# Immobilization of Pectinolytic Enzymes on Nylon 6/6 Carriers
## 1 Introduction  
Pectinolytic enzymes, also known as pectinases, are a heterogeneous group of enzymes catalyzing different steps of the hydrolysis of pectin, a major component of plant cell walls [1]. Based on their mode of action, pectinases are classified as (i) de-esterifying enzymes (pectin esterase), and (ii) de-polymerizing enzymes (hydrolaseses, lyases). De-polymerizing enzymes are further classified based on their specific action site to endo- and exo-polygalacturonases and rhamnoadematuronases [2], or separated as per the nature of action mechanism, primary substrate, and products [3]. Pectinases have a wide range of applications in different industries including food, textile, and paper industries. Microbial pectinases are one of the most commercially produced enzymes, with 25% share in the global food and beverage enzyme market [4]. Pectinases are considered as a sustainable and environmentally-friendly industrial catalyst as they are produced by fungi (_Aspergillus_, _Penicillium_, _Trichoderma_, _Rhizomucor_, etc.) and bacterial (_Bacillus_, _Streptomyces_, _Erwina_) fermentation of different by-products like wheat bran, citrus peels, and sugar beet pulp [2, 5]. Commercial pectinase preparations are widely utilized in the extraction and clarification of fruits and vegetables juices [6]. They are also used for the extraction of vegetable oil [7] and the fermentation of coffee beans and tea leaves [2]. Moreover, along with cellulases, pectinases are an important tool for 'green' enzyme-assisted extraction of bioactive compounds from different plant matrices including citrus peel, carrot pomace, tomato, etc. [8, 9]. Several studies have highlighted the attractiveness of pectinases' immobilization allowing multiple reuses and increasing the efficiency of production processes, easy separation of the enzyme from the final product [10], but also the development of continuous automated processes particularly relevant in juice production and winemaking [11]. Various techniques have been used for the immobilization of different pectinase preparations including adsorption [12; 13; 14], entrapment [15; 16; 17], covalent binding to insoluble carriers [18; 19; 20; 21], as well as to magnetic nanoparticles [22; 23; 24; 25]. Despite the variety of immobilization methods used, the most stable enzyme coupling is achieved using covalent attachment. Commonly, enzymes are coupled via their amino groups, but other functional groups like carboxylic, thiol, sulfhydryl, and other groups can be also used. Carriers for the immobilization of pectinase and other enzymes which are used in food industry must be non-toxic, biocompatible, and insoluble under reaction conditions, and thermally stable [26]. Functional groups for protein binding onto an insoluble carrier are usually generated through chemical activation of the support surface. In addition, linker molecules (e.g., glutaraldehydehyde, carbodiimide, etc.) are often used to enhance enzyme flexibility and activity [27]. For the technological production of immobilized enzymes, important criteria are the durability of the carrier, but also its availability and low cost [26]. Nylon 6/6 is an interesting, inexpensive, and convenient carrier for enzyme immobilization. It is inert, non-toxic, mechanically strong, and readily available in different forms (pellets, discs, threads, fabric, etc.) [28; 29]. The inertness of nylon 6/6 makes it unable to bind proteins as it is, so it should be activated by partial acid hydrolysis or O-alkylation with dimethyl sulfate (DMS) [30]. Nylon 6/6 activation generates free amino (-NH2) and carboxyl (-COOH) groups on its surface, allowing the covalent binding of enzymes using glutaraldehydehyde as a crosslinking agent [31]. In the present study, we used nylon 6/6 pellets and threads as carriers for the immobilization of multi-enzyme pectinase commercial preparation used for degrading fibrous plant material and providing higher juice yields and clarity.