Abstract: Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg−1 protein, 20.0 U mg−1 protein and 11.0 U mg−1 protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl2. Km and Vmax values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co2+, Zn2+, Cu2+, Hg2+, Ba2+, Cd2+, and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry.
Keywords: alkaliphiles; soda lakes; cyclodextrin glycosyltransferase; Amphibacillus sp.; purification; 16S rDNA
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Ibrahim, A.S.S.; Al-Salamah, A.A.; El-Tayeb, M.A.; El-Badawi, Y.B.; Antranikian, G. A Novel Cyclodextrin Glycosyltransferase from Alkaliphilic Amphibacillus sp. NPST-10: Purification and Properties. Int. J. Mol. Sci. 2012, 13, 10505-10522.
Ibrahim ASS, Al-Salamah AA, El-Tayeb MA, El-Badawi YB, Antranikian G. A Novel Cyclodextrin Glycosyltransferase from Alkaliphilic Amphibacillus sp. NPST-10: Purification and Properties. International Journal of Molecular Sciences. 2012; 13(8):10505-10522.
Ibrahim, Abdelnasser S. S.; Al-Salamah, Ali A.; El-Tayeb, Mohamed A.; El-Badawi, Yahya B.; Antranikian, Garabed. 2012. "A Novel Cyclodextrin Glycosyltransferase from Alkaliphilic Amphibacillus sp. NPST-10: Purification and Properties." Int. J. Mol. Sci. 13, no. 8: 10505-10522.