title: Purification and characterization of cyclodextrin β-glucanotransferase from novel alkalophilic bacilli.
abstract: The discovery of novel bacterial cyclodextrin glucanotransferase (CGTase) enzyme could provide advantages in terms of its production and relative activity. In this study, eight bacterial strains isolated from soils of a biodiversity-rich vegetation in Egypt based on their hydrolyzing activity of starch, were screened for CGTase activity, where the most active strain was identified as Bacillus lehensis. Optimization process revealed that the using of rice starch (25%) and a mixture of peptone/yeast extract (1%) at pH 10.5 and 37 °C for 24 h improved the bacterial growth and enzyme activity. The bacterial CGTase was successively purified by acetone precipitation, gel filtration chromatography in a Sephadex G-100 column and ion exchange chromatography in a DEAE-cellulose column. The specific activity of the CGTase was increased approximately 274-fold, from 0.21 U/mg protein in crude broth to 57.7 U/mg protein after applying the DEAE-cellulose column chromatography. SDS-PAGE showed that the purified CGTase was homogeneous with a molecular weight of 74.1 kDa. Characterization of the enzyme exhibited optimum pH and temperature of 7 and 60 °C, respectively. CGTase relative activity was strongly inhibited by Mg(2+), Zn(2+), Al(3+) and K(+), while it was slightly enhanced by 5 and 9% with Cu(2+) and Fe(2+) metal ions, respectively.
authors.affiliation: Genetic Engineering and Biotechnology Research Institute, University of Sadat City (USC), Sadat City, Egypt.
authors.forename: Ashraf F
authors.initials: AF
authors.lastname: Elbaz
authors.forename: Ahmed
authors.initials: A
authors.lastname: Sobhi
authors.forename: Ahmed
authors.initials: A
authors.lastname: ElMekawy
mesh_terms: D000383:Agriculture; D001407:Bacillus / Q000201:enzymology*; D019149:Bioreactors; D002850:Chromatography, Gel; D003911:Dextrans; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D005964:Glucosyltransferases / Q000737:chemistry* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007218:Industrial Microbiology; D007477:Ions; D007700:Kinetics; D008970:Molecular Weight; D012275:Oryza / Q000201:enzymology; D010461:Peptones / Q000737:chemistry; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D003911:Dextrans; D007477:Ions; D010461:Peptones; C025614:sephadex; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase