Recombinant extracellular expression of β-glucosidase in Bacillus subtilis and its potential secretion mechanism.

β-Glucosidase is an important specific β-1,4 glycosidic bond hydrolase. Here, β-glucosidase Bgl2A achieved extracellular secretory expression in Bacillus subtilis mediated by the signal peptide SP, and its extracellular activity was increased 3.35-fold by signal peptide optimization. Interestingly, the recombinant extracellular activity of Bgl2A without signal peptide mediation was 5.05-fold higher than that with SP mediation, and its stability was better at acidic conditions and low temperatures. Combining the results of classical secretory pathway knockout and cell integrity assays, Bgl2A without signal peptide can perform non-classical extracellular secretion in B. subtilis. Notably, the hydrophobic region I266-Q269 is a critical sequence affecting the non-classical secretion of Bgl2A. Mutants I267A and I267E increased the extracellular activity and secretion rate of Bgl2A by 1.42- and 1.58-fold, respectively. In addition, maintaining the hydrophobicity of the amino acids at positions 268 and 269 may be essential for the non-classical secretion of Bgl2A. To our knowledge, this study first reported that SP was not detected at the N-terminus of the intracellular Bgl2A precursor from WBLBBgl-SP mediated by the Sec-type signal peptide SP. This offers a theoretical foundation for enhancing recombinant extracellular expression of β-glucosidase in B. subtilis and understanding the mechanism of non-classical secretion in this organism.