Biochemical caracterization of digestive proteases in Alphitobius diaperinus (Coleoptera: Tenebrionidae) larvae

Autor: Cruz, Wellington Oliveira da
Přispěvatelé: Pontes, Emerson Guedes, Souza, Cristiane Martins de, Santos, Danielle M. P. de Oliveira, Mesquita, Rafael Dias, Sant`Anna, Carlos Mauricio
Jazyk: portugalština
Rok vydání: 2019
Předmět:
Zdroj: Biblioteca Digital de Teses e Dissertações da UFRRJ
Universidade Federal Rural do Rio de Janeiro (UFRRJ)
instacron:UFRRJ
Popis: Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2022-07-27T16:56:46Z No. of bitstreams: 1 2019 - Wellington Oliveira da Cruz.pdf: 14172587 bytes, checksum: 12767101c3271533d8d58b1826cc3777 (MD5) Made available in DSpace on 2022-07-27T16:56:47Z (GMT). No. of bitstreams: 1 2019 - Wellington Oliveira da Cruz.pdf: 14172587 bytes, checksum: 12767101c3271533d8d58b1826cc3777 (MD5) Previous issue date: 2019-08-06 CAPES - Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior Alphitobius diaperinus is considered an important issue in the avian environment. Studying your behavior, eating habits and digestive enzymes may indicate important targets in your control. Control of A. diaperinus is considered difficult, its natural enemies are little known, and the use of chemical insecticides is the main method of control. Insect digestive proteases catalyze the production of peptides and amino acids from a protein diet and they are found in the midgut region of insects. Proteolytic activities in the digestive tract of A. diper?nus larvae and adults were determined using azocasein and specific substrates after feeding on different diets. The crude extract of the digestive tract of A. diaperinus larvae showed activity on azocasein and Abz-Phe-Arg-MCA. It was inhibited by more than 50% by E-64 and activated by 2.0 mM DTT at acid pH. Nine bands were detected with proteolytic activity and molecular mass in the range 10 kDa - 100 kDa through zymogram, using 0.1% gelatin as substrate. Different classes of proteases were identified in the presence of classical protease inhibitors such as E-64, iodoacetamide and iodoacetic acid, PMSF, SBTI, aprotinin and TLCK. A higher level of proteolytic activity was observed at pHs between 5 and 6. Although combinations of serine and cysteine proteases have been observed, acid proteases showed higher levels of proteolytic activity. Calcium ion increased the protease activity at concentrations greater than 2.0 mM. Two-dimensional nano-HPLC and ESI-MS / MS analyzes resulted in the identification of proteases such as tripine, cathepsins, metalloproteases, aminopeptidases, peptidases and dipeptidases. High inhibitory potential of different series of imidazolone on cysteine protease was observed, what can be used as control tools of A. diaperinus. O Alphitobius diaperinus ? considerado uma praga importante em ambiente avi?rio. O estudo do seu comportamento, h?bitos alimentares e enzimas digestivas, podem indicar alvos importantes no seu controle. O controle do A. diaperinus ? considerado dif?cil, seus inimigos naturais s?o pouco conhecidos e sendo o uso de inseticidas qu?mico o principal m?todo de controle. Proteases digestivas de insetos catalisam a produ??o de pept?deos e amino?cidos a partir de uma dieta prot?ica e s?o encontradas na regi?o do intestino m?dio dos insetos. As atividades proteol?ticas no trato digestivo das larvas e adultos de A. diperinus foram determinadas utilizando azocase?na e substratos especificos ap?s alimentarem-se de diferentes dietas. O extrato bruto do homogeneizado do trato digestivo das larvas A. diaperinus mostrou atividade sobre azocase?na e Abz-Phe-Arg-MCA e foi inibida em mais de 50% pelo inibidor da ciste?no protease E-64 e foi ativado por DTT 2,0 mM em pH ?cido. Nove bandas foram detectadas com atividade proteol?tica e massa molecular na faixa 10 kDa ? 100 kDa atrav?s de zimograma, utilizando gelatina 0,1% como substrato. Diferentes classes de proteases foram identificadas na presen?a de inibidores de proteases cl?ssicos como E-64, iodoacetamida e ?cido iodoac?tico, PMSF, SBTI, aprotinina e TLCK. Foi observado maior n?vel de atividade proteol?tica em pHs entre 5 e 6. Embora tenham sido observadas combina??es de proteases de serina e ciste?na, as proteases ?cidas apresentaram n?veis mais altos de atividade proteol?tica. O c?lcio aumentou a atividade proteasica em concentra??es maiores que 2,0 mM. An?lises de nano-HPLC bidimensional e ESI-MS/MS resultaram na identifica??o de proteases como tripsina, catepsinas, metaloproteases, aminopeptidases, peptidases e dipeptidases. Foi observado alto potencial inibit?rio de diferentes s?ries de imidazolonas sobre atividade ciste?no proteases, a qual pode ser utilizada como ferramentas de controle do A. diaperinus.
Databáze: OpenAIRE
Externí odkaz: