Transcriptoma e expressão recombinante de peptidases do fungo Phanerochaete chrysosporium em Pichia pastoris

Abstract

metanol. A atividade enzimática dos clones contendo a construção pPICZαA-DN10508 (aspartil peptidase) mostraram que os clones 1 (16,00 ± 3,27 U/mL) ,2 (14,44 ± 1,12 U/mL), 11 (14,27± 0,315 U/mL), 4 (14,27 ± 0,086 U/mL) e 5 (14,15 ± 2,15 U/mL) obtiveram a maior atividade enzimática. A atividade enzimática dos clones contendo pPICZαA-DN7040 (cisteíno peptidase) não apresentaram resultados promissores. Nas condições avaliadas foi possível induzir a expressão de diferentes classes enzimáticas. A biblioteca de transcritos gerou mais de 33 mil sequências e através dela, foi possível selecionar e expressar duas peptidases (aspartil e cisteíno peptidases) para a expressão heteróloga. Palavras chave: Peptidases recombinantes, Aspartil peptidase, Cisteíno peptidase iii ABSTRACT SIMÕES, F.A.O. Trancriptome and recombinant peptidase expression from the fungus Phanerochaete chrysosporium in Pichia pastoris. 2019. 87p. Dissertation (Master). Faculdade de Ciências Farmacêuticas de Ribeirão Preto -Universidade de São Paulo, Ribeirão Preto, 2019.Fungi can be found in a variety of habitats and have great genetic diversity. These microorganisms produce a variety of enzymes and some of these may work under specific temperature and pH conditions. Phanerochaete chrysosporium can completely degrade lignin. Its genome possess a large repertoire of genes related to lignin metabolism including cytochrome p450, oxidoreductose and hydrolases, which includes peptidase enzymes. Peptidase enzymes are involved in various biological pathways and have industrial applications (textile and food industry). Our research group have described two native peptidases (aspartyl and cysteine peptidase) with great potential for industrial application. Recombinant protein production could provide an efficient way for enzyme production and purification. Pichia patoris expression system is widely used for recombinant protein expression, and it is described as efficient for both production and secretion of recombinant enzymes, it also is able to execute post-translational modifications. The aim of this study was to perform transcriptome analyses of the fungus Phanerochaete chrysosporium to identify and express two heterologous peptidase (aspartic and cysteine peptidase) in P. pastoris for biochemical and functional studies. P. chrysosporium was submitted to different bioprocesses supplemented with feather meal (1%), casein (1%), avicel (1%), or olive oil (1%). Enzymatic activities of peptidases, laccases, amylases, lipases, CMCases, xylanases and pectinases were determined. All activities were detected, except lipase and pectinase activities. Extracts supplemented with feather meal were purified by ion exchange chromatography and the N-terminal sequence of aspartic and cysteine peptidases were determined by Edman degradation. The mycelium was used for RNA extraction and cDNA synthesis. A transcript library was built using cDNA sequences, which generated 33840 sequences. The library was analyzed by UNIPROT and 7327 sequences were identified (52% non-enzymes and 4...