Catalytic and thermodynamic properties of immobilized Bacillus amyloliquefaciens cyclodextrin glucosyltransferase on different carriers (original) (raw)
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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.
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.
The aim of this study was to stabilize an enzymatic preparation with alpha-amylase activity produced by a Bacillus amyloliquefaciens strain in submerged culture. The immobilization techniques used were physical bonding on a porous ceramic support, entrapment in silica gels and entrapment/deposition on ceramics. The immobilized alpha-amylase was characterized in terms of optimal pH and temperature, pH, thermal and storage stability, kinetic parameters in the context of all used immobilization methods. The optimal temperature and pH of the native and immobilized enzymes did not vary significantly. At temperature and pH values lower than the optimum, the relative activities have been higher for the immobilized Bacillus amyloliquefaciens enzyme compared to the native one. The immobilization has led to an enzymatic compound with high stability and good catalytic efficiency, more pregnant in the case of entrapment/deposition.
Bioprocess and Biosystems Engineering, 2012
The a-amylase of Bacillus amyloliquifaciens TSWK1-1 (GenBank Number, GQ121033) was immobilized by various methods, including ionic binding with DEAE cellulose, covalent coupling with gelatin and entrapment in polyacrylamide and agar. The immobilization of the purified enzyme was most effective with the DEAE cellulose followed by gelatin, agar and polyacrylamide. The K m increased, while V max decreased upon immobilization on various supports. The temperature and pH profiles broadened, while thermostability and pH stability enhanced after immobilization. The immobilized enzyme exhibited greater activity in various non-ionic surfactants, such as Tween-20, Tween-80 and Triton X-100 and ionic surfactant, SDS. Similarly, the enhanced stability of the immobilized a-amylase in various organic solvents was among the attractive features of the study. The reusability of the immobilized enzyme in terms of operational stability was assessed. The DEAE cellulose immobilized a-amylase retained its initial activity even after 20 consequent cycles. The DEAE cellulose immobilized enzyme hydrolyzed starch with 27 % of efficiency. In summary, the immobilization of B. amyloliquifaciens TSWK1-1 a-amylase with DEAE cellulose appeared most suitable for the improved biocatalytic properties and stability.
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.
Enzyme and Microbial Technology, 2005
Immobilized enzymes have provided tremendous advantages for the efficient production of biomaterials. There is increasing demand on simple and convenient protein immobilization methods because protein microarray is emerging as a cutting-edge technology for the proteome analysis and diagnosis. It has been shown that a poly-lysine tag facilitates protein purification and refolding processes. This study demonstrates that the same poly-lysine tag can be employed for the immobilization of enzyme on a solid support without deterioration of its enzymatic characteristics. Cyclodextrin glycosyltransferase (CGTase) derived from Bacillus macerans was fused to consecutive 10 lysine residues (CGTK10ase) and electrostatically immobilized on a cation exchanger. Analyses on the binding characteristics, effects of pH and temperature on enzyme stability and operational stability indicate that the poly-lysine tag is also effective for non-covalent immobilization of CGTase. Though the poly-lysine-mediated immobilization is reversible, binding force is strong enough to block protein leakage from the solid support at neutral and basic pH.
International journal of biological macromolecules, 2015
Cyclodextrin glycosyltransferase (CGTase) was covalently coupled to five oxidized polysaccharides differing in structure and chemical nature. The conjugates were evaluated for the retained activity, kinetic and thermodynamic stability. The conjugated CGTase with oxidized dextran (MW 47000) had the highest retained specific activity (70.05%) and the highest half-life (T1/2) at 80°C. Compared to the native enzyme, the conjugated preparation exhibited higher optimum temperature, lower activation energy (Ea), lower deactivation constant rate (kd), higher T1/2, and higher D values (decimal reduction time) within the temperature range of 60-80°C. The values of thermodynamic parameters for irreversible inactivation of native and conjugated CGTase indicated that conjugation significantly decreased entropy (ΔS*) and enthalpy of deactivation (ΔH*). The results of thermodynamic analysis for cyclodextrin production from starch indicated that The enthalpy of activation (ΔH*) and free energy of a...
Immobilization of Bacillus subtilis α-amylase and characterization of its enzymatic properties
Microbiological Research, 1999
Bacillus subtilis I a-amylase was immobilized on different carriers by different method s of immobilization. The immobilized enzymes were prepared by physical adsorption on aminoa lkylsilane-alumina (AS-alumina), ionic binding onto DEAE-cellul ose, covalent binding on chitin, and entrapment in polyacrylamide had the highest activities. The specific activity of the immobili zed enzy mes, calculated on bound protein basis, were 13.5-50% of the original specific activity exhibited by the free enzyme. The optimal pH of the immobilized enzymes was shifted to lower values than for the free enzyme. The optimum reaction tempe rature was determined to be 45°C for the free amylase, whereas that for the immobilized enzymes was shifted to 60-65°C. In all cases , K; values for the hydrol ysis of starch of the immobilized enzymes were higher than that of the native enzyme. Compared to the free form , the immobilized enzymes exhibited improved therm al stability and higher values of activation energy of denaturation.
Biochemical Engineering Journal, 2000
Production of cyclodextrin glucosyltransferase (CGTase) enzymes by six bacterial cultures was investigated. Enzyme production was better in shaken than in surface cultures. Bacillus macerans 3 14 and B. amyloliquifaciens cultured on potato dextrose (PD) medium were the most potent strains used. The former strain which produced @cyclodextrin (P-CD) was chosen for further studies. Addition of corn-steep liquor to PD medium afforded the maximal CD yield; nevertheless, it was Q-CD instead of P-CD. Starch addition directed the organism to produce a mixture of (Y-and P-CD. 5% (by volume) inoculum, 72 h incubation period and 37 "C were the most applicable and led to maximal productivity of CGTase by B. macerans 3 14 cultured on PD medium supplemented with 0.3% CaCl,. The lyophilized crude CGTase exhibited maximal activity at an enzyme concentration of 1.65 mg ml-', 2% soluble starch, 60 "C and pH of 6.
Production of cyclodextrin glycosyltransferase by immobilized Bacillus sp. on chitosan matrix
The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol–sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron micro-scopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml-1 (36 h), 47.50 U ml-1 (36 h) and 68.36 U ml-1 (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml-1 (18 h) on cassava, 79.17 U ml-1 (12 h) on potato and 55.37 U ml-1 (in 6 h and max 77.75 U ml-1 in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.