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Master's Dissertation
DOI
https://doi.org/10.11606/D.60.2020.tde-18122019-171832
Document
Author
Full name
Flávio Antônio de Oliveira Simões
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2019
Supervisor
Committee
Cabral, Hamilton (President)
Braga, Gilberto Ubida Leite
Fuentes, Andrea Soares da Costa
Title in Portuguese
Transcriptoma e expressão recombinante de peptidases do fungo Phanerochaete chrysosporium em Pichia pastoris
Keywords in Portuguese
Aspartil peptidase
Cisteíno peptidase
Peptidases recombinantes
Abstract in Portuguese
Os fungos possuem uma grande diversidade genética e habitam os mais variados hábitats. Esses microrganismos podem ser fontes de enzimas que atuam em condições de temperatura e pH que as tornam interessantes para bioprocessos industriais. O Phanerochaete chrysosporium possui em seu genoma genes do citocromo p450, além de uma grande variedade de oxidases e hidrolases, que incluem as peptidases. As peptidases estão envolvidas em diversas vias biológicas e possuem diversas aplicações industriais (indústria alimentícia, farmacêutica e têxtil). Duas peptidases (aspartil e cisteíno peptidases) produzidas pelo P. chrysosporium já foram identificadas em sua forma nativa por nosso grupo de pesquisa e a expressão recombinante dessas proteínas pode fornecer meios para a caracterização bioquímica e funcional. O sistema Pichia patoris possui um eficiente sistema de secreção de proteínas e é capaz de realizar modificações pós-traducionais, produzindo enzimas recombinantes funcionalmente ativas. O objetivo deste estudo foi realizar o transcriptoma do fungo Phanerochaete chrysosporium para identificação e expressão recombinante de peptidases aspartil e cisteíno para estudos bioquímicos e funcionais. Para isso, o P. chrysosporium foi submetido à diferentes cultivos suplementados com farinha de pena (1%), ou caseína (1%), ou avicel (1%), ou azeite (1%). Foram feitos ensaios de atividade enzimática para peptidase, lacase, amilase, lipase, CMCase, xilanase e pectinase. Foram detectadas atividade para todas as enzimas em todas as condições, exceto pectinase e lipase. Os extratos suplementados com farinha de pena foram purificados em cromatografia de troca iônia e o N-terminal da aspartil e da cisteíno peptidases foi sequenciado via degradação de Edman. O micélio das culturas foi usado para extração do RNA e síntese do cDNA para a construção da biblioteca de transcritos, que gerou 33840 sequências. A biblioteca foi submetida a análise pelo UNIPROT e 7327 sequências foram identificadas (52% não enzimas e 48% enzimas). As classes enzimáticas com maior número de sequências foram das hidrolases, transferases e oxidorredutases, com 34%, 30% e 23%, respectivamente. Quanto as peptidases, 1208 foram anotadas como pertencentes a essa subclasse, sendo aspartil peptidases 16,2%, seguida de ômega peptidases 15,1%, de metalo peptidases 10,8%, serino peptidases 10,4% e cisteíno peptidases, 8,3%. A sequência obtida do N-terminal da aspartil peptidase foi utilizada para busca na biblioteca de transcritos. A busca revelou 5 isoformas da mesma sequência DN10508 (2, 6, 7, 10 e 11) e após a análise a isoforma 10 foi selecionada para expressão heteróloga. A busca da sequência da cisteíno peptidase foi feita através da anotação funcional da biblioteca e duas sequências foram encontradas DN7040, isoforma 1 e 3. A isoforma 3 foi selecionada para expressão heteróloga, após análise dos domínios e da massa teórica da sequência. As sequências selecionadas foram clonadas no vetor pPICZ?A. Os clones de P. pastoris contendo as construções foram submetidos a um screening (20 clones de cada construção) em mini biorreatores Corning, induzido com metanol. A atividade enzimática dos clones contendo a construção pPICZ?A-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 pPICZ?A-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
Title in English
Trancriptome and recombinant peptidase expression from the fungus Phanerochaete chrysosporium in Pichia pastoris
Keywords in English
Aspartic peptidase
Cysteine peptidase
Recombinant peptidases
Abstract in English
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 48% enzymes). The largest class detected we hydrolases, transferases and oxidoreductases, with 34%, 30% and 23%, respectively. As for peptidases, 1208 were noted as peptidases, the highest number of its subclass were aspartic peptidase 16.2%, followed by omega peptidase 15.1%, metallo peptidase 10.8%, serinoe peptidase 10.4% and cysteine peptidase, 8,3%. The sequence obtained from the N-terminal aspartic peptidase was used to search the primary sequence in the transcript library. Five isoforms of the same sequence were find DN10508 (2, 6, 7, 10 and 11) and after sequence analysis isoform 10 was selected for heterologous expression. Cysteine peptidase sequence was found using the functional annotation in the library and two sequences were found DN7040, isoform 1 and 3. Isoform 3 was selected for heterologous expression after domain analysis. Sequences selected for recombinant expression were cloned into the pPICZ?A vector, followed by transformation of Pichia pastoris KM71H cells. Fifty colonies were obtained for pPICZ?A-DN10508 and 55 colonies for pPICZ?A-DN7040. The colonies were screened (20 colonies of each construct) in methanol-induced Corning mini bioreactors. Enzyme activities for the pPICZ?A-DN10508 (aspartyl peptidase) construct showed that colonies 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) and 5 (14.15 ± 2.15 U / mL) had the highest enzymatic activities. Enzymatic activities of clones containing pPICZ?A-DN7040 (cysteine peptidase) showed no promising results. Under these conditions evaluated in the enzymatic assays, 5 out of 7 enzymes were detected. In addition, it was possible to construct a library of transcripts containing more than 33,000 sequences, select and perform recombinant expression of two peptidase using library sequences
 
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Release Date
2021-12-17
Publishing Date
2020-02-28
 
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