2009-07-08 · Activity characteristics and kinetic aspects of a cyclodextrin glycosyltransferase (CGTase) from Bacillus circulans DF 9R were studied. A mixture of α-, β- and γ-cyclodextrins (CDs), glucose, maltose and negligible amounts of longer linear dextrins were produced from gelatinized amylose, amylopectin and starch from different sources.

Some of the enzyme activity was also observed in the periplasmic and the intracellular fractions. When the mature CGTase G1 gene (without signal peptide) was cloned into pQE and pWH expression vectors and transformed into E. coli, almost all of the CGTase activity was detected intracellularly (data not shown).

The kinetic parameters Km and Vmax were 10 mg/ml and 146 µmol/mg min, respectively, for purified CGTase. CGTase can catalyse four reactions: cyclization, coupling, disproportionation, and hydrolysis (Li et al., 2014b), though the hydrolysis activity is relatively weak (Costa et al., 2009). CGTase is a member of glycoside hydrolase family 13 (GH13) (Buchholz & Seibel, 2008 ; Stam et al., 2006 ). 1999-01-01 · Figure 5 shows the profiles of CGTase concentration (A) and activity (B) in cells after induction of proteins. The enzyme concentration (A) and enzyme activity (B) are both shown.

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At temperature higher than 70 °C, activity of CGTase declines sharply may be because the CGTase could be denatured above 70 °C. Relative activity also decreased by about 60% at 80 °C. CGTase activity. The beads were washed with 25 ml of saline, phosphate buffer (0Æ01mol l)1,pH6Æ5) and dis-tilled water under sterile conditions and transferred to 50 ml of fresh medium to start new cycle.

CGTase was expressed by recombinant K. phaffii through pH maintenance in range of 5.5–7.0. β‐CGTase activity increased to 122.0 U/mL after optimization of glycerol, phosphate buffer, pH value, ammonium sulfate, temperature, methanol, and additives based on BSM, establishing a modified defined medium.

The precipitate was collected by centrifugation (27,000 × g for 30 min), dissolved in 25 mM Tris-HCl buffer (pH 7.0) containing 12% ammonium sulfate saturation, and applied to a phenyl-Superose HR 5/5 column (Pharmacia) previously equilibrated with the same buffer. Estimation of CGTase Activity CGTase activity was measured using phenolphthalein β-cyclodextrin complexation method20. Enzyme solution dialyzed against buffer before enzyme assay. When required, different salts such as NaCl, KCl, CaCl2 and NH4Cl were introduced in the reaction mixture in 10-70 mM final A cyclic activity of accumulation and consumption of beta -CD and gamma -CD occurred during the bacterial growth.

The enzymes from the α-amylase family all share a similar α-retaining catalytic mechanism but can have different reaction and product specificities. One family member, cyclodextrin glycosyltransferase (CGTase), has an uncommonly high transglycosylation activity and is able to form cyclodextrins. We have determined the 2.0 and 2.5 Å X-ray structures of E257A/D229A CGTase in complex with

The enzyme displays unusually high amylolytic activity in relation to the cyclization activity. The standard CGTase activity assays described above was used to determine the residual activity of each enzyme (Jeang et al. 2005).

The enzymes from the α-amylase family all share a similar α-retaining catalytic mechanism but can have different reaction and product specificities. One family member, cyclodextrin glycosyltransferase (CGTase), has an uncommonly high transglycosylation activity and is able to form cyclodextrins. We have determined the 2.0 and 2.5 Å X-ray structures of E257A/D229A CGTase in complex with One family member, cyclodextrin glycosyltransferase (CGTase), has an uncommonly high transglycosylation activity and is able to form cyclodextrins. We have determined the 2.0 and 2.5 Angstrom X-ray structures of E257A/D229A CGTase in complex with maltoheptaose and maltohexaose. At this point the total soluble γ-CGTase activity had reached 5.51 U·mL − 1.
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Very interestingly, AmyA, but not AmyB, displayed high transglycosylation activity on maltooligosaccharides and also had significant β-cyclodextrin glycosyltransferase (CGTase) activity. CGTase activity has not been reported for typical α-amylases before. In conclusion, the hydrolyzing activity of CGTase is limited by the hydrophobicity of Phe-183 and Phe-259, as shown by the increased hydrolysis resulting from the replacement of these residues by hydrophilic ones. Furthermore, Phe-183 and Phe-259 have specific roles in the transglycosylation reactions catalyzed by CGTase.

CGTase activity was found in yeast extract supplemented medium and low in tryptophan supplementation. Maximum CGTase activity reached at 24 h of incubation in all media except medium supplemented with ammonium sulphate (Fig. 5). The value of R2 is low between incubation time and cell density, incubation time and CGTase in all nitrogen sources.
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CGTase which showed high specific activity at 80°C without any γ-CGTase activity (Zhang et al. 2017). Takada et al. reported that the γ-CGTase produced by Bacillus clarkii 7364 showed the maximum cyclization activity at pH10.5–11.0, and at 60°C (Takada et al. 2003). Hence, the recombinant γ-CGTase properties, op-

The enzyme displays unusually high amylolytic activity in relation to the cyclization activity.