Coupling of the Penicillium duponti acid protease to ethylene-maleic acid (1 : 1) linear copolymer. Preparation and properties of the water-soluble derivative.

The coupling of the thermostable acid protease (EC 3.4.23.-) of Penicillium duponti K 1014 to ethylene-maleic acid (1 : 1) linear copolymer in the presence of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide at pH 3.0, afforded a soluble enzyme derivative with a protein incorporation yield of 67% under optimal conditions. The protein content of the enzyme-polymer complex, the molecular weights of the reactants, and the mean value of 2.2 lysine residues per mol of enzyme found in amide linkage to the matrix, support a structure consisting of two polymer chains per mol of protease, each chain acylating a single lysine residue of the enzyme. The isoelectric point of the coupled enzyme was found to be 3,47, a value lower than that measured on the free protease (3.81). The specific activity of the bound protease against casein, at pH 3.7 and 30 degrees C, was 34% of that of the free enzyme, and at 75 degrees C increased to 70%. The increased size of the coupled enzyme resulted in an improved retention of activity by ultrafiltration membranes over that observed with free protease, alone or in admixture with ethylene-maleic acid copolymer. A water-soluble, coupled pepsin was prepared in 43% yield on protein basis by using the aminoethylmonoamide of ethylene-maleic acid copolymer and the same water-soluble carbodiimide.