Purification and Characterization of Two Milk-clotting Enzymes from Irpex lacteus

Abstract

Two carboxyl proteinases with high milk clotting activity from Irpex lacteus designated as enzymes A and B were isolated by affinity chromatography with dehydroacetyl-pepstatin as ligand, DEAE-cellulose chromatography and isoelectric focusing. The final preparations were judged homogeneous by polyacrylamide gel electrophoresis. The molecular weights of enzymes A and B were determined by gel filtration and SDS-polyacrylamide gel electrophoresis to be 36,000. The isoelectric points of A and B were 4.9 and 5.3, respectively.

A and B had similar amino acid compositions lacking sulfur-containing amino acids, such as cystine, cysteine and methionine. And both of the enzymes were inhibited by carboxyl proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucine methyl ester (DAN) and 1,2-epoxy-(p-nitrophenoxy) propane (EPNP). These results indicate that enzymes A and B are similar to calf chymosin and other microbial rennets in their active site structure having two different carboxyl groups, although they showed minor differences in regard to enzymatic and molecular properties. Enzymes A and B exhibited almost the same ratio of milk-clotting activity to proteolytic activity as commercial microbial rennets obtained from Mucor pusillus and Mucor miehei.