Li He, Wang Wenjing, Que Longwei, Xiao Yuyang, Fang Zemin, Fang Wei, Xiao Yazhong, Yao Dongbang
School of Life Sciences, Anhui University, Hefei 230601, China; Anhui Key Laboratory of Biocatalysis and Modern Biomanufacturing, Hefei 230601, China; Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, China; School of Environmental Engineering and Chemistry, Luoyang Institute of Science and Technology, Henan Province, Luoyang 471023, China.
School of Life Sciences, Anhui University, Hefei 230601, China; Anhui Key Laboratory of Biocatalysis and Modern Biomanufacturing, Hefei 230601, China; Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, China.
Int J Biol Macromol. 2025 May;308(Pt 2):142251. doi: 10.1016/j.ijbiomac.2025.142251. Epub 2025 Mar 25.
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.
β-葡萄糖苷酶是一种重要的特异性β-1,4糖苷键水解酶。在此，β-葡萄糖苷酶Bgl2A在信号肽SP介导下于枯草芽孢杆菌中实现了细胞外分泌表达，通过信号肽优化其细胞外活性提高了3.35倍。有趣的是，无信号肽介导的Bgl2A重组细胞外活性比有SP介导的高5.05倍，且在酸性条件和低温下其稳定性更好。结合经典分泌途径敲除和细胞完整性检测结果，无信号肽的Bgl2A可在枯草芽孢杆菌中进行非经典的细胞外分泌。值得注意的是，疏水区域I266-Q269是影响Bgl2A非经典分泌的关键序列。突变体I267A和I267E分别使Bgl2A的细胞外活性和分泌率提高了1.42倍和1.58倍。此外，维持268和269位氨基酸的疏水性可能对Bgl2A的非经典分泌至关重要。据我们所知，本研究首次报道在由Sec型信号肽SP介导的WBLBBgl-SP的细胞内Bgl2A前体的N端未检测到SP。这为提高β-葡萄糖苷酶在枯草芽孢杆菌中的重组细胞外表达及理解该生物体中非经典分泌机制提供了理论基础。
