Cloning and Construction of Recombinant palI Gene from Klebsiella oxytoca on pET-32b into E. coli BL21 (DE3) pLysS for Production of Isomaltulose, A New Generation of Sugar

Klebsiella oxytoca produces sucrose isomerase which catalyses the conversion of sucrose to isomaltulose, a new generation of sugar. From the previous study, pall gene from Klebsiella avytoca was succesfully isolated from sapodilla fruit (Manilkara zapota). The full-length pall gene sequence of Kiebsiella oxytoca was cloned in E. coli DH5 alpha. The deduced amino acid sequence shows 498 residues which includes conserved motif for sucrose isomeiisation (RLDRD329)-R-325 and 97% identical to pall gene from Klebsiella sp. LX3 (GeliBank:AAK82938.1). This fragment was succesfullly ligated into the expression vector pET-32b using overlap-extension PCR and cloned in Escherichia coli BL21 (DE3) pLysS. DNA sequencing result shows that pall gene of Klebsiella oxytoca was inserted in-frame in pET-32b. This is the first report on cloning of pail gene from Klebsiella oxytoca.