Pernisine is an extracellular serine protease from the hyperthermophilic archaea Aeropyrum pernix. Development of an efficient recombinant expression system is crucial for development of industrial applications and further characterisation of this enzyme. Several expression vectors for overexpression in E. coli were constructed based on pMCSG7 and pMD204 vectors and codon-optimised pernisine gene. Initial problems with pernisine purification were resolved with translocation of enzyme accumulation into periplasm. Enzyme yield of ~4 mg L-1 production culture was achieved with isolation from the periplasmic fraction and purification of His-tagged enzyme with Ni-NTA column affinity chromatography. Pernisine is matured into an activate form with autocatalytic cleavage of proregion from the N-terminus. Based on the predicted cleavage sites of post-translational maturation unprocessed pernisine (Prn1), enzyme without signal sequence (Prn26) and maturated enzyme without the proregion (Prn92; Prn94) were synthesised. Zymography confirmed successful folding of all recombinant pernisines into an active proteolytic conformation. CD-spectrometry and intrinsic fluorescence emission spectrometry were used to demonstrate high thermal stability of recombinant pernisine and high proteolytic activity at 100 °C was shoved with azocasein assays. We have demonstrated that auto-proteolysis of pernisine samples can be problematic during enzyme production and biochemical characterisation. Binding of calcium ions (Ca2+) results in conformational changes in structure, probably via formation of Ca2+-binding loop. This increases conformational stability and proteolytic activity, thus consequently also the rate of autocatalytic maturation and auto-proteolysis in pernisine solutions.
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