Carbonic Anhydrase Carrying Electrospun Nanofibers for Biocatalysis Applications
Authors: Ünlüer Özlem Biçen , Ecevit Kardelen , Diltemiz Sibel Emir 
Published: November 2020

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
carbonic anhydrase enzyme 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
temperature 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.