Tempeh is an Indonesian traditional fermented food with rich nutrition and bioactive components. Rhizopus sp, especially Rhizopus oligosporus), lactic acid bacteria (Lactobacillus sp.), and yeast are microorganisms involved in Tempeh fermentation. An interesting offer of Bioinformatics (in silico method) as a supporting tool in molecular biology studies has emerged, such as in protein cleavage. This study utilized PeptideCutter application on ExPASy Bioinformatics portal (https://web.expasy.org/peptide_cutter/) to cleave soy proteins glycinin G1, G2, G3, G4, G5, b-conglycinin-a chain, and b chain using available enzymes in the application with two simulations. Simulation I was conducted using enzyme complex produced by Lactobacillus sp. and Rhizopus oligosporus, while simulation II was used enzyme complex produced by Lactobacillus sp., Rhizopus oligosporus, and Klebsiella pneumonia. Simulation I was conducted using enzyme complex produced by Lactobacillus sp. and Rhizopus oligosporus, while simulation II was used enzyme complex produced by Lactobacillus sp., Rhizopus oligosporus, and Klebsiella pneumoniae. A total of 58 peptides was found from the simulation I and higher than simulation I (41 peptides). The bioactive peptides by the cleavages using PeptideCutter tool were dominated with dipeptides and only three peptides were in the form of tripeptides, namely Leu-Leu-Phe (glycinin G1), Val-Val-Phe (glycinin G5), and Arg-His-Lys (b-conglycinin-a chain). Bioactive peptides with antihypertensive and antidiabetic properties were mostly found in this in silico method of soybean
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