Biochemical and biophysical properties of a recombinant serine peptidase from Purpureocillium lilacinum

Autor(es)

Pedezzi, Rafael ; Evangelista, Danilo Elton ; Garzon, Nathalia Gonsales da Rosa ; Simoes, Flavio Antonio de Oliveira ; de Oliveira, Arthur Henrique Cavalcante ; Polikarpov, Igor ; Cabral, Hamilton

• Tipo de documento: Artigo Científico
• Fonte: Biophysical Chemistry; v. 296, p. 9-pg., 2023-02-22.

Resumo

The industrial uses of peptidases have already been consolidated; however, their range of applications is increasing. Thus, the biochemical characterization of new peptidases could increase the range of their biotechnological applications. In silico analysis identified a gene encoding a putative serine peptidase from Purpureocillium lilacinum (Pl_SerPep), annotated as a cuticle-degrading enzyme. The Pl_SerPep gene product was expressed as a recombinant in a Komagataella phaffii (previously Pichia pastoris) expression system. The enzyme (rPl_SerPep) showed optimal pH and temperature of 8.0 and 60 degrees C, respectively. Moreover, rPl_SerPep has a higher thermal stability than the cuticle-degrading enzymes described elsewhere. The structural analysis indi-cated a conformational change in the rPl_SerPep secondary structure, which would allow an increase in catalytic activity at 60 degrees C. Komagataella phaffii secretes rPl_SerPep with the pro peptide in its inactive form. Low-resolution small-angle X-ray scattering (SAXS) analysis showed little mobility of the pro peptide portion, which indicates the apparent stability of the inactive form of the enzyme. The presence of 20 mM guanidine in the reaction resulted in the maintenance of activity, which was apparently a consequence of pro peptide structure flexibilization. (AU)

• Processo FAPESP: 20/14426-3 - Clonagem, expressão e mapeamento dos subsítios de peptidases não clássicas: aplicação biotecnológica na obtenção de peptídeos bioativos
• Beneficiário: Hamilton Cabral
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 15/16084-4 - Prospecção de enzimas no fungo Skopulariopsis koningii, utilizando a técnica de proteoma: Produção Recombinante de Enzimas com Potencial Biotecnológico e Avaliação de Antifúngicos
• Beneficiário: Rafael Pedezzi
• Modalidade de apoio: Bolsas no Brasil - Doutorado