Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis.
The gene for an enzyme with single or dual specificity on complex carbohydrates has been transferred from its native host (Thermoanaerobium brockii), a thermophilic anaerobe, into Escherichia coli and Bacillus subtilis. Most of the gene coding region is in a 2.2-kilobase PstI fragment that is common to the E. coli and B. subtilis chimeric vectors pCPC902 and pCPC903, respectively. Although the T. brockii debranching enzyme secreted from B. subtilis was unglycosylated and had less thermostability, more enzyme was secreted from B. subtilis (0.80 to 1.0 U/ml) than from T. brockii (0.23 U/ml). E. coli did not export any measurable enzyme. From the fermentation broth of B. subtilis containing pCPC903, three active species of the debranching enzyme were separated; two species are possibly protease digestion products of the larger protein (105,000 molecular weight). Whereas the enzyme can cleave all of the alpha-1----6 glucosidic linkages (and none of the alpha-1----4 bonds) in pullulan, it hydrolyzed mostly alpha-1----4 and very few of the alpha-1----6 linkages in starch. Upon hydrolysis of pullulan by the enzyme, only maltotriose was produced, while starch was digested to various-sized oligomers.
D001412:Bacillus subtilis / Q000235:genetics; D001421:Bacteria, Anaerobic / Q000201:enzymology / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes; D006026:Glycoside Hydrolases; C100181:pullulanase