Enzymes have enormous potential to be used as catalysts in a wide range of biotechnological and industrial processes. They offer a distinct advantage due to their specificity, high catalytic efficiency and biodegradability (Bullock, 1995; Munjal and Sawhney, 2002). A common drawback of enzymatic procedures is the consumption of expensive enzyme molecules during the process (Fiorentino et al., 2010). Thus, the industrial exploitation of enzymes is limited due to their non-reusability and instability (Wang et al., 2009). Enzyme immobilization has been found to be a promising way to overcome these limitations and to enhance the catalytic performance of enzymes (Ensuncho et al., 2005; Arica and Bayramoglu, 2006; Bayramoglu et al., 2012). Immobilization of enzyme has several advantages, such as improved stability by protecting the protein from deactivation, high reproducibility, improved thermal and storage stability, significant reduction in the production cost, easy recovery of the enzyme and continuous and repeated use (Cirpan et al., 2003; Yildiz et al., 2013).

Polymer blending is one of the most productive approaches for providing novel and desirable polymeric materials to immobilize enzymes (Tembe et al., 2006). The composite thus prepared, combine the physicochemical attributes of both components and overcome their shortcomings (Gupte and D'Souza, 1999; Jha and D'Souza, 2005). Also this simply-prepared protein–polysaccharide bio-composite film facilitates the enzymes to retain their activity (Forzani et al., 1999, 2000). Biocompatible polymers have their unique advantages for enzyme immobilization, as they aid in retaining the activity of enzyme well by providing desirable micro-environment (Li et al., 2006). Chitosan (2-acetamido-2-deoxy-d-glucose) is an N-deacetylated derivative with chemically modified acetyl group of chitin. Chitosan is widely considered to be a biocompatible, nontoxic and chemically distinctive polysaccharide which occurs as a natural biopolymer in the exoskeleton of crustaceans and in fungal cell wall (Liu et al., 2005). Due to its unique chemical properties, such as excellent membrane-forming ability, high permeability toward water, good adhesion, biocompatibility, non-toxicity, biodegradability, high mechanical strength and susceptibility to chemical modifications due to the presence of reactive amino and hydroxyl functional groups (Cruz et al., 2000), chitosan serves as a matrix for the assembly of biomolecules including enzymes, DNA, and antibodies (Ye et al., 2005; Tan et al., 2009; Xu et al., 2010). Gelatin is also a naturally occurring biopolymer having good adhesion qualities and non-toxicity. It is commonly used as a gelling agent in food, such as marsh mallows, desserts, ice-creams and pharmaceuticals. Also it is commonly used as a biological substrate to culture adherent cells. The composite film consisting of chitosan and gelatin mixed and gelled together, can overcome the brittleness of pure chitosan matrix and its shrinkage. Another advantage of this blend is that both the polymers are biocompatible, transparent, safe, abundant and inexpensive, thereby providing substantial microenvironment for enzyme to be immobilized.

Tyrosinase is a copper containing multifunctional metalloenzyme that catalyzes the hydroxylation of monophenols to o-diphenols (monophenolase or cresolase activity) and the oxidation of the latter to o-quinones (diphenolase or catecholase activity), using oxygen (Labus et al., 2011). The two activities can be used as the basis for widespread biotechnological and industrial applications, such as biosensors to determine the phenolic content in waste water (Atlow et al., 1984), for bioremediation of phenolic effluents and for biotransformation of L-tyrosine to L-DOPA, the preferred drug for Parkinson's disease.

Previously chitosan-gelatin composite formation has been studied by Chen et al. (2003), Basavaraju et al. (2006), and Mi (2005) and its application for food products had been explored by Arvanitoyannis et al. (1998) and Lo'pez-Caballero et al. (2005), but this bio-composite had never been investigated to be used as a matrix for tyrosinase immobilization. However, several workers have endeavored to immobilize tyrosinase onto alginate, polyacrylamide and gelatin gels (Munjal and Sawhney, 2002), ZnO nanoparticles (Li et al., 2006), cellulose support (Labus et al., 2011) and polystyrene microplate (Saini et al., 2014). Moreover, this is the first report of immobilization of tyrosinase from A. niger on any support matrix.

In the present study, tyrosinase from A. niger PA2 had been immobilized onto a bio-composite film consisting of chitosan and gelatin polymers (Figure 1). The prepared films were thoroughly characterized using XRD, FESEM, AFM, and UV-spectroscopy and the efficiency of immobilized tyrosinase was evaluated in terms of stability, reusability and for the production of L-DOPA.

Tyrosinase from A. niger PA2 has been immobilized onto a bio-composite film consisting of chitosan and gelatin polymers. The homogeneity of the film was characterized by X-ray diffraction analyses (Figure 2). The distinct crystalline peaks observed in the diffraction pattern of chitosan were due to the abundance of −OH and −NH₂ groups in the chitosan structure, which could form strong inter and intra molecular hydrogen bonds and thus the chitosan molecules form crystalline regions and gain certain regularity easily (Ramya et al., 2012). While a broad hump observed in XRD pattern of gelatin powder was due to its amorphous morphology. It was also observed that the crystalline peaks of chitosan disappeared as the composite was formed between chitosan and gelatin, which is due to the elimination of hydrogen bonding between amino groups and hydroxyl groups in chitosan (Smitha et al., 2005). Also the broadened hump which was present in gelatin became lower in the composite film, demonstrating an interaction between the two components. To evaluate immobilization efficiency, chitosan-gelatin-Tyr bio-composite films were analyzed by FESEM, AFM and UV-spectroscopy. As observed through FESEM, chitosan-gelatin composite membrane is a cross-linked three-dimensional network with a few smaller sphere particles filled in the mess space (Figure 3). This network like structure could contribute a significant role toward the high enzyme stability within the polymer matrix providing a large surface area for efficient immobilization of enzyme (Liu et al., 2005). When tyrosinase was immobilized onto the film matrix, the network like structure disappeared and instead a regularly distributed aggregated smear was observed. A typical AFM image of the surface of the chitosan-gelatin bio-composite membrane displayed a three-dimensional rough surface (Figure 4), which provided a significantly larger surface area, improving its loading efficiency for the immobilization of biomolecules and other substances and also effectively preventing the leaking of biomolecule from the preparation. The roughness of the film surface is an important aspect that may provide information about the surface modifications after immobilization. It was apparent that the surface of the blank film is less rough than that of immobilized preparation. Increase in the roughness value after adsorption of the enzyme suggests the deposition of enzyme molecules onto the matrix surface (Saini et al., 2014). UV-spectral analysis also depicted the formation of L-DOPA, when the film with immobilized enzyme was immersed in buffer containing L-tyrosine (Figure 5). Soluble enzyme was most stable at 25°C and the stability decreased thereafter after increasing the temperature (Figure 6A), whereas, enzyme following immobilization appears to have increased thermostability at higher temperature i.e., upto 55°C. Munjal and Sawhney (2002) obtained similar results with gelatin gel. They observed temperature optima for free tyrosinase from mushroom at 20°C, while the tyrosinase immobilized onto gelatin gel demonstrated the optima at 40°C. Labus et al. (2011) observed temperature maxima for Agaricus bisporus tyrosinase around 15–25°C, while enzyme immobilized onto cellulose based carrier had shown maxima around 30–40°C. Bayramoglu et al. (2013) obtained the optimum reaction temperature for the immobilized tyrosinase onto biosilica at 45°C, which was 10°C higher than its free counterpart. The comparatively higher thermostability of the immobilized enzyme as compared to the soluble enzyme may possibly be due to its conformational rigidity due to the enzymatic interactions with the matrices (Labus et al., 2011; Bayramoglu et al., 2013) and thus preventing the unfolding (Mateo et al., 2007; Zhang et al., 2011). pH is one of the vital factors influencing the amino acid nature in the protein structure. Free enzyme was observed to be most active at pH 5.0 with a quick drop in its activity at acidic and basic pH. While the pH profile of the immobilized tyrosinase displayed comparatively better activity in a broader pH range (Figure 6B). Labus et al. (2011) observed pH optima with tyrosinase immobilized onto cellulose based carrier around pH 7.0–9.0, while free tyrosinase was most active around pH 6.0–7.0. These results implied that the immobilization led to retention of the enzyme activity at extreme pH due to adsorption of tyrosinase within the support matrix (Monsan, 1978; Bayramoglu and Arica, 2010; Li et al., 2012), thus making this biocatalyst more suitable for industrial exploitation (Bayramoglu et al., 2013). For leaching studies, the ratio of the activities of tyrosinase adsorbed onto the membrane and the activity of free enzyme i.e., before immobilization was calculated. About 72% of activity was retained following immobilization as compared to the free counterpart. It was also expected beforehand that immobilized/free tyrosinase activity ratio should be lower than 1.0 (Labus et al., 2011). Thus, this matrix proves to be a biocompatible composite that provides a natural environment to the enzyme, minimizing it's leaching, retaining the activity and therefore making the immobilization highly productive. Generally there is a gradual decrease in enzyme activity upon storage and immobilization significantly slows down this phenomenon (Figure 7A). The activity of the immobilized tyrosinase decreased more slowly compared to the free tyrosinase under the similar storage conditions. Immobilized enzyme was observed to have 84% activity after 5 days of incubation and was able to maintain more than 72% of its original activity after 10 days, which indicates that the immobilization of the enzymes decreases the likelihood of its denaturation (Zhang et al., 2011). Munjal and Sawhney (2002) also obtained 65–75% retention in enzyme activity with tyrosinase immobilized onto polyacrylamide gel after 14 days of incubation. Bayramoglu et al. (2013) had also observed 80% retention of activity after 14 days of incubation with tyrosinase immobilized onto biosilica. The greater stability of the immobilized enzyme may also be ascribed as an effect of film (Munjal and Sawhney, 2002), which provides a biocompatible microenvironment to the enzyme (Schnapp and Shalitin, 1976). These results are also in agreement with the previous studies done by several groups on enzyme immobilization (Edwards et al., 1999; Bayramoglu et al., 2011; Karagoz et al., 2011; Nicolucci et al., 2011). The reproducibility of the immobilized enzyme preparation is an important attribute for large-scale applications. The immobilized tyrosinase maintained 84% of its activity and produced 1.23 mg ml⁻¹ L-DOPA even following six successive cycles (Figure 7B). Munjal and Sawhney (2002) also obtained 75–85, 60–70, and 45–50% retention in enzyme activity after 5 cycles of reuses with tyrosinase immobilized onto alginate, polyacrylamide and gelatin gel, respectively, whereas, Labus et al. (2011) observed 45–65% retention in Agaricus bisporus tyrosinase activity after 6 reuses when it was immobilized onto cellulose based carriers. Further 80% of the enzyme activity was retained following 7 cycles with tyrosinase immobilized onto polystyrene microplate (Saini et al., 2014). Higher retention of enzyme activity and ability to produce L-DOPA following repetitive usage may be attributed to the effective adsorption of enzyme onto the polymer matrix, thus making it promising and amenable for consistent usage for industrial exploitations.

In the present study, tyrosinase from a fungal source, A. niger PA2 was immobilized onto a composite of two biopolymers: chitosan and gelatin. This is the first report where tyrosinase extracted from A. niger was immobilized onto chitosan-gelatin copolymer matrix and evaluated for its activity and L-DOPA synthesis. Being biocompatible, this unique composite film provided a suitable natural microenvironment, which could successfully offer efficient loading and thus prevent the leaching of the immobilized enzyme. The enzyme adsorbed onto the co-polymer matrix exhibited notably increased thermal and pH stability. The immobilized preparation was stable for 20 days and could be reused for multiple cycles. The reusability of enzyme-co-polymer system may provide cost-effective strategy for large-scale production of L-DOPA. Thus, the proposed blend of the enzyme-biocomposite film may provide a promising and viable system for L-DOPA production and for a wide range of biotechnological applications.

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.