Banca de DEFESA: WELLINGTON OLIVEIRA DA CRUZ

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
DISCENTE : WELLINGTON OLIVEIRA DA CRUZ
DATA : 06/08/2019
HORA: 13:00
LOCAL: Sala 50 do prédio de Química
TÍTULO:

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

PALAVRAS-CHAVES:

Insect, Diets, Inhibitor, Protein

PÁGINAS: 172
GRANDE ÁREA: Ciências Exatas e da Terra
ÁREA: Química
RESUMO:

Alphitobius diaperinus is considered an important pest in avian environment. The study of its behavior, eating habits and digestive enzymes may indicate important targets in its control. The control of A. diaperinus is considered difficult, its natural enemies are little known and to this day there is no efficient and safe method. The digestive proteases of insects catalyze the production of peptides and amino acids of a protein diet and are found in the middle intestine region of the insects' middle intestine. The objective of this work was to characterize the proteases present in the digestive tract of A. diaperinus. Proteolytic activities were determined using azocasein and specific substrates. The crude extract of the homogenized intestine of A. diaperinus showed activity against azocasein and Abz-Phe-Arg-MCA and was inhibited in more than 50% by the cysteine protease inhibitor E-64 and was activated by DTT 2.0 mM at acid pH, which suggests that the substrates are being hydrolysed by cysteine protease. Nine bands were detected with proteolytic activity and molecular weight in the range 10kDa - 100kDa by zimogram, using 0.1% gelatin as substrate. To determine which classes of proteases were present in the homogenized, classical protease inhibitors such as E-64, iodoacetic acid and acetic acid (cysteine protease inhibitors), PMSF, SBTI, Aprotinin (serine protease inhibitor) and TLCK (cysteine and serine protease inhibitor) were used. The presence of proteolytic activity was observed in all pHs analyzed, but between pHs 5 and 6 a greater potential of activity was registered in relation to the other pHs, corroding with the data found in the literature, where the digestion system of A. diaperinus presents more acid pHs. Although combinations of serine and cysteine proteases were observed, proteases with acid character showed higher levels of proteolytic activity in the model studied. The results observed in a zimogram, using 0.1% gelatin as substrate, in the presence of E-64 inhibitor confirm the presence of cysteine protease in the molecular weight range between 20kDa and 30kDa. Ca2+ ions increased enzyme activity in concentrations higher than 2.0mM and 10mM, in 14% and 34%, respectively. Insects maintained in different diets, both in the formulation and in the concentrations used, showed significant difference in the proteolytic activity determined in the larvae of A. diaperinus. The one-dimensional electrophoretic separation of x intestinal proteins from A. diaperinus larvae, followed by separation, band extraction and analysis of two-dimensional nano-HPLC and ESI-MS/MS resulted in the identification of different classes of proteases.

MEMBROS DA BANCA:
Presidente - 1555317 - EMERSON GUEDES PONTES
Interno - 1220404 - CARLOS MAURICIO RABELLO DE SANTANNA
Interno - 1681790 - CRISTIANE MARTINS CARDOSO DE SALLES
Externo à Instituição - DANIELLE MARIA P. DE OLIVEIRA SANTOS - UFRJ
Externo à Instituição - RAFAEL DIAS MESQUITA - UFRJ