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

Author(s)

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

• Document type: Journal article
• Source: Biophysical Chemistry; v. 296, p. 9-pg., 2023-02-22.

Abstract

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)

• FAPESP's process: 20/14426-3 - Cloning, expression and mapping of non-classical peptidase subsites: biotechnological application in obtaining bioactive peptides
• Grantee: Hamilton Cabral
• Support Opportunities: Regular Research Grants
• FAPESP's process: 15/16084-4 - Skopuleriopsis koningii enzyme prospection using proteome: Recombinant production of enzimes with biotecnological potential and evaluation of Antifungal
• Grantee: Rafael Pedezzi
• Support Opportunities: Scholarships in Brazil - Doctorate