Cloning and Expression of Thermostable β-Amylase Gene of Thermoanaerobacterium thermosulfurogenes in Escherichia coli and Bacillus subtilis BR151

Abstract

DNA fragment encoding thermostable beta-amylase gene from Thermoanaerobacterium thermosulfurogenes was amplified by PCR and then cloned into pBluescript II KS/SK, pBT10, pNW33N and pUB110 plasmids. Recombinant plasmids were designated as pBluescript beta, pBT10 beta, pNW33N beta and pUB110 beta respectively. pBluescript beta, pBT10 beta and pNW33N beta recombinant plasmids were transferred into Escherichia coli: and pUB110 beta was electrotransformed into Bacillus subtilis BR151. Insert and PCR analysis of recombinant plasmids from E. coli and B. subtilis confirmed the 1935 bp beta-amylase gene fragment on agarose gel electrophoresis. On LB-starch-agar plates, all recombinant E. coli colonies showed positive zones with 12 staining. Although thermostable beta-amylase gene was cloned in B. subtilis BR151. the enzyme activity was not detected on LB-starch-agar plate. But after renaturation of extracellular proteins from B. subtilis on SDS-Starch-PAGE, beta-amylase enzyme regained enzymatic activity by zymogram technique and thereby confirmed that enzyme was not folding properly in B. subtilis host.