Partition behaviour and purification of a Mucor bacilliformis acid protease in aqueous two-phase systems

Abstract

The partitioning of a Mucor bacilliformis acids 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 20 000 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 was performed 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.