| Summary: | Extracts from ‘kesinai’ (Streblus asper) leaves were investigated as a potential source of enzymes
that can serve as an alternative to calf rennet in cheese making. Different types of extraction
buffers were investigated namely sodium acetate buffer (pH 4.2-5.0), phosphate buffer (pH
6.0-7.0) and Tris-HCl buffer (pH 7.0-9.0). Finally, the milk-clotting enzyme was extracted
using 100 mM Tris-HCl buffer (pH 7.4) with and without 5.0 mg/mL polyvinylpyrrolidone,
0.015 mL/mL Triton X-100 and 2 mM sodium metabisulphite. Purification was carried out
using acetone precipitation, and ion-exchange and size-exclusion chromatographic techniques.
Results showed that 100 mM Tris-HCl buffer (pH 7.4) was the most efficient extraction buffer
among the buffers used in the extraction study. After the final purification step of size-exclusion
chromatography, the enzyme was purified 3.3-fold with 42.3% of recovery. The enzyme
showed an optimum temperature and pH at 60°C and pH 7.4, respectively. The enzyme was
stable up to 70°C for one hour and the partially purified enzyme retained 83% and 96% of its
original activity at pH 6.0 and 8.0, respectively. The molecular weight of the partially enzyme
was estimated to be 75.8 kDa on SDS-PAGE. The milk-clotting activity of ‘kesinai’ enzyme
was found to be lower than that of commercial Mucor rennet.
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