The cyclodextrin glycosyltransferase of Paenibacillus pabuli US132 strain: molecular characterization and overproduction of the recombinant enzyme.
The gene encoding the cyclodextrin glycosyltransferase (CGTase) of Paenibacillus pabuli US132, previously described as efficient antistaling agent and good candidate for cyclodextrins production, was cloned, sequenced, and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34 residues. The enzyme exhibited the highest identity (94%) to the beta-CGTase of Bacillus circulans no. 8. The production of the recombinant CGTase, as active form, was very low (about 1 U/mL) in shake flasks at 37 degrees C. This production reached 22 U/mL after 22 hours of induction by mainly shifting the postinduction temperature from 37 to 19 degrees C and using 2TY instead of LB medium. High enzyme production (35 U/mL) was attained after 18 hours of induction in fermentor using the same culture conditions as in shake flask. The recombinant enzyme showed V(max) and K(m) values of 253 +/- 36 mumol of beta-cyclodextrin/mg/min and 0.36 +/- 0.18 g/L, respectively.
Jemli
Sonia
S
Laboratory of Enzymes and Metabolites of Prokaryotic, Centre of Biotechnology of Sfax, P.O. Box 1177, 3018 Sfax, Tunisia.
Ben Messaoud
Ezzedine
E
Ben Mabrouk
Sameh
S
Bejar
Samir
S
D001407:Bacillus / Q000145:classification* / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000502:physiology; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013045:Species Specificity
D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase