Constitution of different signal peptides for enhanced thermostable alpha amylase secretion in Bacillus subtilis

Abstract

Three signal peptides of alpha amylase genes of three isolated strains: Bacillus licheniformis DA23, Bacillus subtilis D5-2, Bacillus cereus CN1-5 were successfully sequenced. Three predicted "Sec - type" signal peptides have a length varying from 27 (CN1-5) to 33 residues (D5-2). The secretion of alpha amylase of the recombination B. subtilis 168MPgrac strain (pHV33-PgracAmy3BT2) with 71.4 ± 6.3 U/ml and the ratio of α - amylase activities to total amount of protein secretion reached 38.05 U/mg was larger than that of l68MPamy with 53.2 U/ml. Base on analyzed results of PAGE and zymogram about molecular weight, alpha amylases in both strains were the same size, nearly 58 kDa. Three signal peptides were constructed in recombinant pHV33 - Pgrac vectors. To further evaluate the efficiency of these SPs in B.subtilis, α-amylase activity was measured. The extracellular amylase activity of signal peptide SsubtilisD5.2 in 168M was the highest with 76.4 ± 3.7 U/ml in four signal peptide targets. The results indicated that the promoter Pgrac and signal peptide amylase gen SsubtilisD5.2 tested in this study might be used for secretion α - amylase in B.subtilis 168M.