Chitosan CS a deacetylated derivative of chitin was chosen

Chitosan (CS), a deacetylated derivative of chitin, was chosen in this study due to its unique biological characteristics including biodegradability, biocompatibility, non-toxicity, and antimicrobial properties (Klossner, Queen, Coughlin, & Krause, 2008; Kong, Chen, Xing, & Park, 2010; Ravi Kumar, 2000). Furthermore, the successful use of chitosan polymer in many electrospinning studies is well documented (Abdelgawad, El-Naggar, Hudson, & Rojas, 2017; Gudjónsdóttir et al., 2015; Rieger, Birch, & Schiffman, 2016). However, the electrospinnability of CS is generally restricted by its polycationic nature in solution. Indeed, the formation of strong hydrogen bonds inhibits the free movement of polymeric chain, while repulsive forces between ionic groups prevent the formation of sufficient chain entanglements (Duan, Dong, Yuan, & Yao, 2004; Homayoni, Ravandi, & Valizadeh, 2009; Pakravan, Heuzey, & Ajji, 2011). Electrospinning of CS with other polymers is an efficient method to develop properties of its fibers. Polyvinyl alcohol (PVA) is a synthetic polymer being extensively used in biomedical field due to its water solubility, biocompatibility, biodegradability, and significant mechanical properties (Shahzad et al., 2015). The application of PVA in the synthesis of CS-based nanofibers has been reported many times. Recently, electrospun nanofibrous membranes have attracted much attention for enzyme immobilization due to several outstanding properties including large specific surface, providing high enzyme loading, and fine porous structure, making the active sites of enzymes more accessible, as well as reducing the cantharidin synthesis resistance necessary for both high reaction rate and conversion (Huang, Ge, & Xu, 2007; Wong et al., 2014). However, previous studies often used multi-step synthesis methods such as surface modification with cross linkers. El-Aassar (2013) modified poly (An-co-MMA) nanofibers with polyethylenimine before the immobilization of β-galactosidase with glutaraldehyde. Klein et al. (2016) immobilized β-D-galactosidase on chitosan particles modified with genipin, as a crosslinking reagent. Their results showed that the immobilized enzyme maintained 100% of its initial activity after 25 batches of lactose hydrolysis. In another study by Misson, Jin, and Chen, (2015), polystyrene nanofiber (PSNF) was fabricated by electrospinning and functionalized through a chemical oxidation in order to immobilize β-galactosidase. According to their results, the immobilized enzyme increased galactooligosaccharides (GOS) yield from 14 to 28% compared to free enzyme. In previous studies, β-galactosidase was immobilized on the modified chitosan nanoparticles. However, in this study β-galactosidase was immobilized on chitosan/PVA nanofibers fabricated by a one-step electrospinning method for the first time. In the current study, β-galactosidase was added to the polymer solutions and immobilized during electrospinning; hence all the specified amount of enzyme was electrospun. However, according to previous literatures, enzyme immobilization after fabrication of nanofibers might decrease adsorption capacity. In this regard, Misson et al. (2015) showed that the adsorption capacity of immobilized β-Galactosidase on unmodified PSNF was lower (50 mg/g), compared to modified nanofibers (100 mg/g). In this study all the specified amount of enzyme was immobilized on nanofibers. Furthermore, the activity and stability of free and immobilized enzymes could be compared in the same amount.
Materials and methods
Results and discussion
Conclusion Incorporation of enzymes into nano-scale materials has many advantages including high surface area-to-volume ratio of nanomaterials, increased accessible surface area for enzyme incorporation and catalytic efficiency. In this study, β-galactosidase was successfully immobilized onto chitosan (CS)/polyvinyl alcohol (PVA) electrospun nanofibers. Determining the effect of the properties of CS/PVA blend solutions and electrospinning parameters showed that the activity of β-galactosidase nanofiber mats at pH 6.8 and 4 was 57.03% of free enzyme activity and enzyme catalytic activity was not significantly influenced by these factors. Furthermore, immobilized β-galactosidase showed more temperature stability than free enzyme at 50 . Upon 28 days of storage at 4 and 25 , the immobilized enzyme retained 77% and 42% of its initial activity, respectively. The results indicated that β-galactosidase incorporation into CS/PVA blend nanofibers could significantly improve its temperature and storage stability, which are the most important factors in enzyme delivering systems.