Sphingobium Sp Research Articles

The first characterized phage against a member of the ecologically important sphingomonads reveals high dissimilarity against all other known phages

This study describes the first molecular characterization of a bacteriophage infecting a member of the environmentally important Sphingomonadaceae family. Both bacteriophage Lacusarx and its host Sphingobium sp. IP1 were isolated from activated sludge from a wastewater treatment plant. Genome sequencing revealed that the phage genes display little similarity to other known phages, despite a remarkable conservation of the synteny in which the functional genes occur among distantly related phages. Phylogenetic analyses confirmed that Lacusarx represents a hitherto undescribed genus of phages. A classical lysis cassette could not be identified in Lacusarx, suggesting that the genes encoding endolysin, holin, and spanin are host-specific and not found in phages infecting other bacteria. The virus harbors 24 tRNA genes corresponding to 18 different amino acids and furthermore has a significantly different codon usage than its host. Proteomic analysis of Lacusarx revealed the protein components of the phage particle. A lysogeny test indicated that Lacusarx is not a temperate phage.

Characterization of a topologically unique oxygenase from Sphingobium sp. PNB capable of catalyzing a broad spectrum of aromatics.

A Rieske non-heme iron ring-hydroxylating oxygenase (RHO) from Sphingobium sp. PNB involved in the initial oxidation of a wide range of low and high molecular weight polycyclic aromatic hydrocarbons (PAHs) was investigated. The RHO was shown to comprise of the gene products of distantly located ahdA1f-ahdA2f, ahdA3 and ahdA4 genes, which encoded the oxygenase α- and β-subunits, ferredoxin and reductase, respectively. In silico structural analysis of AhdA1f revealed a very large substrate-binding pocket, satisfying the spatial requirements to accommodate high molecular weight substrates. In addition, an atypical substrate access channel was noticed from the topology analysis of the oxygenase. Guided by molecular docking studies, dioxygenation of several PAHs as well as alkyl- and aryl benzenes was examined with the recombinant AhdA1fA2f expressed in Escherichia coli. Chromatographic and mass spectrometric analyses confirmed that AhdA1fA2f displays broad substrate specificity towards a wide range of aromatic hydrocarbons including potential xenobiotics, demonstrating metabolic robustness of strain PNB.

Isolation and characterization of ethoprophos-degrading soil bacterium Sphingobium sp. EP60845

Isolation and characterization of ethoprophos-degrading soil bacterium Sphingobium sp. EP60845

Assigning ecological roles to the populations belonging to a phenanthrene-degrading bacterial consortium using omic approaches.

The present study describes the behavior of a natural phenanthrene-degrading consortium (CON), a synthetic consortium (constructed with isolated strains from CON) and an isolated strain form CON (Sphingobium sp. AM) in phenanthrene cultures to understand the interactions among the microorganisms present in the natural consortium during phenanthrene degradation as a sole carbon and energy source in liquid cultures. In the contaminant degradation assay, the defined consortium not only achieved a major phenanthrene degradation percentage (> 95%) but also showed a more efficient elimination of the intermediate metabolite. The opposite behavior occurred in the CON culture where the lowest phenanthrene degradation and the highest HNA accumulation were observed, which suggests the presence of positive and also negative interaction in CON. To consider the uncultured bacteria present in CON, a metagenomic library was constructed with total CON DNA. One of the resulting scaffolds (S1P3) was affiliated with the Betaproteobacteria class and resulted in a significant similarity with a genome fragment from Burkholderia sp. HB1 chromosome 1. A complete gene cluster, which is related to one of the lower pathways (meta-cleavage of catechol) involved in PAH degradation (ORF 31–43), mobile genetic elements and associated proteins, was found. These results suggest the presence of at least one other microorganism in CON besides Sphingobium sp. AM, which is capable of degrading PAH through the meta-cleavage pathway. Burkholderiales order was further found, along with Sphingomonadales order, by a metaproteomic approach, which indicated that both orders were metabolically active in CON. Our results show the presence of negative interactions between bacterial populations found in a natural consortium selected by enrichment techniques; moreover, the synthetic syntrophic processing chain with only one microorganism with the capability of degrading phenanthrene was more efficient in contaminant and intermediate metabolite degradation than a generalist strain (Sphingobium sp. AM).

Efficient removal of toxic phthalate by immobilized serine-type aldehyde-tagged esterase G

Abstract Esterase G (EstG) from dibutyl phthalate (DBP)-degrading Sphingobium sp. SM42 was immobilized on amine-functionalized supports through aldehyde tag technology. Two different sulfatase motif tags, either LCTPSR (cysteine-type) or MSAPAR (serine-type), each of which is recognized by a specific formylglycine generating enzyme (FGE), were fused to the C-terminus of EstG. The cysteine-specific FGE was derived from Pseudomonas putida KT2440 while Klebsiella sp. SLS5 provided serine-specific FGE. The EstG with serine-type aldehyde tag showed a greater immobilization yield and higher specific activity by 4.8-fold and 1.8-fold, respectively. The immobilized EstG retained over 90% of its original activity after seven cycles of usage, and exhibited significantly improved thermostability by retaining 66% activity after 1 h incubation at 60 °C. Additionally, nearly 100% and over 30% of the DBP in 10 mM and 100 mM solutions, respectively, was degraded by the immobilized EstG within 18 h.

Effects of Bacterial-Feeding Nematode Grazing and Tea Saponin Addition on the Enhanced Bioremediation of Pyrene-Contaminated Soil Using Polycyclic Aromatic Hydrocarbon-Degrading Bacterial Strain

As one of the most widely distributed bacterial predators in the soil, the role of bacterivorous nematodes on the enhanced bioremediation of polycyclic aromatic hydrocarbon-contaminated soils is crucial, but remains to be investigated. A microcosm-level study was conducted to examine the effects of bacterial-feeding nematode grazing and tea saponin (TS) addition on bioremediation of a pyrene-contaminated soil enhanced by the polycyclic aromatic hydrocarbon (PAH)-degrading bacterial strain Sphingobium sp. PHE9. After 180 d of incubation, the highest pyrene dissipation (71.3%) was achieved through a combination of Sphingobium sp. PHE9 inoculation with nematode and TS addition. Meanwhile, high counts of culturable PAH-degrading bacteria, soil enzyme activity, and biodiversity indices were observed under the combined treatment, implying that the microbiological function of the contaminated soil was significantly restored. Additionally, the results of Tenax® extraction with the first-order three-compartment model indicated that rate-limiting factors varied among treatments. The lack of degrading microorganisms was the main rate-limiting factor for the treatments involving TS/nematode addition, and inadequate bioaccessible pyrene was the vital rate-limiting factor in the treatments involving Sphingobium sp. PHE9 inoculation. The proposed combined clean-up strategy proved to be a promising bioremediation technology for aged pyrene-contaminated soils.

Colonization and Maize Growth Promotion Induced by Phosphate Solubilizing Bacterial Isolates.

Phosphorus (P) limits the production of maize, one of the major food crops in China. Phosphate-solubilizing bacteria (PSB) have the capacity to solubilize phosphate complexes into plant absorbable and utilizable forms by the process of acidification, chelation, and exchange reactions. In this study, six bacteria, including one Paenibacillus sp. B1 strain, four Pseudomonas sp. strains (B10, B14, SX1, and SX2) and one Sphingobium sp. SX14 strain, were those isolated from the maize rhizosphere and identified based on their 16S rRNA sequences. All strains could solubilize inorganic P (Ca3(PO4)2, FePO4 and AlPO4), and only B1 and B10 organic P (lecithin). All strains, except of SX1, produced IAA, and SX14 and B1 showed the highest level. B1 incited the highest increase in root length and the second increase in shoot and total dry weight, shoot length, and total P and nitrogen (N), along with increased root length. In addition, by confocal laser scanning microscopy (CLSM), we found that green fluorescent protein (GFP)-labeled B1 mainly colonized root surfaces and in epidermal and cortical tissue. Importantly, B1 can survive through forming spores under adverse conditions and prolong quality guarantee period of bio-fertilizer. Therefore, it can act as a good substitute for bio-fertilizer to promote agricultural sustainability.

Evaluation of bacterial biodegradation and accumulation of phenanthrene in the presence of humic acid

This study evaluated the effect of humic acid (HA) on physicochemical properties of bacterial surfaces and on mass transfer of polycyclic aromatic hydrocarbons (PAHs) from aqueous phase into intracellular bacteria. Due to this process’ potential for bacterial degradation, using Sphingobium sp. PHE3, degradation of phenanthrene (PHE) was compared in HA and non-HA sets. The results showed that approximately 51.1% of PHE at a concentration of 102.0 mg L−1 was biodegraded in the non-HA sets, whereas almost all PHE was biodegraded with HA after 72 h. Interestingly, PHE that accumulated in the intracellular bacteria reached 3.80 mg L−1 for the HA sets, which was significantly higher than that of non-HA. Lipid inclusion bodies appeared when Sphingobium sp. PHE3 was treated with HA. The results were further confirmed by the enhanced bacterial surface sorption capacity for the HA sets. Therefore, we concluded that added HA not only act as carriers and biosurfactants facilitating PHE uptake but also adjust bacteria cell wall properties for internalizing PHE, which ultimately overcame the PHE bioavailability resulting in enhanced biodegradation.

Feasibility of Tea Saponin-Enhanced Soil Washing in a Soybean Oil-Water Solvent System to Extract PAHs/Cd/Ni Efficiently from a Coking Plant Site

Mixed contaminated brownfield sites have brought serious risks to human health and environmental safety. With the purpose of removing polycyclic aromatic hydrocarbons (PAHs) and heavy metals from a coking plant site, an innovative technology for ex-situ washing was developed in the present work. The combination of 15.0 mL L−1 soybean oil and 7.5 g L−1 tea saponin proved an effective method to extract co-pollutants from soil. After two consecutive washing cycles, the efficiency rates of removal for 3-, 4-, 5(+6)-ring, and total PAHs, Cd, and Ni were approximately 98.2%, 96.4%, 92.3%, 96.3%, 94.1%, and 89.4%, respectively. Meanwhile, as evaluated by Tenax extraction method and metal stability indices, the residual PAHs and heavy metals after consecutive washing mainly existed in the form with extremely low bioaccessibility in the soil. Thus, in the soil after two washing cycles, there appeared limited environmental transfer risk of co-pollutants. Moreover, a subsequent precipitation method with alkaline solution and PAH-degrading strain Sphingobium sp. PHE9 inoculation effectively removed 84.6%–100% of Cd, 82.5%–91.7% of Ni, and 92.6%–98.4% of PAHs from the first and second washing solvents. The recovered solvents also exhibited a high recycling effectiveness. Therefore, the combined cleanup strategy proposed in this study proved environmentally friendly, which also played a major role in risk assessment and management in mixed polluted sites.

Characterization of an extradiol dioxygenase involved in the catabolism of lignin-derived biphenyl.

In the catabolism of lignin-derived biphenyl by Sphingobium sp. SYK-6, LigZ catalyzes the cleavage of 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) to a meta-cleavage product (MCP) identified here as 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (DCHM-HOPDA). DCHM-HOPDA is transformed nonenzymatically, likely to a lactone (k = 0.13 ± 0.01 min-1 , pH 7.5). This is hydrolyzed to the dienolate at alkaline pH (apparent pKa ~ 11.3). Only the dienolate is a substrate for LigY, the putative MCP hydrolase. LigZ has higher specificity for OH-DDVA (kcat /Km = 2.20 ± 0.02 × 107 s-1 ·m-1 ) than for protocatechuate (PCA; 6 ± 1 × 102 s-1 ·m-1 ). PCA also inactivates LigZ (partition ratio of 50), but at rates too low to be physiologically relevant. This study provides insight into the bacterial catabolism of lignin and facilitates the study of downstream catabolic enzymes.

Overcoming a hemihedral twinning problem in tetrahydrofolate-dependent O-demethylase crystals by the microseeding method.

A tetrahydrofolate-dependent O-demethylase, LigM, from Sphingobium sp. SYK-6 was crystallized by the hanging-drop vapour-diffusion method. However, the obtained P3121 or P3221 crystals, which diffracted to 2.5-3.3 Å resolution, were hemihedrally twinned. To overcome the twinning problem, microseeding using P3121/P3221 crystals as microseeds was performed with optimization of the reservoir conditions. As a result, another crystal form was obtained. The newly obtained crystal diffracted to 2.5-3.0 Å resolution and belonged to space group P21212, with unit-cell parameters a = 102.0, b = 117.3, c = 128.1 Å. The P21212 crystals diffracted to better than 2.0 Å resolution after optimizing the cryoconditions. Phasing using the single anomalous diffraction method was successful at 3.0 Å resolution with a Pt-derivative crystal. This experience suggested that microseeding is an effective method to overcome the twinning problem, even when twinned crystals are utilized as microseeds.

Characterisation of the phenanthrene degradation-related genes and degrading ability of a newly isolated copper-tolerant bacterium

A copper-tolerant phenanthrene (PHE)-degrading bacterium, strain Sphingobium sp. PHE-1, was newly isolated from the activated sludge in a wastewater treatment plant. Two key genes, ahdA1b-1 encoding polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHDɑ) and xyLE encoding catechol-2,3-dioxygenase (C23O), involved in the PHE metabolism by strain PHE-1 were identified. The PAH-RHD gene cluster showed 96% identity with the same cluster of Sphingomonas sp. P2. Our results indicated the induced transcription of xylE and ahdA1b-1 genes by PHE, simultaneously promoted by Cu(II). For the first time, high concentration of Cu(II) is found to encourage the expression of PAH-RHDɑ and C23O genes during PHE degradation. Applying Sphingomonas PHE-1 in PHE-contaminated soils for bioaugmentation, the abundance of xylE gene was increased by the planting of ryegrass and the presence of Cu(II), which, in turn, benefited ryegrass growth. The best performance of PHE degradation and the highest abundance of xylE genes occurred in PHE-copper co-contaminated soils planted with ryegrass.

Degradation pathways of 1-methylphenanthrene in bacterial Sphingobium sp. MP9-4 isolated from petroleum-contaminated soil

Alkylated polycyclic aromatic hydrocarbons (PAHs) are abundant in petroleum, and alkylated phenanthrenes are considered as the primary PAHs during some oil spill events. Bacterial strain of Sphingobium sp. MP9-4, isolated from petroleum-contaminated soil, was efficient to degrade 1-methylphenanthrene (1-MP). A detailed metabolism map of 1-MP in this strain was delineated based on analysis of metabolites with gas chromatograph–mass spectrometer (GC–MS). 1-MP was initially oxidized via two different biochemical strategies, including benzene ring and methyl-group attacks. Benzene ring attack was initiated with dioxygenation of the non-methylated aromatic ring via similar degradation pathways of phenanthrene (PHE) by bacteria. For methyl-group attack, mono oxygenase system was involved and more diverse enzymes were needed than that of PHE degradation. This study enhances the understanding of the metabolic pathways of alkylated PAHs and shows the significant potential of Sphingobium sp. MP9-4 for the bioremediation of alkylated PAHs contaminated environments.

Effects of dissolved organic matter derived from forest leaf litter on biodegradation of phenanthrene in aqueous phase

Dissolved organic matter (DOM) released from forest leaf litter is potentially effective for the degradation of polycyclic aromatic hydrocarbons (PAHs), yet the inherent mechanism remains insufficiently elucidated. In this study, we investigated the effects of DOM derived from Pinus elliottii and Schima superba leaf litter on the degradation of phenanthrene by the phenanthrene degrading bacterium Sphingobium sp. Phe-1. DOM from different origins and at a large range of concentrations enhanced the degradation rate of phenanthrene. DOM derived from P. elliottii leaf litter decomposed for 12 months used at a concentration of 100mg/L yielded the highest degradation rate (16.9% in 36h) and shortened the degradation time from 48h to 24h. Changes in the composition of DOM during degradation as measured by EEMs-FRI showed that proteins and tyrosine in the DOM supplied readily available nutrients that stimulated biological activity of Phe-1, increasing its growth rate and catechol 2,3-dioxygenase activity. Simultaneously, fulvic acid and humic acid in the DOM enhanced phenanthrene bioavailability by increasing the solubility and mass transfer of phenanthrene, enhancing the uptake kinetics of Phe-1, and increasing the bacteria’s direct access to DOM-associated phenanthrene. Humic acid was co-metabolized by Phe-1, resulting in further stimulation of phenanthrene degradation.

Enhanced Biological Trace Organic Contaminant Removal: A Lab-Scale Demonstration with Bisphenol A-Degrading Bacteria Sphingobium sp. BiD32.

Discharge of trace organic contaminants (TOrCs) from wastewater treatment plants (WWTPs) may contribute to deleterious effects on aquatic life. Release to the environment occurs both through WWTP effluent discharge and runoff following land applications of biosolids. This study introduces Enhanced Biological TOrC Removal (EBTCR), which involves continuous bioaugmentation of TOrC-degrading bacteria for improved removal in WWTPs. Influence of bioaugmentation on enhanced degradation was investigated in two lab-scale sequencing batch reactors (SBRs), using bisphenol A (BPA) as the TOrC. The reactors were operated with 8 cycles per day and at two solids retention times (SRTs). Once each day, the test reactor was bioaugmented with Sphingobium sp. BiD32, a documented BPA-degrading culture. After bioaugmentation, BPA degradation (including both the dissolved and sorbed fractions) was 2-4 times higher in the test reactor than in a control reactor. Improved removal persisted for >5 cycles following bioaugmentation. By the last cycle of the day, enhanced BPA removal was lost, although it returned with the next bioaugmentation. A net loss of Sphingobium sp. BiD32 was observed in the reactors, supporting the original hypothesis that continuous bioaugmentation (rather than single-dose bioaugmentation) would be required to improve TOrCs removal during wastewater treatment. This study represents a first demonstration of a biologically based approach for enhanced TOrCs removal that both reduces concentrations in wastewater effluent and prevents transfer to biosolids.

Structure of a Novel Phosphotriesterase from Sphingobium sp. TCM1: A Familiar Binuclear Metal Center Embedded in a Seven-Bladed β-Propeller Protein Fold.

A novel phosphotriesterase was recently discovered and purified from Sphingobium sp. TCM1 (Sb-PTE) and shown to catalyze the hydrolysis of a broad spectrum of organophosphate esters with a catalytic efficiency that exceeds 10(6) M(-1) s(-1) for the hydrolysis of triphenyl phosphate. The enzyme was crystallized and the three-dimensional structure determined to a resolution of 2.1 Å using single-wavelength anomalous diffraction (Protein Data Bank entry 5HRM ). The enzyme adopts a seven-bladed β-propeller protein fold, and three disulfide bonds were identified between Cys-146 and Cys-242, Cys-411 and Cys-443, and Cys-542 and Cys-559. The active site of Sb-PTE contains a binuclear manganese center that is nearly identical to that of the structurally unrelated phosphotriesterase from Pseudomonas diminuta (Pd-PTE). The two metal ions in the active site are bridged to one another by Glu-201 and a water molecule. The α-metal ion is further coordinated to the protein by interactions with His-389, His-475, and Glu-407, whereas the β-metal ion is further liganded to His-317 and His-258. Computational docking of mimics of the proposed pentavalent reaction intermediates for the hydrolysis of organophosphates was used to provide a model for the binding of chiral substrates in the active site of Sb-PTE. The most striking difference in the catalytic properties of Sb-PTE, relative to those of Pd-PTE, is the enhanced rate of hydrolysis of organophosphate esters with substantially weaker leaving groups. The structural basis for this difference in the catalytic properties between Sb-PTE and Pd-PTE, despite the nearly identical binuclear metal centers for the activation of the substrate and nucleophilic water molecule, is at present unclear.

Monitoring the impact of bioaugmentation with a PAH-degrading strain on different soil microbiomes using pyrosequencing.

The effect of bioaugmentation with Sphingobium sp. AM strain on different soils microbiomes, pristine soil (PS), chronically contaminated soil (IPK) and recently contaminated soil (Phe) and their implications in bioremediation efficiency was studied by focusing on the ecology that drives bacterial communities in response to inoculation. AM strain draft genome codifies genes for metabolism of aromatic and aliphatic hydrocarbons. In Phe, the inoculation improved the elimination of phenanthrene during the whole treatment, whereas in IPK no improvement of degradation of any PAH was observed. Through the pyrosequencing analysis, we observed that inoculation managed to increase the richness and diversity in both contaminated microbiomes, therefore, independently of PAH degradation improvement, we observed clues of inoculant establishment, suggesting it may use other resources to survive. On the other hand, the inoculation did not influence the bacterial community of PS. On both contaminated microbiomes, incubation conditions produced a sharp increase on Actinomycetales and Sphingomonadales orders, while inoculation caused a relative decline of Actinomycetales. Inoculation of most diverse microbiomes, PS and Phe, produced a coupled increase of Sphingomonadales, Burkholderiales and Rhizobiales orders, although it may exist a synergy between those genera; our results suggest that this would not be directly related to PAH degradation.

Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6

There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of β-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. β-Aryl ether units are typically abundant in lignin, corresponding to 50–70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic β-aryl ether (β-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the β-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts.

Biodegradation of endocrine disrupting dibutyl phthalate by a bacterial consortium expressing Sphingobium sp. SM42 esterase

EsteraseG (EstG) from Sphingobium sp. SM42 efficiently degraded the toxic chemical, dibutyl phthalate (DBP). In this study EstG was successfully displayed on the surface of Escherichia coli using the Pseudomonas aeruginosa outer membrane protein, OprF, as a carrier protein. EstG was fused to the C-terminus of OprF at Lys164 resulting in export to the surface of E. coli that was detected by western blot analysis and flow cytometry, as well as immunofluorescence microscopy. The surface displayed EstG exhibited increased thermal stability, retaining 55, 43, and 39% of its original activity after 24h incubation at 45, 55, and 65°C, respectively. In addition, EstG was engineered to be efficiently secreted into the culture media of Bacillus subtilis, which exhibited 8-fold higher esterase activity than cell lysates. A bacterial consortium consisting of recombinant bacteria has high potential for DBP degradation by removing almost 70% of 1mM DBP within 5 days.

Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of β-Aryl Ether Bonds in Lignin

Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50–70% of all interunit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.