Novel Promoter PQ Mediates Expression Of Thermostable α-amylase

Thermosable alpha-amylase (BLA) is one of the most important enzymes, widely applied in sugar and fermentation industry. This study has investigated a novel promoter PQ, by which mediated BLA is overexpressed in Escherchia coli, Bacillus subtilis and Bacillus licheniformis.A DNA fragment containingα-amylase structure gene, amyL with its own signal peptide sequences was obtained by PCR from genomic DNA of B. licheniformis B0204. The recombinant plasmid, pLa-amyL, was constructed by directly inserting the amplified fragment into the cloning vector pLakr. To identify the function of amyL, the recombinant plasmid pLa-Gm-PQ-amyL was constructed by inserting promoter PQ into pLa-amyL and expressed in E. coli. Theα-amylase activity was detected to 16.1 U/mL. The results show that the amyL gene could be expressed in E. coli using promoter PQ with its own signal peptide andα-amylase expressed could secrete into the culture medium.Next, the function and strength of promoter PQ were comparatively investigated in B. subtilis. The two recombinant plasmids harboring amyL, pUB-PQ-amyL and pUB-P43-amyL were constructed using promoters PQ and P43 and transformed into B. subtilis 1A717 (ΔamyA). Different transformants and control were spotted on plate containing starch and the forming areas of the starch-hydrolyzation were different obviously. By shake-flask fermentation, the specific activities of BLA by transformants of B. subtilis 1A717(pUB-PQ-amyL) and B. subtilis 1A717(pUB-P43-amyL) were 280 U/mL and 191 U/ mL, respectively. The results indicate that synthetic promoter PQ mediates 1.47 folds production of BLA comparison to that of the promoter P43 deriving from B. subtilis when using amyL as a report gene.The function and strength of promoter PQ were further determined in B. licheniformis. The two recombinant plasmids pUB-PQ-amyL and pUB-P43-amyL were transformed into B. licheniformis CBB D302. By shake-flask fermentation, B. licheniformis CBB D302 (pUB-PQ-amyL) produced 61% more BLA than that of strain B. licheniformis CBB D302; B. licheniformis CBB D302(pUB-P43-amyL) produced 21% more BLA. The results show that promoter PQ produces more BLA comparison to that of the promoter P43 in B. licheniformis.