A Novel Cyclodextrin Glycosyltransferase from Alkaliphilic Amphibacillus sp. NPST-10: Purification and Properties

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.

Optimization of large-ring cyclodextrin production from starch by amylomaltase fromCorynebacterium glutamicumand effect of organic solvent on product size

“…CD yields produced from CGTase are limited by enzyme product inhibition (Leemhuis et al 2003;Gaston et al 2009) and breakdown of CDs into linear oligosaccharides through the coupling reaction. While CGTase exhibited high coupling activity (Ibrahim et al 2012;Ara et al 2015), coupling activity of CgAM was low or not detectable (Srisimarat et al 2012). In addition, CGTase linearized large cycloamyloses at higher rate, compared to CD7 and CD8 (Terada et al 2001).…”

“…While CGTase exhibited high coupling activity (Ibrahim et al . ; Ara et al . ), coupling activity of Cg AM was low or not detectable (Srisimarat et al .…”

Immobilization of cyclodextrin glucanotransferase on aminopropyl-functionalized silica-coated superparamagnetic nanoparticles

“…NPST-10 used in this study was recently isolated from hypersaline Soda Lakes, located in Wadi Natrun valley in northern Egypt (Ibrahim et al 2012). The bacterium was propagated in rich alkaline agar medium containing 0.02% (w/v) phenolphthalein, as an indicator of CGTase production (Park et al 1989).…”

“…CGTase purification was carried out, as described previously, by adsorption of CGTase to corn starch followed by enzyme elution using β-CD solution (Ibrahim et al 2012). Briefly, corn starch and ammonium sulphate were added to one liter of cell-free supernatant to final concentrations of 5% (w/v) and 1 M, respectively.…”

RECOVERY OF CYCLODEXTRIN GLUCANOTRANSFERASE (CGTase) USING IMMOBILIZED METAL CHELATING AFFINITY CHROMATOGRAPHY

“…obtaining it requires downstream processing, which typically consist of a cascade of recovery steps. Current purification strategies include starch adsorption (Higuti et al, 2013, Atanasova et al, 2011, Kitayska et al, 2011, Vassileva et al, 2007, Yampayont et al, 2006and Martins and Hatti-Kaul, 2002, α-cyclodextrin bound epoxy-activated Sepharose 6B affinity chromatography (Goh et al, 2012, Guru et al, 2012, Qi et al, 2007, Rahman et al, 2006and Sian et al, 2005, ion exchange chromatography (Ibrahim et al, 2012, Savergave et al, 2008, Alves-Prado et al, 2007and Doukyu et al, 2003, hydrophobic interaction chromatography (Shetty et al, 2011 andCharoensakdi et al, 2007) and aqueous two-phase separation (Rosso et al, 2005).…”