The thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications. Due to its properties, A. pernix cannot be cultivated in standard industrial fermentation facilities. Furthermore, pernisine is a demanding target for heterologous expression in mesophilic heterologous hosts due to the relatively complex processing step involved in its activation.We achieved production of active extracellular pernisine in a Streptomyces rimosus host through heterologous expression of the codon-optimised gene by applying step-by-step protein engineering approaches. To ensure secretion of fully active enzyme, the srT signal sequence from the S. rimosus protease was fused to pernisine. To promote correct processing and folding of pernisine, the srT functional cleavage site motif was fused directly to the core pernisine sequence, this way omitting the proregion. Comparative biochemical analysis of the wild-type and recombinant pernisine confirmed that the enzyme produced by S. rimosus retained all of the desired properties of native pernisine. Importantly, the recombinant pernisine also degraded cellular and infectious bovine prion proteins, which is one of the particular applications of this protease. Functional pernisine that retains all of the advantageous properties of the native enzyme from the thermophilic host was successfully produced in a S. rimosus heterologous host. Importantly, we achieved extracellular production of active pernisine, which significantly simplifies further downstream procedures and also omits the need for any pre-processing step for its activation. We demonstrate that S. rimosus can be used as an attractive host for industrial production of recombinant proteins that originate from thermophilic organisms.
COBISS.SI-ID: 5116792
We studied seven known peptides derived from different food proteins, and one hydrophobic peptide derived through in-silico hydrolysis. We investigated their antioxidative, antimicrobial and haemolytic activities and their interactions with model lipid membranes. Our data reveal that peptides with histidines are the most effective for protection against lipid peroxidation, as: P#3 (VHHA)?)?P#5 (LHALLL)?)?P#7 (LLPHH). The most potent peptide towards metal-induced oxidation was P#2 (LQKW), which contained an aromatic tryptophan at its C-terminus. The peptides with negative hydrophobicity, as P#1 (PEL) and P#2 (LQKW), were the most potent according to DPPH radical scavenging. The most potent peptide towards superoxide radicals was the four-amino-acid chain with aromatic amino acids, P#2 (LQKW). The peptides studied did not show haemolytic or antimicrobial activities, except P#4 (AAGGV) against Listeria monocytogenes. These peptides did not interact with model lipid membranes, except P#8, which was designed to span lipid bilayers. Furthermore, we used pernisine as protease for obtaining the new bioactive peptides.
COBISS.SI-ID: 5031288
The contribution was published as invited lecture. Pernisine is subtilisin-like protease from the hyperthermophilic archaeon Aeropyrum pernix. Due to high thermal stability and activity in the presence of denaturants, pernisine emerges as a promising enzyme for use in various industrial applications. Ability to degrade infectious prion aggregates is another applicable characteristic of this protease. Pernisine is composed of signal sequence, proregion and catalytic domain. Signal sequence directs pernisine into extracellular space, where proregion is autocatalytically degraded, leaving activated catalytic domain. Pernisine activation, stability and activity are strongly modulated by calcium ions. Production of pernisine in A. pernix results in insufficient yield for commercial use, hence the need for alternative production strategies. Progress has been made with production of recombinant form of pernisine using Escherichia coli expression system, where codon optimization of pernisine gene was necessary for protein synthesis. Furthermore, successful production of active recombinant pernisine was achieved also in Streptomyces rimosus.
COBISS.SI-ID: 5112184
The hyperthermophilic archaeon Aeropyrum pernix has adapted to optimal growth under high temperatures in saline environments and under oxidizing conditions. In the present study, we focused on the antioxidative activity of proteins from A. pernix K1. Following high temperature methanol and water extractions of the protein from the biomass of A. pernix K1, the total sulphydryl groups and radical scavenging activities were investigated. The total protein in the methanolic extract was 36% lower and showed 10% fewer sulphydryl groups than that from the water extract. However, the radical scavenging activity of the water extract was four-fold greater than for the methanolic extract. The proteins of both of these extracts were separated by two-dimensional electrophoresis, and selected proteins were identified using mass spectrometry. The majority of these identified proteins were intracellular proteins, such as those involved in oxidative stress responses and osmotic stress responses, and proteins with hydrolase and dehydrogenase activities. These proteins are also common to most organisms, and included putative uncharacterized proteins.
COBISS.SI-ID: 4877176
Biological membranes are complex systems that are composed of lipids, proteins and carbohydrates. They are difficult to study, so it is established practice to use lipid vesicles that consist of closed ‘shells’ of phospholipid bilayers as model systems to study various functional and structural aspects of lipid organisation. To define the effects of the structural properties of lipid vesicles on their phase behaviour, we investigated their headgroup and chain length, and the chemical bonds by which their acyl chains are attached to the glycerol moiety of glycerophospholipid species, in terms of phase transition temperature, enthalpy change and calcein permeability. We used differential scanning calorimetry to measure the temperature and enthalpy changes of phase transition, and fluorescence to follow calcein release through the bilayer structure. Our data show that longer acyl chains increase the stability of the lipid bilayers, whereas higher salt concentrations decrease the thermal stability and widen the phase transitions of these lipid bilayers. We discuss the possible reasons for the observed phase transition behaviour.
COBISS.SI-ID: 5081976