A 1.2 purification factor was obtained with the yield of 35.4%, when ammonium sulphate, at a saturation degree of 30–90% (F2), was used. An insignificant activity was detected in the F1 fraction (0–30% of saturation) and no activity was observed in the final supernatant fraction (SF). Therefore, fraction F2 was chosen to be applied to a Sephadex® G75 column. After this step, a 7.7-fold increase was observed in specific activity, with a yield of 33.2%. The chromatograms of the protein elution and the trypsin activity profiles are shown
in Fig. 1A. Etoposide datasheet Other studies that used the same methodology to purify tropical fish trypsins, reported chromatogram profiles which were similar to those obtained in the present research with the Sephadex® G75 column (Bezerra et al., 2001, Bezerra et al., 2005 and Souza et al., 2007). This reinforces the reproducibility of the methodology described by Bezerra et al. (2001) for the purification of trypsin from the viscera of tropical fish. The highest trypsin activity was found in the second protein peak. Therefore, this peak was pooled and applied to a benzamidine–agarose affinity chromatography column. After elution, only one peak with trypsin activity was observed (Fig. 1B). A 24.9-fold increase was observed in specific activity, with a yield of 17.4%. It is known that one of the most important limiting factors for the
commercial use of fish processing waste
as a source of Pexidartinib manufacturer proteases is the strategy of protein purification (Souza et al., 2007). In fact, these methodologies are generally high in cost and time-consuming (Bezerra et al., 2001). However, the procedures, as well as the raw material (fish viscera), used in the present study are of relatively low cost, being therefore easily adapted for processing on an industrial scale. Furthermore, the use of these proteases in some industries, selleck chemicals such as food and detergent, does not require a high degree of purity, which makes the process more economically viable. Using heat treatment (followed by ethanolic precipitation) of alkaline proteases from the crude extract of intestine from Colossoma macropomum, Espósito et al. (2009a) reported the large potential of its fractions as adjuvants in detergent formulations. Moreover, the crude extract was clearer when this process was employed, and the characteristic fish smell was also eliminated. The purified sample showed only one band on SDS–PAGE with a molecular mass of approximately 28.0 kDa (Fig. 2A). According to the literature, fish trypsins have molecular weights between 23 and 28 kDa, which is confirmed for other fish species, such as: L. vitta (23 kDa) ( Khantaphant & Benjakul, 2010), K. pelamis (24 kDa) ( Klomklao et al., 2009a), Sardina pilchardus (25 kDa) ( Bougatef et al., 2007) and Pomatomus saltatrix (29 kDa) ( Klomklao et al., 2007).