Secreted Expression of a Hyperthermophilic α-Amylase Gene from Thermococcus sp HJ21 in Bacillus subtilis

The hyperthermophilic alpha-amylase from Thermococcus sp. HJ21 possesses unique traits (Ca2+-independent thermostability and optimal temperature of 95 degrees C) that make it a great potential candidate for use in the food industry. However, this Archaea isolated from a deep-sea thermal vent requires strict control of culture conditions and produces only small amounts of alpha-amylase. To solve these problems, the alpha-amylase gene was cloned and expressed in Bacillus subtilis, which is an ideal food-grade host for heterologous protein expression. To express high levels of this alpha-amylase in B. subtilis, the promoters P-grac, P-xylA, P43, and P-hag were used to construct four different expression vectors for testing. The vector containing the P-xylA promoter was found to have the highest transcriptional activity and produce the highest amylase activity (19.6 U/ml). To test the secretion efficiency of signal peptides in B. subtilis, three signal peptides were cloned and fused to the alpha-amylase gene (lacking its native signal peptide). The optimal signal peptide was S-amyQ, with a secretion efficiency of approximately 90%. These results indicate that the promoter P-xylA and signal peptide S-amyQ tested in this study may be useful for the expression and secretion of archaeal proteins in B. subtilis. Copyright (C) 2013 S. Karger AG, Basel