In addition to the generic structure-function assays conducted, all purified enzymes were evaluated for their ability to oxidize AFB1. Reactions were incubated for 5 days at room temperature in PBS (pH 7.5) containing 22 µg/mL aflatoxin and 87-17,778 µg/mL enzyme, a catalytic loading of ~400-80,800%. Reactions were quenched with 70% acetonitrile and the remaining AFB1 was evaluated using established HPLC methods. Previous efforts to quantify laccase-aflatoxin activity have tested many pHs, different durations for incubation, a wide range of temperatures, and had varying levels of success. Some studies found almost complete degradation of AFB1 while others had only marginal levels of oxidation. Of our 46 enzymes tested, 4 laccases were observed to achieve greater than 95% oxidation under the reaction conditions and 20 were able to oxidize at least 6% of the AFB1 present (Figure and Figure ). Several clades or subclades within the tree have a high proportion of members that can oxidize AFB1, meaning the reaction can be carried out by structurally distinct laccases. C0STU6 is one of several members from Figure 's Group A that are active on AFB1 and within that clade there is an average percent identity of 63% (Figure ). When compared to P07788 from Group E, which has moderate AFB1 activity and an average intraclade percent identity of 58%, we see that although C0STU6 and P07788 are only 13% identical to each other they can both carry out the same reaction. This has implications for future screening efforts as it implies the entirety of the multi-copper oxidase family has potential for this chemistry and there may be catalytically active clades that have yet to be studied. Another trend within the AFB1 activity data shows that there is a weak correlation between AFB1 activity and ABTS activity, though it is notable that 6 genes were able to degrade AFB1 even though they were below the median ABTS activity (1.93E-3 µmol min -1 mg -1 ) for the dataset. This means almost a third of the AFB1 active laccases showed very low specific activity on ABTS, highlighting the importance of screening for function directly for the function of interest as these enzymes would likely have been missed if ABTS had been used as a prescreening selection criterion. In order to better understand how these oxidases interact with AFB1, we determined that increased emphasis should be applied to representative laccases that could serve as starting points for future design efforts. Two enzymes were selected for further characterization, P07788 and C0STU6. Both had robust levels of expression, were active on AFB1, and represented two highly distinct structural classes of laccases: monomeric and trimeric, respectively (Figure ). Despite P07788 and C0STU6 having differing in vivo multi-or monomeric structures, their active site tertiary structures maintain a relatively high level of similarity with an RMSD of 3.7Å for residues within 8Å of the two catalytic histidines in the T1 site (Figure ). However, their active site primary structures vary greatly, with an active site sequence identity of 31%. When comparing the specific activity of these enzymes against ABTS and AFB1 (Figure ) we see that C0STU6 generally has an activity on ABTS that is approximately double P07788. Previous work by Guo et al showed that their laccase with a catalytic loading of approximately 5% was able to degrade 96% of AFB1 in 12 hours. Laccase C0STU6 was able to degrade 99% of AFB1 in 24 hours with a catalytic loading of 97% while P07788 could only degrade 80% in 144 hours with a catalytic loading of approximately 9% (Figure ). These datum points indicate that P07788 is approximately 3 times less efficient than C0STU6 for degrading AFB1 and C0STU6 is almost 40-fold slower than the laccase from Guo et al. Several factors could account for the differences in activity. For the assay conditions of Guo et al., the pH and temperatures had been optimized for the enzyme which meant it had near ideal conditions for its activity. Their laccase was only 40% of its max efficiency on ABTS at our assay's pH conditions and more than 5-fold slower at our assay's temperature. Because of the nature of our high throughput screen, it was infeasible to optimize every assay for each enzyme's preferred conditions. Future research can focus on optimization of assay conditions for individual laccases of interest. Differences in catalytic efficiency may also be tied to these laccases' origins. Previous work has sought to identify the biological functions of the laccase P07788 , while little research has been devoted to C0STU6's biological function. It is hypothesized that P07788 takes part in a biosynthetic pathway to produce a melanin-like substance which may protect a spore from UV damage. C0STU6 is less well-known because it was isolated from an unknown environmental bacterium and has not been identified beyond its structural and biochemical properties. In order to better understand the reasons for these laccases' differing activities, future work may wish to identify each enzyme's native substrates. Highly different active sites could mean the native substrate for C0STU6 more closely resembles AFB1 or that the enzyme is a general detoxifying laccase, while P07788 is more suited for the intermediate in the melanin-like biosynthetic pathway. If C0STU6 functions to degrade motifs associated with toxic small molecules, it could explain why its active site is more solvent exposed and less pocket-like than P07788. The differences in activity and structure between these laccases present an opportunity for future efforts to elucidate structural features critical for efficient AFB1 oxidation.