Carbonic Anhydrase Carrying Electrospun Nanofibers for Biocatalysis Applications

Background: Enzymes are efficient biocatalysis that catalysis a large number of reactions due to their chemical, regional, or stereo specifities and selectivity. Their usage in bioreactor or biosensor systems has great importance. Carbonic anhydrase enzyme catalyzes the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid. In organisms, the CA has crucial roles connected with pH and CO2 homeostasis, respiration, and transport of CO2/bicarbonate, etc. So, immobilization of the enzyme is important in stabilizing the catalyst against thermal and chemical denaturation in bioreactor systems when compared to the free enzyme that is unstable at high temperatures and extreme pH values, as well as in the presence of organic solvents or toxic reagents. Nano-scale composite materials have attracted considerable attention in recent years, and electrospinning based all-nanocomposite materials have a wide range of applications. In this study, electrospun nanofibers were fabricated and used for the supporting media for carbonic anhydrase enzyme immobilization to enhance the enzyme storage and usage facilities. Objective: In this article, our motivation is to obtain attractive electrospun support for carbonic anhydrase enzyme immobilization to enhance the enzyme reusability and storage ability in biocatalysis applications. Methods: In this article, we propose electrospun nanofibers for carbonic anhydrase carrying support for achieving our aforementioned object. In the first part of the study, agar with polyacrylonitrile (PAN) nanofibers was directly fabricated from an agar-PAN mixture solution using the electrospinning method, and fabricated nanofibers were cross-linked via glutaraldehyde (GA). The morphology, chemical structure, and stability of the electrospun nanofibers were characterized. In the second part of the study, the carbonic anhydrase enzyme was immobilized onto fabricated electrospun nanofibers. Then, enzyme activity, the parameters that affect enzyme immobilization such as pH, enzyme amount, immobilization time, etc. and reusability were investigated. Results: When the scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) analysis results are combined in the characterization process of the synthesized electrospun nanofibers, the optimum cross-linking time is found to be 8 hours using 5 % glutaraldehyde cross-linking agent. Then, thermal stability measurements showed that the thermal stability of electrospun nanofibers has an excellent characteristic for biomedical applications. The optimum temper-ature value was found 37°C, pH 8 was determined as an optimum pH, and 100 ppm carbonic anhydrase enzyme concentration was found to be optimum enzyme concentration for the carbonic anhydrase enzyme immobilization. According to the kinetic data, carbonic anhydrase immobilized electrospun nanofibers acted as a biocatalyst in the conversion of the substrate to the product in 83.98 %, and immobilized carbonic anhydrase enzyme is reusable up to 9 cycles in biocatalysis applications. Conclusion: After applying the framework, we get a new biocatalysis application platform for carbonic anhydrase enzyme. Electrospun nanofibers were chosen as the support material for enzyme immobilization. By using this approach, the carbonic anhydrase enzyme could easily be used in the industrial area by cost-effective advantageous aspects.

A Theoretical Approach for the Electrochemical Characterization of Ciliary Epithelium

The ciliary epithelium (CE) is the primary site of aqueous humor (AH) production, which results from the combined action of ultrafiltration and ionic secretion. Modulation of ionic secretion is a fundamental target for drug therapy in glaucoma, and therefore it is important to identify the main factors contributing to it. As several ion transporters have been hypothesized as relevant players in CE physiology, we propose a theoretical approach to complement experimental methods in characterizing their role in the electrochemical and fluid-dynamical conditions of CE. As a first step, we compare two model configurations that differ by (i) types of transporters included for ion exchange across the epithelial membrane, and by (i) presence or absence of the intracellular production of carbonic acid mediated by the carbonic anhydrase enzyme. The proposed model configurations do not include neurohumoral mechanisms such as P2Y receptor-dependent, cAMP, or calcium-dependent pathways, which occur in the ciliary epithelium bilayer and influence the activity of ion transporters, pumps, and channels present in the cell membrane. Results suggest that one of the two configurations predicts sodium and potassium intracellular concentrations and transmembrane potential much more accurately than the other. Because of its quantitative prediction power, the proposed theoretical approach may help relate phenomena at the cellular scale, that cannot be accessed clinically, with phenomena occurring at the scale of the whole eye, for which clinical assessment is feasible.