation parameter. It has been found, that bifurcation diagrams for competitive and uncompetitive inhibition by a substrate differ in the number and position of bifurcation points. Steady states of multiplicity five have been localised in case of uncompetitive inhibition by the substrate at high affinity of the enzyme to this substrate. Retention of reagents, related to transport properties of a membrane, influences significantly the effectiveness of a process. A specially written software in Delphi™ has been used for the calculations.     

Enzymatic hydrolysis of canola oil with hydrodynamic cavitation

A hydrodynamic cavitation system based on a venturi was used to test the effectiveness of cavitation for enhancing the enzymatic hydrolysis of canola oil using lipase from Candida rugosa. Cavitation led to the production of fine oil-in-water and water-in-oil emulsions with the enzyme in the water phase. Using venturi inlet pressures of up to 8 bar, the yield of fatty acids was only about 60% of the maximum possible. In contrast, a simple stirred batch reactor produced over 90% of the maximum possible yield with reaction rates equal to, or better than, those obtained in a cavitating system. It was concluded that cavitation inhibited the reaction in some way and is not effective for intensification of hydrolysis.     

Inhibitory Effects of Cholesterol Derivatives on DNA Polymerase and Topoisomerase Activities, and Human Cancer Cell Growth

This paper describes the inhibitory activities of cholesterol derivatives such as cholesterol, sodium cholesteryl sulfate, cholesteryl-5α, 6α-epoxide, cholesteryl chloride, cholesteryl bromide, and cholesteryl hemisuccinate (compounds 1–6, respectively) against DNA polymerase (pol), DNA topoisomerase (topo), and human cancer cell growth. Among the compounds tested, compounds 2 and 6 revealed themselves to be potent inhibitors of animal pols, and the IC₅₀ values for pols were 0.84–11.6 and 2.9–148 μM, respectively. Compounds 2, 3 and 6 inhibited the activity of human topo II, with IC₅₀ values of 5.0, 12.5 and 120 μM, respectively. Compounds 2, 3 and 6 also suppressed human cancer cell (promyelocytic leukemia cell line, HL-60) growth, and LD₅₀ values were 8.8, 20.2 and 72.3 μM, respectively, suggesting that cell growth inhibition had the same tendency as the inhibition of topos rather than pols. Compounds 2 and 6 arrested the cells in S and G2/M phases, compound 3 arrested the cells in the G2/M phase, and these compounds also increased sub-G1 phase in the cell cycle. These results suggested that the effect of cell cycle arrest might be effective on both pols and topos activities. From these findings, the action mode of cholesterol derivatives as anti-cancer compounds is discussed.     

酶的兼职功能和催化混杂性

随着酶学研究的不断深入,最近提出了酶的兼职功能和催化混杂性的新概念.酶的兼职功能和催化混杂性在酶类中广泛存在,Neurospora crassa酪氨酰tRNA合成酶、磷酸葡萄糖异构酶(PGI)、gephyrin和细胞色素C等具有典型的兼职功能;而CAL-B、O-乙酰丝氨酸巯基酶、胰蛋白酶和非血红素二铁酶等表现出很好的催化混杂性.     

ON THE BEHAVIOR OF CONSECUTIVE ENZYMATIC REACTIONS IN CYCLICALLY OPERATED REACTORS

The dynamics of a process involving consecutive enzymatic reactions were investigated for a case in which the reactions are carried out in a cyclically operated reactor. Each cycle of the operation protocol involves three phases. During phase I the reactor operates in a semi-batch mode involving input of the reactants. During the second phase, the vessel operates in a batch mode. During phase III the reactor has an output only and a fraction of its contents are emptied before another identical cycle begins.

It was found that there are regimes in the operating parameter space where the system can reach more than one limit cycle (multistability). Using computer software based on the bifurcation theory for forced systems, as well as one- and two-parameter continuation algorithms, the impact of various parameters on the dynamics of the system was investigated. The results are presented in the form of diagrams. Conditions under which formation of the intermediate product in the reaction sequence is maximized were also investigated.

Production of the intermediate product in a limit cycle was compared to that obtained in batch and semi-batch operation. Implications of the proposed operation protocol for process optimization and pollution prevention are discussed.     

Polystyrene-based diazonium salt as adhesive: A new approach for enzyme immobilization on polymeric supports

In this work, a new way for enzyme immobilization was explored and properties of the enzyme immobilized on different polymer films were investigated. In the process, a polystyrene-based diazonium salt (PS-DAS) was synthesized and used as molecular adhesive to immobilize β-glucosidase on the polymeric supports (films of polyethylene, polypropylene and poly(ethylene terephthalate)). The immobilization of β-glucosidase on the polymer surfaces was achieved by sequential depositions of a piece of the polymer films in PS-DAS and the enzyme solutions. The surface modification was investigated by X-ray photoelectron spectroscopy (XPS), water contact angle measurement, and atomic force microscopy (AFM). The activity of the immobilized β-glucosidase was evaluated by measuring its enzymatic activity to the hydrolysis of p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimized reaction conditions (such as pH and temperature), thermal stability, and reusability of the immobilized enzyme on PE films were assayed by using the enzyme-catalyzed reaction. Results show that the polymeric diazonium salt is firmly adhered on the polymer surfaces and the modified surfaces can react with the enzyme to form covalent bonds. The immobilized enzyme shows changes in the optimized pH and temperature for the hydrolysis reaction catalyzed by β-glucosidase. The kinetic parameter (K_m) of the immobilized β-glucosidase is lower than that of its free counterpart. The immobilized enzyme shows significant enhancement in the thermal stability and reasonable reusability. This new approach can be used as a simple and versatile method for protein immobilization.     

辽宁地区潜水蒸发规律研究

黑土中加入有机物料和硫磺粉(S),对越桔叶片的过氧化氢酶、过氧化物酶、硝酸还原酶都产生显著影响,不同处理有较大差异,其中,加入苔藓、苔藓和草炭及只加入1.5kgS-2kgS的处理,叶片的过氧化氢酶活性较高,过氧化物酶和硝酸还原酶活性较低,叶片生长发育良好。     

混交林地土壤物理性质与微生物数量及酶活性的研究

研究了枫香×樟树、楠木×尖叶杜英、椆木×海南红豆、格木×海南红豆、火力楠×阴香、枫香×米老排×降香黄檀、樟树×马占相思混交林林地的土壤物理性质与微生物数量及酶活性.各林地的容重、毛管孔隙、非毛管孔隙、自然含水量、毛管持水量的不同引起其保水性和通气性的差异.细菌是土壤微生物总量的主要组成者.各混交林地的细菌、真菌和放线菌的数量差异大.各混交林地的脲酶、过氧化氢酶和纤维素分解酶活性有一定的差异.放线菌与容重呈显著正相关,而与总孔隙呈显著负相关.脲酶与自然含水量、毛管持水量、毛管孔隙呈显著或极显著正相关.