Enzymatic Degradation of PrPSc by a Protease Secreted from Aeropyrum pernix K1

BACKGROUND: An R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrP(Sc)) from different species (human, bovine, deer and mouse). METHODOLOGY/PRINCIPAL FINDINGS: Degradation of the PrP(Sc) isoform by the R30 fraction and the purified protease was evaluated using the 6H4 anti-PrP monoclonal antibody. Fragments from the N-terminal and C-terminal of PrP(Sc) were also monitored by Western blotting using the EB8 anti-PrP monoclonal antibody, and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrP(Sc), the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 23 kDa. The purified pernisine was active between 58 °C and 99 °C, and between pH 3.5 and 8.0. The temperature and pH optima of the enzymatic activity of the purified pernisine in the presence of 1 mM CaCl(2) were 105 °C ± 0.5 °C and pH 6.5 ± 0.2, respectively. CONCLUSIONS/SIGNIFICANCE: Our study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest the pathological prion protein PrP(Sc)

Functional Insight into the C-Terminal Extension of Halolysin SptA from Haloarchaeon Natrinema sp. J7

Halolysin SptA from haloarchaeon Natrinema sp. J7 consists of a subtilisin-like catalytic domain and a C-terminal extension (CTE) containing two cysteine residues. In this report, we have investigated the function of the CTE using recombinant enzymes expressed in Haloferax volcanii WFD11. Deletion of the CTE greatly reduced but did not abolish protease activity, which suggests that the CTE is not essential for enzyme folding. Mutational analysis suggests that residues Cys303 and Cys338 within the CTE form a disulfide bond that make this domain resistant to autocleavage and proteolysis under hypotonic conditions. Characterization of full-length and CTE-truncation enzymes indicates the CTE not only confers extra stability to the enzyme but also assists enzyme activity on protein substrates by facilitating binding at high salinities. Interestingly, homology modeling of the CTE yields a β-jelly roll-like structure similar to those seen in Claudin-binding domain of Clostridium perfringens enterotoxin (clostridial C-CPE) and collagen binding domain (CBD), and the CTE also possesses collagen-binding activity, making it a potential candidate as an anchoring unit in drug delivery systems

Pročišćavanje i karakterizacija nove izvanstanične haloproteaze Vpr izolirane iz soja bakterije Bacillus licheniformis KB111

Research background. Haloalkaline proteases are one of the most interesting types of commercial enzymes in various industries due to their high specific activity and stability under extreme conditions. Biochemical characterization of enzymes is an important requirement for determining their potential for application in industrial fields. Most of microbial proteases have been isolated from Bacillus spp. In this study, the purification and characterization of an extracellular haloprotease produced from Bacillus sp. KB111 strain, which was previously isolated from mangrove forest sediments, are investigated for industrial applications. Experimental approach. The whole genome of KB111 strain was identified by DNA sequencing. Its produced protease was purified by salting out and anion-exchange chromatography, characterized based on protease activity and stability using a peptide substrate, and identified by LC-MS/MS. Results and conclusions. The strain KB111 was identified as Bacillus licheniformis. The molecular mass of its extracellular protease, termed KB-SP, was estimated to be 70 kDa. The optimal pH and temperature for the activity of this protease were 7 and 50 °C, respectively, while the enzyme exhibited maximal activity in the broad salinity range of 2–4 M NaCl. It was fully stable at an alkaline pH range of 7–11 at 50 °C with a half-life of 90 min. Metal ions such as K+, Ca2+ and Mg2+ could enhance the enzyme activity. Therefore, this protease indicates a high potential for the applications in the food and feed industry, as well as the waste management since it can hydrolyse protein at high alkaline pH and salt concentrations. The amino acid profiles of the purified KB-SP determined by LC-MS/MS analysis showed high score matching with the peptidase S8 of B. licheniformis LMG 17339, corresponding to the mature domain of a minor extracellular protease (Vpr). Amino acid sequence alignment and 3D structure modelling of KB-SP showed a conserved catalytic domain, a protease-associated (PA) domain and a C-terminal domain. Novelty and scientific contribution. A novel extracellular haloprotease from B. licheniformis was purified, characterized and identified. The purified protease was identified as being a minor extracellular protease (Vpr) and this is the first report on the halotolerance of Vpr. This protease has the ability to work in harsh conditions, with a broad alkaline pH and salinity range. Therefore, it can be useful in various applications in industrial fields.Pozadina istraživanja. Haloalkalne proteaze spadaju u najzanimljivije komercijalne enzime koji se koriste u različitim industrijama zbog njihove vrlo specifične aktivnosti i stabilnosti pri ekstremnim uvjetima. Biokemijske značajke enzima bitne su za određivanje njihovog potencijala primjene u industriji. Većina je mikrobnih proteaza izolirana iz bakterija vrste Bacillus spp. U ovom je radu pročišćena i okarakterizirana izvanstanična haloproteaza dobivena iz soja bakterije Bacillus sp. KB111, prethodno izoliranog iz sedimenata šume mangrova za industrijsku primjenu. Eksperimentalni pristup. Genom soja KB111 identificiran je sekvenciranjem DNA. Dobivena je proteaza pročišćena isoljavanjem i ionsko-izmjenjvačkom kromatografijom, okarakterizirana na osnovi njezine aktivnosti i stabilnosti u supstratu, te identificirana pomoću LC-MS/MS. Rezultati i zaključci. Soj KB111 identificiran je kao Bacillus licheniformis. Molekularna masa dobivene izvanstanične proteaze, nazvane KB-SP, procijenjena je na 70 kDa. Optimalna pH-vrijednost za djelovanje ove proteaze bila je 7, a temperatura 50 °C, a njezina je najveća aktivnost postignuta pri koncentraciji soli od 2 do 4 M. Proteaza je bila potpuno stabilna u alkalnom rasponu pH-vrijednosti 7−11 pri 50 °C, s vremenom poluraspada od 90 min. Utvrđeno je da ioni metala, kao što su K+, Ca2+ i Mg2+, mogu pospješiti aktivnost enzima. Stoga je zaključeno da se ova proteaza može uvelike primijeniti u prehrambenoj industriji, proizvodnji stočne hrane, te za obradu otpada, jer može hidrolizirati proteine pri alkalnim pH-vrijednostima i visokoj koncentraciji soli. Profil amino kiselina pročišćene proteaze KB-SP određen pomoću LC-MS/MS imao je veliki stupanj preklapanja s peptidazom S8 iz bakterije B. licheniformis LMG 17339, te je odgovarao zreloj domeni manje izvanstanične proteaze (Vpr). Analizom aminokiselinskog slijeda i 3D modeliranjem strukture proteaze KB-SP utvrđeno je da enzim ima konzerviranu katalitičku domenu, domenu povezanu s proteazom i C-terminalnu domenu. Novina i znanstveni doprinos. Nova izvanstanična proteaza izolirana iz bakterije B. licheniformis je pročišćena, okarakterizirana i identificirana kao jedna od manjih izvanstaničnih proteaza (Vpr), te je ovo prvi izvještaj o njezinoj halotoleranciji. Ova je proteaza aktivna u nepovoljnim uvjetima, u širokom rasponu alkalnih pH-vrijednosti i saliniteta, stoga se može primijeniti u različitim industrijskim granama