A New Pepstatin-Insensitive Thermopsin-Like Protease Overproduced in Peptide-Rich Cultures of Sulfolobus solfataricus

Abstract

In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease), while the less abundant (named SsMTP-1) one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50–90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.

Revealing S-layer Functions in the Hyperthermophilic Crenarchaeon Sulfolobus islandicus

“…3) present in both the N- and C-terminal of M164_1049 suggests that M164_1049 has a membrane topological organization, implying there is a potential connection between M164_1049 and the outermost layer (SlaA). Interestingly, the M164_1049 homolog in a related Sulfolobus species S. solftaricus P2 (SSO1175, sharing 83% of amino acid identify with 96% of query coverage) has been shown to be an N-glycosylated protein (37), and biochemically characterized to function as a multi-domain thermopsin-like protease (38). Our data show that M164_1049 is not required to maintain a structured and intact outermost layer (SlaA) when SlaB is present, as the single ∆ M164_1049 mutant exhibits a very similar S-layer structure as that of the wild-type strain.…”

Exploration of extremophiles for high temperature biotechnological processes

“…The thermopsin-like protease SsMTP-1 from Sulfolobus solfataricus is another example of a heatstable and acid-stable proteolytic enzyme of archaeal origin with well-suited properties. The 75 kDa protease exhibited optimal activity at 70 8C and pH 2.0 and showed a half-life of 50 h at 90 8C [32]. In addition, the serine protease proteolysin from the thermophilic bacterium Coprothermobacter proteolyticus is a prominent candidate for the detergent industry because of its thermostability at elevated temperatures, its optimal activity at alkaline pH and its tolerance towards highly concentrated detergents [33].…”

Estabelecimento de condições de cultivo de leveduras isoladas na Antártica visando à produção de proteases

“…Entretanto, há uma diculdade em correlacionar os diversos trabalhos disponíveis na literatura, em virtude dos diferentes métodos e análises aplicadas (BOSSI; BONIZZATO; ZAPPAROLI, 2006;MUKHTAR, 2009;NEGI;BANERJEE, 2009;YIN et al, 2013;GOGLIETTINO et al, 2014). de incubação nesta temperatura (60°C), verificou-se uma queda de 98,3% na atividade proteolítica, sugerindo uma inativação térmica, conforme reportado por outros autores em relação a enzimas produzidas em baixas temperaturas (GERDAY et al, 2000;DAMARE et al, 2006;RAO et al, 2011;YIN et al, 2013).…”

“…Porém, novos gêneros têm sido explorados quanto à produção de proteases ácidas por muitos cientistas, embora estes relatem uma atividade enzimática ainda baixa (RAY et al, 1992;BOSSI;BONIZZATO;ZAPPAROLI, 2006;MANDUJANO-GONZÁLEZ et al, 2013;GOGLIETTINO et al, 2014).…”

“…Entretanto, embora a produção da maioria das proteases ácidas seja por fungos filamentosos, há uma grande dificuldade em correlacionar os trabalhos disponíveis na literatura envolvendo a atividade proteolítica de diferentes linhagens destes microorganismos, em virtude dos diferentes métodos e análises empregadas ( BOSSI; BONIZZATO;ZAPPAROLI, 2006;MUKHTAR, HAQ, 2009;NEGI;BANERJEE, 2009;YIN et al, 2013;GOGLIETTINO et al, 2014). No caso das leveduras, embora a secreção de enzimas proteolíticas não seja uma propriedade comum, a maioria delas produz aspartil proteases ácidas e algumas poucas espécies produzem serina peptidases e proteases neutras ou alcalinas (CHAROENCHAI et al, 1997;TURKIEWICZ et al, 2003;RUBERTO;MAC CORMACK, 2005;CHI et al, 2009).…”