Molecular docking and dynamics simulation analyses unraveling the differential enzymatic catalysis by plant and fungal laccases with respect to lignin biosynthesis and degradation

Laccase, widely distributed in bacteria, fungi, and plants, catalyzes the oxidation of wide range of compounds. With regards to one of the important physiological functions, plant laccases are considered to catalyze lignin biosynthesis while fungal laccases are considered for lignin degradation. The present study was undertaken to explain this dual function of laccases using in-silico molecular docking and dynamics simulation approaches. Modeling and superimposition analyses of one each representative of plant and fungal laccases, namely, Populus trichocarpa and Trametes versicolor, respectively, revealed low level of similarity in the folding of two laccases at 3D levels. Docking analyses revealed significantly higher binding efficiency for lignin model compounds, in proportion to their size, for fungal laccase as compared to that of plant laccase. Residues interacting with the model compounds at the respective enzyme active sites were found to be in conformity with their role in lignin biosynthesis and degradation. Molecular dynamics simulation analyses for the stability of docked complexes of plant and fungal laccases with lignin model compounds revealed that tetrameric lignin model compound remains attached to the active site of fungal laccase throughout the simulation period, while it protrudes outwards from the active site of plant laccase. Stability of these complexes was further analyzed on the basis of binding energy which revealed significantly higher stability of fungal laccase with tetrameric compound than that of plant. The overall data suggested a situation favorable for the degradation of lignin polymer by fungal laccase while its synthesis by plant laccase.     

The effect of lignin model compound structure on the rate of oxidation catalyzed by two different fungal laccases

Laccases (EC 1.10.3.2) are multicopper oxidases able to oxidize various substrates, such as phenolic subunits of lignin. The substrate range can be widened to non-phenolic units by the use of mediators. Since discovery of the laccase-mediator system, direct reactions of lignin and laccase without mediated electron-transfer have gained much less attention. The objective of this study was to investigate lignin as a substrate for fungal laccases by using lignin model compounds. These model compounds contained guaiacylic and syringylic moieties and also compounds of guaiacylic origin at a higher oxidation level. Some of these compounds are commercially available, but most of them were synthesized. The oxidation reaction rates of the lignin model compounds were studied by monitoring consumption of the co-substrate oxygen, in reactions catalyzed by laccases from two different fungi; Melanocarpus albomyces and Trametes hirsuta, possessing different molecular and catalytic properties. These reaction rate studies were compared to physicochemical properties of the lignin model compounds: relative redox potentials determined using cyclic voltammetry and pK_a-values. Docking of syringylic and biphenylic compounds to the active sites of both laccases was performed and the resulting model complex structures were used to further interpret the reaction rate results. Reaction rates of laccases are mainly affected by the ability of a lignin model compound to be oxidized and the pK_a-value of the substrate seems to be less important. As a consequence, syringylic compounds are oxidized with the highest rates and compounds at a higher oxidation level and redox-potential, such as vanillin, are oxidized at a much lower rate. Both guaiacylic and syringylic type compounds fit well in the active sites of both laccases. Only for a biphenylic compound, steric clashes were observed, and they are likely to have an effect on the reaction rate. When the oxidation rates on the selected model compounds with the two different laccases were compared, the redox-potential difference between laccases T1 copper and the lignin model compound (ΔE) was not the only property that determined the oxidation rate. In the case of lignin model substrates, also the selectivity of a specific laccase, reflected in the k_cat/K_m value, plays an important role.     

Depolymerization of biorefinery lignin by improved laccases of the white-rot fungus Obba rivulosa

Fungal laccases are attracting enzymes for sustainable valorization of biorefinery lignins. To improve the lignin oxidation capacity of two previously characterized laccase isoenzymes from the white-rot fungus Obba rivulosa, we mutated their substrate-binding site at T1. As a result, the pH optimum of the recombinantly produced laccase variant rOrLcc2-D206N shifted by three units towards neutral pH. O. rivulosa laccase variants with redox mediators oxidized both the dimeric lignin model compound and biorefinery poplar lignin. Significant structural changes, such as selective benzylic α-oxidation, were detected by nuclear magnetic resonance analysis, although no polymerization of lignin was observed by gel permeation chromatography. This suggests that especially rOrLcc2-D206N is a promising candidate for lignin-related applications.     

Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound.

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3, 4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both kcat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect kcat and the oxidation rate of dimer I.     

MiR397b regulates both lignin content and seed number in Arabidopsis via modulating a laccase involved in lignin biosynthesis

Plant laccase (LAC) enzymes belong to the blue copper oxidase family and polymerize monolignols into lignin. Recent studies have established the involvement of microRNAs in this process; however, physiological functions and regulation of plant laccases remain poorly understood. Here, we show that a laccase gene, LAC4, regulated by a microRNA, miR397b, controls both lignin biosynthesis and seed yield in Arabidopsis. In transgenic plants, overexpression of miR397b (OXmiR397b) reduced lignin deposition. The secondary wall thickness of vessels and the fibres was reduced in the OXmiR397b line, and both syringyl and guaiacyl subunits are decreased, leading to weakening of vascular tissues. In contrast, overexpression of miR397b‐resistant laccase mRNA results in an opposite phenotype. Plants overexpressing miR397b develop more than two inflorescence shoots and have an increased silique number and silique length, resulting in higher seed numbers. In addition, enlarged seeds and more seeds are formed in these miR397b overexpression plants. The study suggests that miR397‐mediated development via regulating laccase genes might be a common mechanism in flowering plants and that the modulation of laccase by miR397 may be potential for engineering plant biomass production with less lignin.     

A new role for veratryl alcohol: regulation of synthesis of lignocellulose-degrading enzymes in the ligninolytic ascomyceteous fungus, Botryosphaeria sp.; influence of carbon source

A new physiological role for veratryl alcohol in fungi important in the biodegradation of the lignified plant cell wall is presented. Botryosphaeria sp., grown on starch, pectin, cellulose or xylan produced amylase, pectinase, cellulase, xylanase and laccase, whereas glucose and xylose repressed the synthesis of cellulase and xylanase, but not laccase. When cultured on each of these substrates in the presence of veratryl alcohol, laccase activity increased but the activities of amylase, pectinase, cellulase and xylanase significantly decreased. Basal medium containing softwood kraft lignin in the presence of veratryl alcohol induced laccases above constitutive levels. Ethyl alcohol also stimulated laccase production.     

Generation and characterization of transgenic poplar plants overexpressing a cotton laccase gene

Laccases are copper-containing glycoproteins, which are widespread in higher plants as multigene families. To gain more insight in the function of laccases in plants, especially potential role in lignification, we produced transgenic poplar plants overexpressing a cotton laccase cDNA (GaLAC1) under the control of the cauliflower mosaic virus 35S promoter. As compared with untransformed control plants, transgenic plants exhibited a 2.1- to 13.2-fold increased laccase activity, whereas plant growth rate and morphological characters remained similar to control plants. A 2.1–19.6% increase in total lignin content of the stem was found in transgenic plants. Moreover, transgenic plants showed a dramatically accelerated oxidation rate of phenolics, without obvious change in total phenolic content. Our data suggested that GaLAC1 may participate in lignin synthesis and phenolic metabolism in plants. The present work provided a new genetic evidence for the involvement of plant laccases in lignification.     

High redox potential laccases from the ligninolytic fungi Pycnoporus coccineus and Pycnoporus sanguineus suitable for white biotechnology: from gene cloning to enzyme characterization and applications

Aims: Exploitation of natural biodiversity in species Pycnoporus coccineus and Pycnoporus sanguineus to screen for a new generation of laccases with properties suitable for the lignin‐processing sector. Methods and Results: Thirty strains originating from subtropical and tropical environments, mainly isolated from fresh specimens collected in situ, were screened for laccase activity. On the basis of levels of enzyme activity and percentage of similarity between protein sequences, the laccases from strains BRFM 938, BRFM 66 and BRFM 902 were selected for purification and characterization. Each BRFM 938, BRFM 66 and BRFM 902 laccase gene encoded a predicted protein of 518 amino acids; the three deduced proteins showed 68·7–97·5% similarity with other Polyporale laccases. The three laccases (59·5–62·9 kDa with 7–10% carbohydrate content) had high redox potentials (0·72–0·75 V vs normal hydrogen electrode at pH 6), remained highly stable up to 75–78°C and at pH 5–7 mixtures, and were resistant to methyl and ethyl alcohols, acetonitrile and dimethylsulfoxide at concentrations as high as 50% (v/v). The best laccase‐1‐hydroxybenzotriazole systems permitted almost 100% of various polyphenolic dye decolourization and oxidation of adlerol and veratryl alcohol. Conclusions: The three laccases showed complementary biochemical features. BRFM 938 laccase had the highest thermo‐ and pH stability, catalytic efficiency towards 2,2′‐azino‐bis‐[3‐ethylthiazoline‐6‐sulfonate] and resistance to alcoholic solvents. BRFM 66 laccase had the highest rates of dye decolourization and oxidation of nonphenolic compounds. Significance and Impact of the Study: This study identified P. coccineus and P. sanguineus as outstanding producers of high redox potential laccases, easy to purify and scale‐up for industrial production. Three new laccases proved to be suitable models for white biotechnology processes and for further molecular breeding to create a new generation of tailor‐made enzymes.     

Increasing <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> laccase production by culture medium optimization and copper/lignin synergistic induction

Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 2^6–2 where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively.     

Comparison of Fungal Laccases and Redox Mediators in Oxidation of a Nonphenolic Lignin Model Compound

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both k_cat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect k_cat and the oxidation rate of dimer I.     

Novel thermotolerant laccases produced by the white-rot fungus <Emphasis Type="Italic">Physisporinus rivulosus</Emphasis>

The white-rot basidiomycete Physisporinus rivulosus strain T241i is highly selective for degradation of softwood lignin, which makes this fungus suitable for biopulping. In order to promote laccase production, P. rivulosus was cultivated in nutrient-nitrogen sufficient liquid media containing either charcoal or spruce sawdust as supplements. Two laccases with distinct pI values, Lac-3.5 and Lac-4.8, were purified from peptone-spruce sawdust-charcoal cultures of P. rivulosus. Both laccases showed thermal stability at up to 60°C. Lac-4.8 was thermally activated at 50°C. Surprisingly, both laccases displayed atypically low pH optima (pH 3.0–3.5) in oxidation of the commonly used laccase substrates syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine), 2,6-dimethoxyphenol and guaiacol (2-methoxyphenol). Steady-state kinetic measurements pointed to unusually low affinity to guaiacol at low pH, whereas the kinetic constants for the methoxyphenols and ABTS were within the ranges reported for other fungal laccases. The combination of thermotolerance with low pH optima for methoxylated phenol substrates suggests that the two P. rivulosus T241i laccases possess potential for use in biotechnological applications.     

Plant biotechnology for lignocellulosic biofuel production

Lignocelluloses from plant cell walls are attractive resources for sustainable biofuel production. However, conversion of lignocellulose to biofuel is more expensive than other current technologies, due to the costs of chemical pretreatment and enzyme hydrolysis for cell wall deconstruction. Recalcitrance of cell walls to deconstruction has been reduced in many plant species by modifying plant cell walls through biotechnology. These results have been achieved by reducing lignin content and altering its composition and structure. Reduction of recalcitrance has also been achieved by manipulating hemicellulose biosynthesis and by overexpression of bacterial enzymes in plants to disrupt linkages in the lignin–carbohydrate complexes. These modified plants often have improved saccharification yield and higher ethanol production. Cell wall‐degrading (CWD) enzymes from bacteria and fungi have been expressed at high levels in plants to increase the efficiency of saccharification compared with exogenous addition of cellulolytic enzymes. In planta expression of heat‐stable CWD enzymes from bacterial thermophiles has made autohydrolysis possible. Transgenic plants can be engineered to reduce recalcitrance without any yield penalty, indicating that successful cell wall modification can be achieved without impacting cell wall integrity or plant development. A more complete understanding of cell wall formation and structure should greatly improve lignocellulosic feedstocks and reduce the cost of biofuel production.     

棉花漆酶基因在转基因新疆杨中的表达及其对木质素合成的影响

以新疆杨叶柄为外植体,利用农杆菌法将棉花漆酶基因GaLAC1导入新疆杨.PCR,Soutllern杂交证明外源基因已经整合到杨树基因组中.漆酶活性分析表明转基因植株中漆酶活性较非转基因对照显著提高.与对照植株相比,转基因新疆杨茎段中总木质素的含量有不同程度的增加,最高达21.5%.木质素的组织化学染色进一步证实了GaLAC1的过量表达能够导致转基因植株中总木质素含量的增加.实验结果表明GaLAC1参与了植物体内木质素的合成,这是首次成功利用转基因植物证实植物漆酶基因参与木质素合成的报道.     

Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) in kraft lignin depolymerization.

Two laccase isozymes (I and II) produced by the white-rot fungus Trametes versicolor were purified, and their reactivities towards various substrates and lignins were studied. The N-terminal amino acid sequences of these enzymes were determined and compared to other known laccase sequences. Laccase II showed a very high sequence similarity to a laccase which was previously reported to depolymerize lignin. The reactivities of the two isozymes on most of the substrates tested were similar, but there were some differences in the oxidation rate of polymeric substrates. We found that the two laccases produced similar qualitative effects on kraft lignin and residual lignin in kraft pulp, with no evidence of a marked preference for depolymerization by either enzyme. However, the presence of the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) prevented and reversed the polymerization of kraft lignin by either laccase. The delignification of hardwood and softwood kraft pulps with the two isozymes and the mediator was compared; either laccase was able to reduce the kappa number of pulp, but only in the presence of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate).     

Ferulic acid: a key component in grass lignocellulose recalcitrance to hydrolysis

In the near future, grasses must provide most of the biomass for the production of renewable fuels. However, grass cell walls are characterized by a large quantity of hydroxycinnamic acids such as ferulic and p‐coumaric acids, which are thought to reduce the biomass saccharification. Ferulic acid (FA) binds to lignin, polysaccharides and structural proteins of grass cell walls cross‐linking these components. A controlled reduction of FA level or of FA cross‐linkages in plants of industrial interest can improve the production of cellulosic ethanol. Here, we review the biosynthesis and roles of FA in cell wall architecture and in grass biomass recalcitrance to enzyme hydrolysis.