Covalent biofunctionalization of chitosan nanofibers with trypsin for high enzyme stability

Abstract

The electrospun chitosan nanofibers provide excellent material for immobilized proteolytic enzymes, and are biocompatible, nontoxic and hydrophilic matrices with large specific area. This paper deals with an application of electrospun chitosan nanofibers and optimizing conditions for their biofunctionalization by model proteolytic enzyme trypsin. Nanofibers from chitosan were prepared using Nanospider TM technology and covalent immobilization of trypsin followed. Three immobilization techniques preserving biocompatibility and utilizing amine and/or hydroxyl groups of chitosan were optimized and compared to simple adsorption to achieve maximum proteolytic activity per cm 2 of the functionalized chitosan nanofibers (Tryp-NF). Significant differences were observed. Trypsin immobilized by the carbodiimide one-step protocol demonstrated the highest activity of the three procedures, ranging from 132 to 210 IU/cm 2 (i.e., 548-874 IU/mg of nanofibers), depending on the initial amount of trypsin used. Long-term storage stability together with high reusability of Tryp-NF confirmed advantages of the immobilized enzyme. Tryp-NF showed no cytotoxicity toward growth of HeLa cells. The in vivo tests for irritation and skin sensitization demonstrated no undesirable skin reactions.

Mechanism of Action of Electrospun Chitosan-Based Nanofibers against Meat Spoilage and Pathogenic Bacteria

“…Electrospinning of chitosan in the form of nanofibers is a promising process that has attracted much interest lately and has been the subject of recent studies [ 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. The high surface area to weight ratio of the nanofiber mats, their biocompatibility, porosity, small diameter—similar to collagen fibers—and their functional properties make them particularly attractive for various applications such as tissue engineering [ 24 ], wound dressings [ 25 ], controlled drug release and gene delivery [ 26 ], water filtration [ 27 ], enzyme immobilization [ 28 ], as well as biosensors in the scope of diagnosis [ 24 ].…”

Biopolymer Matrices Based on Chitosan and Fibroin: A Review Focused on Methods for Studying Surface Properties

“…Conventional films are produced by casting followed by solvent evaporation. These methods make it possible to include biologically active compounds, enzymes, and other protein compounds in their structure [62,63]. For improve the water-resistance and mechanical properties of proteins and polysaccharides, are used chemical crosslinking methods.…”

Imobilização Enzimática: Conceito E Efeitos Na Proteólise

“…Para o procedimento com enzima imobilizada vários suportes têm sido descritos na literatura tais como, partículas magnéticas, 63,64 microchips, 16 sílica mesoporosa, 65-67 nanofibras 68,69 dentre outros. [68][69][70][71] O emprego destes suportes apresenta vantagens devido à elevada área superficial, o que garante a imobilização de grande quantidade de enzima; fácil preparo e manuseio em relação à separação dos peptídeos obtidos; redução no tempo de digestão e aumento na estabilidade da enzima. 16,[63][64][65][66][67][68] Assim como na abordagem on-line, os métodos off-line também necessitam de um pré-tratamento da amostra antes do contato com a protease.…”