A method for the quantitative analysis of the enzyme complement of commercial rennets

Abstract

A method by which it is possible to quantify with very high accuracy the levels of rennin, bovine pepsin, porcine pepsin and proteases from Mucor miehei (fromase) or M. pusillus (emporase) in a ‘commercial rennet’ containing some or all of these enzymes is described. The method employs chromatography on DEAE cellulose in piperazine buffer (0·02M, pH 5·3) to fractionate the enzymes into 2 groups: (peak A) rennin, emporase, and fromase, eluted at ∼0·2 m-NaCl and (peak B) bovine and porcine pepsins eluted at ∼0·4 m-NaCl. The rennin in peak A is selectively denatured at pH 9·0, 20°C for 60 min and the rennin plus emporase at pH 6·0, 60°C for 20 min, permitting the quantification of emporase and fromase (and rennin by difference) and fromase, respectively. The porcine pepsin content of peak B is quantified by selective denaturation at pH 7·0, 30°C for 3 min. A number of commercial rennet preparations were analysed using this method: full rennet contained 45–60% bovine pepsin and ‘half and half’ contained 20–25% rennin, 25–30% bovine pepsin and 50% porcine pepsin.