Alpha-galactosidase taken from a raw extract of Sesbania marginata legume seeds was purified by partitioning in aqueous two-phase systems (ATPS). Initially, galactomannan/dextran 2,000,000 systems were used for the purification, and the partition coefficients of alpha -galactosidase varied from 1.5 to 4.0. However, mass transport in these systems was poor due to the high viscosity of the employed polymers. Therefore, partitioning in polyethyleneglycol (PEG)/ sodium phosphate systems and the effect of sodium chloride upon the enzyme purification and the yield of alpha -galactosidase were also investigated. The purification achieved in a single-step was 5.7 with a recovery of 144% of alpha -galactosidase, possibly due to the removal of materials which inhibited alpha -galactosidase activity before the purification. The removal of the main protein contaminants and the highest yields were achieved in PEG 4,000/ sodium phosphate + 6% NaCl system at pH 5.0. Further purification by preparative on-exchange chromatography was also developed.

A culture medium for a wild strain of Bacillus cereus was developed for chitosanase production by using an experimental design. The factors having the strongest influence on chitosanase production were ammonium sulfate concentration, aeration, pH and the interaction between the first two parameters. Optimal conditions for chitosan hydrolysis were pH 5.8 and 54 oC; however, hydrolysis activity drastically decreased at pH 7.0. The enzyme was purified (single-electrophoretic band) by partitioning in an aqueous two-phase system (ATPS), followed by cation-exchange chromatography with a 66% yield. Chitosanase was mainly collected in the top phase (K = 129) of a 22% PEG 1,500, 13% phosphate (pH = 5.8) and 12% NaCl (w/w) solution, and the main protein contaminants were evenly distributed between the phases (K = 1.07). The apparent molecular weight and the isoelectric point of the chitosanase, determined by SDS-PAGE electrophoresis and by isoelectric focalization, were 47 kDa and 8.8, respectively.