Optimization and Partial Characterization of Trypsin-Like Protease Enzyme (TLP) from Indonesian Indigenous Lactiplantibacillus plantarum M4 for Digestive Enzyme.

Trypsin is a digestive enzyme crucial for protein breakdown and the activation of other digestive enzymes. Several studies have shown that trypsin deficiency often leads to various health problems, indicating the need to produce its supplements. These supplements can be obtained from various sources, including different strains of Lactiplantibacillus plantarum, which produces a protease similar to trypsin, called trypsin-like protease (TLP). Therefore, this study aims to enhance TLP production by selecting the optimal medium and fermentation conditions to maximize enzyme activity using Response Surface Methodology (RSM) as an analytical tool, followed by partial enzyme characterization. Lactiplantibacillus plantarum M4 used in this study was obtained from fermented Menteng fruit from Banjarmasin, Indonesia. The optimization results using central composite design in RSM showed that the ideal production medium composition was 1.96% glucose, 0.39% yeast extract, 1.97% skim milk, and pH 6.62, with TLP activity of 0.281 ± 0.012 mU/mL. Ultrafiltration 5000 MWCO was used to concentrate the crude enzyme under ideal conditions, leading to an increase in purity of 11,075 times and an activity of 2,467 mU/mL. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of TLP indicated molecular weights of 29 and 19 kDa as well as stability between 30 and 40 °C, with pH 7-8 indicating optimum activity. Protease activity similar to trypsin was affected by the addition of metal ions, such as Zn²⁺, Ca²⁺, and Ethylenediaminetetraacetic Acid (EDTA). Based on these results, TLP production can be increased by adjusting fermentation conditions to support people with deficiency and preserve health.