Prospecting of enzymes in Purpureocillium lilacinum using proteomic technique: recombinant production of enzymes with biotechnological potential and evaluation of antifungals

Author(s)

Rafael Pedezzi

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-08-09

Examining board members

Hamilton Cabral; Eleni Gomes; Arthur Henrique Cavalcante de Oliveira; Maria de Lourdes Teixeira de Moraes Polizeli

• Advisor: Hamilton Cabral

Abstract

The ascomicota fungus Purpureocillium lilacinum can be found in soil, decaying vegetation, insects, nematodes and other places. It has great agricultural importance, since it can be used as biological control by being able to infect and kill nematode crop pests, thanks to its enzymes such as peptidases and chitinases. In addition, the fungus is of medical importance because it can generate infection in humans. The treatment of infectious diseases caused by P. lilacinum is hampered by the fact that it is resistant to \"azolic\" antifungals, the main molecules applied in medical practice today. The present work aimed to molecularly explore the fungus P. lilacinum in relation to its ability to resist antifungal and its ability to produce robust peptidases with potential for biotechnological application. Molecular tools such as transcriptomics and proteomics were applied to access the molecular data of the fungus and enable differential expression analyzes to verify the responses to the antifungal fluconazole and itraconazole and gene annotation to prospect for peptidases. It has been observed that P. lilacinum promotes an increase in expression of antifungal target genes and genes encoding efflux pumps. With this mechanism, there is no accumulation of intracellular antifungal and there is numerical compensation of the enzyme that is being inhibited by the antifungus with more expression of the same. In this way, the fungus can maintain standard ergosterol synthesis. The serine peptidases prospected were recombinantly expressed in Pichia pastoris. There were a serine peptidase (rPl_SerPep) and a metal peptidase (rPl_MetPep). The rPl_SerPep was alkaline, with an apparent optimum temperature of 60 ° C and showed thermostable characteristics. The rPl_MetPep was neutral and with an apparent optimum temperature of 40 ° C. These enzymes have proven to be promising for future biotech application tests. (AU)

• 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