DOI: 10.1002/cssc.201402465

From Gene Towards Selective Biomass Valorization:Bacterial b-Etherases with Catalytic Activity on Lignin-LikePolymersPere Picart,[a] Christoph M�ller,[b] Jakob Mottweiler,[c] Lotte Wiermans,[b] Carsten Bolm,[c]

Pablo Dom�nguez de Mar�a,[b, d] and Anett Schallmey*[a, e]

Introduction

Lignin accounts for 15–30 % of lignocellulose, comprising anaromatic matrix closely associated with cellulose, and covalent-ly attached to hemicelluloses.[1] Lignin is composed of dime-thoxylated (syringyl), monomethoxylated (guaiacyl), and non-methoxylated (p-hydroxyphenyl)phenylpropanoid units—all ofwhich are derived from the corresponding p-hydroxycinnamylalcohols—giving rise to a random variety of ether and C�Cbonds.[2] The b-O-4-aryl ether linkage is the most abundantone (45–60 %, depending on the wood type) followed by b–b,b–5, 5–5, and 5-O-4 linkages.[3]

Lignins hold potential to become major renewable sourcesfor biofuel production, as well as for bulky and fine chemicals,particularly aromatics, which are relatively rare in nature ona large scale.[4] Despite its potential, practical applications oflignin have been scarce, so far, presumably due to its recalci-trance and challenging processing for depolymerization and

valorization, in terms of what forces to apply severe, unselec-tive, and degrading conditions. In this respect, lignin can bedepolymerized by chemical means, such as thermochemicalmethods, for example, pyrolysis (thermolysis), chemical oxida-tion, hydrogenolysis, gasification, and hydrolysis under super-critical conditions.[5] Many of these processing strategies areenergy-consuming and environmentally unfriendly,[6] leading toa complex mixture of degraded and partially repolymerizedproducts ; thus compromising efficient lignin valorization,which ideally would require carefully controlled depolymeriza-tion.[2]

Apart from some recent chemical approaches for the selec-tive lignin fractionation,[7, 8] “white biotechnology” may offersome promising avenues for lignin valorization to enable thecleavage of different bonds under mild conditions. For in-stance, white-rot fungi produce a range of extracellular ligno-lytic enzymes, including peroxidases, such as heme-dependentlignin peroxidases, manganese peroxidases, and versatile per-oxidases, as well as laccases.[9] Laccases (Lac, EC 1.10.3.2) arecopper-containing enzymes with a low redox potential thatenable direct oxidation of phenolic lignin (less than 10 % ofthe total polymer), whereas lignin peroxidase (LiP, EC 1.11.1.14)and manganese peroxidase (MnP, EC 1.11.1.13) have been de-scribed as true ligninases because of their high redox poten-tial.[10] LiP degrades non-phenolic lignin units, which oftencomprise up to 90 % of the total lignin polymer, whereas MnPgenerates Mn3 + , which acts as a diffusible oxidizer on phenolicor non-phenolic lignin units through lipid peroxidation reac-tions.[11] More recently, versatile peroxidases (VP, EC 1.11.1.16)were described as a third type of ligninolytic peroxidases thatcombined the catalytic properties of LiP, MnP, and plant/micro-bial peroxidases to oxidize phenolic compounds.[12, 13] Based ontheir intrinsic radical-based mechanism, all of these enzymesproceed by unselective mechanisms, causing random lignin

Microbial b-etherases, which selectively cleave the b-O-4 arylether linkage present in lignin, hold great promise for futureapplications in lignin valorization. However, very few membershave been reported so far and little is known about these en-zymes. By using a database mining approach, four novel bacte-rial b-etherases were identified, recombinantly produced in Es-cherichia coli, and investigated together with known b-etheras-

es in the conversion of various lignin and non-lignin-typemodel compounds. The resulting activities revealed the signifi-cant influence of the substituents at the phenyl ring adjacentto the ether bond. Finally, b-etherase activity on polymericsubstrates, measured by using a fluorescently labeled syntheticlignin, was also proven; this underlined the applicability of theenzymes for the conversion of lignin into renewable chemicals.

[a] Dr. P. Picart, Prof. Dr. A. SchallmeyInstitute of Biotechnology, RWTH Aachen UniversityWorringerweg 3, 52074 Aachen (Germany)E-mail : a.schallmey@uva.nl

[b] C. M�ller, L. Wiermans, Dr. P. Dom�nguez de Mar�aInstitut f�r Technische und Makromolekulare Chemie (ITMC)RWTH Aachen University, Worringerweg 1, 52074 Aachen (Germany)

[c] J. Mottweiler, Prof. Dr. C. BolmInstitute of Organic Chemistry, RWTH Aachen UniversityLandoltweg 1, 52056 Aachen (Germany)

[d] Dr. P. Dom�nguez de Mar�aPresent address: Sustainable Momentum, SLUAp. Correos 3517, 35004, Las Palmas de Gran Canaria (Spain)

[e] Prof. Dr. A. SchallmeyPresent address: BiocatalysisVan’t Hoff Institute for Molecular SciencesUniversity of Amsterdam, Science Park 9041098 XH Amsterdam (The Netherlands)

Supporting Information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cssc.201402465.

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depolymerization and probably the repolymerization of re-leased monolignols as well.[2]

Remarkably, non-radical ligninolytic enzymes, such as b-etherases, should deliver a more specific and effective alterna-tive for lignin cleavage and valorization. Because the major in-termolecular lignin linkage is the arylglycerol-b-aryl etherbond,[3] one pathway proposed for biochemical lignin catabo-lism entails the enzymatic cleavage of such linkages. By usingb-etherases, which cleave b-aryl ether bonds,[14] valuable indus-trially useful low-molecular-mass lignins that retain aromaticrings would be selectively achieved. Surprisingly, until now fewstudies have focused on the biocatalytic characterization andapplication of b-etherases in lignin valorization. Their naturaloccurrence has scarcely been reported, for example, for an as-comycete believed to be a member of the genus Chaetomi-um[15] and for the soil a-proteobacterium Sphingobium pauci-mobilis SYK-6.[14] The extracellular fungal b-etherase was shownto cleave the b-aryl ether bonds in two lignin model dimers,guaiacylglycerol b-guaiacyl ether (GGE) and guaiacylglycerol-b-O-4-methylumbelliferone (GOU), as well as in a polymeric syn-thetic lignin.[15] However, the gene coding for this b-etherasehas not been identified, so far.

In S. paucimobilis SYK-6, the roles of the genes ligE, ligF, andligP, which encode enantioselective b-etherases, have alsobeen described. These enzymes are members of the gluta-thione transferase superfamily (GSTs; EC 2.5.1.18) and cleavethe b-aryl ether bond upon glutathione (GSH) consump-tion.[16–19] GSTs form a superfamily of enzymes involved ina broad range of detoxifying processes, including xenobiot-ics.[20, 21] GST genes present widely divergent sequences group-ed into several classes, but the roles of these enzymes are notyet completely understood.[22] The b-aryl ether degradation

pathway of S. paucimobilis SYK-6 has been studied by usinglignin model compounds,[23–26] indicating the necessary addi-tional presence of at least four nicotinamide adenine dinucleo-tide (NADH)-dependent stereospecific alcohol dehydrogenases(LigD, LigL, LigN, and LigO), which catalyze the initial oxidationof the a-hydroxyl group in guaiacyl-a-veratrylglycerol (GVL) tothe corresponding ketone (Figure 1). Subsequently, an unusualreductive ether cleavage through a b-etherase reaction takesplace. LigE, LigF, and LigP catalyze nucleophilic attack by thetripeptide GSH on the carbon atom at the b position of sub-strates containing b-aryl ether linkages, such as bGVG, to pro-duce guaiacol and a GSH-conjugated aromatic compound(Figure 1). The reported b-etherases are stereoselective andattack the two enantiomers of racemic bGVG selectively.Whereas LigF produces GS-b(R)VG from b(S)GVG, LigE and LigPare selective for b(R)GVG to provide GS-b(S)VG.[26] LigG is in-stead a GSH lyase that, in the presence of GSH, catalyzes thio-ether cleavage with high stereoselectivity to produce GSSGand VG that can be utilized as a growth substrate by the bac-terium.[25, 26] The further conversion of the GSH conjugate pro-duced by b-etherases LigE and LigP (GS-b(S)VG) is, however,still unknown. It was suggested by Gall et al. that both enan-tiomers of GS-bVG could be interconverted by a racemase, andthus, only LigG would be required to cleave the thioether inGS-bVG.[26]

So far, a more detailed description of the enzymatic proper-ties of b-etherases has only been reported for LigF,[24, 25] forwhich preliminary catalytic studies on polymer-like lignin sub-strates have been conducted with modest success.[24] Interest-ingly, many genes in the National Center for Biotechnology In-formation (NCBI) database have been annotated as putative b-etherases, according to sequence homology to ligF, ligE and

Figure 1. Postulated catalytic pathway for GVL degradation in S. paucimobilis SYK-6. NADH-dependent LigD, LigL, LigN, and LigO oxidize the Ca-hydroxylgroup of the GVL diastereomers. LigE, LigF, and LigP are b-etherases that cleave the two GVG enantiomers produced, yielding two enantiomeric GSH conju-gates (GS-bVG). LigG is the glutathione lyase that uses GSH to liberate veratrylglycerone from GS-b(S)VG through the formation of glutathione disulfide(GSSG).

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ligP genes, but the properties of the encoded enzymes andtheir substrate specificities have not yet been studied. Consid-ering the potential practical importance that a b-etherase tool-box might have for lignin valorization, as well as for other in-novative processes for asymmetric synthesis, herein we de-scribe the identification and biocatalytic characterization ofnovel b-etherases from Novosphingobium sp. PP1Y and Novo-sphingobium aromaticivorans DSM1244. For the first time, theproperties and substrate specificity of each b-etherase havebeen studied in detail by using various lignin b-O-4 modelcompounds, as well as a fluorogenic synthetic lignin polymer(4MU-DHP), to ensure that enzymes may actually catalyzecleavage reactions in macromolecules as well.

Experimental Section

Identification of novel b-etherases

b-Etherase encoding sequences were identified by screening thepublicly available GenBank nr database (release 193) of the NCBIby using the basic local alignment search tool (BLAST) algorithm.The database was screened by using the amino acid sequences ofb-etherases LigF (GenBank: BAA02031.1), LigE (GenBank:BAA02032.1), and LigP (GenBank: BAK67935.1) from Sphingobiumsp. SYK6. Protein sequences obtained for each b-etherase were an-alyzed and aligned by using ClustalX,[27] and phylogenetic treeswere constructed by using the neighbor-joining method.[28] Pro-teins closely related to LigF (LigF-NS, GenBank: YP_004533905.1;and LigF-NA, GenBank: YP_497364.1), LigE (LigE-NS, GenBank: YP_004533906.1; and LigE-NF, GenBank: YP_497675.1) and LigP (LigP-SC, GenBank: YP_001616516.1) were selected for further studies.

Strains and plasmids

The genes coding for LigF, LigF-NS, LigF-NA, LigE, LigE-NS, LigE-NA,LigP, and LigP-SC were synthesized by Geneart GmbH (Regens-burg/Germany) with codon optimi-zation for E. coli, and cloned intoa pET28a(+) vector by using re-striction sites NdeI and HindIII, re-sulting in the addition of a N-ter-minal His tag. Expression of all en-zymes was performed with E. coliBL21(DE3). Strains for all experi-ments were grown in terrific brothmedium (TB) with kanamycin(50 mg L�1) to an optical density atl= 600 nm (OD600) of about 0.6 at37 8C. After induction with 0.1 mm

isopropyl-b-d-thiogalactopyrano-side (IPTG), expression was per-formed overnight at 20 8C.

Enzyme purification

For the purification of enzymes,overexpressed cells were resus-pended in binding buffer (20 mm

sodium phosphate buffer, pH 7.4,500 mm NaCl, 20 mm imidazole)and lysed by sonication. After re-

moval of cell debris by centrifugation, the resulting cell lysate wasapplied on a Ni2 +–nitrilotriacetic acid (NTA) column for purificationof the His-tagged enzymes with an �KTA purification system (GEHealthcare, USA) at a flow rate of 1 mL min�1. His-tagged enzymeswere eluted from the column by using elution buffer (20 mm

sodium phosphate buffer, pH 6.5, 500 mm NaCl, 500 mm imida-zole). Protein fractions were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Coomassiestaining to check the presence and purity of recombinant en-zymes. Fractions containing a purified enzyme were combined, de-salted by using PD-10 columns (GE Healthcare), and quantified bythe Bradford assay.[29]

Synthesis of model substrates

Several lignin-type substrates were used (Figure 2). Substrates 1, 2,3, and 4 were synthesized as described in the Supporting Informa-tion. Substrate 5 was purchased from Tokyo Kasei Kogyo Co.(Tokyo, Japan), whereas substrates 6 and 7 were obtained fromSigma Aldrich. For fluorogenic substrates (Figure 3), a-O-(b-methyl-umbelliferyl)acetovanillone (MUAV; 8) was synthesized accordingto the protocol reported by Weinstein and Gold.[30] Fluorescentlylabeled synthetic lignin (DHP-MUAV; 9) was prepared as follows:solution A (60 mL; coniferyl alcohol (50 mg), 8 (50 mg), and horse-radish peroxidase (3 mg) in 50 % DMSO) and solution B (60 mL;30 % H2O2 (3.73 mL) and H2O (56.25 mL)) were slowly dropped into100 mm potassium phosphate buffer (15 mL), pH 6.5, and stirredfor 16 h at room temperature. Afterwards, more peroxidase (2 mg)was added and the reaction mixture was stirred for additional 6 h.The resulting precipitate was collected by centrifugation, washedthree times with 10 % isopropanol, and dispersed in distilled water.Lyophilization was performed to yield DHP-MUAV.[31] All substratesused for enzyme assays in this study were dissolved in pure DMSObefore use.

Figure 2. Lignin model substrates and non-lignin ether compounds studied herein: guaiacyl-a-veratrylglycerone(1; GVG), b-guaiacyl-a-veratrylethanone (2 ; GVE), b-(2,6-methoxyphenoxy)-a-veratrylglycerone (3 ; 2,6-MP-VG), b-(3,5-methoxyphenoxy)-a-veratrylglycerone (4 ; 3,5-MP-VG), guaiacylglycerol-b-guaiacyl ether (5 ; GGE), 3-phenoxy-2-butanone (6 ; PB), and phenoxyacetone (7; PA).

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Activity assays

The b-etherase activity of each purified enzyme towards 1 and 2was determined by quantifying the amount of released guaiacolby HPLC. For 3 and 4, 2,6-dimethoxyphenol (2,6MP) and 3,5-dime-thoxyphenol (3,5MP), respectively, were formed instead of guaiacoland measured by HPLC as well. The quantification of guaiacol,2,6MP, and 3,5MP was performed by using calibration curves of re-spective commercial standards (Sigma–Aldrich). The assay mixture(1 mL) contained 20 mm glycine/NaOH buffer, pH 9, 0.2 mm sub-strate, 1 mm GSH (reduced), and 10 mg purified b-etherase. Reac-tions were carried out at 25 8C and stopped by the addition ofmethanol (final concentration, 25 % v/v) after different incubationtimes. Precipitated protein was removed by centrifugation (15 000gfor 5 min), and the supernatant was analyzed on a Prominencemodular HPLC system (Shimadzu) equipped with a Nucleosil 100-5C18 column (4.6 � 150 mm; Macherey–Nagel, Germany). A mixtureof water (49 %), acetonitrile (50 %), and phosphoric acid (1.0 %) wasused as mobile phase with a flow rate of 1.0 mL min�1. All com-pounds were detected at l= 280 nm, except for 4, which was de-tected at l= 310 nm. Retention times of the different substratesand products are given in Table S1 in the Supporting Information.One unit was defined as the amount of enzyme that degraded1 mmol of substrate min�1. Specific activity was expressed as unitsper milligram of protein. Temperature and pH profiles of the en-zymes were determined with 1 by using the assay conditions men-tioned above, but varying the temperature between 20 and 65 8Cor the pH between 4.5 and 12.5. Reactions were stopped after15 min of incubation at the respective temperature and theamount of released guaiacol was determined by HPLC, as men-tioned above, to calculate the b-etherase activity.

Stereoselectivity measurements

To analyze the enantioselectivities of each b-etherase towards 1,0.2 mm substrate was incubated for 60 min with each purifiedenzyme (10 mg) in a reaction mixture (1 mL) containing 20 mm gly-cine/NaOH buffer, pH 9, and 1 mm GSH (reduced). Reactions werecarried out at 25 8C and stopped by the addition of methanol (finalconcentration, 25 % v/v) after different incubation times. Precipitat-ed protein was removed by centrifugation (15 000g for 5 min), andthe supernatant was analyzed on a Prominence modular HPLCsystem (Shimadzu) equipped with a NUCLEOCEL DELTA S column(4.6 � 250 mm; Macherey–Nagel, Germany). As a mobile phase,a mixture of hexane (74.5 %), ethanol (24.5 %), and acetic acid(1.0 %) was used with a flow rate of 0.5 mL min�1. The enantiomerswere detected at l= 310 nm, and their retention times are givenin Table S1 in the Supporting Information. The assignment of enan-tiomers was performed according to the reported order of elutionof enantiomers of 1 on a CHIRALCEL OD-H column (Daicel; thesame cellulose tris(3,5-dimethylphenylcarbamate) chiral stationaryphase as the NUCLEOCEL DELTA S column used in this study).[18]

Fluorometric assays

The b-etherase activity of each enzyme towards 8 was measuredfluorometrically in a plate reader using an excitation wavelength ofl= 360 nm and an emission wavelength of l= 450 nm.[14] Each re-action mixture (0.2 mL) contained 100 mm 8, 1 mm GSH (reduced),and appropriate concentrations of each enzyme in optimal pH andtemperature conditions for activity. Formation of 4-methylumbelli-ferone (4MU) was monitored continuously over 30 min by using anInfinite M1000 fluorometer (Tecan, Switzerland) calibrated withcommercial 4MU (Sigma Aldrich) as the standard. Specific activitywas expressed as units per milligram of protein. Kinetic parametersKM, vmax, and kcat. for 8 were determined at optimum pH and tem-perature of the enzymes with 25, 50, 75, 100, 125, 150, 175, and200 mm concentrations of 8 at a GSH (reduced) concentration of1 mm. The b-etherase activity towards synthetic lignin 9 was ana-lyzed in a similar manner to that described for 8. Here, substrate 9(0.2 mg) was cleaved with each purified b-etherase (20 mg) underoptimal conditions for activity and the release of 4MU was moni-tored continuously every 60 s over 3 h. Substrate 9 was applied asa stock solution in DMSO with a 30 % final DMSO concentration inthe reaction mixture. Samples of 9 with and without b-etherasetreatment were also analyzed by ESI-MS. Mass spectra were record-ed on a Varian model 500-MS instrument with electrospray ioniza-tion through direct injection by using 80 V in positive mode.

Results and Discussion

Database mining for novel b-etherases

To identify further potential enzymes that exhibit b-etheraseactivity, the publicly available GenBank nr database of the NCBI(release 193) was screened by BLAST with the amino acid se-quences of known b-etherases LigE (GenBank: BAA02032.1),LigF (GenBank: BAA02031.1), and LigP (GenBank: BAK67935.1)of Sphingobium sp. SYK6. Several hits sharing up to 87 % simi-larity with the three b-etherases used for the screening wereidentified. When the amino acid sequences of the retrieved pu-tative b-etherases were compared to LigF and LigE, the highestsimilarity was observed for LigF-NS from Novosphingobium sp.PP1Y (GenBank: YP_004533905.1), LigF-NA from N. aromatici-

Figure 3. Chiral HPLC chromatograms of rac-GVG (top) and enantiomers ob-tained after incubation with GSH and various b-etherases (middle andbottom).

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vorans DSM 12444 (GenBank: YP_497364.1), LigE-NS from No-vosphingobium sp. PP1Y (GenBank: YP_004533906.1), and LigE-NA from N. aromaticivorans DSM 12444 (GenBank: YP_497675.1), with 66, 59, 78, and 61 % sequence identity, respec-tively (Figure S1 in the Supporting Information).[32] In contrast,low sequence identity was observed for putative homologuesof LigP. More specifically, LigP displayed only 36 % sequenceidentity to LigP-SC from Sorangium cellulosum So Ce56 (Gen-Bank: YP_001616516.1) and LigP-PL from Parvibaculum lava-mentivorans DS-1 (GenBank: YP_001413220.1; data not shown).In addition to LigE-NS, LigE-NA, LigF-NS, and LigF-NA, manyother sequences were found in the databases that displayed41 to 48 % sequence identity to LigE or LigF from Sphingobiumsp. and were annotated as putative b-etherases. Phylogeneticanalysis of the sequences revealed that enzymes LigF, LigE,and LigP clustered together with LigF-NS, LigF-NA, LigE-NS,LigE-NA, LigP-SC, and LigP-PL, whereas the other putative b-etherases were located on different clades in the phylogenetictree (Figure S2 in the Supporting Information). Hence, LigF-NS,LigF-NA, LigE-NS, and LigE-NA were chosen in this study to-gether with already known LigF, LigE, and LigP for a detailedbiochemical characterization of the (putative) b-etherases todetermine substrate specificities towards several dimeric ligninmodel compounds, as well as a synthetic lignin polymer. Addi-tionally, the more distantly related LigP-SC from S. cellulosumSo Ce56 was also selected for characterization.

Recombinant production and biochemical characterizationof novel b-etherases

The three b-etherase genes (ligF, ligE, and ligP) from the soilproteobacterium Sphingobium sp. SYK6, as well as novel b-etherase genes ligF-NS and ligE-NS from Novosphingobium sp.PP1Y, ligF-NA and ligE-NA from N. aromaticovorans DSM 12444,and ligP-SC from Sorangium cellulosum So Ce56, were obtainedas synthetic genes with codon optimization for E. coli. Eachgene was cloned into vector pET28a(+), resulting in N-terminalHis-tag fusion, under control of a T7 promoter and transferredinto E. coli BL21(DE3) cells. All eight genes were overexpressedin E. coli BL21(DE3) and the resulting enzymes were purifiedthrough their N-terminal His tag by affinity chromatography.All proteins were obtained in high purity (as confirmed bySDS-PAGE analysis) with yields of 75 to 150 mg L�1 cell culture(Figure S3 in the Supporting Information).

To evaluate whether the putative enzymes LigF-NS, LigF-NA,LigE-NS, LigE-NA, and LigP-SC displayed real b-etherase activity,all enzymes were incubated with substrate 1 (Figure 2) andGSH at 25 8C and pH 7. HPLC analysis showed the accumula-tion of guaiacol, as well as the probable GSH conjugate prod-uct, GS-GV, from incubations with LigE, LigE-NS, LigE-NA, LigF,LigF-NS, LigF-NA, and LigP (Figure S4 A in the Supporting Infor-mation). Conversely, LigP-SC exhibited no observable etherbond cleavage, similar to blank experiments. Within 3 h of re-action, all active b-etherases led to a guaiacol production ofapproximately 50 % of initial substrate concentration, and in-terestingly none of the tested b-etherases was able to com-pletely hydrolyze 1, even if an excess of enzyme was applied

and the incubation time was prolonged to 16 h. These resultssuggested that the novel b-etherases LigF-NS, LigF-NA, LigE-NS, and LigE-NA were also stereospecific, as reported earlierfor LigF, LigE, and LigP.[23, 25, 26] If confirmed, apart from ligninvalorization, novel opportunities for asymmetric synthesis bymeans of these enzymes could also be envisaged.

To confirm the enantioselectivity of the novel b-etherases,reactions with 1 were also analyzed by HPLC with a chiralcolumn. Thus, the highly selective conversion of the GVG enan-tiomers for all applied b-etherases that exhibited differentenantiopreference was observed (Figure 3). Similar to LigF,LigF-NS and LigF-NA exclusively attack the S enantiomer ofGVG (b(S)GVG), whereas LigE-NS and LigE-NA are highly selec-tive for the R enantiomer (b(R)GVG), as reported previously forLigE and LigP.[26]

Furthermore, to identify optimal reaction conditions of thepurified enzymes, b-etherase activity towards 1 was deter-mined at different temperatures and pH values. As a result, allactive b-etherases in our study exhibited the highest activity attemperatures between 20 and 30 8C (Figure S5 in the Support-ing Information) and at alkaline pH in the range from 8.5 to 10(Figure S6 in the Supporting Information). Furthermore, someof the enzymes lost activity when incubated at temperatureshigher than 45 8C, with the exception of LigE, LigF, and LigF-NA, which still displayed activity up to 60 8C. Interestingly, allb-etherases exhibited a clear pH optimum of pH 9 to 9.5, butseveral of them were still active at pH 6 or pH 12; this suggestsa potential practical application for crude biorefinery-based ef-fluents (e.g. , combined with different pretreatment conditions).These results are consistent with recent work on LigF, whichshowed that this enzyme exhibited an optimal pH of 10 forthe highest activity and the enzyme remained active when in-cubated at temperatures up to 60 8C.[24]

Substrate specificity of the novel b-etherases towards di-meric model compounds

To better compare the activities of different b-etherases, specif-ic activities of all active enzymes towards 1 were determinedunder optimal pH and temperature conditions (Table 1). Re-sults revealed that LigF-NA and LigE were the most active b-etherases on substrate 1 (specific activities of 2.9 and2.2 U mg�1, respectively). On the other hand, the specific activi-ty of LigP towards 1 was only 6.2 mU mg�1, which was 400-foldlower than that of LigF-NA.

To gain insights into the substrate specificities and reactivi-ties of the novel b-etherases, lignin model compounds 2–5, aswell as structurally related aryl ethers 6 and 7, were assessedas substrates. When an achiral, side-chain-truncated modelsubstrate (2) was used in the presence of GSH, full conversionwas achieved by each enzyme, except for LigP-SC, whichshowed no activity (Figure S4B in the Supporting Information).Consumption of 2 was again accompanied by the productionof guaiacol, the probable GSH adduct GS-VE, and an unidenti-fied compound (retention time 3.52 min;[33] Figure S4 B in theSupporting Information). Notably, the specific activity of thepurified enzymes with 2, except for LigF, was significantly

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lower than that observed for 1 (Table 1). Thus, the side chainindeed seems to have a significant influence on the b-etheraseactivity of the wild-type enzymes.

Furthermore, substrates 3 and 4, which were similar to 1,but with different substitution patterns at the aromatic ringnext to the ether bond, were tested in the cleavage reaction(Figure 2). Ether bond cleavage of 3 and 4 resulted in the re-lease of 2,6MP and 3,5MP, respectively. The reactions with 3(Figure S4 C in the Supporting Information) showed up to 50 %conversion and simultaneous accumulation of 2,6MP, as well asthe probable GSH adduct GS-VG for each tested b-etherase,except LigP-SC, which again exhibited no activity. The resultingspecific activities on 3 were much higher than those on theother substrates, in which LigF-NA exhibited the highest activi-ty value of 6.8 U mg�1 (Table 1). In contrast, the reactions with4 (Figure S4 D in the Supporting Information) revealed lowconversions in the presence of LigF and LigF-NA, whereas theother enzymes did not display any activity. The resulting spe-cific activities of LigF and LigF-NA towards 4 were a factor of100 lower than those for 3.Overall, enzyme LigF-NA provedto be the most active b-etheraseamong the seven enzymes usedin this study for the conversionof dimeric lignin model sub-strates. Moreover, LigF-NA exhib-its high activity over a broad pHand temperature range withmore than 50 % relative activityat a pH between 7.0 and 10.5,and 34 % residual activity afterincubation at 50 8C for 15 min.This makes LigF-NA an attractivealternative to b-etherase LigF forapplication in lignin depolymeri-zation.

With regard to the still unex-plored mechanism of b-etheras-es, our results suggest a stronginfluence of the phenyl substitu-ents on enzyme activity. Thus,activating groups at the orthoposition (e.g. , in the case of

2,6MP-VG, two methoxy groups)enhance the reactivity of theenzyme, whereas deactivatingones (e.g. , two methoxy groupsat the meta position, as in thecase of 3,5MP-VG) would lowerthe enzymatic degradation activ-ities. As a logical consequence,all enzymes display higher spe-cific activities for 3 than for 1 or4 (with low or no activity ob-served). Whether that is causedby unfavorable substrate recog-

nition of 4 due to steric hindrance or by electronic effectscannot be assessed, but requires further investigation.

Additionally, the fluorogenic lignin model compound (8) wasassessed as a substrate for our novel etherases.[14, 24, 25] Etherbond cleavage of 8 results in the release of fluorescent 4MU,which facilitates enzyme activity determination (Figure 4).Thus, kinetic parameters for each enzyme in that reaction weredetermined at varying substrate concentrations and a fixedGSH concentration of 1 mm. Surprisingly, in this case, LigE-NAexhibited the highest etherase activity towards 8 with a vmax of32.5 mU, a KM of 24 mm, and a kcat. of 1.0 min�1, whereas thelowest b-etherase activity was observed for LigE with a vmax of0.01 mU, a KM of 54 mm, and a kcat. of 3.4 � 10�4 min�1 (Table 2).These results are in sharp contrast to the specific activities ob-tained by using substrates 1 to 4. First, the resulting specificactivities with 8 are several orders of magnitude lower thanthose of the other lignin model compounds (except 4).Second, the b-etherase LigE-NA, which was one of the en-

Table 1. Specific activities of each b-etherase determined in reactions with various lignin model substrates.

Substrate Specific activity [mU mg�1]LigF LigF-NS LigF-NA LigE LigE-NS LigE-NA LigP LigP-SC

1 530 300 2920 2240 140 150 6.2 ND[a]

2 1250 240 1920 480 34 65 5.0 ND[a]

3 2604 370 6790 5930 2780 2670 100 ND[a]

4 40 ND[a] 45 ND[a] ND[a] ND[a] ND[a] ND[a]

8 3.68 0.17 3.13 0.007 0.012 26.94 0.041 ND[a]

9 0.052 0.0038 0.039 0.0016 0.0001 0.072 0.0023 ND[a]

[a] ND: not detected.

Figure 4. Schematic representation of 4MU released from 8 and 9 after cleavage of the b-aryl ether linkage by theaction of b-etherases. Released 4MU emits fluorescence that was measured by using an excitation wavelength ofl= 360 nm and an emission wavelength of l = 450 nm.

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zymes with the lowest specific activity on substrates 1 to 4,now displayed the highest specific activity (Table 1). Additional-ly, LigE, which exhibited rather high specific activities with sub-strates 1 and 3, was the least active on 8. Hence, overall, theabsolute activities of already known and newly identified b-etherases are strongly substrate dependent.

It was reported previously that substrate specificity of LigE,LigF, and LigP was restricted to Ca-carbonyl-containing ligninmodel compounds, whereas the corresponding hydroxyl-con-taining substrates were not converted.[16–19] To investigatewhether the new b-etherases LigF-NS, LigF-NA, LigE-NS, andLigE-NA exhibited similar behavior, reactions with 5, which hada hydroxyl group in the Ca position instead of a carbonylgroup, were performed. As a result, none of these enzymescould cleave the b-aryl ether linkage in 5 ; this confirmed theirdependence on a carbonyl group at the Ca position (data notshown).

To test whether b-etherase activity of the bacterial GST en-zymes was restricted to lignin-derived compounds containingtwo aromatic rings next to the b-O-4 ether linkage, conversionof commercially available aryl ethers 6 and 7, containing onlyone aromatic ring, was investigated (Figure 2). However, evenwhen excess b-etherase was added, none of the enzymescould cleave the tested substrates (data not shown); this indi-cated a high specificity for lignin-derived b-O-4 ether com-pounds.

From model components to polymeric substrates: Activityof b-etherases on fluorogenic polymers

The b-etherases reported herein are particularly active on di-meric b-O-4 aryl ether model compounds that mimic thechemical structure of b-O-4 aryl ether linkages occurring in nat-ural lignins.[34] However, it must be emphasized that the activi-ty towards these model compounds does not directly implyactivity of the b-etherases on more challenging polymeric lig-nins (the desired substrates for lignin valorization). Thus, toassess whether our purified b-etherases were also able tocleave the b-O-4 aryl ether linkage in lignin-like polymers, thefluorescently labeled synthetic lignin 9 (Figure 4) was synthe-sized by dehydrogenative polymerization of coniferyl alcoholand 8.[35] This fluorescently labeled polymer allowed a straight-forward and highly sensitive detection of b-etherase activity

on a polymeric substrate, as reported earlier for cell-free ex-tracts of LigF and LigDEF.[35] To ensure that polymer 9 did notcontain any traces of dimeric 8, both substrates were dissolvedin DMSO and analyzed by HPLC under the same conditions;this indicated successful polymerization (Figure S7 in the Sup-porting Information). Moreover, polymer 9 was further charac-terized through ESI-MS, and showed a highly uniform mass dis-tribution (major peak at m/z 1347; Figure S8 A in the Support-ing Information). For etherase reactions, polymer 9 was incu-bated with each enzyme under optimal pH and temperatureconditions by using 30 % v/v DMSO as a cosolvent to assurecomplete dissolution of the polymer in the reaction media.Gratifyingly, b-etherases remained fully active under these co-solvent conditions (data not shown). Results obtained fromfluorescence measurements clearly proved that all tested b-etherases also cleaved the b-aryl ether linkage in the lignin-likepolymer 9 ; this indicated their suitability for lignin polymerdegradation (Table 1). Again, LigF and LigE-NA exhibited thehighest specific activity on substrate 9 (0.052 and0.072 mU mg�1, respectively). After 24 h incubation of 9(0.2 mg) with LigF-NA (10 mg), roughly 0.1 mg of 4MU had beenreleased. To further assess how the polymer was enzymaticallycracked, reaction products after etherase treatment of 9 werealso analyzed by ESI-MS. Remarkably, polymer 9 was fractionat-ed into different novel fragments with smaller masses (majorpeaks at m/z 288, 750, 860, and 1038), showing that the en-zymes were active on the polymeric structure (Figure S8 B inthe Supporting Information). To the best of our knowledge,this is the first confirmation that b-etherases cleave lignin-likepolymers. Clearly, much more research is needed to character-ize the produced fractions to understand the enzymatic per-formance and to allow a practical application of such a biocata-lytic approach in lignin valorization.

For the efficient degradation of natural lignins by using b-etherases, efficient oxidation of Ca-hydroxyl groups in ligninpolymers will also be crucial. Recently, Reiter et al. publishedan integrated biocatalytic concept for lignin degradation in-volving an alcohol dehydrogenase (LigD), a glutathione lyase(LigG), and a b-etherase (LigF), which showed, however, onlylow activities towards lignin polymers.[24] Complementing this,our results herein broaden the potential scope of b-etherasesfor lignin valorization. Activities observed for wild-type en-zymes are still far from practical applications. However, the useof widely established methods for protein design may lead tothe provision of highly active and selective biocatalysts forlignin valorization. In this respect, the combination of an ade-quate medium engineering to dissolve lignin (e.g. , enzyme-friendly biobased solvents) together with active variants ap-pears to be necessary to envisage future practical applications.

Conclusions

The use of b-etherases for lignin processing appears to be stillin its infancy, but holds great potential for the development ofselective methods for lignin cleavage and further valorizationof the achieved fractions. Our quest for homologous b-ether-ase sequences in public sequence databases revealed novel

Table 2. Kinetic parameters of each b-etherase in the conversion of 8.

b-etherase vmax

[mU]KM

[mm]kcat.

[min�1]

LigF 5.02 30.89 0.162LigF-NS 0.26 60.06 0.006LigF-NA 4.92 47.57 0.138LigE 0.01 53.81 3.43 � 10�4

LigE-NS 0.021 63.91 6.7 � 10�4

LigE-NA 32.53 24.09 1.014LigP 0.067 74.49 0.0021LigP-SC ND[a] ND[a] ND[a]

[a] ND: not detected.

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promising biocatalysts able to catalyze selective ether bondcleavage both in lignin model components and in lignin-likepolymeric fractions; this opens up options for designing pow-erful variants for lignin processing. Because these are biocata-lysts, the reaction conditions are extremely mild, and therefore,rule out the possibility of degrading the remnant fractions oflignin, which allows their further valorization. A key aspect forthe successful implementation of b-etherases (and other en-zymes) in lignin valorization is the complete dissolution oflignin polymers in the reaction media. Herein, for biocatalyticcharacterization purposes, DMSO has been used as a cosolvent.For future practical applications, other biobased (neoteric) sol-vents, such as deep eutectic solvents, appear to be morepromising alternatives, because they may be enzyme-friendlysolvents with straightforward downstream processing ap-proaches and adequate environmental footprints. Last, but notleast, apart from lignin, the development of novel enzymesable to cleave ether bonds with high stereoselectivity mightalso lead to new synthetic applications in asymmetric synthe-sis. In a nutshell, b-etherases hold the potential to become rel-evant practical biocatalysts in forthcoming years.

Acknowledgements

This work was performed as part of the Cluster of Excellence“Tailor-Made Fuels from Biomass”, which is funded by the Excel-lence Initiative of the German Research Foundation to promotescience and research at German universities. Furthermore, finan-cial support from DFG training group 1166 “BioNoCo” (“Biocataly-sis in Non-conventional Media”) is gratefully acknowledged aswell.

Keywords: biocatalysis · biomass · cleavage reactions ·lignins · enzymes

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Received: May 26, 2014Revised: July 15, 2014Published online on September 3, 2014

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