Pernisine is a subtilisin-like serine proteinase from the hyperthermophilic archaeon Aeropyrum pernix K1. Due to its exceptional temperature stability and ability to degrade infectious prion proteins, pernisine has a high potential for industrial applications. The practical use of pernisine is limited by the low yield of this proteinase in the parent organism, where it is less than 0.1 mg per liter of culture. One of the suitable host organisms for the production of recombinant proteins are Bacillus subtilis bacteria due to their known strong promoters and efficient protein secretion systems, which means that B. subtlis may represent a promising expression system for the production of pernisine. B. subtilis bacteria naturally produce the proteinase subtilisin, which is homologous to pernisine, at levels up to 1 g per litre of culture. In the master's thesis, we examined production of recombinant pernisine in B. subtilis. We compared three different promoters (PaprE, Pveg and Pylb) for controlling pernisine expression. Among the promoters tested, Pylb appeared the most suitable, as around 40 % higher pernisine activity was detected in medium compared to the other two promoters. We have also isolated the produced pernisine from culture medium and evaluated its activity and stability. Using CD spectrometry and fluorescence emission spectrometry we confirmed that recombinant pernisine isolated from B. subtilis has similar high temperature stability and activity as recombinant pernisine produced with Eschericha coli bacteria.
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