Abstract
BackgroundLaccases (EC 1.10.3.2) are multi-copper oxidoreductases with great biotechnological importance due to their high oxidative potential and utility for removing synthetic dyes, oxidizing phenolic compounds, and degrading pesticides, among others.MethodsA real-time stability study (RTS) was conducted for a year, by using enzyme concentrates from 3 batches (L1, L3, and L4). For which, five temperatures 243.15, 277.15, 298.15, 303.15, 308.15, and 313.15 K were assayed. Using RTS data and the Arrhenius equation, we calculated the rPOXA 1B accelerated stability (AS). Molecular dynamics (MD) computational study results were very close to those obtained experimentally at four different temperatures 241, 278, 298, and 314 K.ResultsIn the RTS, 101.16, 115.81, 75.23, 46.09, 5.81, and 4.83% of the relative enzyme activity were recovered, at respective assayed temperatures. AS study, showed that rPOXA 1B is stable at 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K; with t1/2 values of 230.8, 46.2, and 12.6 months, respectively. Kinetic and thermodynamic parameters supported the high stability of rPOXA 1B, with an Ed value of 41.40 KJ mol− 1, a low variation of KM and Vmax, at 240.98 ± 5.38, and 297.53 ± 3.88 K, and ∆G values showing deactivation reaction does not occur. The MD indicates that fluctuations in loop, coils or loops with hydrophilic or intermediate polarity amino acids as well as in some residues of POXA 1B 3D structure, increases with temperature; changing from three fluctuating residues at 278 K to six residues at 298 K, and nine residues at 314 K.ConclusionsLaccase rPOXA 1B demonstrated experimentally and computationally to be a stable enzyme, with t1/2 of 230.8, 46.2 or 12.6 months, if it is preserved impure without preservatives at temperatures of 240.98 ± 5.38, 277.40 ± 1.32 or 297.53 ± 3.88 K respectively; this study could be of great utility for large scale producers.
Highlights
Laccases (EC 1.10.3.2) are multi-copper oxidoreductases with great biotechnological importance due to their high oxidative potential and utility for removing synthetic dyes, oxidizing phenolic compounds, and degrading pesticides, among others
The objectives of this work were, i) to propose the design of a more statistically based stability study, which would allow estimating at each sampling time with the same precision and confidence level the average enzyme activity (UL− 1) of rPOXA 1B laccase and support the stability study of other enzymes and proteins of non-pharmaceutical use [39], ii) use this statistical foundation to determine the real-time and accelerated stability of the recombinant rPOXA 1B enzyme from P. ostreatus produced in P. pastoris and iii) relate the real-time-stability with the in silico behaviour of the enzyme during molecular dynamics simulations
Assuming equal variances for each temperature, a combined variance estimator was used to obtain an estimate of enzyme activity variability; with an estimate of 96.6 for rPOXA 1B UL− 1 standard deviation
Summary
Laccases (EC 1.10.3.2) are multi-copper oxidoreductases with great biotechnological importance due to their high oxidative potential and utility for removing synthetic dyes, oxidizing phenolic compounds, and degrading pesticides, among others. The laccases catalytic centre contains four copper atoms distributed over CuT1, CuT2 and CuT3 sites, located between cupredoxin-like domains 1 and 3 [1, 18]. Site CuT1 is mononuclear and coordinated with D3 residues. CuT1 is considered as the indicator of the redox potential of the enzyme [7, 19] because it captures electrons from the substrate and transferring them to the trinuclear copper centre (TNC) of the enzyme [20]. Copper ions coordinate with the D1 and D3 domains located residues, where the reduction of O2 to H2O occurs [1, 19, 21]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
