Separation and partial purification of collagenolytic protease from peacock bass (Cichla ocellaris) using different protocol: Precipitation and partitioning approaches

Abstract This comparative study provides a useful protocol for the separation and partial purification of collagenolytic proteases obtained from peacock bass, by using precipitation and partitioning. In the precipitation process, 30% acetone (PF = 1.76; Y = 198%) and ethanol (PF = 2.56; Y = 272%) were used, while 30–60% fraction of (NH4)2SO4 was the most efficient in the separation process (Y = 201%; PF: 4.28).For the use of the three-phase partitioning (TPP), the sample was submitted to different proportions of t-butanol (1.0:0.5, 1.0:1.0, 1.0:1.5 and 1.0:2.0; v/v) and fixed proportion of (NH4)2SO4 (30%). The assay with the best Y (232%) of the collagenolytic protease in the interphase was in the ratio of 1.0:0.5, which also resulted in higher PF (5.07) of this same phase. The formation of the two-phase aqueous partitioning (ATPS) was obtained by mixing polyethylene glycol(1500, 4000 and 8000), according to a 24-factorial design. The highest values of PF (8.24) and Y (116%) were obtained at pH 8.0, 12.5% (w/w) PEG 8000, and 10.0% (w/w) phosphate. PEG-collagenolytic was physicochemically characterized and exhibited optimal state at a temperature of 55 °C and pH 7.5, which was activated by Ca2+ and inhibited by Zn2+; this activity was reduced when exposed to PMSF and TLCK. The PEG-collagenolytic showed three bands of protein, with molecular weights ranging from 10.0 to 60.3 kDa. The protease hydrolyzed native and fish skin collagen. Therefore, separation of the target molecule by precipitation and partitioningmay be a viable alternative. Furthermore, ATPS presented physicochemical characteristics compatible with the industrial interest.