Optimization of Protease Enzyme from Kesinai (Streblus asper) for Milk Coagulation

Abstract

Streblus asper (Kesinai) leaves contain proteases that can be used to coagulate milk. The enzyme was extracted by using phosphate buffer at pH 7.2 and the extracted enzyme activity was tested for protease assay. Response Surface Methodology (RSM) using Two-Level Factorial and Central Composite Design (CCD) was used in screening and optimization of serine protease extraction from kesinai (Streblus asper) leaves respectively. The effect of independent variables, namely ratio of sample to buffer (1-5), weight of sample (10-50 gram) and mixing time (2-6 minutes) on proteases activity from kesinai leaves was investigated. The screening results showed that ratio sample to weight had the most significant effect (p<0.0001) on protease activity for enzyme extraction. The study showed that the control of ratio of sample to buffer, weight of sample and mixing time able to obtain the highest extract of enzyme. 50 g of sample, 1:1 ratio of sample to buffer at 2 min mixing time was established for serine proteases extraction from kesinai leaves. Studies continued with encompassing variation of milk clotting time and protein concentration by using the optimized enzyme extract. Extract of kesinai leaves had protease activity that degrades proteins by hydrolysis of peptide bonds. The effects of coagulation temperature and enzyme concentration on milk coagulating activities (MCA) and protein concentration were evaluated. MCA and protein concentration were optimized at temperature 90 °C and enzyme concentration 290 μL.