Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst (original) (raw)
Related papers
Enzyme and Microbial Technology, 2006
This paper presents the immobilization of the Thermoanaerobacter cyclomaltodextrin glucanotransferase (CGTase) enzyme into cross-linked 6% agarose beads activated by high density of linear aldehyde groups (glyoxyl-agarose) that allow the establishment of multi-attachment enzymesupport bonds. The immobilization conditions were 25 • C, pH 10 and 5 h of contact time. The immobilization yield was almost 100% and the activity recovery was ca. 32%. The biocatalyst at 85 • C was capable of producing cyclodextrins (CDs) from dextrin or soluble starch (both at 1% (w/v)) at a greater rate than the soluble enzyme. In addition, the biocatalyst maintained 90% of its initial activity after 5 h at 85 • C. The maximum conversion of dextrin to -CD and ␥-CD (total mass of produced CDs/substrate initial mass × 100) was 29% both for the soluble and immobilized enzymes. Using starch as substrate the maximum starch conversion to -CD and ␥-CD was 29% and 38%, for the immobilized and soluble enzyme, respectively. The -CD selectivity yield [mass of -CD produced/(mass of -CD produced + mass of ␥-CD produced) × 100] increased from 67.9% for the free enzyme to 85.4% for the immobilized CGTase.
Evaluation of supports and methods for immobilization of enzyme cyclodextringlycosyltransferase
Applied Biochemistry and Biotechnology, 2003
An experimental design with factorial planning was used for the immobilization of the enzyme cyclodextringlycosyltransferase (CGTase) from Bacillus firmus (strain no.37) to select the best combination of support, method of immobilization, and conditions that gives primarily higher average values for the specific immobilized enzyme activity, and secondarily, higher average values for the percentage of protein fixation. The experimental design factors were as follows: supports—controlled-pore silica, chitosan, and alumina; immobilization methods—adsorption, and two covalent bonding methods, either with γ-aminopropyltriethoxysilane or hexamethylenediamine (HEMDA); conditions—7°C without agitation and 26°C with stirring. The best combination of factors that lead to higher average values of the response variables was obtained with immobilization of CGTase in silica with HEMDA at 7°C. However, immobilization in chitosan at 7°C gave the highest immobilized CGTase specific activity, 0.25 µmole of β-CD/(min·mg protein). Physical adsorption gave low specific enzyme activities, and, in general, a high load of enzyme leads to lower specific enzyme activity.
Comprehensive study on transglycosylation of CGTase from various sources
Heliyon, 2021
Transglycosylation is the in-vivo or in-vitro process of transferring glycosyl groups from a donor to an acceptor, which is usually performed by enzymatic reactions because of their simplicity, low steric hindrance, high regionspecificity, low production cost, and mild processing conditions. One of the enzymes commonly used in the transglycosylation reaction is cyclodextrin glucanotransferase (CGTase). The transglycosylated products, catalyzed by CGTase, are widely used in food additives, supplements, and personal care and cosmetic products. This is due to improvements in the solubility, stability, bioactivity and length of the synthesized products. This paper's focus is on the importance of enzymes used in the transglycosylation reaction, their characteristics and mechanism of action, sources and production yield, and donor and acceptor specificities. Moreover, the influence of intrinsic and extrinsic factors on the enzymatic reaction, catalysis of glycosidic linkages, and advantages of CGTase transglycosylation reactions are discussed in detail.
Organic solvent and pH induced alteration of product specificity of CGTase
Biotechnology and Bioprocess Engineering, 1998
Cyclodextrin glucanotransferase [CGTase, E.C.2.4.1.19] is an extracellular enzyme, which catalyzes the formation of a-, j3-, ;-CDs from starch. Their proportions of formations depend on enzyme sources and reaction conditions. To understand what determines the product specificity of CGTases, we examined the alteration of product specificity of CGTase from Bacillus macerans by organic solvents and pH. At acidic pH range less than pH 6 where the enzyme was unstable, the ratio of a-//~-CD production was increased 4 times more than that at neutral pH range. As we increased the concentration of 2-butanol, a-//3-CD ratio was proportionally increased but/ratio remained constant. The a-/~-CD ratio of products was increased in the reaction media which yielded low products.
Journal of Molecular Catalysis B: Enzymatic, 2015
Bacillus amyloliquefaciens cyclodextrin glucosyltransferase (CGTase) was immobilized on 35 supports by different methods of immobilization. The immobilized enzymes were prepared by physical adsorption on chitin, ionic binding onto Amberalite IRA-45, covalent binding on Duolite XAD761, and entrapment in polyacrylamide had the highest recovered activity. The immobilized preparations retained 12.08-43.5% of the original specific activity exhibited by the free enzyme. Compared to the free enzyme, the immobilized preparations exhibited higher optimum temperature, lower activation energy, lower deactivation constant rate, higher half-life (T 1/2) values, and resistance to chemical denaturation. The values of thermodynamic parameters for irreversible inactivation indicated that immobilization significantly decreased entropy (S*) and enthalpy of deactivation (H*). The immobilized enzyme displayed higher K m and lower V max values. After using for 10 cycles, the retained catalytic activity were 30.0, 35.0, 69, and 54% of the initial values of the immobilized enzyme on chitin, Amberlite IRA-45, Duolite XAD761, and polyacrylamide respectively.
Journal of Molecular Catalysis B: Enzymatic, 2007
A novel mutant enzyme namely H43T CGTase can produce up to 39% ␥-cyclodextrin (␥-CD) compared to the native enzyme which produces only 10% ␥-CD. The effect of the reaction conditions on ␥-CD production was studied using this mutant CGTase. The effects of substrate-buffer combination, starch pretreatment and concentration, pH, additives and finally the use of a debranching enzyme improved the ␥-CD ratio further. The tapioca-acetate pair gave the highest conversion (16% conversion) among four types of starch and four buffer system combinations. Gelatinized starch was preferred compared to raw tapioca starch in producing a high percentage of ␥-CD and conversion rate. Higher pH especially pH 8-9 led to a higher proportion of ␥-CD, and was relatively more apparent when the concentration of starch was increased. Forty-six percent ␥-CD was produced using 2.5% gelatinized tapioca starch at pH 8. Pullulanase enzyme was found to be useful in reducing the viscosity of tapioca starch paste thus increasing the efficiency of utilization of starch by CGTase by at least 20-to 30-fold. Up to 48% ␥-CD can be produced when 4% pullulanase-pretreated tapioca starch was reacted with the CGTase mutant. It was also found that the supplementation of the reaction mixture with glucose, toluene, or cyclododecanone improved the ␥-CD yield by 42.2, 46.4, 43.4, and 43.4%, respectively. All the parameters involved have been shown to affect the product specificity of the mutant H43T CGTase transglycosylation mechanism.
Acta Scientiarum. Technology, 2013
The enzyme cyclomaltodextrin-glucanotransferase (CGTase) is a transglicosidase able to convert corn starch into cyclodextrin (CD). CDs are widely applied in industry given the ability to form inclusion complexes with a great variety of organic molecules. Regarding the optimum pH of CGTase, values reported in the literature vary according to the enzyme producing microorganism, being 8.0 the optimum pH of CGTase produced by Bacillus firmus Strain No. 37. This work studied the influence of the pH of culture medium with different concentration of nutrients on the production of the enzyme CGTase by Bacillus firmus Strain No. 37. For this purpose, the microorganism was grown in three culture media with different concentrations of carbon and nitrogen. The pH control was performed by adding sodium carbonate. The fermentation process was analyzed by the following methods: Bradford (1976) method to determine soluble proteins, DNS method to analyze sugars, and the method of complexation with β-CD to analyze the enzyme activity. The best result for CGTase enzyme activity was 0.22 U mL -1 , obtained with medium containing 2.0% soluble corn starch and yeast extract, and pH 8.3.
Biologia, 2012
The cyclodextrin glycosyltransferase (CGTase) of the recombinants Escherichia coli pAD26 cells immobilized on cotton was optimally produced by statistical methodology. Primarily, carbon and nitrogen sources were selected by one-factor-at-a-time method. Wheat starch, Casamino acid, Edamin and Hy-soy were identified as the best nutrients. These sources were secondly confirmed by Plackett-Burman design (fifteen variables were studied with sixteen experiments), as the most significant components with respect to CGTase production. In the third step, concentration of most significant factors and their interaction were optimized with a Box-Behnken experimental design. Under the optimized conditions (agitation 200 rpm, yeast extract concentration 20 g/L, wheat starch concentration 10 g/L and Hy-soy concentration 2.5 g/L), CGTase yield 145.11 U/mL was 3.6 and 23 folds higher than those obtained by the use of the initial conditions (39.77 U/mL) and free cells (6.37 U/mL), respectively.
THE EGYPTIAN JOURNAL OF EXPERIMENTAL BIOLOGY (Botany), 2018
To l b a M o h a me d Na g wa A h m e d A b d a l l a Optimization and characterization of c yclodextrin gl ycos yltransferase produced by Thermoactinomyces vulgaris TA1 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 DEAEcellulose anion exchange chromatography yielded 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%, respectively while ethylenediamine tetraacetic acid (EDTA), β-CD and dichloro-diphenyl-trichloroethane (DDT) decreased the relative activity to 68, 46, and 13%, respectively. Moreover, SDS and β-Mercaptoethanol totally inhibited the CGTase activity at concentrations of 1 and 10mM, respectively. The purified CGTase had Km value of 0.633 mg/ml and Vmax value of 135.14 mg/ml.min.