Date of Award:


Document Type:


Degree Name:

Doctor of Philosophy (PhD)


Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Sciences


Rodney J. Brown


Commercial microbial milk clotting enzyme preparations were purified by immunoaffinity chromatography using purified antibody covalently coupled to porous glass beads as the column matrix. Commercial enzyme preparation diluted in 1 mM sodium acetate buffer at pH 5.0 was then biospecifically adsorbed to the column matrix by end-over-end mixing of the glass-antibody complex in the enzyme solution for 12 h at 5°C. The antibody bound enzyme adsorbed glass beads were soaked in .2 M glycine or ethanolarnine at pH 7.0 to block uncoupled reactive sites on the matrix. Following this, the column was washed with 1 mM sodium acetate buffer at pH 7 .0, followed by additional wash with .5 M NaCl, until absorbance at 280 nm returned to baseline. Elution of adsorbed enzyme was achieved with .2 M sodium acetate at pH 3.0, .2 M acetate at pH 3.5, containing .15 M NaCl and .5 M acetate at pH 4.0 containing .5 M NaCL At the same protein concentration, immunoaffinity chromatography purified enzymes had higher clotting activity than the commercial enzyme preparations. Amino acid analysis and OPA proteolysis tests of TCA soluble peptides liberated from casein hydrolysis showed purified enzymes to exhibit lower general proteolytic activity.

Immunological reactivity of Mucor enzymes with calf rennet was determined with antibodies produced by intramuscular injections of Mucor miehei protease, Mucor pusillus protease and calf rennet emulsified in Freund's adjuvant into three New Zealand White rabbits . Harvested antisera were heated at 56°C for 30 min to inactivate complement factors and contaminating proteins then centrifuged at 1700 x g for 30 min. Ouchterlony double immunodiffusion method was used to test for presence of antibodies in the antisera, and for cross immunoreactivity. Antibodies against M. miehei were cross reactive with M. pusillus antigen and M. pusillus antibodies cross reacted with M. miehei antigen. Immunodiffusion assay did not show cross reactivity of calf rennet antibodies with either M. miehei antigen or M. pusillus antigen. Antibodies against the Mucor enzymes did not show cross reactivity with calf rennet antigen.

Although their actions in milk differ, proteolytic enzyme preparations from M. miehei and M. pusillus are both used as calf rennet substitutes in cheese manufacture. Differences in the characteristics of the two Mucor enzyme preparations exist, even though they exhibit some immunological homology . From our results, at least one antigenic factor is common to both enzyme preparations.