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The high-molecular weight fraction of olive mill wastewater (HMW-OMW), a byproduct of olive oil biorefinery, was used at the reactor level as the basal medium for production of laccase and Mn-dependent peroxidase (MnP) by Trametes ochracea. Three reactor systems, namely stirred tank reactors equipped with either Rushton turbines or marine impeller and draft tube (STR and STR-MD, respectively) and an air-lift reactor (ALR) were compared for this purpose. Although inocula were supplied as intact pellets, in both STR-based systems fungal growth evolved rapidly into a dispersed form while the ALR enabled the maintenance of the pellet growth mode. STR was deemed to be the most promising system since it best supported the production MnP activity on the HMW-OMW-based medium and its performance in laccase production did not differ from that observed with the STR-MD. Among the stirring regimes considered (250, 400, 500 and 600 rpm), the best production in the STR was observed at 500 rpm and 1.0 vvm for both laccase (8850 ± 270 IU L -1 on day 15) and MnP (17,027.4 ± 87.2 IU L -1 on day 13). When the inocula were supplied to the STR in homogenized form, the MnP production peak (16,856 ± 1070 IU L -1 ) was attained 8 days earlier than the previous condition and that of laccase was nearly doubled (14,967 ± 907 IU L -1 ). When compared with literature data, T. ochracea MnP production and productivity on the HMW-OMW-based medium were the highest reported for a wild-type fungal strain., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Laccases catalyze the one-electron oxidation of a broad range of substrates coupled to the 4 electron reduction of O2 to H2O. Phenols are typical substrates, because their redox potentials (ranging from 0.5 to 1.0 V vs. NHE) are low enough to allow electron abstraction by the T1 Cu(II) that, although a relatively modest oxidant (in the 0.4-0.8 V range), is the electron-acceptor in laccases. The present study comparatively investigated the oxidation performances of Trametes villosa and Myceliophthora thermophila laccases, two enzymes markedly differing in redox potential (0.79 and 0.46 V). The oxidation efficiency and kinetic constants of laccase-catalyzed conversion of putative substrates were determined. Hammett plots related to the oxidation of substituted phenols by the two laccases, in combination with the kinetic isotope effect determination, confirmed a rate-determining electron transfer from the substrate to the enzyme. The efficiency of oxidation was found to increase with the decrease in redox potential of the substrates, and the Marcus reorganisation energy for electron transfer to the T1 copper site was determined. Steric hindrance to substrate docking was inferred because some of the phenols and anilines investigated, despite possessing a redox potential compatible with one-electron abstraction, were scarcely oxidised. A threshold value of steric hindrance of the substrate, allowed for fitting into the active site of T. villosa laccase, was extrapolated from structural information provided by X-ray analysis of T. versicolor lac3B, sharing an identity of 99% at the protein level, thus enabling us to assess the relative contribution of steric and redox properties of a substrate in determining its susceptibility to laccase oxidation. The inferred structural threshold is compatible with the distance between two phenylalanine residues that mark the entrance to the active site. Interaction of the substrate with other residues of the active site is commented on.

The phytotoxicity of olive-mill wastewater (OMW) has been suggested to be mainly due to its phenolic components. This study investigated the impact of three different low-cost dephenolization treatments on the wastewater phytotoxicity. To this aim, germinability of maize (Zea mays L.) seeds sown on a sandy-loamy soil which had been spread with different volumes (from 40 to 160m(3)ha(-1)) of either biologically-treated OMW or relative incubation control was determined. Biological treatments included either Panus tigrinus liquid cultures or incubation with commercial laccase (1UIml(-1)) or an innovative sequential combination of laccase and P. tigrinus cultures. All treatments markedly reduced phytotoxicity and promising results were obtained with commercial laccase. In fact, germinability and mean germination times in soil spread with laccase-treated OMW, did not significantly differ from those observed in soil irrigated with tap water (control) up to OMW volumes of 120m(3)ha(-1). Although the highest phenol reduction (ca. 81%) was obtained by the sequential use of laccase and P. tigrinus, the feasibility of the enzyme treatment is undoubtedly more convincing under the technological point of view.

Extracellular laccase from Panus tigrinus CBS 577.79 was produced in a bubble-column reactor using glucose-containing medium supplemented with 2,5-xylidine under conditions of nitrogen sufficiency. The main laccase isoenzyme was purified to apparent homogeneity by ultra-filtration, anion-exchange chromatography and gel filtration that led to a purified enzyme with a specific activity of 317 IU (mg protein)-1 and a final yield of 66%. Laccase was found to be a monomeric protein with a molecular mass of 69.1 kDa, pI of 3.15 and 6.9% N-glycosylation of the high mannose type. Temperature and pH optima were 55 degrees C and 3.75 (2,6-dimethoxyphenol as substrate). At 50 and 60 degrees C, the enzyme half-lives were 281 and 25 min, respectively. The P. tigrinus laccase oxidized a wide range of both naturally occurring and synthetic aromatic compounds: the highest catalytic efficiencies were for 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid and 2,6-dimethoxyphenol (5.99x10(6) and 3.07x10(6) M-1 s-1, respectively). Catalytic rate constants for typical N-OH redox mediators, such as 1-hydroxybenzotriazole (2.6 s-1), violuric acid (8.4 s-1) and 2,2,6,6-tetramethylpiperidin-N-oxide radical (7.8 s-1), were found to be higher than those reported for other high redox potential fungal laccases.

Due to its very low moisture content, the solid waste from the two-phase olive oil extraction process (dry olive mill residue, DOR) can be easily stored and used as a readily available basal liquid culture medium via its reconstitution with water. Five white-rot fungi (namely Phanerochaete chrysosporium NRRL 6361, Lentinus tigrinus CBS 577.79, Pleurotus pulmonarius CBS 664.97, Phlebia radiata DABAC 9 and Botryosphaeria rhodina DABAC P82), previously selected in a screening program, were compared for their ability to grow and to produce laccase on non-supplemented aqueous extracts of the dry olive mill residue (ADOR). The most effective strains, namely L. tigrinus and P. radiata, grown in a 3-l bubble-column reactor on 25% (w/v) ADOR, produced 6602 and 6001 nkatal l-1 with respective mean volumetric productivities (MVP) of 68 and 83 nkatal l-1 h-1 and production yields of 375 and 418 nkatal g-1 COD consumed, respectively. These results show that non-supplemented ADOR might constitute the basis of a growth medium allowing promising productions of laccase amenable to further enzyme optimization. [ABSTRACT FROM AUTHOR]

Abstract: This study investigated the efficiency of four different immobilised laccase preparations in the mediator-assisted oxidation of a non-phenolic lignin model compound. To this aim, Trametes villosa laccase was either covalently bound onto Eupergit® C or activated carbon, or entrapped within copper or calcium alginate. The benchmark reaction, consisting in the side-chain oxidation of 4-methoxybenzyl alcohol (4-MBA) to 4-methoxybenzaldehyde, was taken as the criterion to compare the efficiency of the immobilised laccases, and to assess the possible advantages of their use in alternative to the native enzyme. For each laccase preparation, four mediators (including HBT, HPI, VLA and TEMPO) were comparatively investigated. Comparisons were made by using the same amount of activity (i.e. 10U) for each laccase preparation, and showed that the native enzyme generally led to higher p-anisaldehyde yields than the immobilised laccases. The only exception was observed with Cu-alginate-laccase, which led to a 85% conversion of 4-MBA in the presence of HBT as the mediator. When testing the reusability of this immobilised system, a significant catalytic efficiency was maintained along three consecutive reaction cycles. [Copyright &y& Elsevier]

This review summarizes all the research efforts that have been spent to immobilize laccase and tyrosinase for various applications, including synthetic and analytical purposes, bioremediation, wastewater treatment, and must and wine stabilization. All immobilization procedures used in these areas are discussed. Considerations on the efficacy of immobilized copper oxidases and products, in addition to their kinetic parameters are also discussed. The available data indicate that the immobilization of laccase into cationic polymer cross-linked with epichlorohydrin appears to be a promising procedure for industrial applications. The development of laccase and tyrosinase-based biosensors to monitor a wide range of compounds appears to be at a mature stage of technology. [Copyright &y& Elsevier]

Abstract: The main aim of this study was to assess the ability of producing extracellular phenoloxidases (EPO) of fungi isolated from olive brine wastewater (OBW), the effluent from the debittering process of table olives. Five out of twenty isolates displayed EPO-producing ability as assessed by selective agar plate assays and were all halotolerant. Among them, Citeromyces matritensis (syn. Candida globosa) and Aspergillus fumigatus concomitantly produced laccase and Mn-peroxidase (MnP) activities. Candida boidinii and Candida bombi, instead, only produced laccase and Candida diddensiae only released MnP. Both C. matritensis and A. fumigatus, grown in shaken cultures on OBW, maintained their EPO-producing ability and removed phenols by 52.3 and 82.3%, respectively, after 10d incubation in the non-supplemented effluent. These results might suggest the possible use of these strains in the treatment of other saline phenol-rich effluents, such as pickling and tannery wastewater. [Copyright &y& Elsevier]

Abstract: The ability of stationary and shaken Lentinus tigrinus CBS 577.79 liquid cultures to degrade a mixture of polycyclic aromatic hydrocarbons (PAHs) in N-rich (i.e., malt extract glucose, MEG) and in N-limited (low-N Kirk’s medium, LNKM) media was investigated. Best results were obtained in shaken cultures where PAHs were degraded by 91% and 97% in MEG and LNKM, respectively; in stationary cultures, on the contrary, the degradation was never higher than 50%. Laccase activity was predominant on MEG while Mn-peroxidase (MnP) was preferentially produced in LNKM. The identification of degradation products showed the presence of several PAH derivatives, such as quinones, dicarboxylated and ring fission derivatives, presumably derived from the action of lignin-modifying enzymes. The presence of some degradation products (e.g., hydroxylated derivatives of anthrone and phenanthrene 9,10-dihydrodiol) suggested the possible involvement of cytochrome P-450-epoxide hydrolase system, the active form of which was found in 7-day-old cultures on MEG. In vitro experiments showed that the MnP from L. tigrinus had wider PAH substrate range and higher oxidation ability than the laccase produced by the same strain. [Copyright &y& Elsevier]

Abstract: Dry olive mill residue (DOR), the by-product of the two-phase extraction process, is very rich in organic matter and nutritionally relevant cations. For this reason, the agronomic use of this waste has been suggested although DOR exhibits significant phytotoxicity. The objective of this study was to investigate the impact of Paecilomyces farinosus on both organic matter modification and detoxification of this waste. Humification ratio in DOR colonized by the fungus for 20 weeks was increased by about 65% with respect to the abiotic control and humification index reached 0.38, a value that characterizes well-humified materials. High performance size-exclusion chromatography of humic acids from fungal cultures showed a marked increase in both weight-averaged and number-averaged molecular weights with respect to abiotic controls. Water-soluble phenols were reduced by 45% in 20-week-old P. farinosus cultures on DOR and mass-balance ultra-filtration showed that the relative abundance of the molecular weight fraction of phenols above 30kDa increased from 31 to 72% suggesting the occurrence of polymerization. Experiments performed with alfalfa grown on soils containing 2.5% (w/w) of abiotic controls and fungal-treated DOR showed that phytotoxicity was totally suppressed in the waste that underwent fungal treatment. [Copyright &y& Elsevier]

Abstract: The growth medium for Panus tigrinus laccase production was studied in shaken culture through response surface methodology. The impact of five crucial variables on enzyme production, including glucose and nitrogen concentrations and three distinct putative inducers, namely, copper sulphate, 2,5-xylidine and olive-mill wastewater was thoroughly investigated. Among them, 2,5-xylidine remarkably stimulated enzyme production while Cu2+ ions within the tested concentration range (0–0.25mM) had an opposite effect. The maximal laccase activity (about 2IU/mL on day 11) was observed in the medium containing glucose and nitrogen at concentrations of 12.5g/L and 20mM, respectively, and 0.25mM 2,5-xylidine as the sole inducer. A 35-fold increase in laccase activity was obtained with respect to the same medium where 2,5-xylidine had been omitted. With the medium thus optimised, further experiments were performed in a 3-L stirred-tank reactor (STR) leading to a laccase activity of 2200IU/L on day 9 with a productivity of 10.2IU/(Lh) and a specific activity of 26.7IU/mg protein. RT-PCR analyses of total RNA from both non-induced and putatively induced cultures conducted in STR showed that the transcript levels of only one out of two constitutively expressed laccase genes were markedly increased by the presence of 0.25mM 2,5-xylidine in the optimised medium. [Copyright &y& Elsevier]

The possible use of olive-mill wastewater (OMW) as a growth medium for the production of extracellular laccase and manganese peroxidase (MnP) from the white-rot fungus Panus tigrinus (P. tigrinus) CBS 577.79 was studied using a properly formulated OMW-based medium (2-fold diluted OMW supplemented with 0.5% sucrose and 0.1% yeast extract) either in a stirred-tank or an air-lift reactor. Solid-state fermentation (SSF) was also performed in a rotary drum reactor using maize stalks moistened with the OMW-based medium. Highest levels of laccase and manganese peroxidase activity were obtained in the stirred-tank reactor (4600±98 U l−1 on day 13) and in the air-lift reactor (410±22 on day 7), respectively. Based on total enzyme activities, SSF appears to be more suitable than LSF but the latter exhibits better volumetric productivities. [Copyright &y& Elsevier]