Screening, Partial Purification and Stability Studies of Cyclodextrin glycosyl transferases from Bacillus amyloliquefaciens Strain L4-6

Abstract

Cyclodextrin glycosyl transferases (CGTases) are produced by a wide range of bacteria, mainly Bacillus species by submerged fermentation. CGTase production is highly dependent on the strain, medium composition and culture conditions. The present investigation was undertaken with an objective to screen, isolate, identify the microorganism producing CGTase and its purification with improved production and purification fold. Among the 37 colonies screened for production of CGTase, 07 isolates were selected [RS 1-7 having zone diameter in the range 18-27 mm and optimum exhibited by RS-4 (Sample B-27 mm)] based on their hydrolytic zones, microscopic charecteristics and CGTase production capabilities and further streaked onto Horikoshi-phenolphthalein (PHP) plates for futher studies. The isolated sample B subjected to morphological and strain level characterization by 16S r-DNA based molecular technique was found to be Bacillus amyloliquefaciens strain L4-6 (GenBank Accession Number: KC464454.1) based on nucleotide homology and phylogenetic analysis. The purified enzyme was stable at a wide pH range from 4 to 12 and was optimally active at pH 8. The enzyme exhibited optimum activity at a temperature of 60°C was stable for 1 h. With increase in substrate concentration from 40-200 μg/ml, the enzyme activity of CGTase (μg β-CD /min/ mg protein) was not enhanced significantly, which increases proportionally with an increase in substrate concentration (200-300 μg/ml) and reached the plateau stage. A potent CGTase was obtained with a purification fold, specific activity and yield value of 41.65, 3000.68 U/mg and 2.23 % respectively by ammonium salt precipitation, followed by Sephadex G-25 and DEAE Cellulose coloumn chromatography. The apparent molecular weight of the CGTase was found to be 81 kDa by SDS PAGE.