Structural and functional properties of trypsin under conditions of immobilization on poly-N,N-dimethylaminoethyl methacrylate

Abstract

The structural and functional properties of trypsin have been studied upon its immobilization on poly-N,N-dimethylaminoethylmethacrylate (pDMAEMA). Trypsin, which belongs to the group of serine proteases, plays a key role in many biological processes and is used in various industries such as biotechnology and medicine. Despite its widespread use, trypsin has a number of disadvantages, including instability and inability to be reused, which reduces the overall efficiency of processes and shortens the shelf life of preparations. A way of solving the above mentioned problems is to immobilize enzymes to increase their stability, half-life and shelf life. In this work, the object of study was trypsin, which was immobilized on pDMAEMA, a weak cationic polyelectrolyte with unique properties, such as the transition from hydrophilic to hydrophobic state under the influence of external stimuli, such as temperature and pH. The process of synthesizing pDMAEMA involved the use of N,N-dimethylaminoethyl methacrylate and N,N-dimethylformamide, which were pre-purified by vacuum distillation. The reaction was carried out at 60 °C for five hours with the addition of the initiator azo-bis-isobutyronitrile. The resulting polymer was isolated by lyophilization to constant sample weight. Trypsin immobilization was carried out in two steps: preparation of trypsin solution in polymer buffer solution with addition of cysteine and incubation of the obtained solution with resorcinol solution at 50 °C. The process was completed by full drying of the prepared drug. Various methods including catalytic activity measurement, IR spectroscopy and molecular docking were used to study the characteristics of immobilized trypsin. The results showed that immobilization of trypsin on PDMAEMA increased its catalytic activity by ~1.5-fold, which was attributed to the formation of a specific microenvironment and improved conditions for the reaction to proceed. Analysis of IR spectra confirmed the presence of hydrogen bonds and hydrophobic interactions between trypsin and pDMAEMA, indicating the importance of these types of physicochemical interactions in the immobilization process. Molecular docking enabled the identification of key amino acids involved in the formation of the complex and confirmed the presence of salt bridges. Thus, this work demonstrates the potential of using PDMAEMA as a carrier for trypsin immobilization, which opens new opportunities for the application of this complex in biotechnology and medicine.