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.