Characterisation of a thermoalkali-stable cyclodextrin glycosyltransferase from the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii.
The thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii produces an extracellular CGTase when grown on starch at 55 degrees C and pH 9.0. The gene encoding this CGTase was cloned and successfully expressed in Escherichia coli. It encodes a protein consisting of 721 amino acids with a signal sequence of 34 amino acids. On SDS-polyacrylamide gels, the purified CGTase from A. gottschalkii displayed the expected molecular mass of 78 kDa. The recombinant enzyme was purified with a yield of 13.5% and displayed a specific activity of 210 units/mg. This CGTase, which represents the first report of a CGTase from an anaerobic thermoalkaliphile, was active at a broad range of temperature and pH, namely 55-70 degrees C and pH 5-10. It completely converted amylose, amylopectin and native starch to cyclodextrins, preferentially alpha-cyclodextrin. With a longer incubation period, the alpha-cyclodextrin to beta-cyclodextrin ratio declined. Variations in substrate type and concentration influenced the product pattern. Increasing the substrate concentration (0.5-20.0%) and glucans containing branching points (alpha-1,6 glycosidic linkages) shifted the product pattern to: beta-cyclodextin > alpha-cyclodextrin > gamma-cyclodextrin. In addition to these cyclodextrins, larger cyclodextrins (>8 glucose units) were formed in the initial reaction period. The CGTase was stabilised against thermal inactivation by calcium ions and high substrate concentrations; and 5 mM of CaCl(2) shifted the apparent melting point of the enzyme from 60 degrees C to 69 degrees C.
Thiemann
Volker
V
Institute of Technical Microbiology, Technical University Hamburg-Harburg, Kasernenstrasse 12, 21073 Hamburg, Germany.
Dönges
Catharina
C
Prowe
Steffen G
SG
Sterner
Reinhard
R
Antranikian
Garabed
G
D000595:Amino Acid Sequence; D000687:Amylopectin / Q000378:metabolism; D000688:Amylose / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D002122:Calcium Chloride / Q000494:pharmacology; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D016937:Gram-Positive Endospore-Forming Rods / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D047391:alpha-Cyclodextrins / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000378:metabolism; D047408:gamma-Cyclodextrins / Q000032:analysis / Q000378:metabolism
D001426:Bacterial Proteins; D003067:Coenzymes; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D047391:alpha-Cyclodextrins; D047392:beta-Cyclodextrins; D047408:gamma-Cyclodextrins; D013213:Starch; D000688:Amylose; D000687:Amylopectin; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; C031215:betadex; D002122:Calcium Chloride; C032613:alpha-cyclodextrin