An alkaliphilic cyclodextrin glycosyltransferase from a new Bacillus agaradhaerens WN-I strain isolated from an Egyptian soda lake: Purification and properties
Alkaliphilic bacteria were isolated from soil and water samples obtained from Egyptian soda lakes in the Wadi Natrun area. Screening for cyclodextrin glycosyltransferase- producing alkaliphilic bacteria resulted in the isolation of 15 positive strains. Strain WN-I was selected as the best producer of CGTase. 16S rDNA sequence analysis and DNA-DNA hybridization identified the isolate as Bacillus agaradhaerens. The enzyme was purified to homogeneity up to 21 fold by starch adsorption and anion exchange chromatography with a yield of 26.40%. The pure enzyme was a monomer with an estimated molecular weight of 85 kDa. The enzyme was stable, at 25°C, over a pH range of 5.0 to 11, with a maximum activity at pH 9.0. The enzyme activity exhibited an optimum temperature of 55°C and was stable at 40°C for at least 1 h. Thermal stability was improved in the presence of maltodextrin, starch or CaCl2. The enzyme was slightly stimulated by CaCl2, KC1 and BaCl2 but was completely inhibited in the presence of FeCl2 and strongly inhibited by ZnCl2 and CoCl2 and to a lower extent by CuCl2, EDTA, 2-mercaptoethanol, and dithiothritol. The enzyme produced mainly β-CD (71.20% of the total cyclodextrin amount). The enzyme had higher cyclyzation activity (1.9 fold higher) toward Paselli starch than soluble starch.
Key words: Alkaliphiles, soda lakes, cyclodextrin glycosyltransferase, Bacillus agaradhaeren, purification, 16S rDNA.