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 s ubstitute for bovine chymosin in cheese manufacture, was accomplished in various aqueous two-phase systems in order to investigate how chang es in factors such as PEG (poly(ethylene glycol)) molecular weight, pH and sodium chloride concentration, can modify the partition coefficie nt value. PEG-Reppal and PEG-phosphate systems were evaluated, in the presence of contaminating material from the solid substrate fermentati on of the microorganism. When PEG-phosphate systems were analysed it w as found that K-AP depended strongly on the PEG molecular weight (at p H 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 d istribution 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). Furth ermore, 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 foun d in PEG 3350-phosphate systems at pH 3.0 and NaCl 1.0 mol kg(-1) (K-A P > 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. Furt hermore, direct extraction of the fermented mass was peformed using a PEG 20 000-Reppal-NaCl system (K-AP = 14, K-CP = 0.19, PF (purificatio n factor) = 5.9). The enzyme can be recovered in the PEG 20 000-rich p hase 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 purific ation of an enzyme produced by solid-state fermentation.