Thermitase (EC 3.4.21.66) is a thermostable endo-protease with the ability to convert various food relevant substrates into low-molecular weight peptides. A thermitase produced by Laceyella sacchari strain DSM43353 was found to have a mature amino acid sequence nearly identical to that of the original thermitase isolated from Thermoactinomyces vulgaris. The DSM43353 thermitase gene sequence contains a pro-peptide including parts of an 19 inhibitor motif. Expression of the thermitase gene in the Lactococcus lactis P170 expression system allowed secretion of stable thermitase in an auto-induced fermentation setup at 30 degrees C. Thermitase accumulated in the culture supernatant during batch fermentations and was easily activated at 50 degrees C or by prolonged dialysis. The activation step resulted in an almost complete degradation of endogenous L lactis host proteins present in the supernatant. Mature activated product was stable at 50 degrees C and functional at values between pH 6 and pH 11, suggesting that substrate hydrolysis can be performed over a broad range of pH values. The L lactis based P170 expression system is a simple and safe system for obtaining food compatible thermitase in the range of 100 mg/L. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved.