Development of a technique for the adsorption immobilization of trypsin on ion-exchange resins
Abstract
Trypsin (EC 3.4.21.4) is the most popular enzyme in industry and biomedicine. However, the impossibility of reuse and the complexity of recovery make its large-scale industrial use ineffective and expensive. This problem can be solved by immobilizing trypsin on ion-exchange materials. The aim of this study was investigation of the regularities of the adsorption immobilization of trypsin on ion-exchange resins for creation of a heterogeneous preparation based on it, available for use in domestic laboratories and industry.
A technique for the immobilization of trypsin on different types of ion exchange resins: cationic resins with functional groups –SO3H (KU-2, KU-2-8chS, IMAC-HP111), anionic resins with active groups
–N+(CH3)3 (AV-17-2P, Purolite A100), secondary and tertiary aliphatic amino groups and pyridine groups (AB-16-GS), secondary, tertiary and quaternary aliphatic amino groups (EDE-10-P) was proposed. The preparation of ion exchange resins and the sorption immobilization of trypsin were carried out according to standard methods. The protein content in the immobilized trypsin preparations was measured according to a modified Lowry method, the protease activity of the samples was determined using azocasein as the substrate, and the esterase activity was determined using N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as the substrate.
The obtained preparations of trypsin immobilized on ion-exchange resins can become the basis for solving a number of problems arising in the production of sorbents for waste water treatment, in the separation and purification of various substances in the chemical industry, as well as in the study of nutrient flows and fertilization in the fields.
It was found that the optimal ratio of the protein content (mg per g of the carrier), the total protease activity (units per ml of solution) and the specific protease activity (units per mg of protein) was observed when trypsin was immobilized on the EDE-10-P carrier with a phosphate buffer, pH 11.0 and NaOH-KCl buffer, pH 12.0. The optimal ratio of protein content, total esterase activity and specific esterase activity was obtained by adsorption of trypsin on AV-16-GS with NaOH-KCl buffer, pH 12.0.
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