Use of Polyurethane-Based Materials in Enzyme Immobilization

1. Polyurethanes and Biomedical Applications

Polyurethanes are a type of polymer containing repeating urethane groups (1). Urethane bonds form through the reaction of isocyanates and polyols. During the preparation and curing of polyurethanes, various reactions occur alongside urethane bond formation, creating diverse linkages such as allophanate, biuret, acyl urea and isocyanurate, leading to increased branching and cross-linking (2, 3). It is common practice to blend polyurethane materials with other polymers to enhance properties such as quality, strength and even biocompatibility. Polymer composite materials, being produced from two or more components with significantly different physical or chemical properties, possess superior properties distinct from individual components. Each material used remains separate and distinct within the composite structure (4). Furthermore, the preparation of polyurethane-based materials using polyols obtained from renewable sources (polysaccharides, proteins, antioxidants, etc.) is currently being extensively researched (Figure 1). [caption id="attachment_105153" align="aligncenter"] Figure 1. Synthesis of bio-based polyurethane materials using renewable sources (5)[/caption]   Polyurethanes, which possess diverse structure and properties, are among the well-known high-biocompatibility materials today. Along with their broad range of applications, there are critical considerations related to the material (6). There should be no solvents, monomers, chain extenders or other chemicals that could cause toxic effects in the body. They should possess properties such as durability, elasticity, elastomer-like character, fatigue resistance, compliance and biocompatibility during healing (7, 8). Polyurethanes bearing many of these properties are used in various biomedical fields including tissue engineering, wound dressings, cardiovascular therapy, drug delivery systems and enzyme immobilization (9).

2. Enzyme Immobilization

Enzymes are known as catalysts characterized by high specificity and activation, performing various processes on biomolecules very effectively and efficiently (10). Due to their high substrate specificity, ease of production and environmental friendliness, they are used by various fields such as biomedical, food and industrial sectors (11). The widespread use of enzymes in working fields has led to the development of new techniques to improve enzyme properties. The most important of these techniques is enzyme immobilization, which is based on binding enzymes to a solid support that is insoluble in the reaction medium (12). The greatest advantage of immobilization compared to other methods is that it can significantly improve enzyme stability under various reaction conditions and increase reusability by making them resistant to sequential catalytic cycles (13). As matrix or support systems for enzyme immobilization, inert polymers and inorganic materials are generally used. There are certain properties that an ideal carrier matrix should possess. These can be listed as cost-effectiveness, inertness, stability, ability to enhance enzyme activity, renewability, prevention of non-specific adsorption and bacterial contamination. In this article, we focus on the use of polyurethanes in enzyme immobilization due to their high mechanical properties and biocompatibility. Studies prepared using polyurethanes in enzyme immobilization are presented under the heading "Applications of Polyurethane-Based Materials in Enzyme Immobilization". [caption id="attachment_105154" align="aligncenter"] Figure 2. Schematic representation showing the detailed procedure for preparation of Fe(III)-PU/AOPAN/β-CD nanofiber membrane with immobilized laccase (14)[/caption]   Results obtained from various experiments show that the laccase enzyme was successfully immobilized on the nanofiber membrane. The immobilization amount of laccase reached a high value of 186.34 mg/g and exhibited excellent catalytic activity. Additionally, immobilized laccase showed significant resistance to changes in parameters such as temperature and pH compared to free laccase. When compared with other carriers used for enzyme immobilization, the PU/AOPAN/β-CD nanofiber membrane exhibited much higher mechanical properties, morphological stability and regeneration capacity. Considering these results, we can say that the PU/AOPAN/β-CD composite nanofiber membrane is an innovative and high-performance carrier support material for enzyme immobilization (14). In their study, Bresolin and colleagues aimed to immobilize lipase enzyme onto polyurethane foams (PUF) prepared using the green synthesis method. First, they obtained PUF through enzymatic glycerolysis between coconut oil and glycerol. They then used it as a support material for lipase immobilization. The immobilization efficiency was found to be approximately 94%. Both immobilized and free lipase were tested in different solvents (methanol, ethanol, propanol), pH values, temperatures and in the presence of various ions (Na+, Mg2+, Ca2+). While free lipase activity lost 80% at different pH conditions, immobilized lipase lost 22%. Under high temperature conditions, free enzyme activity decreased by 50% while immobilized lipase retained 95% of its activity. Additionally, in the presence of solvents, free lipase was more tolerant to methanol and ethanol while immobilized lipase showed greater tolerance to propanol. [caption id="attachment_105155" align="aligncenter"] Figure 3. Schematic representation of synthesized polyurethane materials (16)[/caption] Following the experiments, immobilized lipase retained 95.298.6% of its initial activity even after 25 days of storage. Furthermore, the best results were achieved with 90% immobilization efficiency and maltogenase adsorption method on AuNPs. Additionally, AuNPs showed less participation in clustering compared to Ag-NPs. Taking all these into account, PU metal conjugates can be considered suitable materials for enzyme immobilization (16). Cui and colleagues developed an effective method to immobilize lipase on PUF with different modifications. First, PUF surface was treated with HCl to expose active groups, and lipase immobilized on PUF-HCl showed higher activity compared to PUF. Subsequently, PUF-HCl was modified with AA/polyethylenimine (PEI). PEI is a polymer containing primary, secondary and tertiary amino groups with strong ion exchange capacity under a wide range of conditions and the ability to react with different parts on an enzyme or support. Since the isoelectric point of lipase is at pH 5.4, it was easily immobilized with PUF-HCl-AA/PEI. The procedures performed are shown in Figure 4. According to the results obtained, the highest activity was observed in PUF-HCl-AA/PEI. The activity observed in PUF-HCl-AA/PEI was found to be 2.41 times the initial ratio. Finally, immobilized lipase was used for the synthesis of vitamin A palmitate. It was observed that immobilized lipase could be used up to 18 times in a 3 L bioreactor with a conversion rate exceeding 90% over 12 hours. In conclusion, this study demonstrates that a moderate polyurethane micro-environment is important for increasing the activity and stability of immobilized lipase (17). Facin and colleagues investigated different methods of Thermomyces lanuginosus lipase immobilization on PUF. PU polymer was synthesized using polyether and toluene diisocyanate as monomers and lipase was immobilized. Before and after immobilization, the PU matrix was characterized by various methods. The immobilized enzyme exhibited stability over 24 hours within pH (7 and 9) and temperature (24, 50 and 60°C) ranges and storage stability, retaining 80% activity over 30 days. Results from this study showed that the best results in terms of immobilization efficiency were obtained through covalent binding using the capture method and dopamine as a ligand. Similar Vmax and Km results for both free and immobilized enzyme showed that immobilization did not affect enzyme affinity. Additionally, regarding temperature, pH, solvent and storage stability, lipase immobilized on PU-based support gave similar results to free enzyme. The application and use of immobilized enzyme in soybean oil hydrolysis allowed five reuse cycles. Therefore, although PU does not improve enzyme properties against temperature and pH, it can be considered as a low-cost matrix for immobilization (18). Cipolatti and colleagues synthesized PU with polyethylene glycol (PEG) using mini-emulsion polymerization and presented it as a support material for enzyme immobilization. To examine the effects of molar weight and PEG concentration in using enzyme as support material, three PEGs with different molecular weights (400, 4000 and 6000 Da) were used. Subsequently, Thermomyces lanuginosus lipase (TLL) was immobilized. The TLL-PU-PEG6000 immobilized system showed high catalytic results. It demonstrated stability at 50°C and over a wider pH range and led to high ethyl ester production. The relationship between enzyme immobilization and PEG molecular weight is shown in Table 1. In conclusion, since TLL-PU-PEG 6000 has the highest values in ethyl ester production, it can be considered a potential low-cost catalyst for ethanolysis reactions in solvent-free systems (19). Creczynski-Pasa and colleagues in their study immobilized purified horseradish peroxidase (HRP) by functionalizing with PEG NPs. The effect of immobilization on the catalytic activity was evaluated by processing and morphological analyses. The immobilization process of HRP on PU-PEG NPs, as expected, increased the reusability of the enzyme. Additionally, after 50 days of storage, 50% of the original activity was retained. Figure 5 is a schematic representation of HRP immobilized on PU-PEG. In this study, dopamine recovery with the modified carbon electrode ranged from 97% to 107%, which is of great importance in terms of the total test time of approximately 2 minutes, ease of preparation and usability up to 400 assays. Finally, we can say that PU-PEG NPs are a suitable material for enzyme immobilization and the use of carbon electrode for dopamine assay is extremely advantageous even at low detection limits (20). Hooda and colleagues in their study immobilized chitinase purified from Vigna mungo and N-acetyl B-glucosaminidase purified from Canavalia ensiformis on a PU/zinc oxide NPs composite matrix with conjugation efficiencies of 0.785 and 96.19 respectively and retention of specific activity. Structural and morphological analyses confirmed the presence of ZnO NPs and enzymes on the PU matrix. The synthesized PU/ZnO/Chitinase/NAGase conjugate was used as an optical biosensor for chitin determination in wheat grains by determining optimum temperature, pH and substrate concentrations. The detection limit was found to be 0.01 mM with linearity of 0.1 to 10 mM. The recovery percentage of added chitin was found to be approximately between 95% and 96.5%. This method showed good correlation with the 3,5-dinitrosalicylic acid method. PU/ZnO/Chitinase/NAGase showed good thermal and stock stability and was found to be reusable 10 times without significant loss of activity. [caption id="attachment_105159" align="aligncenter"] Figure 6. Co-immobilization of Chitinase/NAGase on PU/nano ZnO composite. The reaction between urethane groups on PU and amine and/or hydroxyl groups on the enzyme is shown in the box (21)[/caption]   In summary, two chitinolytic enzymes, chitinase and NAGase, were co-immobilized on PU/ZnO nano-hybrid support and used to develop and implement an absorption-based optical biosensor for determining chitin contents in stored wheat grains. Broader pH and temperature range for activity, acceptable values for kinetic parameters and improved thermal and storage stability of PU/ZnO/Chitinase/NAGase conjugates as compared with free enzymes validated the suitability of the support. The effectiveness of conjugates in detecting chitin in stored wheat flour is evidenced by significant improvements in detection limit, acceptable values and variation coefficient and determination coefficient for analytical recovery. Overall, results for chitin determination were consistent, reliable and reproducible. We can say that this method can be suitably used without any complex equipment and on a routine basis to control the quality of grain products (21). In their study, Migliardini and colleagues, starting from a biomimetic approach, developed an interesting CO2 capture strategy by immobilizing carbonic anhydrase (CA, EC 4.2.1.1) on PUF. Biomimetic uses microorganisms that sequester from metabolic pathways or using an enzyme such as carbonic anhydrase. In the study conducted, the CA enzyme called SspCA was purified from Sulfurihydrogenibium yellowstonense. SspCA was immobilized onto PUF and a bioreactor containing PU-SspCA was used for experimental tests aimed at verifying CO2 retention capacity under conditions close to a power plant application. In this bioreactor, a gas phase containing CO2 was brought into contact with a liquid phase under conditions where CO2 present in the gas phase was absorbed and efficiently converted to bicarbonate by CA. The enzyme immobilized on PUF retained activity at 100°C and was stable for up to 50 hours. Additionally, it maintained stability for 1 month at room temperature. Moreover, PU-SspCA showed good CO2 capture performance when used in the bioreactor reported in the article. Considering these results, thermostable PU-SspCA represents a strong candidate for CO2 hydration through biomimetic approach (22).

4. Discussion and Conclusion

Enzymes are biomolecules with wide-ranging applications from the biomedical field to the industrial field. However, high usage costs, inability to be reused and susceptibility to environmental conditions bring new strategies to ensure enzyme stabilization and improve application conditions. As seen from the literature studies examined in this article, immobilization of enzymes onto polyurethane matrices or support materials is a very important method in stabilizing enzymes. The high mechanical properties and biocompatibility of polyurethanes make the use of immobilized enzymes in industrial and biomedical fields favorable. For this reason, studies on enzyme immobilization using polyurethane materials alone or as composites with various polymers or nanomaterials continue to increase.

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  Prof. Dr. Burhan Ateş İnönü University Faculty of Arts and Sciences Department of Chemistry Gamze Dik İnönü University Faculty of Arts and Sciences Department of Chemistry Ahmet Ulu İnönü University Faculty of Arts and Sciences Department of Chemistry