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| β-Galactosidase catalyzes the hydrolysis of lactose and is widely used in dairy processing. The thermostable β-galactosidase obtained from Geobacillus stearothermophilus has several advantages and it has potential applications in the dairy industry. In this work, this enzyme was secreted by Bacillus subtilis using the general secretion (Sec) pathway. A signal peptide library was constructed with 20 selected signal peptide coding sequences, and it was used for probing the secretory capacity of the thermostable β-galactosidase in Bacillus subtilis 168. In the 20 recombinants constructed, expression of the target enzyme was observed in 4 plasmids carrying the signal peptide coding sequences from Bacillus subtilis AmyX, NprE, OppA and YweA, respectively. The target enzyme fused with N-terminal signal peptide from NprE achieved extracellular secretion at a level of 64.0 IU/mL, which accounts for 29.6% of the total target enyzme synthesized. This is the first report of the thermostable β-galactosidase from Geobacillus stearothermophilus by a Sec signal peptide in Bacillus subtilis. The results indicated the applicability of the signal peptide library constructed. Yet the low secretion capacity of the target enzyme also suggested that, in dealing with such kind of substrate, the Sec pathway might have no advantage over the twin-arginine translocation pathway. |
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| Keywords:Genetic Engineering; β-Galactosidase; Bacillus subtilis; Secretion; Signal peptide |
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