ИСТИНА

Enzyme-based biosensors play an important role in food industry, clinical analysis, environment biotechnology, etc. In recent years a lot of studies have been carried out on the development of so-called third generation biosensors, in which no mediator involved in the direct electron transfer (DET) between the electrode and the redox enzyme and the electrode acts as an electron-donor substrate. This design is responsible for either the repeated use of the biosensor or under continuous-flow conditions for the monitoring of different industrial processes and water quality control. Most studied and commercially available horseradish peroxidase (HRP) is poorly suited for this purpose as the carbohydrate moiety hinders the DET and also because of fast inactivation by hydrogen peroxide resulted in low operational stability. In contrast, investigated in our laboratory anionic tobacco peroxidase (TOP) exhibits a higher stability to inactivation with H2O2, a wider optimum pH range, and a higher efficiency in DET processes [1]. However, to use in practice, a high product yield is required. Heterologous expression is frequently used for production of recombinant enzymes in amounts sufficient for practical uses. Fast growth and high cell concentrations can be obtained in bacterial cultivation processes. High-level expression of non-glycosylated TOP has been achieved in Escherichia coli [2]. However, the recombinant TOP (rTOP) was accumulated in inclusion bodies in an insoluble form, and special refolding procedure was required to reactivate the enzyme. Nevertheless, previously reported conditions for rTOP refolding did not provide the sufficient amount of rTOP for practical uses. The refolding of peroxidases is a very complex process, which involves the formation of native disulfide bridges and incorporation of heme and Ca2+ ions. The refolding yield is limited to a large extend due to the incorrect folding and formation of aggregates. Therefore, it was necessary to optimize a broad range of critical parameters. Extensive screening experiments were performed to increase a yield of rTOP. Optimization of the refolding conditions (such as pH value, protein dilution, and concentration of DTT, GSSG, hemin, urea, and glycerol) and of subsequent purification procedure resulted in an increased active enzyme yield by up to 83%. Thus, optimized refolding system was characterized by higher yields and minimum product losses in the purification step.