Secretory Production of Mature Protein-Glutaminase from Bacteroides helcogenes in Bacillus subtilis

Abstract

The recombinant production of the protein-glutaminase (PG) from Bacteroides helcogenes (PGB) was investigated in the three Bacillus subtilis strains: 168, RIK1285 and the isolated strain 007. B. subtilis 007 produced the highest PG activity (7.6 ± 0.7 µkat LCulture supernatant−1) in shake flask cultivations and, thus, was used for further investigations. As a wild-type strain, B. subtilis 007 exhibited high extracellular proteolytic activity and formed large amounts of foam in a bioreactor cultivation. While the proteolytic activity would be favorable for the extracellular cleavage of PGB’s propeptide, foaming is undesirable and should be prevented. Therefore, the influence of the two foam-demolishing substances anti-foaming agent 204 and rapeseed oil on the PGB production was investigated in bioreactor cultivations. A PGB activity of 11.8 ± 1.1 µkat LCulture supernatant−1 was obtained using anti-foaming agent 204, while rapeseed oil led to a lower maximal PGB activity of 7.6 ± 0.2 µkat LCulture supernatant−1. PGB was partially purified from the culture supernatant by fractionated ammonium sulphate precipitation followed by hydrophobic interaction chromatography. A final yield of 14.8% was obtained, while the specific PGB activity increased from 6.7 to 28.6 nkat mg−1. Alternatively, PGB was purified from culture supernatant by cross-flow filtration and subsequent heating step (1 h, 60 °C) to inactivate the extracellular peptidases. Thereby, the total proteolytic activity was decreased to below 4% while the specific PGB activity increased to 32.1 ± 2.0 nkat mg−1. The PGB preparation obtained was applied in gluten deamidation experiments which showed that PGB deamidated a 1% (w/v) gluten suspension by 95 ± 2%.