Abstract
Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from a thermophilic anaerobic bacterium, Thermoanaerobacter sp. P4, was purified by ammonium sulfate precipitation followed by α-cyclodextrin epoxy activated-sepharose 6B column chromatography. Enzyme was purified 141 fold and had the specific activity of 143.8 U/mg proteins. Purification yields after ammonium sulfate precipitation and affinity chromatography were 25.8 and 17.8%, respectively. SDS-PAGE analysis showed that enzyme was purified successfully and had a single band. Molecular weight of the enzyme was determined as 68.7 kDa. The enzyme had optimum cyclization activity at 80 to 90°C and hydrolyzing activity at 90°C and maintained 87 and 95% of these activities at 95°C, respectively. Optimal pH was found as 7.0. It retained full activity at 80°C for 4 h. Enzyme was strongly inhibited by HgSO 4 and AgNO 3 . Addition of 1 mM CaCl 2 increased the enzymatic activity up to 7%. This novel enzyme could be a good candidate for industrial applications according to its characteristic found in the current study. Key words: Cyclodextrin glycosyltransferase, cyclodextrin production, Thermoanaerobacter sp. P4, thermophilic, enzyme purification, enzyme characterization.
Highlights
Cyclodextrin glycosyltransferase (CGTase, EC2.4.1.19) is a starch converting extracellular enzyme which belongs to α-amylase family of glycoside hydrolases (Bertoldo and Atranikian, 2002; van der Maarel et al, 2002)
P4, was purified by ammonium sulfate precipitation followed by α-cyclodextrin epoxy activated-sepharose 6B column chromatography
Thermophilic anaerobic bacterium was isolated from an oil field (Raman/Turkey) and identified as Thermoanaerobacter sp
Summary
Cyclodextrin glycosyltransferase (CGTase, EC2.4.1.19) is a starch converting extracellular enzyme which belongs to α-amylase family (family 13) of glycoside hydrolases (Bertoldo and Atranikian, 2002; van der Maarel et al, 2002) It is a unique enzyme capable of producing cyclodextrins (CDs) through intramolecular transglycosylation reaction called cyclization (Ibrahim et al, 2005; Tonkova, 1998). CDs are cyclic oligosaccharides consisting of mainly six, seven and eight glucose units named as α, β and γ-CD, respectively, linked via α(1-4) glucosidic bonds (Biwer et al, 2002; van der Ween et al, 2000) Due to their hydrophobic cavity and hydrophilic outer surface, they are able to form inclusion (host-guest) complexes with a variety of organic and inorganic molecules, changing the chemical and physical properties of the guest molecules (Del Valle, 2004; Singh et al, 2002). Inclusion complex formation makes CDs attractive for the use in wide industrial applications including food, pharmacy, agriculture, cosmetics, analytical chemistry (Brewster and Loftsson, 2007; Del Valle, 2004; Loftsson and Duchene, 2007; Song et al, 2009; Szente and Szejtli, 2004)
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