Objective  Conditions to achieve high-efficiency secretory expression of the thermophilic α-cyclodextrin glucosyltransferase (α-CGTase) in Bacillus subtilis RIK1285 were explored to understand the role played by signal peptide.

  Method  A library based on 173 signal peptides from B. subtilis was constructed to identify the highest secretion efficiency candidate. Using the saturation mutagenesis of segments in the selected signal peptide, the secretion expression of α-CGTase was enhanced.

  Result  There were 9 signal peptides with high expression efficiency identified from the library. Among them, the signal peptide citH showed the highest secretion efficiency. Subsequently, the saturation mutagenesis of Gly2, Asn3, and Thr4 of citH produced a mutant, G2R-N3K-T4L-CGT, with an extracellular activity of α-CGTase as high as 14.2 U·mL⁻¹ and a secretion efficiency increased by 47.9% over the non-mutated signal peptide (9.6 U·mL⁻¹), which was 21.5 times higher than that of the wild-type Geobacillus caldoxylosilyticus CHB1 (0.66 U·mL⁻¹). The purified α-CGTase had a maximal activity at pH 6.0 and 60 ℃, was thermally stable within 50 ℃, and could be activated by Mg²⁺ and Ca²⁺.

  Conclusion  The important effect signal peptide had on the high-efficiency expression of cyclodextrinase in B. subtilis was verified. The result provided a new and valuable reference for studies on the expression of extraneous proteins in the bacteria.