Cloning of the cyclomaltodextrinase gene from Bacillus subtilis high-temperature growth transformant H-17.
The cyclomaltodextrinase gene from Bacillus subtilis high-temperature growth transformant H-17 was cloned on separate PstI, BamHI, and EcoRI fragments into the plasmid vector pUC18, but was expressed in an inactive form in the host, Escherichia coli DH5 alpha. High level constitutive expression of the gene product was also detrimental to the E. coli host, which led to structural instability of the recombinant plasmid. The cyclomaltodextrinase gene was cloned on a 3-kb EcoRI fragment into the plasmid vector pPL708, and the fragment was structurally maintained in the host B. subtilis YB886. The cloned gene product was synthesized in an enzymatically active form in the B. subtilis host; however, expression was at a low level. Subcloning of the 3-kb EcoRI fragment into pUC18 and transformation into E. coli XL1-Blue (F' lacIq) 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 the Bacillus temperature phage SPO2 promoter of pPL708 may increase expression of this gene.
Krohn
B M
BM
Food Science and Human Nutrition Department, University of Florida, Gainesville 32611.
Lindsay
J A
JA
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D015151:Immunoblotting; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D014170:Transformation, Genetic
D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase