Laccases hold great potential for biotechnological applications, particularly in environmental pollutant remediation. Laccase activity is governed by the solvent environment, and ionic liquids (ILs) emerge as a versatile solvent for activation or stabilization of enzymes. Herein, effects of cholinium-based ILs formulated with carboxylic acids, inorganic acid, and amino acids as anionic species, on the catalytic activity of laccase from Trametes versicolor were investigated by experimental and computational approaches. Experimental results showed that laccase activity was enhanced by 21.39 % in 0.5 M cholinium dihydrogen citrate ([Cho][DHC]), in relation to the laccase activity in phosphate buffer medium. However, cholinium aminoate ILs negatively affected laccase activity, as evidenced by the partial deactivation of laccase in both cholinium glycinate and cholinium phenylalaninate, at concentrations of 0.1 M and 0.5 M, respectively. Molecular dynamics studies revealed that the enhancement of laccase activity in [Cho][DHC] might be attributed to the highly stabilized and compact structure of laccase, facilitating a better internal electron transfer during the laccase-substrate interactions. Enhanced catalytic performance of laccase in [Cho][DHC] was postulated to be driven by the high accumulation level of dihydrogen citrate anions around laccase's surface. [Cho][DHC] holds great promise as a cosolvent in laccase-catalyzed biochemical reactions.
Free laccase and fungal biomass from white-rot fungi were compared in the thermokinetics study of the laccase-catalyzed decolorization of an azo dye, i.e., Trypan Blue. The decolorization in both systems followed a first-order kinetics. The apparent first-order rate constant, k1', value increases with temperature. Apparent activation energy of decolorization was similar for both systems at ∼ 22 kJ mol(-1), while energy for laccase inactivation was 18 kJ mol(-1). Although both systems were endothermic, fungal biomass showed higher enthalpy, entropy, and Gibbs free energy changes for the decolorization compared to free laccase. On the other hand, free laccase showed reaction spontaneity over a wider range of temperature (ΔT = 40 K) as opposed to fungal biomass (ΔT = 15 K). Comparison of entropy change (ΔS) values indicated metabolism of the dye by the biomass.
Laccases, oxidative copper-enzymes found in fungi and bacteria were used as the basis in the design of nona- and tetrapeptides. Laccases are known to be excellent catalysts for the degradation of phenolic xenobiotic waste. However, since solvent extraction of laccases is environmentally-unfriendly and yields obtained are low, they are less preferred compared to synthetic catalysts. The histidine rich peptides were designed based on the active site of laccase extracted from Trametes versicolor through RCSB Protein Data Bank, LOMETS and PyMol software. The peptides were synthesized using Fmoc-solid phase peptide synthesis (SPPS) with 30-40% yield. These peptides were purified and characterized using LC-MS (purities >75%), FTIR and NMR spectroscopy. Synthesized copper(II)-peptides were crystallized and then analyzed spectroscopically. Their structures were elucidated using 1D and 2D NMR. Standards (o,m,p-cresol, 2,4-dichlorophenol) catalysed using laccase from Trametes versicolor (0.66 U/mg) were screened under different temperatures and stirring rate conditions. After optimizing the degradation of the standards with the best reaction conditions reported herein, medications with phenolic and aromatic structures such as ibuprofen, paracetamol (acetaminophen), salbutamol, erythromycin and insulin were screened using laccase (positive control), apo-peptides and copper-peptides. Their activities evaluated using GC-MS, were compared with those of peptide and copper-peptide catalysts. The tetrapeptide was found to have the higher degradation activity towards salbutamol (96.8%) compared with laccase at 42.8%. Ibuprofen (35.1%), salbutamol (52.9%) and erythromycin (49.7%) were reported to have the highest degradation activities using Cu-tetrapeptide as catalyst when compared with the other medications. Consequently, o-cresol (84%) was oxidized by Tp-Cu while the apo-peptides failed to oxidize the cresols. Copper(II)-peptides were observed to have higher catalytic activity compared to their parent peptides and the enzyme laccase for xenobiotic degradation.
In this study, laccase was immobilized on nylon 6,6/Fe(3+) composite (NFC) nanofibrous membrane and used for the detoxification of 3,3'-dimethoxybenzidine (DMOB). The average size and tensile strength of the NFC membrane were found to be 60-80 nm (diameter) and 2.70 MPa, respectively. The FTIR results confirm that the amine (N-H) group of laccase was attached with Fe(3+) particles and the carbonyl (C=O) group of NFC membrane via hydrogen bonding. The half-life of the laccase-NFC membrane storage stability was increased from 6 to 11 weeks and the reusability was significantly extended up to 43 cycles against ABTS oxidation. Enhanced electro-oxidation of DMOB by laccase was observed at 0.33 V and the catalytic current was found to be 30 µA. The DMOB-treated mouse fibroblast 3T3-L1 preadipocytes showed maximum (97 %) cell inhibition at 75 µM L(-1) within 24 h. The cytotoxicity of DMOB was significantly decreased to 78 % after laccase treatment. This study suggests that laccase-NFC membrane might be a good candidate for emerging pollutant detoxification.
Laccases are industrially attractive enzymes and their applications have expanded to the field of bioremediation. The challenge of today's biotechnology in enzymatic studies is to design enzymes that not only have a higher activity but are also more stable and could fit well with the condition requirements. Laccases are known to oxidize non-natural substrates like polycyclic aromatic hydrocarbons (PAHs). We suppose by increasing the hydrophobicity of laccase, it would increase the chance of the enzyme to meet the hydrophobic substrates in a contamination site, therefore increasing the bioremediation efficacy of PAHs from environment. In this attempt, the applications of evolutionary trace (ET), molecular surface accessibility and hydrophobicity analysis on laccase sequences and laccase's crystal structure (1KYA) are described for optimal design of an enzyme with higher hydrophobicity. Our analysis revealed that Q23A, Q45I, N141A, Q237V, N262L, N301V, N331A, Q360L and Q482A could be promising exchanges to be tested in mutagenesis experiments.