Regulation of Cell Membrane Permeability Enhanced the Non-Classical Secretion of γ-Cyclodextrin Glycosyltransferase in Bacillus subtilis

Abstract

γ-Cyclodextrin glycosyltransferase (γ-CGTase, EC 2.4.1.19) is an essential enzyme required in the production of γ-cyclodextrin, which shows huge prospects in the food, medicine, materials, and chemical industries. In this study, γ-CGTase from Bacillus sp. G-825-6 STB17 was successfully cloned and expressed in Bacillus subtilis WB600. The final extracellular activity of γ-CGTase can reach 45.34 U/mL with the deletion of the signal peptide, which was about 11.3 times of the initial level of γ-CGTase secreted by the general pathway. By monitoring the whole cultivation process, secretion was divided into two stages, which were dominated by cell membrane changes and apoptosis. The measurement of lactate dehydrogenase and the results of fluorescence microscopy demonstrated that the cell membrane permeability changed significantly in the middle stage of fermentation, proving that it played a crucial role in the non-classical secretion of γ-CGTase. Furthermore, the addition of Triton X-100, a non-ionic surfactant, remarkably enhanced the secretion level of γ-CGTase by 21.5%, which was caused by the increase in cell membrane permeability. This work is the first to obtain the extracellular expression of CGTase via the non-classical secretion pathway in B. subtilis and provides a new strategy to enhance extracellular expression by regulating the cell membranes.