Enhanced extracellular expression of α-Amylase DL3-4-1 in Bacillus subtilis via systematic screening of optimal signal peptides

Amylases have been widely applied for starch saccharification in the food, paper, pharmaceutical, and textile industries. In the present study, α-amylase DL-3-4−1 from B. amyloliquefaciens was successfully expressed in B. subtilis. The enzymatic activity exhibited high stability in a pH range from 4.0 to 11.0 and moderate thermostability. Furthermore, excellent stability was observed when incubated with metal ions, detergents, inhibitors, and organic solvents. Resistance against ethanol, urea, and guanidine−HCl was also observed. More importantly, a strategy to enhance the extracellular expression of α-amylase DL-3-4−1 was developed using 173 Sec-type signal peptides (SPs) as a library based on a fast and sequence-independent method. Fourteen SPs were identified with significantly increased yields of DL-3-4−1 via a high-throughput assay. Specifically, the enzymatic activity of the α-amylase-producing strain containing the signal peptide SP5D10 (YomL) was 3.74-fold higher than that of the strain with the wild type signal peptide (87.30 U/OD). In brief, the maximum specific activity of an α-amylase-producing strain containing the YomL signal peptide was 326.45 U/OD. This is the first report of an α-amylase that exhibits ethanol, urea, and guanidine−HCl tolerance. Hence, this α-amylase represents a promising candidate for future use in industry.