Transformation of cyclodextrin glucanotransferase (CGTase) from aqueous suspension to fine solid particles via electrospraying (original) (raw)
Related papers
IOP Conference Series: Materials Science and Engineering
Cyclodextrin glucanotransferase (CGTase) is an enzyme that convert starch into cyclodextrin (CD) by transglycosylation reaction. The CD has been used in various industries due to the unique characteristics. However, the production of CD is usually restricted due to the instability of the enzyme which easily denatured during the reaction process. Thus, enzyme immobilization technique was applied and optimization of reaction conditions was conducted to enhance the amount of CD production. In this study, CGTase was immobilized on polyvinylidiene difluoride (PVDF) hollow fiber membrane by adsorption. The optimization of the reaction conditions using response surface methodology (RSM) on the production of CD was studied. Under optimized conditions (2.8% w/v of soluble potato starch concentration, 45.2°C of reaction temperature and pH 5.6), the production of CD was 5.65 mg/mL, about 2-fold compared to the value before optimization process. Therefore, the immobilized CGTase on hollow fiber...
Journal of Molecular Catalysis B: Enzymatic, 2003
The extreme thermophilic cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. was covalently attached to Eupergit C. Different immobilization parameters (incubation time, ionic strength, pH, ratio enzyme/support, etc.) were optimized. The maximum yield of bound protein was around 80% (8.1 mg/g support), although the recovery of -cyclodextrin cyclization activity was not higher than 11%. The catalytic efficiency was lower than 15%. Results were compared with previous studies on covalent immobilization of CGTase.
Effect of Reaction Conditions on the Synthesis of Cyclodextrin (CD) by Using Immobilized Enzyme
Jurnal Teknologi, 2018
The production of cyclodextrin (CD) over the years has been increasing due to the numerous applications in industries such as in food, cosmetic, pharmaceutical and agricultural industries. However, cyclodextrin glucanotransferase (CGTase) which involved in the enzymatic reaction on the production of CD is unstable and easily denatured at extreme conditions resulted in low CD production. Hence, the enzyme immobilization technique is introduced to overcome these problems and subsequently increase the production of CD. In the present study, the CGTase was immobilized on hollow fiber membrane to increase the production of CD during the reaction. The effect of reaction conditions (types of starch, concentration of starch, temperature and pH) of the immobilized enzyme on the production of CD were investigated. Among the three types of starch tested, the soluble potato starch was the most suitable substrate for the production of CD with 4.13 mg/mL. In addition, by using 3% (w/v) of the soluble potato starch, the production of CD was 5.22 mg/mL. The optimal reaction temperature and pH were found to be at 40°C and pH 6 with 5.21 mg/mL and 4.62 mg/ml of CD, respectively. The immobilized enzyme exhibited a 1.3-3-fold increase in CD production compared to the free enzyme. Therefore, the hollow fiber membrane is suitable to be used as a support for enzyme immobilization with the high production of CD.
Electronic Journal of Biotechnology, 2014
Background: Cyclodextrin glucanotransferase (CGTase) from Amphibacillus sp. NPST-10 was covalently immobilized onto amino-functionalized magnetic double mesoporous core-shell silica nanospheres (mag@d-SiO 2 @m-SiO 2 -NH 2 ), and the properties of the immobilized enzyme were investigated. The synthesis process of the nanospheres included preparing core magnetic magnetite (Fe 3 O 4 ) nanoparticles, coating the Fe 3 O 4 with a dense silica layer, followed by further coating with functionalized or non-functionalized mesoporous silica shell. The structure of the synthesized nanospheres was characterized using TEM, XRD, and FT-IR analyses. CGTase was immobilized onto the functionalized and non-functionalized nanospheres by covalent attachment and physical adsorption. Results: The results indicated that the enzyme immobilization by covalent attachment onto the activated mag@d-SiO 2 @m-SiO 2 -NH 2 , prepared using anionic surfactant, showed highest immobilization yield (98.1%), loading efficiency (96.2%), and loading capacity 58 µg protein [CGTase]/mg [nanoparticles]) which were among the highest yields reported so far for CGTase. Compared with the free enzyme, the immobilized CGTase demonstrated a shift in the optimal temperature from 50°C to 50-55°C, and showed a significant enhancement in the enzyme thermal stability. The optimum pH values for the activity of the free and immobilized CGTase were pH 8 and pH 8.5, respectively, and there was a significant improvement in pH stability of the immobilized enzyme. Moreover, the immobilized CGTase exhibited good operational stability, retaining 56% of the initial activity after reutilizations of ten successive cycles. Conclusion: The enhancement of CGTase properties upon immobilization suggested that the applied nano-structured carriers and immobilization protocol are promising approach for industrial bioprocess for production of cyclodextrins using immobilized CGTase.
Biotechnology Progress, 2006
Covalent immobilization of cyclodextrin glycosyltransferase on glyoxyl-agarose beads promotes a very high stabilization of the enzyme against any distorting agent (temperature, pH, organic solvents). For example, the optimized immobilized preparation preserves 90% of initial activity when incubated for 22 h in 30% ethanol at pH 7 and 40°C. Other immobilized preparations (obtained via other immobilization protocols) exhibit less than 10% of activity after incubation under similar conditions. Optimized glyoxyl-agarose immobilized preparation expressed a high percentage of catalytic activity (70%). Immobilization using any technique prevents enzyme inactivation by air bubbles during strong stirring of the enzyme. Stabilization of the enzyme immobilized on glyoxyl-agarose is higher when using the highest activation degree (75 µmol of glyoxyl per milliliter of support) as well as when performing long enzyme-support incubation times (4 h) at room temperature. Multipoint covalent immobilization seems to be responsible for this very high stabilization associated to the immobilization process on highly activated glyoxyl-agarose. The stabilization of the enzyme against the inactivation by ethanol seems to be interesting to improve cyclodextrin production: ethanol strongly inhibits the enzymatic degradation of cyclodextrin while hardly affecting the cyclodextrin production rate of the immobilized-stabilized preparation.
Journal of Molecular Catalysis B: Enzymatic, 2012
In this work, the enzymatic properties of immobilized cyclodextrin glycosyltransferase (CGTase) of Thermoanaerobacter sp. were investigated and compared with the soluble form of the enzyme. CGTase was immobilized on mesoporous silica microspheres synthesized using polyethylene glycol 400 as swelling agent, silanized with 3-aminopropyltrimethoxysilane (APTMS), and activated with glutaraldehyde prior to immobilization. This innovative approach for support preparation produced high yields of immobilization (83%) and activity recoveries (73%), which are the highest so far reported for CGTase. The soluble enzyme (CGTase) and its immobilized form (ImCGTase) showed similar values for the optimal pH activity, while optimal reaction temperatures were found to be 100 • C and 80 • C, respectively. The immobilized enzyme showed similar values for K m and thermal stabilities with the soluble form, while its V max was lower. The immobilized enzyme was tested in repeated batches in order to simulate recovery and reuse, keeping about 60% of the initial catalytic activity after 15 cycles, showing its good chemical and mechanical resistance.
Applied and Environmental Microbiology, 2006
The cyclodextrin glucanotransferase (CGTase) gene (cgt) from Bacillus circulans 251 was cloned into plasmid pYD1, which allowed regulated expression, secretion, and detection. The expression of CGTase with a-agglutinin at the N-terminal end on the extracellular surface of Saccharomyces cerevisiae was confirmed by immunofluorescence microscopy. This surface-anchored CGTase gave the yeast the ability to directly utilize starch as a sole carbon source and the ability to produce the anticipated products, cyclodextrins, as well as glucose and maltose. The resulting glucose and maltose, which are efficient acceptors in the CGTase coupling reaction, could be consumed by yeast fermentation and thus facilitated cyclodextrin production. On the other hand, ethanol produced by the yeast may form a complex with cyclodextrin and shift the equilibrium in favor of cyclodextrin production. The yeast with immobilized CGTase produced 24.07 mg/ml cyclodextrins when it was incubated in yeast medium supplemented with 4% starch.
Comptes rendus de l'Académie bulgare des sciences: sciences mathématiques et naturelles
A new recombinant cyclodextrin glucanotransferase JCGT8-5 (CGTase, EC 2.4.1.19) produced by E. coli BL21(DE3) cells was purified by ultrafiltra- tion, starch adsorption and gel filtration with a yield of 34% and displayed a specific activity 35683 U mg−1. The purified recombinant CGTase exhibited molecular weight of 75.5 kDa estimated by SDS-PAGE. It was active at 60– 65 ◦C, stable at a broad pH range (5.0–11.0) and retained more than 50% of its original activities after a heat treatment at 70 ◦C for 1 h without additives. The enzyme produced high amounts of cyclodextrins (CDs) from raw starch (12.0–12.2 mg ml−1) and the products formed were 22% -cyclodextrin (CD), 1% �-CD and 77% �-CD after 2 h incubation at 60 ◦C, without adding any selective agents.
Reuse of Cyclodextrin Glycosyltransferase through Immobilization on Magnetic Carriers
Enzyme Engineering, 2013
CGTase from Bacillus lehensis isolated from wastewater of a cassava flour mill in the state of São Paulo, Brazil was immobilized after being partially purified using precipitation with ammonium sulfate. The partially purified CGTase was immobilized by covalent binding on a magnetite support, which was silanized with 3-aminopropyltrimethoxysilane and activated with glutaraldehyde, resulting in a yield of 16.27% and final activity of 17.54% of the initial activity. The physicochemical properties and kinetics of cyclodextrin glycosyltransferase from Bacillus lehensis were determined. The optimum temperature of the immobilized CGTase was higher than that of the soluble enzyme (70°C and 55°C, respectively). The optimum pH of the immobilized enzyme was the same as that of the soluble enzyme (pH 8.0). In the pH and thermo stability assays, 50% of enzyme activity was maintained after 24 h and 26.22% of the initial activity was retained after 160 min, respectively. The Michaelis-Menten constant was 0.82 mg.ml-1 and maximum velocity was 45.45 mol.ml −1 .min −1. In the reuse study of the immobilized enzyme after four cycles, 16.66% of the initial catalytic activity was maintained, demonstrating the ability to recover and reuse the enzyme immobilized on magnetite.