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The iLOV protein belongs to a family of blue-light photoreceptor proteins containing a lightoxygen- voltage sensing domain with a noncovalently bound flavin mononucleotide (FMN) as its chromophore. Owing to advantages such as its small size, oxygen-independent nature, and pH stability, iLOV is an ideal candidate over other reporter fluorescent proteins such as GFP and DsRed. Here, for the first time, we describe the feasibility of applying LOV domain-based fluorescent iLOV as a metal sensor by measuring the fluorescence quenching of a protein with respect to the concentration of metal ions. In the present study, we demonstrated the inherent copper sensing property of the iLOV protein and identified the possible amino acids responsible for metal binding. The fluorescence quenching upon exposure to Cu(2+) was highly sensitive and exhibited reversibility upon the addition of the metal chelator EDTA. The copper binding constant was found to be 4.72 ± 0.84 micrometer. In addition, Cu(2+)-bound iLOV showed high fluorescence quenching at near physiological pH. Further computational analysis yielded a better insight into understanding the possible amino acids responsible for Cu(2+) binding with the iLOV protein.

The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future.

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