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- Title
Optimization and characterization of cyclodextrin glycosyl- transferase produced by Thermoactinomyces vulgaris TA1.
- Authors
Alsharawi, Samar Zaim; El-Shatoury, Einas Hamed; Mohamed, Sahar Tolba; Abdalla, Nagwa Ahmed
- Abstract
A cyclodextrin glycosyltransferase (CGTase)-producing thermophilic actinomycete was isolated from chicken wastes and identified by biochemical methods and partial 16S rDNA sequence as Thermoactinomyces vulgaris. The nutritional and physical conditions for CGTase production were optimized, and the expressed CGTase enzyme was purified by two steps. In the first step, CGTase was partially purified by 80% ammonium sulfate precipitation with purification fold 9.33 and total activity 938.16U. In the second step, purification by DEAE-cellulose ion exchange chromatography yieled a purification fold of 21.20 with 396.8 U total activity. The resulting CGTase enzyme was homogenous on SDS-PAGE gel with a molecular weight of about 66 KDa. The optimum temperature for purified CGTase was 65°C with 52.8% of its relative activity retained after incubation at 85°C for 1 h. The CGTase enzyme exhibited maximum activity at pH 7.5 and was stable at a pH range from 6 to 9.5. Anhydrous CaCl2 and ZnSO4•7H2O at concentration 1 &10 mM increased CGTase relative activity to 130.04 and 128.42%, respectively. Conversely, KCl decreased the relative activity to 11.78% and anhydrous FeCl3 totally inhibited CGTase activity at concentrations of 1 and 10mM. In addition, α-cyclodextrin (CD) and γ-CD slightly increased the relative activity of CGTase to 102 and 100.72 %, while ethylenediamine tetraacetic acid (EDTA), β-CD and dichloro-diphenyl-trichloroethane (DDT) decreased the relative activity to 68, 46 and 13 %. Moreover, SDS and β –Mercaptoethanol totally inhibited the CGTase activity at concentrations of 1 and 10mM. The purified CGTase had Km value of 0.633 mg/ml and Vmax value of 135.14 mg/ml.min.
- Subjects
BACILLUS subtilis; FERMENTATION; GLUCOSIDASES; CYCLODEXTRINS; ANTIOXIDANTS
- Publication
Egyptian Journal of Experimental Biology (Botany), 2018, Vol 14, Issue 1, p143
- ISSN
1687-7497
- Publication type
Article
- DOI
10.5455/egyjebb.20180321064543