title: β-cyclodextrin production by the cyclodextrin glucanotransferase from Paenibacillus illinoisensis ZY-08: cloning, purification, and properties.
abstract: The gene encoding the cyclodextrin glucanotransferase (CGTase, EC2.4.1.19) of Paenibacillus illinoisensis was isolated, 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 deduced amino acid sequence of the CGTase from P. illinoisensis ZY-08 exhibited highest identity (99 %) to the CGTase sequence from Bacillus licheniformis (P14014). The four consensus regions of carbohydrate converting domain and Ca(2+) binding domain could be identified in the sequence. The CGTase was purified by using cold expression vector, pCold I, and His-tag affinity chromatography. The molecular weight of the purified enzyme was about 74 kDa. The optimum temperature and pH of the enzyme were 40 °C and pH 7.4, respectively. The enzyme activity was increased by the addition of Ca(2+) and inhibited by Ba(2+), Cu(2+), and Hg(2+). The K m and V max values calculated were 0.48 mg/ml and 51.38 mg of β-cyclodextrin/ml/min. The ZY-08 and recombinant readily converted soluble starch to β-cyclodextrin but ZY-08 did not convert king oyster mushroom powder and enoki mushroom powder. However the recombinant CGTase converted king oyster mushroom powder and enoki mushroom powder to β-cyclodextrin.
authors.affiliation: Department of Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea.
authors.forename: Yong-Suk
authors.initials: YS
authors.lastname: Lee
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhou
authors.forename: Dong-Ju
authors.initials: DJ
authors.lastname: Park
authors.forename: Jie
authors.initials: J
authors.lastname: Chang
authors.forename: Yong-Lark
authors.initials: YL
authors.lastname: Choi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D021382:Protein Sorting Signals; D016415:Sequence Alignment; D013379:Substrate Specificity; D047392:beta-Cyclodextrins / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D047392:beta-Cyclodextrins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase