Feruloyl esterase immobilization in mesoporous silica particles and characterization in hydrolysis and transesterification.

BACKGROUND: Enzymes display high reactivity and selectivity under natural conditions, but may suffer from decreased efficiency in industrial applications. A strategy to address this limitation is to immobilize the enzyme. Mesoporous silica materials offer unique properties as an immobilization support, such as high surface area and tunable pore size. RESULTS: The performance of a commercially available feruloyl esterase, E-FAERU, immobilized on mesoporous silica by physical adsorption was evaluated for its transesterification ability. We optimized the immobilization conditions by varying the support pore size, the immobilization buffer and its pH. Maximum loading and maximum activity were achieved at different pHs (4.0 and 6.0 respectively). Selectivity, shown by the transesterification/hydrolysis products molar ratio, varied more than 3-fold depending on the reaction buffer used and its pH. Under all conditions studied, hydrolysis was the dominant activity of the enzyme. pH and water content had the greatest influence on the enzyme selectivity and activity. Determined kinetic parameters of the enzyme were obtained and showed that Km was not affected by the immobilization but kcat was reduced 10-fold when comparing the free and immobilized enzymes. Thermal and pH stabilities as well as the reusability were investigated. The immobilized biocatalyst retained more than 20% of its activity after ten cycles of transesterification reaction. CONCLUSIONS: These results indicate that this enzyme is more suited for hydrolysis reactions than transesterification despite good reusability. Furthermore, it was found that the immobilization conditions are crucial for optimal enzyme activity as they can alter the enzyme performance.

Optimization of a dual-functional biocatalytic system for continuous hydrolysis of lactose in milk.

In this study, an amino-functionalized silica matrix encapsulating ß-galactosidase was first synthesized in situ, with subsequent covalent anchoring of lysozyme to the outer part of the amino-grafted matrix. Fourier transform infrared (FTIR) spectra verified that ß-galactosidase was successfully encapsulated. Meanwhile, the co-immobilized enzymes were demonstrated to retain suitable enzymatic activities and outstanding operational stability during successive reaction cycles. Furthermore, when used for lactose removal from skim milk, the packed-bed column system achieved both a high lactose hydrolysis rate and microbial inactivation ratio during 30 days of continuous operation. Notably, this system exhibited favorable stability during 60 days of continuous hydrolysis of lactose in solution and thus may be appropriate for further development for use in industrial lactose removal from milk.

Immobilization of invertase in conducting polymer matrices.

This paper reports a novel approach in the electrode immobilization of an enzyme, invertase, by electrochemical polymerization of pyrrole in the presence of enzyme. The polypyrrole/invertase and polyamide/polypyrrole/invertase electrodes were constructed by the entrapment of enzyme in conducting matrices during electrochemical polymerization of pyrrole. This study involves the preparation and characterization of polypyrrole/invertase and polyamide/polypyrrole/invertase electrodes under conditions compatible with the enzyme. It demonstrates the effects of pH and temperature on the properties of enzyme electrode. Enzyme leakage tests were carried out during reuse number studies. The preparation of enzyme electrodes was done in two different electrolyte/solvent systems. The enzyme serves as a sucrose electrode and retains its activity for several months.

Electrochemically mediated electrodeposition/electropolymerization to yield a glucose microbiosensor with improved characteristics.

A procedure is described that provides for electrochemically mediated deposition of enzyme and a polymer layer permselective for endogenous electroactive species. Electrodeposition was first employed for the direct immobilization of glucose oxidase to produce a uniform, thin, and compact film on a Pt electrode. Electropolymerization of phenol was then employed to form an anti-interference and protective polyphenol film within the enzyme layer. In addition, a stability-reinforcing membrane derived from (3-aminopropyl)trimethoxysilane was constructed by electrochemically assisted cross-linking. This hybrid film outside the enzyme layer contributed to the improved stability and permselectivity. The resulting glucose sensor was characterized by a short response time (<4 s), high sensitivity (1200 nA/mM x cm2), low interference from endogenous electroactive species, and working lifetime of more than 50 days.

Comparison of chitin and Amberlite IRA-938 for alpha-galactosidase immobilization.

Watermelon alpha-galactosidase (EC 3.2.1.22) was immobilized on a natural (chitin) and a synthetic anion-exchange (Amberlite IRA-938) support by covalent coupling methods. The procedure entails the activation of supports with 1,1'-carbonyldiimidazole (CDI), followed by immobilization of the enzyme on to these supports without and with a spacer arm; gamma-aminobutyric acid (GABA). Optimization of activation was performed by changing the CDI concentrations and coupling efficiencies. The comparison of two immobilization techniques for both chitin and Amberlite IRA-938 was made by comparing different enzyme concentrations against enzyme activity yield. Furthermore, the storage stability of the immobilized enzymes was also investigated and chitin immobilized alpha-galactosidase was found to be better. Although the activity yield of immobilized enzymes were the same for both supports, the short storage stability of immobilized enzyme on Amberlite IRA-938 is currently a drawback to its applications.