Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples

Autor(es)

Coelho, Diego F. ; Saturnino, Thais Peron ; Fernandes, Fernanda Freitas ; Mazzola, Priscila Gava ; Silveira, Edgar ; Tambourgi, Elias Basile

• Tipo de documento: Artigo Científico
• Fonte: BIOMED RESEARCH INTERNATIONAL; v. 2016, p. 6-pg., 2016-01-01.

Resumo

Given the importance of protease's worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimumat 45 degrees Cand pH 9 (Glycine-NaOH 100 mM). We also quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0.072 and 0.494 mg.mL(-1) of azocasein, resp.) and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%), which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise. (AU)

• Processo FAPESP: 11/20733-7 - Desenvolvimento de um Extrator Centrífugo Anular e sua Aplicação na Purificação de Bromelina de Resíduos Agroindustriais por Sistemas Bifásicos Aquosos não Convencionais
• Beneficiário: Diego de Freitas Coêlho
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 12/14533-8 - Caracterização de um sistema bifásico aquoso não convencional e sua aplicação na purificação de bromelina de resíduos agroindustriais
• Beneficiário: Elias Basile Tambourgi
• Modalidade de apoio: Auxílio à Pesquisa - Regular