Construction of Escherichia coli strain, producing di-adenylate cyclase and its application for cyclic di-AMP synthesis

英语. Diadenylate cyclase represents enzyme, catalyzing synthesis of important cyclic 3&#039

-diadenosine monophosphate (AMP) and two molecules of adenosine triphosphate (ATP). Diadenylate cyclase has been revealed at different bacteria such as Bacillus subtilis, Bacillus thuringiensis, Thermotoga maritima and others. Reaction product, catalyzed by diadenylate cyclase, appears cyclic di-AMP, influencing important physiological processes. Thus, cyclic di-AMP can be used as antiviral madicine. The article deals with creation of diadenylate cyclase-producing strain and evaluation of the possibility of use of the enzyme for receiving of cyclic di-AMP for medicinal preparations development. A new recombinant strain of Escherichia coli pBtdac, producing diadenylate cyclase Bacillus thuringiensis has been constructed. The amount of the diadenylate cyclase produced by the strain is 32 mg per 1L of culture broth. The substrate specificity and stability of the produced enzyme has been studied. Application of the purified diadenylate cyclase for preparative (600 mg) cyclic di-AMP synthesis has been demonstrated.

Цель данного исследования – создание отечественного штамма – продуцента ДАЦ и изучение возможности использования этого фермента для получения цикло-ди-АМФ в препаративных количествах. Сконструирован новый рекомбинантный штамм Escherichia coli pBtdac, продуцирующий диаденилатциклазу Bacillus thuringiensis. Количество диаденилатциклазы, продуцируемой этим штаммом, составляет 32 мг в 1 л культуральной жидкости. Изучена субстратная специфичность и стабильность полученного фермента. Продемонстрировано применение очищенной диаденилатциклазы для препаративного (600 мг) синтеза цикло-ди-АМФ.

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