Peptidase-lipase bifunctional enzyme expressed in pichia pastoris

The protein engineering is a shortcut to natural evolution, one of the possible paths is the creation of hybrid enzymes or also called chimeras. These hybrids are the combination of two or more protein sequences, in order to improve or generate new functionalities. The peptidases and lipases are hydrolases capable of hydrolyze various substrates and have combined applications in different fields, as in the food, detergent, leather industries and bioremediation. End-to-end technique was employed to fuse the hydrolases sequences, the main advantage of its use is the independence of protein conformations and structures when compared to other techniques, making the process simpler. The chimera sequence was composed of hydrolases from Fusarium oxysporum. The peptidase was based on the work of Di Pietro et al (1998), which presented a subtilase, and the lipase sequence was based on the similarity with Thermomyces lanuginosus lipase. The position of each enzyme in the chimera was decided based on the in silico analysis of similar structures, where a His-tag was attached to the N-terminal region of the peptidase followed by a linker composed of five amino acids (GGAGG), and then the lipase sequence. The chimera gene was synthetized by GenScript® in pPiczαA expression vector for Pichia pastoris yeast. Please click Additional Files below to see the full abstract

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

O fungo ascomiceto Purpureocillium lilacinum pode ser encontrado em solo, vegetação em decomposição, insetos, nematoides e outros locais. Apresenta grande importância agrícola, visto que pode ser utilizado como controle biológico por ser capaz de infectar e matar nematoides pragas de culturas, graças às suas enzimas, como peptidases e quitinases. Além disso, o fungo apresenta importância médica, pois é capaz de gerar infecção em humanos. A terapêutica das doenças infecciosas causadas por P. lilacinum é dificultada pelo fato de ele apresentar resistência a antifúngicos \"azólicos\", principais moléculas aplicadas na clínica médica atualmente. O presente trabalho teve como objetivo explorar molecularmente o fungo P. lilacinum com relação à sua capacidade de resistir a antifúngicos e à sua capacidade de produzir peptidases robustas, com potencial de aplicação biotecnológica. Ferramentas moleculares como transcriptômica e proteômica foram aplicadas para acessar os dados moleculares do fungo e viabilizar análises de expressão diferencial para verificar as respostas aos antifúngicos fluconazol e itraconazol e anotação gênica para prospecção de peptidases. Foi observado que P. lilacinum promove um aumento de expressão dos genes alvo dos antifúngicos e dos genes que codificam bombas de efluxo. Com esse mecanismo, não há acúmulo de antifúngico intracelular e há compensação numérica da enzima que está sendo inibida pelo antifúngico com mais expressão da mesma. Dessa forma, o fungo consegue manter a síntese de ergosterol normalizada. As enzimas prospectadas e expressas de maneira recombinante em Pichia pastoris foram uma serino peptidase (rPl_SerPep) e uma metalo peptidase (rPl_MetPep). A rPl_SerPep se mostrou alcalina, com temperatura ótima aparente de 60 °C e termoestável. Já a rPl_MetPep se mostrou ativa em pH neutro e com temperatura ótima aparente de 40 °C. Essas enzimas se mostraram promissoras para futuros testes de aplicação biotecnológica.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

β-frutofuranosidases em coleópteros: caracterização funcional e produção heteróloga de uma β-frutofuranosidase de Sphenophorus levis

The sugar cane represents one of the most Important agricultural segments in Brazil, which is the largest producer and exporter of sugar in the world as well the second largest ethanol producer. The Sphenophorus levis (Curculionidae) is an important sugarcane pest which lacks effective methods of control. The larvae of this insect feed the sugar cane plant decreasing productivity and causing the plant death. In view of identify the insect digestive arsenal enzymes, a transcriptome study was previously performed from S. levis larvae. Thereby, an invertase (P-fructofuranosidases class) coding sequence was identified and characterized. Considering the scarcity of functional studies on insect P-fructofuranosidases and their apparent non-occurrence among coleopterans, the aim of the present study was to investigate the occurrence and characterize the P-fructofuranosidase transcript identified. To validate that the P-fructofuranosidase sequence (herein denominated Sl-fi-fruct) is indeed encoded by the S. levis genome, PCRs were performed using genomic DNA extracted from the larval fat body as well as DNA from the midgut with microbial content. Amplification of Sl-fi-fruct gene using larval fat body DNA indicated its presence in the insect's genomic DNA. Quantitative PCR (qRT-PCR) analyses indicated that the production of mRNA only occurs in the midgut and reaches the greatest expression level in 30-day-old larvae, which is the expected pattern for digestive enzymes. Chromatography of glycosidases from S. levis midguts showed two enzymes acting as P-fructofuranosidase, indicating the presence of a Sl-fi-fruct isoform or a P-fructofuranosidase from insect intestinal microbiota. Moreover, it was found that a-glucosidases do not act on sucrose hydrolysis. Phylogenetic analyses indicated this enzyme to be similar to enzymes found in other coleopteran and lepidopteran P-fructofuranosidases, but also closely similar to bacterial enzymes, suggesting potential horizontal gene transfer. Despite this, the enzyme seems to be restricted to different groups of bacteria, which suggests distinct origin events. The Sl-fi-fruct gene was cloned in Pichia pastoris to produce the recombinant enzyme (rSl-P-fruct). Molecular weight of the recombinant protein was about 64 kDa, indicating possible glycosylation, since the theoretical weight was 54.8 kDa. The substrate specificity test revealed that rSl-P-fruct hydrolyzes sucrose and raffinose, but not melibiose or maltose, thereby confirming invertase activity. The pH curve revealed greatest activity at pH 5.0, demonstrating rSl-P-fruct to be an acidic P-fructofuranosidase. The enzymatic characterization was done and the optimum temperature was 50 °C, thermal resistance at 36 °C and pH maximum resistance at 6.0. The Michaelis-Menten curve showed Km=20.02 μM, Kcat=520.9 s-1 and Vmax=105.7 μM.s-1. 5 mM of SDS and MgCl2 cause inhibition of rSl-β-fruct activity. The present study expands the concept of the occurrence of P-fructofuranosidase in insects. Despite the few descriptions of this gene in the animal kingdom, it is possible to state that P-fructofuranosidase is crucial to the establishment of some insects throughout their evolutionary history, especially members of the Lepidoptera and Coleoptera clades. Considering the rSl-P-fruct potential to industrial application, they are promising if the thermal properties are improved.Universidade Federal de Sao CarlosO coleóptero Sphenophorus levis é uma Importante praga da cana-de-açúcar, para o qual ainda não existe um método de controle adequado. Considerando a Importância da cultura canavieira no Brasil, maior produtor e exportador de açúcar e segundo maior produtor de etanol no mundo, um estudo transcriptômico das larvas do inseto foi realizado anteriormente a este trabalho para a identificação do seu arsenal digestivo. Dentre as prováveis enzimas digestivas, foram identificadas sequências que codificam uma enzima da classe das P-frutofuranosidases. O sequenciamento do clone de cDNA foi totalizado e verificou-se a presença de sinal de poliadenilação e cauda poli-A característica de eucariotos, bem como uma região codificadora para um provável peptídeo sinal para secreção da enzima. A comparação da sequência proteica deduzida com o banco de dados do NCBI aponta maior similaridade com invertases bacterianas. A amplificação do gene da P-frutofuranosidase de S. levis (Sl-fi-fruci) por PCR a partir de DNA genômico, livre de contaminação bacteriana, sugere que S. levis realmente codifica uma P-frutofuranosidase. Comparando-se sequências de aminoácidos de P-frutofuranosidases de coleópteros, observam-se regiões conservadas entre membros da família Curculionidae. O ensaio bioquímico das glicosidases intestinais de S. levis mostrou que existem provavelmente duas P-frutofuranosidases responsáveis pela digestão de sacarose. Portanto, o inseto pode apresentar uma isoforma da Sl-fí-fruct ou se beneficiar de uma invertase produzida pela própria microbiota. As análises de expressão via PCR quantitativo em tempo real revelaram que o gene é expresso em intestino médio, nas fases de alimentação do inseto, característica de uma enzima digestiva. As análises filogenéticas indicaram que Sl-P-fruct é similar a outras P-frutofuranosidases de lepidópteros e coleópteros, mas se apresenta mais próxima das enzimas bacterianas. Essa proximidade se dá a grupos distintos de bactérias, quando comparada com enzimas de lepidópteros, levando-nos a propor um evento de transferência horizontal independente do que ocorreu em lepidópteros. A fase aberta de leitura (ORF) codificadora da enzima, excluindo o peptídeo sinal, foi clonada no plasmídeo pPICZa-A para sua produção heteróloga em Pichia pastoris. A enzima produzida (rSl-P-fruc) apresentou-se glicosilada, com massa molecular aproximada de 65 kDa. Os ensaios de especificidade ao substrato confirmaram que a enzima é uma P-frutofuranosidase. A rSl-P-fruc apresentou pH de maior atividade próximo a 5,0 e temperatura de atividade máxima próxima a 50 °C. Os ensaios de termoestabilidade e resistência térmica sugerem uma baixa capacidade térmica para rSl-P-fruc, que se desnatura com facilidade. Os sais Dodecil Sulfato de Sódio (SDS) e MgCl2 provocam inibição da rSl-P-fruc na concentração de 1 mM. As constantes cinéticas estimadas para a rSl-P-fruct foram Km = 20,02 mM, Vmax = 105,7 |iM.s-1 e kcat = 520,9 s-1. O presente estudo expande o conceito da ocorrência de P-frutofuranosidases em coleópteros. Apesar dos poucos relatos desse gene no reino animal, é possível afirmar que a P-frutofuranosidase é importante e vem sendo mantida entre algumas espécies de lepidópteros e coleópteros. Tratando-se das potencialidades que a rSl-P-fruct apresenta para uma aplicação industrial, observa-se um potencial positivo caso haja melhorias em sua estabilidade térmica

A transcriptomic survey of Migdolus fryanus (sugarcane rhizome borer) larvae.

Sugarcane, a major crop grown in the tropical and subtropical areas of the world, is produced mainly for sucrose, which is used as a sweetener or for the production of bioethanol. Among the numerous pests that significantly affect the yield of sugarcane, the sugarcane rhizome borer (Migdolus fryanus, a cerambycidae beetle) is known to cause severe damage to the crops in Brazil. The absence of molecular information about this insect reinforces the need for studies and an effective method to control this pest. In this study, RNA-Seq technology was employed to study different parts of M. fryanus larvae. The generated data will help in further investigations about the taxonomy, development, and adaptation of this insect. RNA was extracted from six different parts (head, fat body, integument, hindgut, midgut, and foregut) using Trizol methodology. Using Illumina paired-end sequencing technology and the Trinity platform, trimming and de novo assembly was performed, resulting in 44,567 contigs longer than 200 nt for a reunion of data from all transcriptomes, with a mean length of 1,095.27 nt. Transcripts were annotated using BLAST against different protein databanks (Uniprot/Swissprot, PFAM, KEEG, SignalP 4.1, Gene Ontology, and CAZY) and were compared for similarity using a Venn diagram. Differential expression patterns were studied for select genes through qPCR and FPKM comprising important protein families (digestive peptidases, glucosyl hydrolases, serine protease inhibitors and otopetrin), which allowed a better understanding of the insect's digestion, immunity and gravity sensorial mechanisms

Classification of Mf_all trascriptome based on GO terms “Molecular Function”, “Biological Process,” and “Cellular Component”.

<p>The graph indicates an abundance of a specific category of genes in a main category.</p

Oligonucleotides used in qPCR experiments.

<p>Oligonucleotides used in qPCR experiments.</p

Glycosyl Hydrolase families found in gut transcriptomes of <i>M</i>. <i>fryanus</i> larvae.

<p>GH18 (Chitinase) is the most abundant, followed by GH2 (β-glucuronidase), GH1 (β-glucosidase), and GH31 (α-glucosidase).</p

Distribution of the 1% of contigs classified as unknown.

<p>DUF was attributed as available in the PFAM system.</p

Dissected parts of <i>M</i>. <i>fryanus</i> larvae body.

<p>(A) whole larva. (B) intestinal tube: B1, foregut; B2, midgut; B3, hindgut. (C) integument. (D) fat body. (E) head.</p