Diego de Freitas Coelho

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

Given the importance of proteases 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 optimum at 45°C and 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.

L-Asparaginase Purification

L-asparaginase (L-ASNase) is the gold standard enzyme used to treat acute lymphoblastic leukemia. This disease primarily affects children; however, treatment increases survival from 20% to 90%. As a bioproduct, it is obtained via a biotechnological process and its purification usually consists of several steps that account for up to 80% of the total production costs. This review discusses available strategies for the purification of L-ASNase and highlights a process with fewer steps, and consequently, lower cost and higher yield. This process emphasizes the possibility of using a novel aqueous two-phase system extraction process to purify L-ASNase.

Poly(N-Isopropylacrylamide)-co-Acrylamide Hydrogels for the Controlled Release of Bromelain from Agroindustrial Residues of Ananas comosus.

This works reports the purification of bromelain extracted from Ananas comosus industrial residues by ethanol purification, its partial characterization from the crude extract as well as the ethanol purified enzyme, and its application onto poly(N-isopropylacrylamide)-co-acrylamide hydrogels. Bromelain was recovered within the 30-70 % ethanol fraction, which achieved a purification factor of 3.12-fold, and yielded more than 90 % of its initial activity. The resulting purified bromelain contained more than 360 U · mg(-1), with a maximum working temperature of 60 °C and pH of 8.0. Poly(N-isopropylacrylamide)-co-acrylamide hydrogels presented a swelling rate of 125 %, which was capable of loading 56 % of bromelain from the solution, and was able to release up to 91 % of the retained bromelain. Ethanol precipitation is suitable for bromelain recovery and application onto poly(N-isopropylacrylamide)-co-acrylamide hydrogels based on its processing time and the applied ethanol prices.

Studies of stability and characterization this enzyme bromelain in pineapple (Ananas comosus)

Background Bromelain is the generic name given to the set of derived endopeptidases belonging to members of the Bromeliaceae family, which belongs to the pineapple (Ananas comosus), being able to break the peptide bond, separating proteins and amino acids [1]. Bromelain possesses a wide range of therapeutic benefit as property of facilitating digestion of proteins, meat softening, ability to facilitate blood clotting [2] And economic importance related to the food industry and textiles and production of drugs resulting in an increase of its value [3]. Thus, the aim of this work is to evaluate the stability of the enzyme in relation to different temperatures and pH to make feasible the purification of the same.