An alkaliphilic cyclodextrin glycosyltransferase from a new Bacillus agaradhaerens WN-I strain isolated from an Egyptian soda lake: Purification and properties

Abstract

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.