I am working with Novozyms 51003. It is a M. thermophila laccase expressed in Aspergillus sp. I collected the uv-vis spectrum for the commercial sample diluted in sodium acetate buffer. No 600 nm band were detected (typically related to Type I copper, which gives laccases their blue color). I've done some research in literature, but currently have not found reportes of it. The commercial enzyme solution is a brown solution, but as it is commercial, there are no specific informations regarding composition, so I don't know if this brown color is laccase-related, or if it is related to the solution and supress the expected blue color. Perhaps the method used was not adequated. I wonder if anyone could help me. Much appreciated.
Could the brown color perhaps be phenolic product added to stabilize the enzyme?
Does the enzyme show activity?
BRENDA only lists one laccase and also information to the M thermophilus enzyme with literature references (https://www.brenda-enzymes.org/enzyme.php?ecno=1.10.3.2)
The enzyme shows high activity towards ABTS oxidation. The brown color might be related to some stabilizer, however, shouldn't the 600 nm peak be present at the uv-vis spectrum? I've found no reports that Myceliophthora thermophila laccase is a white or yellow one, so it would be expected that the signal of Type I copper to be detected.
Maybe the stabilizer, or what ever it is, is masking the copper band. As copper is a quite decent oxidizer and activated by light, it may be a protective screen to avoid oxidation of the enzyme.
You are at a large university. You could perhaps ask for an atomic absorption spectroscopy or some other technique to ascertain the presence of copper.
Thank you very much for your reply. Your thoughts are really helpful. We'll work on spectroscopy.
Spectroscopic analysis were performed. Indeed, the brown color is related to stabilizers. Filtration and buffer exchange/washing helped to "clean" and a blue-ish solution was obtained. 600 nm peak and 330 nm should were present in the UV/vis scanning, as expected for these enzymes. Also, EPR spectroscopy allowed to assign T1 and T2 copper sites. Thank you very much for such helpful considerations!
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