PARTITION BEHAVIOR AND PURIFICATION OF A MUCOR-BACILLIFORMIS ACID PROTEASE IN AQUEOUS 2-PHASE SYSTEMS

The partitioning of a Mucor bacilliformis acid protease, a potential substitute for bovine chymosin in cheese manufacture, was accomplished in various aqueous two-phase systems in order to investigate how changes in factors such as PEG (poly(ethylene glycol)) molecular weight, pH and sodium chloride concentration, can modify the partition coefficient value. PEG-Reppal and PEG-phosphate systems were evaluated, in the presence of contaminating material from the solid substrate fermentation of the microorganism. When PEG-phosphate systems were analysed it was found that K-AP depended strongly on the PEG molecular weight (at pH 5.0, an increase in PEG molecular weight from 600 to 20000 leads to a decrease in the K value from > 50 to 0.1). A dependence between K-AP and system pH was also noticed, this effect being important at lower/intermediate PEG molecular weight. When PEG 1540 was used a V-shaped distribution of K-AP, values was obtained, with a minimum at pH 5.0 (K = 1.40) and maxima at pH values of 3.0 (K> 40) and 5.8 (K = 14). Furthermore, the addition of NaCl led to an increase in K-AP (for PEG 3350/phosphate at pH 5.0, K increased from 1.1 to > 35 when 1.0 mol kg(-1) NaCl was added). Suitable conditions for enzyme purification were found in PEG 3350-phosphate systems at pH 3.0 and NaCl 1.0 mol kg(-1) (K-AP > 35, K-CP = 0.10) and PEG-Reppal at pH 3.0, NaCl 1.5 mol kg(-1) (K-AP = 13, K-CP = 0.32). In these systems, proteinaceous and particulate contaminating materials precipitated and adsorbed at the interphase, thus yielding a clear upper phase containing the purified enzyme. Furthermore, direct extraction of the fermented mass was peformed using a PEG 20 000-Reppal-NaCl system (K-AP = 14, K-CP = 0.19, PF (purification factor) = 5.9). The enzyme can be recovered in the PEG 20 000-rich phase and back-extracted by adding salt (K-AP = 0.25, K-CP = 1.10, PF = 1.7). This method provides a simpler process for leaching and purification of an enzyme produced by solid-state fermentation.