Получение и изучение медико-биологических свойств меченного технецием-99м противомикробного препарата норфлоксацина гидрохлорида

Проведены исследования по созданию стандартного реагента для получения меченного 99mТс норфлоксацина гидрохлорида (НФГ). Оценку влияния компонентов реакционной смеси на радиохимическую чистоту получаемого препарата проводили методом тонкослойной хроматографии. На экспериментальных животных (кроликах) с моделью воспаления различной локализации показана функциональная пригодность меченого антибиотика для диагностики воспалительных процессов

Cloning, expression, charakterization and immobilization of carbonyl reductase from Candida parapsilosis

The carbonyl reductase from Candida parapsilosis DSMZ 70125 strain (CPCR) is a very versatile biocatalyst with a broad substrate spectrum. Attempts for cloning, expression and identification of this enzyme had failed so far, which might be due to failures in the quite old literature data. Recent characterization of the commercially available wild type CPCR (wtCPCR) showed that certain biochemical characters were in good agreement with the data of an ADH known as CpSADH (secondary alcohol dehydrogenase from C.parapsilosis), which had been described and cloned by a Japanese research group from a different type strain of C.parapsilosis (IFO 1396 strain). In this work first a recheck of the features of CPCR was done as a basis for a new attempt to identify amino acids and nucleic acid sequence. For this, using the available partial amino acids sequences of the wtCPCR, the gene coding for an alcohol dehydrogenase was amplified by PCR based genome walking from the genomic DNA of C.parapsilosis DSMZ 70125 strain (“CPCR” type strain). With primers deduced from the published sequence it was possible to amplify the gene coding for the CpSADH also from the genomic DNA of “CPCR” type strain of C.parapsilosis. Amplification of two alcohol dehydrogenases from the “CPCR” type strain of C.parapsilosis showed that at least two ADHs were present in the same C.parapsilosis strain (DSMZ 70125 strain) though it was not known which one of the two was the “Real CPCR”. With biochemical characterization, substrate spectrum study, molecular modeling and experimental validation, it was possible to identify that the CpSADH was the “Real CPCR” with the broad substrate spectrum as had been described in literature data for the wtCPCR. The recombinant CpSADH identified as the “Real CPCR” was successfully expressed in Escherichia coli and after IMAC purification the enzyme reached a very high specific activity and purity. Enzymatic syntheses with the purified recombinant CpSADH were carried out in one-phase system, aqueous-organic two-phase system, gel-stabilized two-phase system [with polyvinyl alcohol (PVA) beads] and also with immobilized enzyme on epoxy-carriers (Sepabeads) in two-phase system. To avoid stoichiometric amounts, substrate-coupled cofactor regeneration was carried out with 2-propanol as co-substrate in all the systems. The highest product concentration was obtained in a liquid two-phase system with 25% (v/v) hexane as substrate reservoir. The activity and stability of the enzyme was studied in different systems. It was found that the enzyme exhibited a higher stability in a two-phase system when immobilized covalently on epoxy-amino Sepabeads than when used as soluble form in the two-phase system. Volumetric productivity obtained with the enzyme immobilized on epoxy-amino Sepabeads was better than PVA-immobilized ones