## 3 Optical Properties of Laccase
### Fluorescence Spectroscopy  
Laccases have been observed to possess intrinsic fluorescence that largely depends on tryptophan residues, which can be excited either directly or by energy transfer from tyrosine residues. [PERSON] and [PERSON] [66], in a pivotal study on lacase extracted from _Rlus vernicifera_ lacquer, evidenced this behavior by considering the dependence of the emission spectra on the excitation wavelengths. When the excitation wavelength is equal to 305 nm, it is absorbed only by tryptophan, and the maximum of the emission spectra is located at 338 nm. When the excitation wavelength is equal to 280 nm, it is absorbed by tryptophan and tyrosine and the maximum of the emission spectrum shifts to shorter wavelengths with a significant increase in the fluorescence signal intensity. A similar behavior is observed when the excitation wavelength is equal to 250 nm that is absorbed only by tyrosine. In Ref. [66], the changes in quantum yield induced by reduction processes and dependent from excitation wavelength have been also reported.  
Using the same lacase cited above, [PERSON] et al. [67] confirmed that the presence of O\({}_{2}\) influences the reactivity of the enzyme and its excitation and emission spectra. This is because the presence of oxygen influences the oxidation state of the enzyme [67].  
The fundamental studies carried out in Refs. [66; 67] evidenced the potentiality of fluorescence spectroscopy in characterizing different lacases. [PERSON] et al. [54; 68] investigated lacase from the phytopathogenic fungus _Sclerotinia sclerotorium_ using this spectroscopic approach. The previously cited enzyme presents interesting characteristics; in fact, it can be classified as a yellow lacase by considering its UV-Vis absorption spectral features, but EPR measurements indicate a similarity with blue lacase. In Figure 5, the visible emission spectrum, obtained with an excitation wavelength equal to 330 nm, showing a maximum located at 440 nm, is reported. In this figure, an excitation spectrum with a band at 330 nm is also shown when the fluorescence emission is monitored at 420 nm. This band is a typical characteristic of type-3 copper. Further details on the intriguing optical properties of this enzyme can be found in Refs [54; 68].  
In the previously mentioned study by [PERSON] et al. [64], the authors showed the emission and excitation spectra of different blue lacases (see Figure 6). In this case, the excitation spectra clearly show, in almost all of the cases, a band at around 330 nm that is  
Figure 5: Fluorescence spectra of lacase from fungus _Sclerotinia sclerotorium_. The emission spectrum shows a visible signal with the maximum located at 440 nm, while the excitation spectrum shows a peak located at 280 nm and a band centered at 330 nm. The lacase solutions were prepared using 2-(N-Morpholino) ethanesulfonic acid 4-Morpholineethanesulfonic acid (MES) buffer (reprinted with permission from Ref. [68]).  
typical of type-3 copper atoms, as above reported. The emission spectra present a maximum of around 440 nm. More recently, the fluorescence properties of lacase from _Trametes versicolor_ were investigated by [PERSON] et al. [69]. In particular, the fluorescence emission spectra were collected in the 350-500 nm wavelength region following the excitation of tryptophan residues (\(\lambda_{\text{exc}}\) = 280 nm), and changes induced by different pH levels were also investigated.  
Fluorescence spectroscopy has been successfully adopted by several authors for characterizing different aspects of lacases [33; 70; 71]. In recent years, interest in the application of this technique has particularly been addressed to fungal and bacterial lacases [72; 73].  
As outlined in Section 5, the fluorescence signal has been adopted for developing a certain number of optical biosensing devices.