Autolysis-induced extracellular production of intracellular carboxylesterase EstGtA2 using multiple-protease-deficient Bacillus subtilis strains

EstGtA2, an intracellular carboxylesterase from Geobacillus thermodenitrificans, can catalyze the high-yield monoacylglycerol (MAG), which is commercially produced by the chemical glycerolysis of triacylglycerol and has applications in multiple industries including foods, pharmaceuticals, and cosmetics. EstGtA2 may be employed industrially for the synthesis of highly pure MAG through the development of an affordable large-scale production system. We examined the extracellular production of EstGtA2 using multiple-protease-deficient Ba-cillus subtilis strains with the expectation of its release by autolysis and suppression of degradation in the culture supernatant. The enhanced autolytic activity of the multiple-protease-deficient strain contributed to the high productivity of EstGtA2. Evaluation of EstGtA2 stability in the presence of culture supernatants of single -protease-deficient B. subtilis strains revealed that AprE plays a critical role in the degradation of EstGtA2. The productivities of culture supernatants of eight-and nine-protease-deficient strains were comparable to those of the homogenized cell-free extract of the wild-type strain. We demonstrated that both enhanced autolytic activity and suppression of degradation by the disruption of protease genes were effective in producing extracellular EstGtA2 in B. subtilis.

Automated summary

EstGtA2, an intracellular carboxylesterase from Geobacillus thermodenitrificans, can be industrially employed for the synthesis of high-purity monoacylglycerol (MAG) through the development of an affordable large-scale production system. We investigated the extracellular production of EstGtA2 using multiple-protease-deficient Bacillus subtilis strains and found that enhanced autolytic activity and suppression of degradation were effective in producing extracellular EstGtA2. Evaluation of EstGtA2 stability revealed the critical role of the proteinase AprE in its degradation. The productivity of culture supernatants of multiple-protease-deficient strains was comparable to that of the wild-type strain's cell-free extract.