Regularities of adsorption immobilisation of inulinase on fibrous polyelectrolytes AK-22, AK-22-1, K-1, K-4, K-5
Abstract
Inulinases (EC 3.2.1.7 and 3.2.1.80) are hydrolytic enzymes which break down β-1,2-fructose bonds. These biocatalysts are widely used in the industrial production of fructose, fructose syrups, and fruc-tooligosaccharides. However, the application of the enzymes in free forms has several disadvantages: they have low thermal stability, it is difficult to separate the catalyst molecules and the final product, and they are cannot be reused. The indicated problems can be solved by immobilising the enzymes on insoluble carriers.Therefore, the aim of our research was to study the patterns of the adsorption immobilisation of inulinase on fibrous polyelectrolytes.
The study of the adsorption immobilisation of inulinase was performed on fibrous polyelectrolytes AK-22, AK-22-1, K-1, K-4, K-5. We investigated the effect of the duration of the sorption process on the amount of immobilised protein and the activity of the heterogeneous catalyst. Adsorption reaches its maxi-mum after 4 hours, when the concentration of the inulinase solution is 1 ∙ 10-7 mmol/l and after 2 hours, when the concentration is 5 ∙ 10-7 and 1 ∙ 10-6 mmol/l. The highest catalytic ability of the immobilised enzyme cor-responds to the largest amount of absorbed protein. The activity of a heterogeneous biocatalyst is from 44.5 to 71.2 % of the activity of the native enzyme.
The study determined that at the initial moment of immobilisation, the active formation of protein associates takes place. As a result its sorption increases sharply, and a peak appears on the kinetic curve. However, over time, the enzyme is released into the solvent phase, and a decrease is observed on the kinetic curve. A desorption process at this stage is possible due to the low interaction energy between the sorbate and the sorbent molecules and, consequently, the weak binding of the protein molecules to the carrier polymer. Then, over time, the adsorption becomes irreversible. Thus, the structure of the formed polyelectrolyte com-plexes, as well as their composition changed with an increase in the duration of the reaction: during the re-structuring of the complexes, some of the protein molecules are released into the solution. Associates of in-ulinase macromolecules (at the concentrations of 1∙10-7, 5∙10-7, and 1∙10-6 mmol/l), are apparently sorbed only on the surface of the carrier, which ensures the availability of active centres of the enzyme and, as a conse-quence, high catalytic activity of the immobilised enzyme preparation.
IR spectroscopy demonstrated that inulinase immobilisation occurs due to the implementation of weak hydrogen bonds and electrostatic interactions of the enzyme and the carrier polymer.
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