Production and characterization of a milk-clotting enzyme from Aspergillus oryzae MTCC 5341

Microbial milk-clotting enzymes are valued as
calf rennet substitutes in the cheese industry. Aspergillus
oryzae MTCC 5341 was identified to produce the highest
milk-clotting activity during screening of 16 fungal strains.
Solid state fermentation using wheat bran along with 4%
defatted soy flour and 2% skim milk powder as substrate
was optimal for growth of A. oryzae and production of the
enzyme. Nearly 40,000 U/g bran of milk-clotting activity
was present at the end of 120 h. The enzyme could be
recovered by percolating the bran with 0.1 M sodium
chloride for 60 min at 4°C. The decolorized enzyme
preparation had high ratio of milk clotting to proteolytic
activity. Affinity precipitation with alginate and subsequent
elution with 0.5 M sodium chloride containing 0.2 M CaCl2
resulted in an enzyme preparation with specific activity of
3,500 U/mg and 72% yield. Optimum pH and temperature
for activity of the enzyme were characterized as 6.3 and
55°C, respectively. Milk-clotting enzyme showed differential
degree of hydrolysis on casein components. High ratio
of milk clotting to proteolytic activity coupled with low
thermal stability strengthens the potential usefulness of
milk-clotting enzyme of A. oryzae MTCC 5341 as a
substitute for calf rennet in cheese manufacturing.