Two-step purification and partial characterization of keratinolytic proteases from feather meal bioconversion by Bacillus sp. P45

This study aimed to purify and partially characterize a keratinolytic protease produced by Bacillus sp. P45 through bioconversion of feather meal. Crude protease extract was purified using a sequence of an aqueous two-phase system (ATPS) in large volume systems (10, 50, and 500 g) to increase obtaining purified enzyme, followed by a diafiltration (DF) step. Purified protease was characterized in terms of protein profile analysis by SDS-PAGE, optimum temperature and pH, thermal deactivation kinetics at different temperatures and pH, and performance in the presence of several salts (NaCl, CaCl2, MnCl2, CaO, C8H5KO4, MgSO4, CuSO4, ZnSO4, and FeCl3) and organic solvents (acetone, ethanol, methanol, acetic acid, diethyl ether, and formaldehyde). ATPS with high capacities resulted in purer protease extract without compromising purity and yields, reaching a purification factor up to 2.6-fold and 6.7-fold in first and second ATPS, respectively, and 4.0-fold in the DF process. Recoveries were up to 79% in both ATPS and reached 84.3% after the DF step. The electrophoretic analysis demonstrated a 25–28 kDa band related to keratinolytic protease. The purified protease’s optimum temperature and pH were 55 ◦C and 7.5, respectively. The deactivation energy (Ed) value was 118.0 kJ/mol, while D (decimal reduction time) and z (temperature interval required to reduce the D value in one log cycle) values ranged from 6.7 to 237.3 min and from 13.6 to 18.8 ◦C, respectively. Salts such as CaCl2, CaO, C8H5KO4, and MgSO4 increased the protease activity, while all organic solvents caused its decrease. The results are useful for future studies about ATPS scale-up for enzyme purification and protease application in different industrial processes

Influence of heating, protein and glycerol concentrations of film-forming solution on the film properties of Argentine anchovy (Engraulis anchoita) protein isolate

This study was conducted to evaluate the influence of thermal treatment (74, 82, and 90 _C), glycerol (30%, 35%, and 40%, w/w) and protein concentrations (3.0%, 3.5%, and 4.0% w/w) of film-forming solution on the properties of Argentine anchovy (Engraulis anchoita) protein isolate (API) films produced by casting. The API presented 88.8% of proteins, 5.5% moisture, 1.3% lipids, 1.0% ash and 53.3% of polar amino acids. The DSC of protein isolate was observed at maximum temperature of 62.2 _C and DH 6.4 J/g. The thickness, water vapor permeability, color difference and opacity of the films were not affected by the experimental variables studied (p > 0.05). The lowest solubility, elongation, and highest tensile strength of the films occurred at low temperature, low protein and glycerol concentrations (p < 0.05). Micrographs obtained by scanning electron microscopy of the films showed homogeneous surfaces at low temperature