Immobilization

The term immobilized enzyme refers to enzymes that are physically confined or localized in a certain defined region of space with retention of their catalytic activities[1]. In this discussion this refers to the linking or bonding of an enzyme to a solid support that will be used as the anode for the bio fuel cell.

There are 4 basic ways that enzymes can be immobilized to a solid surface [2].
 * 1) A suitable reaction to activate the enzyme for the immobilization process is performed prior to binding. This approach often suffers from significant loss of activity, because the protein is modified by highly reactive chemical compounds that are often not strictly group specific and may alter catalytically or structurally essential residues. Also intra- and intermolecular cross-linking has to be considered.
 * 2) The support is modified and activated. The native enzyme is bound in a subsequent step under well-defined conditions using the natural reactivity of the molecule. This is the most prominent technique to covalently bind enzymes to carrier surfaces.
 * 3) A bi- or multi-functional coupling agent is used to mediate between carrier and enzyme functional groups. This can also lead to intra- and intermolecular enzyme cross-linking.
 * 4) The enzyme is modified by recombinant DNA techniques to generate a protein with “(bio)specific” groups, so that it can adsorb onto special carriers using (bio)affinity binding.

A detailed flow chart is shown below A literature example (young lee et al. 2011) uses DNA wrapped electrodes in the presence of EDS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to crosslink the enzyme to the DNA and preserves the active sight of the enzymes. Usually NHS (N-hydroxysuccinimide) would have to be used as well but because the anode is wrapped in DNA it does not need to be used. This study led to the conclusion that using glucose oxidase cross-linked to the DNA wrapped carbon nanotube anode produced a power density maximum of 760 μW cm−2. This power output was maintained for about a week despite power losses due to pseudo physiological conditions[3].

Another paper uses organic micro media to immobilize the enzymes, their studies show that almost all the immobilized enzymes retained there catalytic active site. This included the glucose oxidase that is used most commonly in biofuel cells[4].

From these results they inferred that immobilization of the enzyme to the solid surface was increased when this micro organic media was used. This media most likely activates the carboxyl terminus so that it can initiate immobilization without destroying the catalytic active site. They also completed stability testing by altering pH and temperature. Results of their findings are shown below.

Another group used the functionalization of the solid support to immobilize the enzymes effectively. The following was proposed:

“the enzyme was first immobilized by physical adsorption via selective multi-point anionic exchange involving the largest region of the enzyme containing all enzyme subunits. Then, an additional long incubation of the immobilized derivative under alkaline conditions was performed in order to promote an intense intra-molecular multi-point covalent attachment between amino groups of the adsorbed enzyme and the very stable glyoxyl groups on the support.”

Their findings stated that the immobilization of the enzyme was increased by 85 fold when comparing it to a regular epoxy solid support. An absorption method is shown below that is not very selective and the number of immobilized enzymes was not very high.

Below is the method that was used by the paper, It shows the multistep method to immobilizing enzymes to a solid support.

With new and improved methods of immobilizing enzymes to solid support, functionalized solid supports or media that accelerate the bonding or cross-linking, we are able to increase the amount of enzymes that retain their active site once immobilized. This will improve the power density output as well as the current density output of miniature implantable devices that are powered by enzymatic fuel cells.

References


 * 1) Beatriz, B. Batista-Viera, F. Immobilization of enzymes and cells. 2006. Chemistry and Materials Science, 22: 15-30.
 * 2) Wilhelm Tischer, Frank Wedekind. Immobilized Enzymes: Methods and Applications.1999. Topics in Current Chemistry. 200: 2685-2688.
 * 3) Jin Young Lee, Hyun Yong Shin, Seong Woo Kang, Chulhwan Park, Seung Wook Kim. Improvement of electrical properties via glucose oxidase-immobilization by actively turning over glucose for an enzyme-based biofuel cell modified with DNA-wrapped single walled nanotubes. 2011. Biosensors and Bioelectronics, 26: 2685-2688.
 * 4) Xiongjun Zhu , Covalent immobilization of enzymes within micro-aqueous organic media. 2011. Journal of molecular catalysts B: enzymatic. 72: 145-149.
 * 5) Juan M. Bolivara, Cesar Mateoa, Heterofunctional supports for the one-step purification, immobilization and stabilization of large multimeric enzymes: Amino-glyoxyl versus amino-epoxy supports. 2010. Process Biochemistry. 45: 1692–1698.