The N-terminus of 1,4-α-glucan branching enzyme plays an important role in its non-classical secretion in Bacillus subtilis

Abstract

1,4-α-Glucan branching enzyme (GBE; EC 2.4.1.18), which plays an important role in starch modification, has been mostly expressed in Escherichia coli. The application of GBE in food industry is limited by the low yield of enzyme and production of endotoxins in E. coli. In this study, we first found that GBE from Geobacillus thermoglucosidans STB02 (Gt-GBE) could be highly expressed in the Bacillus subtilis which is a food grade strain. The pathway of secretion was non-classical independent of signal peptides and was related to cell lysis. Bioinformatics analysis combined with site-directed mutagenesis was used to explore the influence of the N-terminal structure on its secretion. We found that the extracellular activity of Gt-GBE was increased about 17.29% and the secretion rate was also greatly improved through truncating the loop structure at the N-terminus. Besides, it was found that there was an optimal hydrophobicity, which increased the extracellular activity of Gt-GBE by 12.90% through the substitution of N-terminal amino acids with hydrophobic residues. In summary, we achieved high-efficiency non-classical secretion of Gt-GBE in B. subtilis and found an important role of the N-terminus in this secretion pathway, which provides a new perspective for improving the expression of proteins in B. subtilis.