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.
Highlights
Cyclodextrin glycosyl transferase (CGTase, EC 2.4.1.19) is an important industrial enzyme, unique in its ability to convert starch and related glycans into non-reducing, cyclic malto-oligosacchrarides called cyclodextrins (CDs) via a cyclization reaction, an intramolecular transglycosylation reaction [1].it is an important hydrolytic enzyme that carries out reversible intermolecular coupling and disproportionation of maltooligosaccharides [1,2]
We present isolation of novel cyclodextrin glycosyltransferase (CGTase) producing alkaliphilic bacterium from hyper saline soda lakes, purification and characterization of the CGTase, in addition to some studies on cyclodextrins production by the purified enzyme
Screening of alkaline water and sediment samples collected from Wadi Natrun hyper saline soda lakes for isolation of CGTase producing alkaliphilic bacteria, using rich alkaline agar medium containing
Summary
Cyclodextrin glycosyl transferase (CGTase, EC 2.4.1.19) is an important industrial enzyme, unique in its ability to convert starch and related glycans into non-reducing, cyclic malto-oligosacchrarides called cyclodextrins (CDs) via a cyclization reaction, an intramolecular transglycosylation reaction [1]. Among the three types of cyclodextrins, β-CD is of high interest due to the size of its non-polar cavity which is suitable to encapsulate several guest molecules; its low solubility in water which facilitates its separation from the reaction mixture. Alkaliphilic microorganisms have attracted much interest in the past few decades because of their ability to produce extracellular enzymes that are active and stable at high pH values [9,10]. We present isolation of novel CGTase producing alkaliphilic bacterium from hyper saline soda lakes, purification and characterization of the CGTase, in addition to some studies on cyclodextrins production by the purified enzyme
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