Lignin-degrading Enzymes Research Articles

Identification and Characterization of a New Serratia proteamaculans Strain That Naturally Produces Significant Amount of Extracellular Laccase.

Natural biodegradation processes hold promises for the conversion of agro-industrial lignocellulosic biomaterials into biofuels and fine chemicals through lignin-degrading enzymes. The high cost and low stability of these enzymes remain a significant challenge to economic lignocellulosic biomass conversion. Wood-degrading microorganisms are a great source for novel enzyme discoveries. In this study, the decomposed wood samples were screened, and a promising γ-proteobacterial strain that naturally secreted a significant amount of laccase enzyme was isolated and identified as Serratia proteamaculans AORB19 based on its phenotypic and genotypic characteristics. The laccase activities in culture medium of strain AORB19 were confirmed both qualitatively and quantitatively. Significant cultural parameters for laccase production under submerged conditions were identified following a one-factor-at-a-time (OFAT) methodology: temperature 30°C, pH 9, yeast extract (2 g/l), Li+, Cu2+, Ca2+, and Mn2+ (0.5 mM), and acetone (5%). Under the selected conditions, a 6-fold increase (73.3 U/L) in laccase production was achieved when compared with the initial culturing conditions (12.18 U/L). Furthermore, laccase production was enhanced under alkaline and mesophilic growth conditions in the presence of metal ions and organic solvents. The results of the study suggest the promising potential of the identified strain and its enzymes in the valorization of lignocellulosic wastes. Further optimization of culturing conditions to enhance the AORB19 strain laccase secretion, identification and characterization of the purified enzyme, and heterologous expression of the specific enzyme may lead to practical industrial and environmental applications.

Genome Functional Analysis of the Psychrotrophic Lignin-Degrading Bacterium Arthrobacter sp. C2 and the Role of DyP in Catalyzing Lignin Degradation.

In the cold regions of China, lignin-rich corn straw accumulates at high levels due to low temperatures. The application of psychrotrophic lignin-degrading bacteria should be an effective means of overcoming the low-temperature limit for lignin degradation and promoting the utilization of corn straw. However, this application is limited by the lack of suitable strains for decomposition of lignin; furthermore, the metabolic mechanism of psychrotrophic lignin-degrading bacteria is unclear. Here, the whole genome of the psychrotrophic lignin-degrading bacterium Arthrobacter sp. C2, isolated in our previous work, was sequenced. Comparative genomics revealed that C2 contained unique genes related to lignin degradation and low-temperature adaptability. DyP may participate in lignin degradation and may be a cold-adapted enzyme. Moreover, DyP was proven to catalyze lignin Cα-Cβ bond cleavage. Deletion and complementation of the DyP gene verified its ability to catalyze the first-step reaction of lignin degradation. Comparative transcriptomic analysis revealed that the transcriptional expression of the DyP gene was upregulated, and the genetic compensation mechanism allowed C2ΔDyP to degrade lignin, which provided novel insights into the survival strategy of the psychrotrophic mutant strain C2ΔdyP. This study improved our understanding of the metabolic mechanism of psychrotrophic lignin-degrading bacteria and provided potential application options for energy-saving production using cold-adapted lignin-degrading enzymes.

Investigations on the Fusants From Wide Cross Between White-Rot Fungi and Saccharomyces cerevisiae Reveal Unknown Lignin Degradation Mechanism.

The degradation of lignocellulose by fungi, especially white-rot fungi, contributes a lot to carbon cycle, bio-fuel production, and many other bio-based applications. However, the existing enzymatic and non-enzymatic degradation mechanisms cannot be unequivocally supported by in vitro simulation experiment, meaning that additional mechanisms might exist. Right now, it is still very difficult to discover new mechanisms with traditional forward genetic approaches. To disclose novel lignin degradation mechanisms in white-rot fungi, a series of fusants from wide cross by protoplast fusion between Pleurotus ostreatus, a well-known lignin-degrading fungus, and Saccharomyces cerevisiae, a well-known model organism unable to degrade lignocellulose, was investigated regarding their abilities to degrade lignin. By analyzing the activity of traditional lignin-degrading enzyme, the ability to utilize pure lignin compounds and degrade corn stalk, a fusant D1-P was screened out and proved not to contain well-recognized lignin-degrading enzyme genes by whole-genome sequencing. Further investigation with two-dimension nuclear magnetic resonance (NMR) shows that D1-P was found to be able to degrade the main lignin structure β-O-4 linkage, leading to reduced level of this structure like that of the wild-type strain P. ostreatus after a 30-day semi-solid fermentation. It was also found that D1-P shows a degradation preference to β-O-4 linkage in Aβ(S)-threo. Therefore, wide cross between white-rot fungi and S. cerevisiae provides a powerful tool to uncover novel lignocellulose degradation mechanism that will contribute to green utilization of lignocellulose to produce bio-fuel and related bio-based refinery.

Laccase-mediator system for enzymatic degradation of carbonaceous matter in the sequential pretreatment of double refractory gold ore from Syama mine, Mali

The sequential bio-treatment of refractory carbonaceous gold ore is a promising solution to recover gold effectively by environmentally friendly technology, which includes bio-oxidation of sulfide and biodegradation of carbonaceous matter by lignin-degrading enzymes. There are several drawbacks in enzyme treatment using cell-free spent medium (CFSM), including lignin peroxidase and manganese peroxidase from Phanerochaete chrysosporium, in particular the poor stability of enzyme activities. In the present work, laccase-mediator system (LMS) was applied for the degradation of carbonaceous matter in real gold ore to improve the efficiency of gold extraction as well as handling. The LMS was intended to be a great alternative process of CFSM with utilizing purified laccase in the presence of 1-hydroxybenzotriazole as a mediator. The application of LMS provided several advantages including not only greater stability, greater efficiency to degrade carbonaceous matter, better handling, much saving the treatment time, but also wider availability in laccase. In addition, replacing bio-oxidation with ferric chloride leaching as the dissolution path of sulfides facilitated avoiding the formation of jarosite and saving the required time. The gold recovery by cyanidation was improved from 41.5 ± 0.3% for the starting material to 81.3 ± 3.9% (n = 2) for the solid residues after the modified sequential pretreatment. This is correspondent to 86.3% of gold recovery for the extractable maximum gold excluding the enclosed gold in acid-insoluble silicates. The improved process involving LMS can be proposed with valuable advantages to fit a sustainable metallurgical technology of gold ores.

Enzymatically triggered delignification through a novel stable laccase: A mixed in-silico /in-vitro exploration of a complex environmental microbiota

Laccases have been broadly applied as a multitasking biocatalyst in various industries, but their applications tend to be limited by easy deactivation, lack of adequate stability, and susceptibility under complex conditions. Identifying stable laccase as a green-biocatalyst is crucial for developing cost-effective biorefining processes. In this direction, we attempted in-silico screening a stable metagenome-derived laccase (PersiLac1) from tannery wastewater in a complex environment. The laccase exhibited high thermostability, retaining 53.19% activity after 180 min at 70 °C, and it was stable in a wide range of pH (4.0–9.0). After 33 days of storage at 50°C, pH 6.0, the enzyme retained 71.65% of its activity. Various metal ions, inhibitors, and organic solvents showed that PersiLac1 has a stable structure. The stable PersiLac1 could successfully remove lignin and phenolic from quinoa husk and rice straw. In the separate hydrolysis and fermentation process (SHF) after 72 h, hydrolysis was obtained 100% and 73.4% for quinoa husk and rice straw, and fermentation by the S. cerevisiae was be produced 41.46 g/L and 27.75g/L ethanol, respectively. Results signified that the novel lignin-degrading enzyme was confirmed to have great potential for industrial application as a green-biocatalyst based on enzymatically triggered to delignification and detoxify lignocellulosic biomass.

Whole-Genome Sequencing Reveals Lignin-Degrading Capacity of a Ligninolytic Bacterium (Bacillus cereus) from Buffalo (Bubalus bubalis) Rumen

The buffalo is an amazing ruminant. Its ability to degrade lignin, which has been recently reported, is most likely due to unique rumen microorganisms with lignin-degradation potential. Our goal was to explore the lignin-degradation potential of ruminal microorganisms, in which ligninolytic enzyme encoding genes were involved to provide ideas for revealing the mechanism of lignin degradation by buffalo. In this study, a bacterium strain identified as Bacillus cereus AH7-7 was isolated from the buffalo (Bubalus bubalis) rumen. After whole-genome sequencing, the results demonstrated that B. cereus AH7-7 had laccase, cytochrome P450 and vanillin alcohol oxidase-encoding genes. Sixty-four genes of B. cereus AH7-7 were involved in multiple aromatic metabolic pathways, such as phenylalanine metabolism and aminobenzoate degradation. A positive reaction resulting in guaiacol medium indicated that laccase secretion from B. cereus AH7-7 increased with time. A biodegradation experiment revealed that a significant reduction in kraft lignin content (25.9%) by B. cereus AH7-7 occurred at the end of 6 days of incubation, which confirmed its lignin-degradation capacity. Overall, this is the first report showing that B. cereus AH7-7 from the buffalo rumen can degrade lignin, and revealing the encoding genes of lignin-degrading enzymes from genome level.

Effects of different forms of nitrogen addition on microbial extracellular enzyme activity in temperate grassland soil

BackgroundNitrogen (N) deposition alters litter decomposition and soil carbon (C) sequestration by influencing the microbial community and its enzyme activity. Natural atmospheric N deposition comprises of inorganic N (IN) and organic N (ON) compounds. However, most studies have focused on IN and its effect on soil C cycling, whereas the effect of ON on microbial enzyme activity is poorly understood. Here we studied the effects of different forms of externally supplied N on soil enzyme activities related to decomposition in a temperate steppe. Ammonium nitrate was chosen as IN source, whereas urea and glycine were chosen as ON sources. Different ratios of IN to ON (Control, 10:0, 7:3, 5:5, 3:7, and 0:10) were mixed with equal total amounts of N and then used to fertilize the grassland soils for 6 years.ResultsOur results show that IN deposition inhibited lignin-degrading enzyme activity, such as phenol oxidase (POX) and peroxidase (PER), which may restrain decomposition and thus induce accumulation of recalcitrant organic C in grassland soils. By contrast, deposition of ON and mixed ON and IN enhanced most of the C-degrading enzyme activities, which may promote the organic matter decomposition in grassland soils. In addition, the β-N-acetyl-glucosaminidase (NAG) activity was remarkably stimulated by fertilization with both IN and ON, maybe because of the elevated N availability and the lack of N limitation after long-term N fertilization at the grassland site. Meanwhile, differences in soil pH, soil dissolved organic carbon (DOC), and microbial biomass partially explained the differential effects on soil enzyme activity under different forms of N treatments.ConclusionsOur results emphasize the importance of organic N deposition in controlling soil processes, which are regulated by microbial enzyme activities, and may consequently change the ecological effect of N deposition. Thus, more ON deposition may promote the decomposition of soil organic matter thus converting C sequestration in grassland soils into a C source.

Melioration of enzymatic ethanol production from alkali pre-treated paddy straw promoted by addition of surfactant

Along with the cellulase enzyme, xylanase plays an efficient role in the production of biofuel from agricultural wastes by degrading the xylan sugar present in the hemicellulose of cell wall. This study aims to improve the sugar production from biomass by the use of different enzymes with surfactant. The objective of this study is to compare sugar production and bioethanol production from sodium hydroxide along polyethylene glycol pre-treated paddy straw (Oryza sativa L.) with different combination of xylanase and cellulose enzymes along with lignin-degrading laccase enzyme. In results, 10.87 g/l of ethanol with saccharification of 64.51% ± 0.90 was obtained when xylanase and laccase were used, while 18.40 ± 0.56 g/l of ethanol with saccharification of 84.01%±1.09 was obtained when cellulase and laccase enzymes were used. Maximum bioethanol production was found to be 19.20 ± 0.26g/l, which was obtained by combination of xylanase, cellulase and laccase enzymes together at 37 °C after 36 h.

Genomic Analysis of Stropharia rugosoannulata Reveals Its Nutritional Strategy and Application Potential in Bioremediation.

Stropharia rugosoannulata is not only a popular edible mushroom, but also has excellent potential in bioremediation. In this study, we present a high-quality genome of a monokaryotic strain of the S. rugosoannulata commercial cultivar in China. The assembly yielded an N50 length of 2.96 Mb and a total size of approximately 48.33 Mb, encoding 11,750 proteins. The number of heme peroxidase-encoding genes in the genome of S. rugosoannulata was twice the average of all of the tested Agaricales. The genes encoding lignin and xenobiotic degradation enzymes accounted for more than half of the genes encoding plant cell wall degradation enzymes. The expansion of genes encoding lignin and xenobiotic degradation enzymes, and cytochrome P450 involved in the xenobiotic metabolism, were responsible for its strong bioremediation and lignin degradation abilities. S. rugosoannulata was classified as a litter-decomposing (LD) fungus, based on the analysis of the cell wall degrading enzymes. Substrate selection for fruiting body cultivation should consider both the nutritional strategy of LD and a strong lignin degradation ability. Consistent with safe usage as an edible mushroom, the S. rugosoannulata genome does not contain genes for known psilocybin biosynthesis. Genome analysis will be helpful for understanding its nutritional strategy to guide fruiting body cultivation and for providing insight into its application in bioremediation.

Improvement of laccase activity by silencing PacC in Ganoderma lucidum.

Ganoderma lucidum is a representative white-rot fungus that has great potential to degrade lignocellulose biomass. Laccase is recognized as a class of the most important lignin-degrading enzymes in G. lucidum. However, the comprehensive regulatory mechanisms of laccase are still lacking. Based on the genome sequence of G. lucidum, 15 laccase genes were identified and their encoding proteins were analyzed in this study. All of the laccase proteins are predicted to be multicopper oxidases with conserved copper-binding domains. Most laccase proteins were secreted enzymes in addition to Lac14 in which the signal peptide could not be predicted. The activity of all laccases showed the highest level at pH 3.0 or pH 7.0, with total laccase activity of approximately 200 U/mg protein. Silencing PacC resulted in a 5.2 fold increase in laccase activity compared with WT. Five laccase genes (lac1, lac6, lac9, lac10 and lac14) showed an increased transcription levels (approximately 1.5-5.6 fold) in the PacC-silenced strains versus that in WT, while other laccase genes were downregulated or unchanged. The extracellular pH value was about 3.1, which was more acidic in the PacC-silenced strains than in the WT (pH 3.5). Moreover, maintaining the fermentation pH resulted in a downregulation of laccase activity which is induced by silencing PacC. Our findings indicate that in addition to its function in acidification of environmental pH, PacC plays an important role in regulating laccase activity in fungi.

Catabolic profiling of selective enzymes in the saccharification of non-food lignocellulose parts of biomass into functional edible sugars and bioenergy: An in silico bioprospecting.

Objectives:The research aims to analyze the catabolic strength of different hydrolytic enzymes in assessing the biological conversion potential of lignocellulose parts of agricultural biomass wastes into functional edible sugars and biofuels.Materials and Methods:The enzymes’ hydrolytic properties—versatile peroxidase, manganese peroxidase, and lignin peroxidase were used to identify their complexing strength with the lignin substrate, whereas endoglucanase cel12A, acidocaldarius cellulase, and Melanocarpus albomyces endoglucanase were tested on the cellulose gel substrate. Because the biodegradation properties are heavily influenced by the “enzyme-substrate complexing energy level,” proper molecular optimization and energy minimization of the enzymes and substrates were carried out, as well as the identification of the enzyme’s active sites prior to complexing.comprehensive molecular dynamic simulation was run to study their—alpha carbon, root-mean-square deviation (Å), molecular surface area (Å2), root-mean-square fluctuation (Å), radius of gyration (nm), hydrogen bonds with hydrophobic interactions, and solvent accessible surface area (Å2) values for 50 ns. The simulated data mining was conducted using advanced programming algorithms to establish the final enzyme-substrate complexing strength in binding and catalysis.Results:Among the lignin-degrading enzymes, versatile peroxidase shows promising catalytic activity with the best docking pose and significant values in all the dynamic simulation parameters. Similarly, Melanocarpus albomyces endoglucanase shows the best activity in all aspects of molecular docking and dynamics among the cellulose-degrading enzymes.Conclusion:The lignin content of biomass wastes can be degraded into cellulose and hemicellulose using lignin-degrading enzymes. The cellulose can be further degraded into glucose and xylose sugars following the cellulose-degrading enzyme activity. These sugars can be further degraded into biofuel through anaerobic fermentation. Systematic bioconversion of the lignocellulosic components can ensure sustainable biomass management, creating an alternative food and energy source for human beings to face the challenges of global hunger where the enzymes can pave the way.

Laccase-Coated Polyethersulfone Membranes for Organic Matter Degradation and Removal

Natural organic matter (NOM) removal from water is getting progressively significant for water treatment plants not only to improve drinking water aesthetics such as taste and smell, but also to avoid disinfection by-products (DBPs) formed during disinfection by chlorine. This study applies the catalytic properties of the wood degrading laccase enzyme produced by white rot fungi (WRF) on breaking down and removing organic matter in drinking water. White rot fungi isolates were collected and examined for their ability to degrade humic acid (HA), a NOM model compound. Highly permeable polyethersulfone (PES) membrane was prepared following the phase inversion process and used as material to support the immobilization of the lignin-degrading enzymes extracted from Perenniporia sp. and Polyporaceae sp. for NOM degradation and removal. A 52 % humic acid removal was recorded for the Polyporaceae sp. The addition of laccase substrate 4-hydroxybenzoic acid showed a great impact on the hydrophilicity of the membranes as a decrease in contact angle measurements of <60 was achieved. Moreover, modified membrane’s immobilization yield and enzyme activity also improved. The modified membrane achieved a rejection of greater than 90 % for the model compound. Enzyme activity was a function of contact time and substrate type. The attained results revealed that catalytic membranes can be an efficient alternative for NOM removal and membrane fouling alleviation during water treatment.

Lignin degradation by microorganisms: A review.

Lignin is an abundant plant-based biopolymer that has found applications in a variety of industries from construction to bioethanol production. This recalcitrant branched polymer is naturally degraded by many different species of microorganisms, including fungi and bacteria. These microbial lignin degradation mechanisms provide a host of possibilities to overcome the challenges of using harmful chemicals to degrade lignin biowaste in many industries. The classes and mechanisms of different microbial lignin degradation options available in nature form the primary focus of the present review. This review first discusses the chemical building blocks of lignin and the industrial sources and applications of this multifaceted polymer. The review further places emphasis on the degradation of lignin by natural means, discussing in detail the lignin degradation activities of various fungal and bacterial species. The lignin-degrading enzymes produced by various microbial species, specifically white-rot fungi, brown-rot fungi, and bacteria, are described. In the end, possible directions for future lignin biodegradation applications and research investigations have been provided.

A novel Streptomyces strain isolated by functional bioprospecting for laccases

In discovering novel laccase enzymes with interesting catalytic and stability properties, there is still scope for improvement in exploring new sites in Algeria. The bio-prospecting approach allowed the isolation of a novel strain, Streptomyces sp. HBD30 from the Agrioun River, northern Algeria. Following a functional approach, two laccases (LacI and LacII) were purified and characterised, showing appealing properties as thermostable enzymes, and oxidised typical phenolic and non-phenolic substrates with a higher affinity towards syringaldazine, followed by 2,6-dimethoxyphenol and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid). Further analyses revealed that the Streptomyces sp. HDB30 genome contained an array of genes coding for carbohydrate active enzymes (CAZome) and lignin-degrading enzymes to degrade lignocellulose. In addition to enlarging the repertoire of thermostable laccases, this study uncovered the metabolic potential of a new Streptomyces strain, which is useful for a wide range of applications.

Enzymatic pretreatment of steam-exploded birch wood for increased biogas production and lignin degradation

Lignocellulose is readily available biomass for biogas production; however, due to its rigid structure, it requires pretreatment to obtain a maximum energy extraction. In this study, steam explosion (SE) (220 °C and 10 minute retention time) has been employed to increase the biogas production potential from birch wood. Although the biogas production increased by over two times after SE, the SE of birch wood negatively affects the structure of C5/C6 sugars and doubled the concentration of non-degradable lignin in all the samples. In this work, SE birch wood has been further pretreated by novel lignin-degrading enzymes cocktail to convert lignin into degradable sugars and increase the biogas production rate. The proposed hybrid pretreatment could increase the biogas production by up to 25% (from 450.5 mL/g VS to 566 mL/g VS), and reduced the lignin concentration by up to 48%.

Effect of manganese peroxidase on the decomposition of cellulosic components: Direct cellulolytic activity and synergistic effect with cellulase

Herein, it was unearthed that manganese peroxidase (MnP) from Phanerochaete chrysosporium, a lignin-degrading enzyme, is capable of not only directly decomposing cellulosic components but also boosting cellulase activity. MnP decomposes various cellulosic substrates (carboxymethyl cellulose, cellobiose [CMC], and Avicel®) and produces reducing sugars rather than oxidized sugars such as lactone and ketoaldolase. MnP with MnII in acetate buffer evolves the MnIII-acetate complex functioning as a strong oxidant, and the non-specificity of MnIII-acetate enables cellulose-decomposition. The catalytic mechanism was proposed by analyzing catalytic products derived from MnP-treated cellopentaose. Notably, MnP also boosts cellulase activity on CMC and Avicel®, even considering the cellulolytic activity of MnP itself. To the best of the authors’ knowledge, this is the first report demonstrating a previously unknown fungal MnP activity in cellulose-decomposition in addition to a known delignification activity. Consequently, the results provide a promising insight for further investigation of the versatility of lignin-degrading biocatalysts.

Oxic and Anoxic Organic Polymer Degradation Potential of Endophytic Fungi From the Marine Macroalga, Ecklonia radiata.

Cellulose and chitin are the most abundant polymeric, organic carbon source globally. Thus, microbes degrading these polymers significantly influence global carbon cycling and greenhouse gas production. Fungi are recognized as important for cellulose decomposition in terrestrial environments, but are far less studied in marine environments, where bacterial organic matter degradation pathways tend to receive more attention. In this study, we investigated the potential of fungi to degrade kelp detritus, which is a major source of cellulose in marine systems. Given that kelp detritus can be transported considerable distances in the marine environment, we were specifically interested in the capability of endophytic fungi, which are transported with detritus, to ultimately contribute to kelp detritus degradation. We isolated 10 species and two strains of endophytic fungi from the kelp Ecklonia radiata. We then used a dye decolorization assay to assess their ability to degrade organic polymers (lignin, cellulose, and hemicellulose) under both oxic and anoxic conditions and compared their degradation ability with common terrestrial fungi. Under oxic conditions, there was evidence that Ascomycota isolates produced cellulose-degrading extracellular enzymes (associated with manganese peroxidase and sulfur-containing lignin peroxidase), while Mucoromycota isolates appeared to produce both lignin and cellulose-degrading extracellular enzymes, and all Basidiomycota isolates produced lignin-degrading enzymes (associated with laccase and lignin peroxidase). Under anoxic conditions, only three kelp endophytes degraded cellulose. We concluded that kelp fungal endophytes can contribute to cellulose degradation in both oxic and anoxic environments. Thus, endophytic kelp fungi may play a significant role in marine carbon cycling via polymeric organic matter degradation.

An efficient strategy to improve enzymatic hydrolysis of naked oat straw pretreated by Irpex lacteus.

The objective of this study was aiming at developing an efficient strategy to promote enzymatic hydrolysis of naked oat straw and deciphering the potential mechanism. Irpex lacteus and Phlebia acerina were employed to inoculated on the naked oat straw for 4weeks which the changes of fiber components, fermentation losses, lignin-degrading enzymes production pattern were determined weekly. Furthermore, the 72h enzymatic hydrolysis of ultimately fermented naked oat straw were also evaluated. The acid detergent lignin was degraded at about 25% along with the moderate dry matter and cellulose loss which both showed selective degradation. The lignin-degrading enzymes production patterns of the two fungi were different which lignin peroxidase was not detected in Irpex lacteus treatment. In addition, the activities of cellulolytic enzymes were higher in Phlebia acerina treatment. After 72h enzymatic hydrolysis, the reducing sugar content and hydrolysis yield pretreated by Irpex lacteus was 12.92g/L and 69.49%, respectively. It was much higher than that in sterilized substrate and Phlebia acerina treatment. Meanwhile, the hydrolysis yields of glucose, sum of xylose and arabinose were all improved by Irpex lacteus which were 30.96% and 25.62%, respectively, and showed significant enhancements compared to control and Phlebia acerina treatment. Irpex lacteus is one of effective white rot fungi which could promote the enzymatic hydrolysis of naked oat straw obviously.

Isolation of Fungi from a Textile Industry Effluent and the Screening of Their Potential to Degrade Industrial Dyes.

Six fungal strains were isolated from the textile industry effluent in which they naturally occur. Subsequently, the fungal strains were identified and characterized in order to establish their potential decolorizing effect on textile industry effluents. The strains of interest were selected based on their capacity to decolorize azo, indigo, and anthraquinone dyes. Three of the strains were identified as Emmia latemarginata (MAP03, MAP04, and MAP05) and the other three as Mucor circinelloides (MAP01, MAP02, and MAP06), while the efficiency of their decolorization of the dyes was determined on agar plate and in liquid fermentation. All the strains co-metabolized the dyes of interest, generating different levels of dye decolorization. Plate screening for lignin-degrading enzymes showed that the MAP03, MAP04, and MAP05 strains were positive for laccase and the MAP01, MAP02, and MAP06 strains for tyrosinase, while all strains were positive for peroxidase. Based on its decolorization capacity, the Emmia latemarginata (MAP03) strain was selected for the further characterization of its growth kinetics and ligninolytic enzyme production in submerged fermentation under both enzyme induction conditions, involving the addition of Acetyl yellow G (AYG) dye or wheat straw extract, and no-induction condition. The induction conditions promoted a clear inductive effect in all of the ligninolytic enzymes analyzed. The highest level of induced enzyme production was observed with the AYG dye fermentation, corresponding to versatile peroxidase (VP), manganese peroxidase (MnP), and lignin peroxidase (LiP). The present study can be considered the first analysis of the ligninolytic enzyme system of Emmia latemarginata in submerged fermentation under different conditions. Depending on the results of further research, the fungal strains analyzed in the present research may be candidates for further biotechnological research on the decontamination of industrial effluents.

A plant host, Nicotiana benthamiana, enables the production and study of fungal lignin-degrading enzymes

Lignin has significant potential as an abundant and renewable source for commodity chemicals yet remains vastly underutilized. Efforts towards engineering a biochemical route to the valorization of lignin are currently limited by the lack of a suitable heterologous host for the production of lignin-degrading enzymes. Here, we show that expression of fungal genes in Nicotiana benthamiana enables production of members from seven major classes of enzymes associated with lignin degradation (23 of 35 tested) in soluble form for direct use in lignin activity assays. We combinatorially characterized a subset of these enzymes in the context of model lignin dimer oxidation, revealing that fine-tuned coupling of peroxide-generators to peroxidases results in more extensive C-C bond cleavage compared to direct addition of peroxide. Comparison of peroxidase isoform activity revealed that the extent of C-C bond cleavage depends on peroxidase identity, suggesting that peroxidases are individually specialized in the context of lignin oxidation. We anticipate the use of N. benthamiana as a platform to rapidly produce a diverse array of fungal lignin-degrading enzymes will facilitate a better understanding of their concerted role in nature and unlock their potential for lignin valorization, including within the plant host itself.