Copper Stability and Specific Considerations for MCO Production

The degree of enzyme purity will ultimately affect fuel cell performance, particularly when enzyme preparations are used to form immobilized films on electrode surfaces in DET reactions. Contaminating proteins that do not provide electron transfer effectively foul the electrode. When enzyme immobilization techniques are specific to the enzyme, then enzyme purity may not be as much as an issue, but rarely the immobilization technique is absolutely specific to the cathodic or anodic enzyme. For example, an attractive immobilization strategy is to link a particular enzyme to an electrode via its cofactor (e.g., flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), etc.) [59]. The cofactor is linked to the electrode material first and then the apoenzyme is allowed to naturally bind to the cofactor all other proteins in the enzyme preparation that cannot bind the cofactor remain unbound and can be removed. Enzymes used in fuel cells are not so unique, and proteins in the immobilizing preparation may use the same cofactor but not the same fuel during fuel cell analysis or operation. 

Metal coordination is essential to the function of electron transfer in MCOs. Accordingly, metal binding and metal complex stability during protein synthesis and subsequent purification is a critical factor [61 ]. A few questions outline the metal complex issues How efficient is copper loading during protein expression When the metal is in complex with the protein, how does its coordination affect the purification of the enzyme and its stability How stable are the copper centers during purification, storage, and when used in fuel cell applications  

In Nature, microorganisms have developed copper transport systems to complete delivery of copper to target proteins [61]. Isolation of MCO enzymes produced in the native organism will best ensure that the holoenzyme has full complement of copper 

Supplementing culture media, crude extracts, storage, and assay buffers with copper salts can assist copper complex formation in MCO preparations. In many cases, copper can be added to bacterial and fungal culture supernatants to increase MCO activity and holoenzyme yield [62-67]. Free copper ions are toxic to microorganisms, so copper concentrations must remain low enough during cultivation so that growth is not inhibited [61]. 

Copper salt solubility varies widely and the buffer combinations must be considered carefully in the protocols. For example, under physiological conditions, copper phosphate is minimally soluble. Phosphate is typically included in cell lysis and protein storage buffers, so addition of any additional copper salts to the protein suspensions containing phosphate must be carefully considered to limit precipitates. Furthermore, excess copper and copper precipitates can be troublesome in 

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