Recombinant expression of Laceyella sacchari thermitase in Lactococcus lactis

•We identified and cloned the full length thermitase gene from Laceyella sacchari.•Thermitase was expressed in Lactococcus lactis using the P170 Expression System.•In batch fermentations the yield of thermitase was 100 mg/L in the culture supernatant.•Activated and pure thermitase was obtained by a simple heat treatment.•The protease is stable at 50 °C and functional at pH values between pH 6 and pH 11.

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 I9 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 °C. Thermitase accumulated in the culture supernatant during batch fermentations and was easily activated at 50 °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 °C and functional at pH 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.