Alkaliphilic bacteria were isolated from soil and water samples obtained from Egyptian soda lakes (Wadi Natrun area, Egypt). Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria resulted in isolation of 10 positive strains. Strain KSU-A11 was selected as the best CGTase producer (2.1 U/ml). 16S rDNA sequence analysis identified the KSU-A11strain as Bacillus agaradhaerens. CGTase was partially purified using starch adsorption technique. The partially purified CGTase was immobilized on chitin by covalent binding tecnique using cross linking reaction with high immobilization yield (85%). The properties of the free and immobilized CGTase were determined. The optimum pH of the immobilized enzyme was slightly higher than that of the free enzyme at pH 10 and 10.5, respectively. In addition, both free and immobilized enzyme retained 94 to 100% of its initial activity over a wide pH range (pH 6.0 to 11.0). The enzymatic activity of both free and immobilized CGTase was highest at temperature 50°C; however, the relative activities of the immobilized CGTase were slightly higher than those of the free enzyme. Furthermore, investigation of thermostability of the enzyme indicated that the immobilization process of CGTase on chitin significantly protected the enzyme against thermo-inactivation. Kinetic parameters, Km and Vmax, values for free and immobilized enzymes were estimated and while there was no change in the Vmax value (83.3 μmol/min. mg) for both free and immobilized CGTase, the Km of the enzyme increased from 14.28 to 20 mg/ml upon immobilization. The immobilization of the enzyme showed high operational stability by retaining almost 50% of the initial activity after nine uses.
Key words: Cyclodextrin glycosyltransferase, Bacillus agaradhaerens, immobilization, chitin, alkaliphiles.
Abbreviations: CGTases, Cyclodextrin glucosyltransferases; CDs, cyclodextrins;PCR, polymerase chain reaction; BLAST, Basic Local Alignment Search Tool; NCBI,National Center For Biotechnology Information.
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