Periplasmic production of pernisine in Escherichia coli and determinants for its high thermostability
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BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Periplasmic production of pernisine in Escherichia coli and determinants for its high thermostability Miha Bahun 1 & Kevin Hartman 1 & Nataša Poklar Ulrih 1,2 Received: 14 April 2020 / Revised: 17 June 2020 / Accepted: 19 July 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Pernisine is a subtilisin-like serine proteinase secreted by the hyperthermophilic archaeon Aeropyrum pernix. The significant properties of this proteinase are remarkable stability and ability to degrade the infectious prion proteins. Here we show the production of pernisine in the periplasm of Escherichia coli. This strategy prevented the aggregation of pernisine in the cytoplasm and increased the purity of the isolated pernisine. The thermostability of this recombinant pernisine was significantly increased compared with previous studies. In addition, several truncated pernisine variants were constructed and expressed in E. coli to identify the minimally active domain. The catalytic domain of pernisine consists of the αẞα structurally similar core flanked by the N-terminal and C-terminal outer regions. The deletion of the C-terminal α helix did not affect the pernisine activity at 90 °C. However, the complete deletion of the C-terminal outer region resulted in loss of proteolytic activity. The pernisine variant, in which the N-terminal outer region was deleted, had a reduced activity at 90 °C. These results underline the importance of the Ca2+ binding sites predicted in these outer regions for stability and activity of pernisine. Key points • Aggregation of produced pernisine was prevented by translocation into periplasm. • Thermostability of mature pernisine was increased. • The outer regions of the catalytic core are required for pernisine thermostability. Keywords Pernisine . Aeropyrum pernix . Proteinase . Periplasmic production . Thermostability
Introduction Hyperthermophilic microorganisms are a known source of extremely thermostable enzymes (Vieille and Zeikus 2001). Archaeon Aeropyrum pernix is a hyperthermophilic model organism that was originally isolated from a coastal sulfataric vent in Japan (Sako et al. 1996). Due to its known genome sequence and its easy cultivability, several studies have Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10791-w) contains supplementary material, which is available to authorized users. * Nataša Poklar Ulrih [email protected] 1
Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
2
Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CIPKeBiP), Ljubljana, Slovenia
investigated the enzymes produced by A. pernix. So far, several heat-stable proteases of A. pernix have been characterized: intracellular pernilase (Croocker et al. 1999), extracellular aeropyrolysin (Sako et al. 1997), and extracellular pernisine (Catara et al. 2003). Among them, pernisine is of particula
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