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

Author(s)

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

• Document type: Journal article
• Source: BIOMED RESEARCH INTERNATIONAL; v. 2016, p. 6-pg., 2016-01-01.

Abstract

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)

• FAPESP's process: 11/20733-7 - Development of Anular Centrifugal Contactor and its Application to Purification of Bromelain from Agroindustrial Waste through unconventional Aqueous Two-Phase Systems
• Grantee: Diego de Freitas Coêlho
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 12/14533-8 - Characterization on unconventional aqueous two-phase system and its application in bromelain purification from agroindustrial wastes
• Grantee: Elias Basile Tambourgi
• Support Opportunities: Regular Research Grants