Cloning of the cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17

Abstract

The cyclomaltodextrinase gene fromBacillus subtilis high-temperature growth transformant H-17 was cloned on separatePstI,BamHI, andEcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host,Escherichia coli DH5α. High level constitutive expression of the gene product was also detrimental to theE. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kbEcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the hostB. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in theB. subtilis host; however, expression was at a low level. Subcloning of the 3-kbEcoRI fragment into pUC18 and transformation intoE. coli XL1-Blue (F′lacI^q) indicated that the cyclomaltodextrinase gene was cloned with its own promoter, since expression of the gene occurred in the absence of IPTG. Subcloning of the cyclomaltodextrinase gene downstream from theBacillus temperate phage SPO2 promoter of pPL708 may increase expression of this gene.