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

Vishwanatha, Kurutahalli S., Appu Rao, A. G., Singh, Sridevi Annapurna
Applied microbiology and biotechnology 2010 v.85 no.6 pp. 1849-1859
Aspergillus oryzae, calcium chloride, calves, casein, cheese industry, cheesemaking, decolorization, dried skim milk, enzyme activity, enzymes, hydrolysis, milk clotting, pH, proteolysis, rennet, rennet substitutes, screening, sodium chloride, solid state fermentation, soy flour, temperature, thermal stability, wheat bran
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.