Invertase immobilized system

Immobilization of invertase on the Dowex resin was performed as follows The anion exchanger (100 mg dry wt) was previously equilibrated in 22 mL of deionized water (pH adjusted to 5.5 by dropping 1M HC1), and the suspension was left for 24 h under agitation (100 rpm) at 32°C. Then, 3 mL of original solution of Bioinvert was added, and the system was left for 4 h under the same conditions. The complex Dowex/invertase was then centrifuged (2880y, 30 min), and the protein content in the supernatant was measured. The separated Dowex/invertase complex was rinsed until no protein was detectable in the supernatant, and the final suspension was stored at 4°C in deionized water (pH 5.5). 

The activation energy (Ea), calculated through the inclination of log v x T 1 (Eqs. 6 and 7), and the thermodynamic parameters (AH, AS, and AG), calculated through Eqs. 3-5, for both forms of invertase are presented in Table 2. As can be seen, the values of those parameters were quite similar for both soluble and immobilized invertase. This fits very well with the previous assumption that the immobilization technique has little effect on enzyme structure. It could be speculated that the reaction catalyzed by either soluble or insoluble invertase is constituted by identical thermodynamic systems. 

The immobilization of invertase on aluminium hydroxide (2) was one of the earliest reports of adsorption technology. The use of aminoacylase adsorbed on DEAE-Sephadex for producing L-amino acids from a racemic mixture of their corresponding ethyl esters (4) was the first industrial application of an immobilized enzyme system. The basic disadvantage of this convenient technique is that binding is weak and the enzyme slowly leaches out. However, for many purposes, this slow leakage is not an important handicap. Immobilizing enzymes by adsorption has been extensively reviewed (5, 6, 27). Some special approaches are described (1, 28-30). 

The principal application of lectins in bioanalytical systems involves the reversible immobilization of glucose oxidase, invertase, and peroxidase on Con A-Sepharose. Such lectin-based affinity media have also been utilized for immobilization of glycoenzymes. Woodward (18) shows that cellobiase is not desorbed by its substrate cellobiose and product glucose from the support matrix. 

Enzyme thermistors have also found applications in more research-related topics, such as the direct estimation of the intrinsic kinetics of immobilized bio-catalysts [64]. Here, the enzyme thermistor offered a rapid and direct method for the determination of kinetic constants (K , Km and Vm) for immobilized enzymes. For the system being investigated, saccharose and immobilized invertase, the results obtained with the enzyme thermistor and with an independent differential reactor system were in very good correlation, within a flow-rate range of 1 to 1.5 ml/min. 

Stefuca V, Gemeiner P, Kurillova L, Danielsson B, Bales V (1990) Application of the enzyme thermistor to the direct estimation of intrinsic kinetics using the saccharose-immobilized invertase system. Enzyme Microb Technol 12 830 - 835 Stefuca V, Welwardova A, Gemeiner P, Jakubova A (1994) Application of enzyme flow microcalorimetry to the study of microkinetic properties of immobilized biocatalyst. Biotech-nolTech 8 497-502... 

Immobilization of cholesterol esterase and cholesterol oxidase made it possible to measure the total cholesterol content in blood serum [72-74]. Nevertheless, the long-term stability of the bienzyme system was limited by the degradation of the cholesterol esterase activity. Last, a PPy film containing up to three enzymes, namely GOD, invertase, and mutarotase, has been developed for the determination of saccharose [91]. 

Docolomansky, P., Breier, A., Gemeiner, P., and Ziegelhoifer, A., Screening of binding properties of con-A immobilized on bead cellulose by flow microcalorimetry using invertase and anti-con-A antibody as reporting systems. Anal. Lett., 1995, vol. 28, no. 15, pp. 2585-2594. 

D-glucose and the three-enzyme system GOx, mutarotase and invertase for sucrose estimation. A common format was adopted to facihtate design and operation, in this case immobilization method, the fact that all enzymes used were oxidases and that a common detection principle, reoxidation of H2O2 generated product, was chosen (except for ascorbic acid which was estimated directly). Pectin, a natural polysaccharide present in plant cells, was used as a novel matrix to enhance enzyme entrapment and stabilization in the sensors. Interferences related to electrochemi-caUy active compounds present in fruits under study were significantly reduced by inclusion of a suitable cellulose acetate membrane diffusional barrier or by enzymatic inactivation with ascorbate oxidase. Enzyme sensors demonstrated expected response with respect to their substrates, on analyte average concentration of 5 mM. 

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