Stability

**Enzyme Precipitate Coating**
One key problem with enzymatic fuel cells (EFC) is the short lifetime of the immobilized enzyme that can deactivate the cell after several days of use. Enzyme Precipitate Coating (EPC) is a method developed by a team led by Mike Fischback, to increase the stability and therefore the lifetime of a EFC. This method coats a specific enzyme onto the anode of the cell, which prevents the protein from easily denaturing. EPC technique is performed in three steps: covalent attachment, enzyme precipitation, and chemical cross-linking [1].

A EFC was built using a monolayer of GOx (Glucose Oxidase) as the immobilized enzyme that was bound to a carbon nanotube surface via covalent attachment. To precipitate the enzyme out, ammonium sulphate was added. A cross link was performed between the precipiated enzymes and the covalently bound enzymes' CNT and entangled throughout the carbon paper. To form the overall electrode, Nafion was used as a binder for the CNT onto carbon paper [1].



The cross linked enzymes form numerous pore sizes that increases mass transport, and active sites available on the enzyme. In order to run implantable bio-devices, a continuous and unremitting wattage is necessary. EPC will prevent the enzyme from being denatured. The increased stability comes from the extra chemical bonds on the enzyme surface that adds resistance to the denaturation of the protein structure [1].

The cell was operated using 200 mM glucose solution, and 10 mM benzoquinone as a mediator and ran over time, measuring voltage consistency. The fuel cell with EPC coating was tested at a fixed voltage of 0.18V. Fluctuations in current density occurred in the first three hours, but stabilized after the next 42 hours (with only a 15% drop over time). A total of 45 hours was run under ambient conditions producing a relatively stable current. This technique is new and therefore requires more research and experimentation. Future developments in EPC could result in extended periods of power generation [1].

**The Protein Engineering Glucose Dehydrogenase**
Glucose dehydrogenase is more effective than glucose oxidase, in terms of oxidation at the bioanode. It exhibits 10-times greater in catalytic activity and is a stable enzyme with high substrate specificity. When harbored with pyrroloquinolinequinone as the prosthetic group (PQQGDH), this combination is highly effective since it’s oxygen insensitive yet maintains a high catalytic efficiency. However, the application of PQQGDH is limited due to it’s thermal stability [2].

Yuhashi et al. (2004) engaged in the protein engineering of PQQGDH in order to develop a stable enzyme suitable for glucose oxidation. One of particular interest was a Ser415Cys mutant (S415C), whose stability was greatly improved through protein engineering. The alteration of the residue at the 415th position did not affect the catalytic efficiency of the PQQGDH enzyme. It maintains a high catalytic efficiency and is even stable at 70⁰C. In fact, this S415C mutant is one of the most stable cofactor-binding GDH available [2].



The thermal stability of PQQGDH with Ser415Cys was increased due to the disulfide bond at the dimer interface. It was 30 times more stable than it’s wild-type enzyme at 55⁰C without any decrease in the catalytic activity. Upon heating at 70⁰C, the engineered enzyme retained 90% of the GDH activity [3].

= = =References=
 * 1) Fischback M, Kwon KY, Lee I, Shin, SJ, Park HG, Kim BC, Kwon Y, Jung HT, Kim J, Ha S. 2011. Enzyme precipitate coatings of Glucose Oxidase onto Carbon paper for biofuel cell applications. Biotechnology and Bioengineering 109 (2): 318-324.
 * 2) Yuhashi, N., Tomiyamy, M., Okuda, J., Igarashi, S., Ikebukuro, K., Sode, K. 2004. Development of a Novel Glucose Enzyme Fuel Cell System Employing Protein Engineered PQQ Glucose Dehydrogenase. Biosensors & Bioelectronics (20): 2145-2150.
 * 3) Igarashi, S., Sode, K. 2003. Stabilization of Quaternary Structure of Water-Soluble Quinoprotein Glucose Dehydrogenase. Molecular Biotechnology (24) 97-103.