Peroxidases bound to the growing lignin polymer produce natural like extracellular lignin in a cell culture of Norway spruce

ORIGINAL RESEARCH published: 18 Oc obe 2016 doi: 10.3389/ pls.2016.01523 F on ie s in Plan Science | www. on ie sin.o g 1Oc obe 2016 | Volume 7 | A icle 1523 Edi ed by: Jacqueline G ima-Pe ena i, Cen e Na ional de la Reche che Scien i ique, F ance Re iewed by: Rosa M. Cusido, Uni e si y o Ba celona, Spain B igi e Chabbe , F ench Na ional Ins i u e o Ag icul u al Resea ch, F ance *Co espondence: Teemu H. Tee i [email p o ec ed] †P esen Add ess: Anna Kä könen, G een Technology, Na u al Resou ces Ins i u e Finland (Luke), Helsinki, Finland; Liisa Kaa ina Simola, Ri a ika u 9 B, 00170 Helsinki, Finland Special y sec ion: This a icle was submi ed o Plan Me abolism and Chemodi e si y, a sec ion o he jou nal F on ie s in Plan Science Recei ed: 22 June 2016 Accep ed: 27 Sep embe 2016 Published: 18 Oc obe 2016 Ci a ion: Wa inowski T, Kou aniemi S, Kä könen A, Sundbe g I, Toikka M, Simola LK, Kilpeläinen I and Tee i TH (2016) Pe oxidases Bound o he G owing Lignin Polyme P oduce Na u al Like Ex acellula Lignin in a Cell Cul u e o No way Sp uce. F on . Plan Sci. 7:1523. doi: 10.3389/ pls.2016.01523 Pe oxidases Bound o he G owing Lignin Polyme P oduce Na u al Like Ex acellula Lignin in a Cell Cul u e o No way Sp uce Tino Wa inowski1, Sanna Kou aniemi2, Anna Kä könen1 †, Ila i Sundbe g1, Me ja Toikka 3, Liisa Kaa ina Simola4 †, Ilkka Kilpeläinen3and Teemu H. Tee i 1* 1Depa men o Ag icul u al Sciences, Viikki Plan Science Cen e, Uni e si y o Helsinki, Finland, 2Depa men o Food and En i onmen al Chemis y, Uni e si y o Helsinki, Finland, 3Labo a o y o O ganic Chemis y, Depa men o Chemis y, Uni e si y o Helsinki, Finland, 4Depa men o Biosciences, Uni e si y o Helsinki, Finland Lignin, an impo an componen o plan cell walls, is a polyme o monolignols de i ed om he phenylp opanoid pa hway. Monolignols a e oxidized in he cell wall by oxida i e enzymes (pe oxidases and/o laccases) o adicals, which hen couple wi h he g owing lignin polyme . We ha e in es iga ed he cha ac e is ics o he polyme iza ion eac ion by p oducing lignin polyme s in i o using di e en oxida i e enzymes and analyzing he s uc u es o med wi h NMR. The abili y o he enzymes o oxidize high-molecula -weigh compounds was es ed using cy och ome cas a subs a e. The esul s suppo an idea ha lignin s uc u e is la gely de e mined by he concen a ion a ios o he monolignol (coni e yl alcohol) and polyme adicals in ol ed in he coupling eac ion. High a e o he lignin polyme oxida ion compa ed o monolignol oxida ion leads o a na u al- like s uc u e. The high ela i e a e can be achie ed by an open ac i e si e o he oxida i e enzyme, close p oximi y o he enzyme wi h he polyme ic subs a e o simply by high enzyma ic ac i i y ha consumes monolignols apidly. Monolignols, which a e oxidized e icien ly, can be seen as compe i i e inhibi o s o polyme oxida ion. Ou esul s indica e ha , a leas in a No way sp uce (Picea abies L. Ka s .) cell cul u e, a g oup o apoplas ic, polyme -oxidizing pe oxidases bind o he lignin polyme and a e esponsible o p oduc ion o na u al-like lignin in cell suspension cul u es in i o, and also in i o. The pe oxidases bound o he ex acellula lignin had he highes abili y o bind o a ious cell wall polyme s in i o. Ex acellula lignin con ains pec in- ype suga s, making hem possible a achmen poin s o hese ca ionic pe oxidases. Keywo ds: dehyd ogena ion polyme , lignin-ca bohyd a e complex, lignin polyme iza ion, oxida i e enzymes, pe oxidase, polyme oxida ion INTRODUCTION Lignin is a complex, hyd ophobic componen in he plan cell wall o wa e -conduc ing essels and acheids, as well as suppo -gi ing scle enchyma cells. I gi es cell walls esilience agains bo h mechanical s ess and biological pe ils, and con ibu es o he calo i ic alue o wood as a uel. Howe e , o pulp and pape manu ac u e as well as in bio uel p oduc ion lignin is an Wa inowski e al. Polyme Oxida ion De e mines Lignin S uc u e FIGURE 1 | The s uc u es o sp uce lignin monome s coni e yl alcohol (A) and p-couma yl alcohol (B), as well as ou p incipal subs uc u es in so wood lignin: 5-5-O-4 (C), β-O-4 (D), β-5 (E), and β-β(F). R deno es a con inuing lignin molecule, o a hyd ogen a om, i he molecule ends a ha poin . Simila ly, R’ deno es ei he a con inuing lignin molecule o a phenylp opanoid side chain. obs uc ion ha needs o be emo ed o b oken down in p ocesses ha a e bo h economically and en i onmen ally challenging. Some lignin s uc u es a e mo e suscep ible o deg ada ion han o he s. Achie ing con ol o lignin con en as well as subuni and linkage composi ion would hus c ea e signi ican bene i s (G abbe , 2005). Lignin is a polyme o monolignols, which a e de i a i es o he phenylp opanoid pa hway. The p incipal monolignol in gymnospe ms is coni e yl alcohol, supplemen ed wi h a small quan i y o p-couma yl alcohol (Figu e 1;Boe jan e al., 2003). Monolignols a e oxidized in he apoplas in o esonance-s abilized adicals by he ac ion o oxida i e enzymes, which can be ei he pe oxidases (class III; EC 1.11.1.7; dono :hyd ogen pe oxide oxido educ ase) o laccases (p-diphenol:oxygen oxido educ ase, EC 1.10.3.2). The adicals hen couple in mu o o o m he lignin polyme ( e iewed by Vanholme e al., 2010; Wang e al., 2013). Bo h pe oxidases and Abb e ia ions: DHP, dehyd ogena ion polyme ; HRP, ho se adish pe oxidase; pI, isoelec ic poin ; PMSF, phenylme hylsul onyl luo ide. laccases a e p esen in he apoplas as nume ous isoenzymes wi h wide subs a e speci ici ies (Kou aniemi e al., 2005, 2015; Be he e al., 2011; Tu lapa i e al., 2011; No o-Uzal e al., 2013; Ba os e al., 2015). I is now known ha bo h laccases and pe oxidases con ibu e o lignin biosyn hesis a leas in A abidopsis (A abidopsis haliana;Be he e al., 2011; No o- Uzal e al., 2013; Zhao e al., 2013; Shige o and Tsu sumi, 2016). Monolignol adicals can couple in a ious ways o c ea e di e en subs uc u es in lignin, which, in p inciple, leads o a andom s uc u e in lignin. The ou main subs uc u es a e β- O-4, β-5, β-βand 5-5-O-4 (dibenzodioxocin) linkages (Figu e 1; Adle , 1977; Ka hunen e al., 1995). Howe e , i has been shown ha he ela i e abundances o hese subs uc u es a y in di e en pa s o he cell wall. Fo example, in No way sp uce (Picea abies (L.) Ka s .), he 5-5-O-4 s uc u e is en iched in he inne mos pa o he acheid seconda y cell wall (Kukkola e al., 2003). The pa ame e s which de e mine he abundance o lignin subs uc u es ha e been unde ac i e e alua ion. The classical (combina o ial) model o lignin polyme iza ion s a es ha he s uc u e o he polyme is de e mined by he chemis y F on ie s in Plan Science | www. on ie sin.o g 2Oc obe 2016 | Volume 7 | A icle 1523 Wa inowski e al. Polyme Oxida ion De e mines Lignin S uc u e o he coupling eac ion, whe e p o eins play no ole beyond p oduc ion o phenolic adicals. Polyme iza ion would hus be a s ochas ic p ocess (Ralph e al., 2004). The al e na i e hypo hesis abou di igen p o ein-media ed coupling o monolignols in lignin o ma ion (Gang e al., 1999; Da in and Lewis, 2005) has no been suppo ed by scien i ic da a, al hough a ecen pape sugges s ha di igen p o eins ha e a ole in lignin o ma ion in he Caspa ian s ip o oo endode mis (Hosmani e al., 2013). Fac o s al eady shown o a ec he ypes o bonds c ea ed include concen a ion o monolignols (Adle , 1977; Te ashima e al., 1995; Sy jänen and B unow, 2000) and pH (Ämmälah i and B unow, 2000). Mo eo e , he ca bohyd a e ma ix o he cell wall has been sugges ed o in luence he s uc u e o lignin, as sphe ical lignin pa icles we e de ec ed in he middle lamella wi hin he andom pec in ne wo k, whe eas lamella lignin pa icles we e de ec ed in he seconda y cell wall ha con ains uni o mly o ien ed cellulose mic o ib ils (Donaldson, 1994; Wi e al., 2005). Fo ma ion o a co alen bond be ween a monolignol and he g owing lignin polyme equi es he p esence o an unpai ed elec on in bo h en i ies, meaning ha also he polyme mus be oxidized du ing he p ocess. In na u al lignin, g ow h o he polyme akes place in an end-wise manne , i.e., monolignols a e sequen ially added o he polyme (B unow e al., 1998). Oxida ion o he polyme may happen by a adical ans e be ween he monolignol adical and he polyme , bu his mechanism has been shown o be ine icien (Sasaki e al., 2004). Also, a shu le mechanism o e he plasma memb ane in ol ing, o example, manganese ions ha would oxidase monolignols/lignin polyme in he apoplas has been sugges ed ( ¯ Onne ud e al., 2002). Al e na i ely, he enzymes may oxidize lignin di ec ly. I has been shown ha popla (Populus alba) has a cell wall-bound pe oxidase capable o di ec ly oxidizing high-molecula -weigh lignin polyme s (Sasaki e al., 2004). In i o his enzyme p oduces om monolignols and hyd ogen pe oxide a dehyd ogena ion polyme (DHP), which has s uc u al simila i ies wi h na u al lignins. The abili y o pe oxidases o oxidize lignin di ec ly was concluded o be impo an in he p oduc ion o na u al lignin polyme s (Sasaki e al., 2004). As such, he 5-5-O-4 subs uc u es a e indica i e o lignin polyme oxida ion, since hese subs uc u es a e no ound in lignin oligome s and a e ound a educed amoun s in in i o-p oduced DHPs (Ralph, 1999). In his s udy, we ha e used a No way sp uce issue cul u e as a model sys em, as he cells p oduce ex acellula lignin in he cul u e medium (Simola e al., 1992; Kä könen e al., 2002; Kou aniemi e al., 2007; Kä könen and Kou aniemi, 2010). S uc u ally, his polyme esembles na u al lignins (B unow e al., 1990, 1993, 1998; Kou aniemi e al., 2005). A high numbe o pe oxidase isoenzymes and one laccase a e p esen in he cul u e medium (Kä könen e al., 2002; Kou aniemi e al., 2015), esembling in his sense he de eloping xylem o No way sp uce (Fage s ed e al., 2010). In e es ingly, a speci ic subse o ca ionic pe oxidases and ano he laccase isoenzyme a e bound o he ex acellula lignin (Kou aniemi, 2007; Kou aniemi e al., 2015). Pu i ied cul u e medium laccase and pe oxidases ha e a high a ini y o coni e yl alcohol and a e able o o m high-molecula -weigh DHP om coni e yl alcohol in i o (Kä könen e al., 2002; Kou aniemi e al., 2005, 2015). In he p esen wo k, we ha e used oxida i e enzymes isola ed om he cul u e medium and om he ex acellula lignin o p oduce DHPs o s uc u al s udies. Fungal laccases om lignin- deg ading ungi T ame es hi su a and Melanoca pus albomyces we e also u ilized as hese enzymes a e known o be able o oxidize polyme ic lignin (Kiiskinen e al., 2002; Ri s ieg e al., 2002). As pec in- ype suga s we e de ec ed o be bound o ex acellula lignin, we assessed binding o he enzymes ex ac ed om he ex acellula lignin and o he sou ces o cell wall componen s and polyme s esembling hem. We sugges ha he ex acellula lignin-bound ac ion o he sec e ed suspension cul u e pe oxidases is specialized in lignin polyme iza ion and, mo e gene ally, ha he concen a ion a io o polyme and monome adicals is a key ac o ha de e mines lignin s uc u e. MATERIALS AND METHODS Tissue Cul u ing and Enzyme Ha es ing The No way sp uce issue cul u e line A3/85, which o ms ex acellula lignin in he cul u e medium (Simola e al., 1992), was main ained and p opaga ed on a solid nu ien medium and ans e ed o liquid medium o lignin p oduc ion (Kä könen e al., 2002; Kou aniemi e al., 2005). Ex acellula lignin was collec ed by cen i uga ion and he soluble enzymes om he cul u e medium we e ha es ed by concen a ing he medium by ul a il a ion. P io o p o ein ex ac ion, ex acellula lignin was washed se e al imes wi h wa e un il no pe oxidase ac i i y was eleased. The polyme -bound p o eins we e ex ac ed wice (1 h each, a +4◦C, wi h slow mixing) wi h 20 mM MES- bu e , pH 6.5 supplemen ed wi h 1 M NaCl, 1 mM CaCl2, and 1 mM phenylme hylsul onyl luo ide (PMSF). P o eins we e concen a ed by ul a il a ion (Pall S i ed Cell Omega 10K sys em) and excess sal was emo ed using a PD-10 desal ing column (GE Heal hca e) equilib a ed wi h he same bu e excep ha NaCl concen a ion was 50 mM. The ex ac ed p o eins we e sepa a ed in an isoelec ic ocusing gel and s ained o pe oxidase ac i i y wi h 0.83 mM 3,3′-diaminobenzidine in 50 mM Na- ci a e bu e , pH 5.0 con aining 10 mM CaCl2and 0.5 mM H2O2 (Kä könen e al., 2002). Pu i ica ion o Ex acellula Lignin-Bound Pe oxidases and Isola ing he Co esponding cDNA Molecules Du ing p o ein pu i ica ion, pe oxidase ac i i y in he ac ions was sc eened on a mic opla e o ma using 0.67 mM ABTS (2,2-azino-bis(3-e hylbenzo hiazoline-6-sul onic acid), Sigma- Ald ich) and 0.4 mM H2O2in 50 mM Na-ace a e bu e , pH 5.0, wi h de ec ion a 412 nm in a Mul iscan MCC/340 pla e eade (Labsys ems). The pe oxidase ac ion was pa ially pu i ied essen ially as desc ibed in Kou aniemi e al. (2005). B ie ly, he ex ac ed p o ein ac ion was b ough o 2 M ammonium sul a e concen a ion and cen i uged a 10,000 ×g o 20 min. The soluble ac ion was loaded in o a Phenyl Sepha ose 6 FF column F on ie s in Plan Science | www. on ie sin.o g 3Oc obe 2016 | Volume 7 | A icle 1523 Wa inowski e al. Polyme Oxida ion De e mines Lignin S uc u e (GE Heal hca e) equilib a ed wi h 20 mM MES pH 6.0, 2 M ammonium sul a e and 1 mM CaCl2. P o eins we e elu ed wi h a s ep g adien o he same bu e wi hou ammonium sul a e. F ac ions con aining pe oxidase ac i i y we e loaded on o a Concana alin A Sepha ose column (GE Heal hca e) equilib a ed wi h 20 mM MES pH 6.0, 1 M ammonium sul a e, 1 mM CaCl2, MnCl2and MgCl2each and elu ed wi h 500 mM me hyl α-D- mannopy anoside (Fluka) in he same bu e . Elu ed p o eins we e concen a ed wi h a Omega 10K S i ed Cell Sys em (Pall Li e Sciences) and loaded in o a Supe dex 75 size exclusion ch oma og aphy column equilib a ed wi h 20 mM MES pH 6.0, 50 mM NaCl, 1 mM CaCl2. Pe oxidases om he size exclusion s ep we e loaded in o a SP Sepha ose High Pe o mance ca ion exchange column (GE Heal hca e) equilib a ed wi h 20 mM MES pH 6.0, 50 mM NaCl, 1 mM CaCl2. Basic pe oxidases we e elu ed wi h a linea g adien o 300 mM NaCl in he same bu e and concen a ed as abo e. The pool o basic pe oxidases was sepa a ed in a 10% SDS- PAGE gel. P o eins in he 32–42 kDa ange we e excised om he gel, subjec ed o in-gel diges ion wi h ypsin and alkyla ed. The yp ic agmen s we e sequenced wi h LC-MS/MS andem mass spec ome y as desc ibed in Pou anen e al. (2001). Degene a e p ime s (5′-GGWGTCGTGTCRTGTGCWGAC ATTCTC-3′, 5′-CTCATCTCAMTITTTCARGCTCA-3′, 5′-CTG GTRCTGTAGGAYTCAAC-3′) we e designed based on ob ained pep ide sequences (CADIL, LISLFQAQ, VESYSTSTHAFFK) showing simila i y o pe oxidases. To al and mRNA we e isola ed om he issue-cul u ed sp uce cells and e e se ansc ibed o cDNA as desc ibed in Kou aniemi e al. (2005). Pa ial pe oxidase cDNA sequences we e ampli ied wi h a polyme ase chain eac ion using he degene a e p ime s. Rapid ampli ica ion o cDNA ends (3′RACE) was done using oligo-dT as he 3′ p ime . Full-leng h 5′RACEs we e pe o med wi h SMART RACE cDNA Ampli ica ion Ki (BD Biosciences Clon ech). Gene speci ic e e se p ime s we e designed based on he 5′un ansla ed egions o he genes and used in 3′RACE (BD Biosciences Clon ech) o ob ain he ull-leng h sequences. Th ee ull-leng h cDNAs, each coding o a ew sequenced pep ides, we e eco e ed. P ime s used o cloning a e shown in Supplemen a y Table 1. P oduc ion o DHP Two di e en enzyme samples om he No way sp uce issue cul u e we e used in he DHP syn hesis, o al soluble p o eins om he cul u e medium and ex ac ed lignin-bound p o eins (Table 1). In addi ion, DHP was p oduced wi h ho se adish pe oxidase (HRP) and lignin-deg ading ungal laccases om T. hi su a and M. albomyces (Table 1). Bo h hyd ogen pe oxide (34 mM, 5 ml) and coni e yl alcohol (Fluka, 34 mM, 5 ml) we e simul aneously injec ed a a cons an a e (250 µl h−1) using a mic o pump (Ha a d Appa a us) du ing a 20 h-pe iod in o 5 ml o enzyme solu ion con aining a ce ain amoun o coni e yl alcohol-oxidizing ac i i y (Table 3 o ac i i y amoun s) in a s i ed lask a oom empe a u e. A e injec ion, he eac ion was allowed o p oceed o addi ional 4 h. Wi h laccases, he hyd ogen pe oxide was omi ed and eplaced wi h he bu e . Polyme s we e collec ed by cen i uga ion (2500 g, 15 min), TABLE 1 | Pe oxidase and laccase samples used in his s udy. Abb e ia ion Desc ip ion Sou ce/ e e ences Pa-SL Picea abies o al soluble cul u e medium p o eins Kä könen e al., 2002 Pa-BD Picea abies o al p o eins ha bind he ex acellula lignin Kou aniemi, 2007; his s udy HRP Ho se adish pe oxidase ype II Sigma-Ald ich MP-11 Mic ope oxidase Sigma-Ald ich Mal LAC Melanoca pus albomyces laccase Kiiskinen e al., 2002 Thi LAC T ame es hi su a laccase Ri s ieg e al., 2002 washed h ee imes wi h wa e and lyophilized. All eac ions we e pe o med in 50 mM sodium ace a e, pH 5.0, supplemen ed wi h 2.5 mM CaCl2. pH 5.0 was chosen o app oxima e he pH o he sp uce cell cul u es du ing ex acellula lignin o ma ion (pH 4.2–4.8; Kä könen e al., 2002) and, on he o he hand, he pH op imum o he isola ed and o al cul u e medium pe oxidases (pH 4.0–6.5; Kou aniemi e al., 2005, and unpublished da a). As pH a ec s he s uc u e o DHPs p oduced (Ämmälah i and B unow, 2000), DHP p oduc ion by HRP was also conduc ed a pH 5.0. S uc u al Analysis o DHPs and Lignin Milled wood lignin (MWL) was p epa ed acco ding o Bjö kman (1956) wi h some modi ica ions. Sawdus was i s ex ac ed in a Soxhle appa a us wi h ace one (48 h) and ace one-wa e 9:1 (4 h), hen d ied unde acuum. D y sawdus was milled in a ball mill (F i sch Pul e ise e 6) a 320 pm o 50 h and he wood powde was ex ac ed wi h dioxane-wa e 9:1 (2 h) in a cooled ul asonic ba h. Dioxane-wa e was e apo a ed wi h a o a apo nea ly o he d yness, glacial ace ic acid was added and he mix u e was s i ed a +50◦C o 2 h. The il e ed ace ic acid solu ion was pou ed in o wa e whe eupon lignin p ecipi a ed. The p ecipi a ed lignin was collec ed by cen i uga ion, washed 1–2 imes wi h wa e and d ied. Fo NMR analyses MWL, isola ed ex acellula lignin and DHPs we e ace yla ed. In a ypical p ocedu e, he lignin sample (5–50 mg) was suspended in o 10 ml o ace anhyd ide/py idine (1:1, / ). A e s i ing o 12 h a oom empe a u e unde a gon, an excess o e hanol was added and he sol en was e apo a ed in a o a apo . Successi e e hanol addi ion and e apo a ion s eps we e pe o med un il all esidual py idine, ace ic acid and e hyl ace a e we e emo ed om he sample. The d y, ace yla ed lignin was dissol ed in o 0.7 ml o CDCl3 o deu e a ed DMSO (Camb idge Iso ope Labo a o ies). The NMR spec a we e measu ed wi h Va ian Uni y Ino a 600 spec ome e (1H equency 600 MHz). A modi ied e sion o Q-HSQC (Heikkinen e al., 2003), QQ-HSQC (Koskela e al., 2005) was applied o ob ain quan i a i e 2D NMR spec a. The spec al wid h was 6000 Hz in 1H and 15,000 Hz in 13C. The numbe o scans was 256 (1024 da a poin s) and he numbe o ime inc emen s 128 (hype complex me hod) wi h a epe i ion a e o 3 s, yielding measu emen ime o ∼52 h o each spec um. The spec a we e ze o- illed o 2048∗1024 F on ie s in Plan Science | www. on ie sin.o g 4Oc obe 2016 | Volume 7 | A icle 1523 Wa inowski e al. Polyme Oxida ion De e mines Lignin S uc u e da a poin s and apodized wi h squa ed bell window unc ions in bo h dimensions p io o Fou ie ans o ma ion. All spec a we e p ocessed and analyzed wi h he s anda d VNMR so wa e (Agilen Technologies). The ela i e amoun s o he main s uc u al uni s o lignin samples we e calcula ed as pe cen age alues om he sum o in eg al alues o β-O-4, β-5, β-βand 5-5-O-4 subs uc u es (Heikkinen e al., 2003). In o de o gi e equal weigh o he di e en bond ypes ha occu in he polyme , s anda d sco es we e calcula ed. Fo each bond ype, he s anda d sco e zhas a e age alue o 0 and a iance o 1 ac oss he samples. Enzyme Ac i i y Measu emen s Coni e yl alcohol pe oxidase was de e mined a 262 nm wi h 100 µM coni e yl alcohol (Fluka) and 250 µM H2O2 in 45 mM Na-ci a e bu e , pH 5.0 (Kä könen e al., 2002). Coni e yl alcohol oxidase ac i i y was measu ed a 262 nm using 70 µM coni e yl alcohol in 45 mM Na-ace a e bu e , pH 4.5, supplemen ed wi h 125 U ml−1ca alase (Sigma C-100). Measu emen s we e done in iplica e, and εo 14,000 M−1 cm−1 o coni e yl alcohol was used o quan i a i e calcula ions (Kä könen e al., 2002). The abili y o pe oxidases o oxidize a high-molecula -weigh subs a e, cy och ome c om bo ine hea (Sigma-Ald ich), was measu ed by ollowing a dec ease in he abso bance o he educed e ocy och ome ca 550 nm (ε=18,500 M−1cm−1; Hodges and Leona d, 1974; Sasaki e al., 2004). Fi s , cy och ome cwas educed wi h 10 mM Na-di hioni e o 30 min. Di hioni e was emo ed by passing he sample h ough a PD-10 column (GE Heal hca e) equilib a ed wi h degassed 50 mM Na-ci a e bu e , pH 5.0. Oxida ion o cy och ome cwas measu ed in he same bu e a oom empe a u e using 40 µM cy och ome cand 1.2–4.6 nka coni e yl alcohol pe oxidase ac i i y (soluble cul u e medium and lignin-bound p o eins) o 10 µg HRP (∼5500 nka coni e yl alcohol pe oxidase ac i i y). Non-enzyma ic oxida ion was i s ollowed o a ew minu es, and pe oxidase ac ion was s a ed wi h addi ion o 50 µM H2O2. The a e o non-enzyma ic oxida ion was sub ac ed om he pe oxidase-ca alyzed eac ion. Resul s we e no malized by calcula ing he a io o cy och ome cpe oxidase ac i i y o coni e yl alcohol pe oxidase ac i i y (Cy Cp x/CAp x). Mic ope oxidase (MP-11, Sigma-Ald ich), which con ains a heme o cy och ome cmolecule wi h amino acids 11–21 s ill a ached, was used as a posi i e con ol (34 nka coni e yl alcohol pe oxidase ac i i y / eac ion), as i ep esen s an enzyme wi h a wide-open ac i e si e capable o accommoda ing high-molecula -weigh subs a es. The abili y o he M. albomyces and T. hi su a laccases o oxidize DHP p oduced wi h HRP (see below) was de e mined by measu ing changes in oxygen concen a ion wi h he Oxyg aph oxygen elec ode (Hansa ech Ins umen s). The measu emen s we e done in an ai -sa u a ed 50 mM Na-ace a e bu e , pH 5.0, supplemen ed wi h 2.5 mM CaCl2, o which a ixed amoun o laccase (1.2 nka coni e yl alcohol oxidase ac i i y) and 1 mg o suspended DHP we e added ( o al olume 1 ml). The measu ing cell was kep unde magne ic s i ing o p e en sedimen a ion o he polyme . Enzyme Binding Assays Binding assays we e pe o med o s udy he abili y o pe oxidases and laccases o bind o lignin and o lignin-like polyme s (MWL, ex acellula lignin, deglycosyla ed ex acellula lignin and DHP p oduced wi h HRP), o o anionic polysaccha ides (polygalac u onic acid and algina e). The es ed enzyme samples included o al soluble p o eins om he cul u e medium, ex ac ed lignin-bound p o eins and HRP, as well as ungal laccases om M. albomyces and T. hi su a. Con ol eac ions con ained he equal amoun o enzymes wi hou any polyme , and we e ea ed simila ly o he samples. Binding assays we e conduc ed a pH 4.5 o app oxima e he pH o he cul u e medium du ing he o ma ion o ex acellula lignin. The enzyme sample (2.5 nka o coni e yl alcohol (pe )oxidase ac i i y) was dilu ed wi h he binding bu e (50 mM sodium a a e, pH 4.5, 50 mM NaCl, 4 mM CaCl2, 1 mM PMSF, 0.01% (w/ ) BSA) o a olume o 0.8 ml. Polyme (15–50 mg depending on a ailabili y; howe e , as polysaccha ides o m a la ge gel- olume when mixed wi h Ca2+ions, a lowe amoun o polysaccha ide polyme s was used) was added and he sample was incuba ed o 2 h a +6◦C wi h a slow mixing. A e cen i uga ion (21,000 g, 4 min) he pelle was washed i e imes wi h he binding bu e , and he polyme -bound enzymes we e elu ed by ex ac ing wice wi h 0.7 ml o he ex ac ion bu e (20 mM MES, pH 6.0, 1 M NaCl, 4 mM CaCl2, 1 mM PMSF, 0.01% BSA). The ex ac ions we e pe o med in condi ions simila o binding wi h a 1-h incuba ion and he ex ac s we e pooled oge he . Coni e yl alcohol (pe )oxidase ac i i ies we e measu ed om he supe na an a e he binding (unbound ac i i y) and he ex ac ion s eps (bound ac i i y). The polyme s o binding assays we e p epa ed as ollows: ex acellula lignin was ha es ed om he suspension cul u e by cen i uga ion, washed b ie ly wi h wa e and hen 5 imes wi h he ex ac ion bu e (see abo e) o emo e he na i e, lignin-bound p o eins. A e washing wice wi h wa e , lignin was lyophilized and g ound o a ine powde in a mo a . This ex acellula lignin sample was u he deglycosyla ed by ea ing he polyme (250 mg suspended in 50 mM Na- ace a e, pH 5.0) wi h a mix u e o se e al glycosyl hyd olases (desc ibed in Vi kki e al., 2008) a 37◦C o 48 h. A 24 h, 5 mg o pec olyase (Sigma-Ald ich) was added o he eac ion o inc ease he deg ada ion o pec ic polysaccha ides. A e hyd olysis, enzymes we e inac i a ed by boiling o 5 min and he ex acellula lignin was washed wice wi h wa e , wice wi h 1 M NaCl, and inally h ee imes wi h wa e . The ex acellula lignin con ol was ea ed simila ly excep ha he enzymes we e eplaced wi h bu e only. DHP and MWL we e washed, lyophilized and g ound o a ine powde in a mo a simila ly o he ex acellula lignin sample. De e mina ion o he Ca bohyd a e Componen o Ex acellula Lignin Ex acellula lignin was washed wi h wa e and hen ex ac ed wi h ace one o emo e low-molecula -weigh phenolic compounds, lyophilized and u he d ied in a acuum o en a F on ie s in Plan Science | www. on ie sin.o g 5Oc obe 2016 | Volume 7 | A icle 1523 Wa inowski e al. Polyme Oxida ion De e mines Lignin S uc u e 40◦C o 1 h. The sample (5 mg; in duplica es om h ee sepa a e issue cul u es) was deg aded by acid me hanolysis essen ially as desc ibed in Sundbe g e al. (1996). Released monosaccha ides we e ime hylsilyla ed be o e gas ch oma og aphic sepa a ion, which was pe o med on HP 6890N GC equipped wi h a lame ioniza ion de ec o (Agilen Technologies) using a DB-1 (30 m, 0.25 mm i.d., 0.25 µm ilm hickness) capilla y column om Agilen Technologies. O en empe a u e p o ile was as ollows: 150◦C o 3 min; +1◦C/min un il 155◦C; hold o 1 min; +2◦C/min un il 200◦C; +20◦C/min un il 325◦C. A abinose, xylose, hamnose, glucu onic acid, galac u onic acid, mannose, galac ose and glucose we e used as s anda ds and so bi ol as an in e nal s anda d o quan i ica ion. RESULTS Cloning o cDNA Molecules Encoding Ex acellula Lignin-Bound Pe oxidases The No way sp uce issue cul u e used in ou s udy sec e es p o eins in o he cul u e medium; pa o hese a e bound o he ex acellula lignin and can be ex ac ed wi h a bu e con aining 1 M NaCl. An isoelec ic ocusing gel s ained o pe oxidase ac i i y showed ha some o he ex ac ed p o eins wi h an isoelec ic poin (pI) ∼10 had pe oxidase ac i i y (Kou aniemi, 2007). These pe oxidases we e pa ially pu i ied, and amino acid sequences o se e al in e nal pep ides we e de e mined using LC-MS/MS andem mass spec ome y (Supplemen a y Table 2). Based on he sequence da a, cDNA molecules encoding he pep ides we e isola ed. Th ee ull leng h cDNAs we e ob ained, each coding o a ew dis inc amino acid sequences ob ained du ing pep ide sequencing (Supplemen a y Figu e 1). The h ee cDNA sequences we e designa ed as PaPx16,PaPx17, and PaPx18 (EMBL: AM293546, AM293547, and AM293548, espec i ely). PaPx16 and PaPx17 a e bo h ansla ed in o 341 amino acids. They a e close homologs, he encoded amino acid sequence di e ing a eigh posi ions. The PaPx18 cDNA codes o a polypep ide o 310 amino acids. All h ee cDNAs encode a p edic ed signal sequence. The p edic ed pIs o ma u e PaPx16, PaPx17, and PaPx18 a e 9.1, 9.2, and 8.4, espec i ely. The lowe pI compa ed wi h he one de e mined om an isoelec ic ocusing gel is p obably due o he bound Ca2+ions and he heme, which inc ease he pI o he co ec ly olded pe oxidase by up o 2 pI uni s (Welinde e al., 2002) We sea ched he co esponding sequences om he No way sp uce genome (Nys ed e al., 2013). The closes homolog o PaPx16 and PaPx17 is MA_3486g0010, and o PaPx18 MA_10432379g0020. Bo h o he genes ha e some exp ession in ea ly wood (phloem+cambium+xylem; Nys ed e al., 2013). Pe oxidase and Laccase Isoenzymes Di e in Polyme Oxida ion Fo ma ion o adicals on lignin oligo- and polyme s is a p e equisi e o he polyme g ow h. The e o e, pe oxidases ha a e in ol ed in lignin polyme iza ion should also be able o oxidize polyme ic lignin. DHP p oduced om coni e yl alcohol wi h HRP was p epa ed as a nea -au hen ic subs a e, bu TABLE 2 | Oxida ion o cy och ome cby pe oxidases (Cy C p x) o dehyd ogena ion polyme (DHP) by laccases as a pe cen age o oxida ion o coni e yl alcohol using soluble cul u e medium pe oxidases (Pa-SL), lignin-bound pe oxidases (Pa-BD), ho se adish pe oxidase (HRP), mic ope oxidase (MP), T. hi su a laccase (Thi LAC) and M. albomyces laccase (Mal LAC); n=3. P o ein sample Cy C p x oxida ion (% o DHP oxida ion (% o coni e yl alcohol oxida ion) coni e yl alcohol oxida ion) Pa-SL 1.8 ±0.2 Pa-BD 0.19 ±0.05 HRP 0.018 ±0.002 MP 35.5 ±1.5 Thi LAC 64.9 ±5.5 Mal LAC 13.8 ±1.6 we could no eliably apply he UV-Vis spec oscopic me hod o Sasaki e al. (2004) due o a limi ed amoun o enzyme a hand. Ins ead, we used cy och ome cas a p oxy high- molecula -weigh subs a e o pe oxidases (Sasaki e al., 2004). We compa ed he abili y o soluble cul u e medium p o eins, ex ac ed lignin-bound p o eins and HRP o oxidize cy och ome c. Mic ope oxidase consis ing o a heme wi h 11 amino acids a ached was used as a posi i e con ol, and i was he mos e icien o all es ed enzymes in cy och ome coxida ion, eaching up o 35% o he oxida ion a e o coni e yl alcohol (Table 2). Ou o sp uce na i e pe oxidases, soluble cul u e medium pe oxidases showed he highes oxida ion a e, 1.8% o he a e o coni e yl alcohol. This was ca. 10- old highe han ha o ex ac ed lignin-bound pe oxidases, which was again 10- old highe han ha o HRP (Table 2). Wi h laccases, assessmen o DHP oxida ion was possible by measu ing oxygen consump ion. O he wo laccases s udied he e, he a e o DHP oxida ion o T. hi su a laccase was ca. 5 imes highe han o he M. albomyces laccase when compa ed wi h he a e o coni e yl alcohol oxida ion (Table 2). Polyme -Oxidizing Enzymes P oduce Na u al-Like Lignin In Vi o To s udy he s uc u al simila i y o DHPs syn hesized wi h a ious pe oxidases and laccases, he dis ibu ion o he bond ypes in he esul ing polyme s we e de e mined by quan i a i e 2D NMR. S anda d sco es we e calcula ed o each esul in o de o gi e an equal weigh o each bond ype, and Pea son co ela ion coe icien s we e calcula ed o he bond dis ibu ion ob ained o No way sp uce MWL (Table 3). The Pea son co ela ion coe icien p o ides a single measu e, anging om −1 o 1, ha desc ibes he simila i y o he DHP o MWL. As lignin is a he e ogeneous polyme ha is composed o di e en s uc u al uni s, i s s uc u e can be desc ibed using he pe cen age alues o di e en monome uni linkages (Table 3). MWL con ained a high p opo ion o β-O-4 (66.7%) and o 5-5- O-4 subs uc u es (6.5%), co esponding well wi h ea lie esul s (Heikkinen e al., 2003). Ex acellula lignin had an equally high p opo ion o 5-5-O-4 while he con en o β-O-4 was sligh ly lowe . The DHP p oduced wi h 200 nka (measu ed as F on ie s in Plan Science | www. on ie sin.o g 6Oc obe 2016 | Volume 7 | A icle 1523 Wa inowski e al. Polyme Oxida ion De e mines Lignin S uc u e TABLE 3 | S uc u al composi ion o na u al and syn he ic lignin samples de e mined using Q-HSQC and QQ-HSQC. Sample Ac i i y Pea son Yield β-O-4% β-5% β-β% 5-5-O-4% NORWAY SPRUCE LIGNIN MWLa–+1.0 – 66.7 ±1.0 z= +2.8 20.1 ±0.4 z= −2.5 6.5 ±0.1 z= −3.0 6.5 ±0.1 z= +2.4 NORWAY SPRUCE EXTRACELLULAR LIGNIN AND POLYMERS PRODUCED WITH ENZYMES ISOLATED FROM THE TISSUE CULTURE ELa–+0.7 – 50.7 ±0.6 z= +0.6 24.8 ±0.2 z= −1.7 18.6 ±0.0 z= +0.2 6.2 ±0.2 z= +2.2 DHP/Pa-BD 200 nka +0.9 68 ±5% 54.2 z= +1.1 26.8 z= −1.3 15.6 z= −0.6 3.4 z= +0.8 DHP/Pa-BD 5 nka −1.0 5 ±2% 39.4 ±0.3 z= −1.0 38.7 ±0.6 z= +0.9 20.7 ±0.3 z= +0.8 1.3 ±0.0 z= −0.4 DHP/Pa-SL 200 nka −0.9 41 ±6% 44.3 ±0.0 z= −0.3 34.7 ±0.2 z= +0.2 19.1 ±0.4 z= +0.4 1.9 ±0.1 z=0.0 DHP/Pa-SL 5 nka −1.0 4 ±2% 40.2 ±1.2 z= −0.9 37.6 ±1.3 z= +0.7 21.1 ±0.3 z= +0.9 1.1 ±0.2 z= −0.5 POLYMERS PRODUCED WITH ENZYMES FROM NON-LIGNIN-FORMING SOURCES DHP/HRP 20,000 nka – 58 ±3% Insoluble p oduc DHP/HRP 5000 nka – 58 ±4% Insoluble p oduc DHP/HRPa1100 nka −0.9 41 ±7% 45.4 ±1.1 z= −0.2 35.4 ±1.0 z= +0.3 18.2 ±0.6 z= +0.1 1.0 ±0.0 z= −0.5 DHP/HRP 200 nka −0.9 39 ±3% 44.2 ±0.7 z= −0.3 36.5 ±1.1 z= +0.5 18.4 ±0.1 z= +0.2 0.9 ±0.0 z= −0.6 DHP/HRP 5 nka – No eco e able p oduc DHP/Mal 300 nka −1.0 28 ±2 41.4 ±0.2 z= −0.7 37.6 ±0.3 z= +0.7 20.0 ±0.1 z= +0.6 0.9 ±0.0 z= −0.6 DHP/Thi 300 nka +0.7 48 ±2 52.1 ±0.9 z= +0.8 31.9 ±0.4 z= −0.3 14.9 ±0.4 z= −0.8 1.1 ±0.0 z= −0.5 DHP/HRP+Thi 150 +150 nka +0.6 39 ±4 52.0 ±0.8 z= +0.7 32.0 ±0.0 z= −0.3 15.1 ±0.1 z= −0.7 1.0 ±0.0 z= −0.5 DHP/HRP+Mal 150 +150 nka −1.0 29 ±4 42.6 ±0.2 z= −0.6 37.2 ±0.3 z= +0.7 19.3 ±0.2 z= +0.4 0.9 ±0.0 z= −0.6 Ac i i y column indica es he o al coni e yl alcohol (pe )oxidase ac i i y used in he p oduc ion o DHPs. Pea son column gi es a Pea son co ela ion o he s anda d sco es o a sample wi h milled wood lignin (MWL). Yield column gi es he yields o DHP syn hesis as a pe cen age o he maximum heo e ical yield. The inal ou columns gi e he p opo ions o he ou main lignin subs uc u es (β-O-4, β-5, β-βand 5-5-O-4) as pe cen ages o he o al 2D NMR signal om hese s uc u es and s anda d sco es (z) calcula ed om hese alues. EL, ex acellula lignin; DHP, dehyd ogena ion polyme ; Pa-BD, o al p o eins ex ac ed om ex acellula lignin; Pa-SL, o al soluble p o eins om he cul u e medium; HRP, ho se adish pe oxidase; Mal, Melanoca pus albomyces laccase; Thi, T ame es hi su a laccase. aResul s published p e iously in Kou aniemi e al. (2005). coni e yl alcohol pe oxidase ac i i y) o enzymes ex ac ed om he ex acellula lignin esembled MWL mo e han any o he DHP, and ga e he highes yield o he polyme (Table 3). Ve y high ac i i y (5000 and 20,000 nka ) o HRP p oduced good yields o he polyme , bu hese DHPs we e esis an o dissolu ion o NMR, possibly indica ing c osslinking eac ions aking place. The soluble cul u e medium enzymes (200 nka ) p oduced DHPs ha we e s uc u ally di e en om he MWL samples, con aining less β-O-5 and 5-5-O-4 linkages (Table 3). A simila esul was ob ained wi h HRP (200 nka ) and lowe ac i i ies (5 nka ) o he ex acellula lignin-ex ac ed enzymes. The second bes s uc u al co ela ion wi h MWL a e he DHP p oduced wi h he ex acellula lignin-ex ac ed enzymes (a 200 nka ) was obse ed in DHP p oduced using T. hi su a laccase (Table 3) wi h he abili y o oxidize lignin polyme s e icien ly (Table 2). In e es ingly, he DHP p oduced wi h T. hi su a laccase was mo e simila o MWL han ha p oduced wi h M. albomyces laccase, he enzyme wi h a lowe polyme -oxidizing ac i i y. DHPs p oduced wi h mix u es o equal amoun s o each laccase and HRP (150 nka each) showed ha he T. hi su a laccase domina ed o e HRP he s uc u e o he esul ing polyme . Wi h HRP and M. albomyces laccase he polyme was simila o hose p oduced wi h ei he enzyme alone. Ex acellula Lignin Con ains Pec ic Polysaccha ides NMR analyses o ex acellula lignin ha e shown he p esence o ca bohyd a es (I. Kilpeläinen, unpublished da a). These lignin- associa ed ca bohyd a es emained bound o lignin a e washing wi h bu e ed sal solu ion (1 M NaCl), sugges ing he p esence o co alen linkages be ween he suga uni s and lignin. The ca bohyd a e componen o he ex acellula lignin was s udied in mo e de ail using gas ch oma og aphy a e deg ada ion o he ca bohyd a e componen by acid me hanolysis. The o al ca bohyd a e con en in he ex acellula lignin was 166 ±27 µg/mg d y weigh (Table 4). Galac u onic acid (GalA), a abinose (A a) and galac ose (Gal) we e de ec ed in la ge amoun s, while hamnose (Rha), xylose (Xyl), glucose (Glc) and mannose (Man) we e p esen a lowe le els. The abundance o GalA sugges s he p esence o pec ic polysaccha ides, mainly homogalac u onan (Albe sheim e al., 2011; A modjo e al., 2013). Howe e , he p esence o Rha, A a and Gal also poin o he p esence o hamnogalac u onan I, which con ains a backbone o epea ing α-GalA-(1-2)-α-L-Rha-(1-4) disaccha ide, which is subs i u ed wi h side chains con aining mos ly A a and Gal (A modjo e al., 2013). The de ec ed Man and Glc sugges he p esence o (galac o)glucomannan, which is he main hemicellulosic polysaccha ide in so woods (Rowell e al., 2005). Ex acellula Lignin-Ex ac ed Pe oxidases Bind o Lignin-like Polyme s To de e mine which componen o he ex acellula lignin he pe oxidases a e bound o, we s udied he abili y o he o al soluble cul u e medium p o eins and he o al p o eins ex ac ed om he ex acellula lignin o bind di e en phenolic and ca bohyd a e polyme s. The polyme s included ex acellula lignin be o e and a e enzyma ic deg ada ion o he ca bohyd a e componen , DHP p oduced om coni e yl F on ie s in Plan Science | www. on ie sin.o g 7Oc obe 2016 | Volume 7 | A icle 1523 Wa inowski e al. Polyme Oxida ion De e mines Lignin S uc u e TABLE 4 | Suga composi ion o he ca bohyd a e ac ion o he ex acellula lignin, de e mined a e acid me hanolysis and ime hylsilyla ion using gas ch oma og aphy (a e age ±s anda d e o o he mean, n=5). Monosaccha ide µg (mg d y weigh )−1 Galac u onic acid 60.1 ±7.6 A abinose 57.4 ±7.8 Galac ose 32.3 ±5.2 Glucose 5.3 ±1.6 Rhamnose 3.9 ±0.6 Mannose 2.1 ±0.8 Xylose 1.7 ±0.4 Fucose 1.5 ±0.3 Glucu onic acid 0.9 ±0.5 alcohol wi h HRP, MWL and wo anionic ca bohyd a e polyme s, calcium polygalac u ona e and calcium algina e. Ca ionic pe oxidases ex ac ed om he ex acellula lignin had a signi ican a ini y o all lignin polyme s es ed (Figu e 2). The binding was g ea es o ex acellula lignin i sel , wi h 55 ±3% o he ac i i y bound, bu signi ican binding was also obse ed o all o he polyme s es ed, wi h binding be ween 17 and 29%. Enzyma ic deg ada ion o he ex acellula lignin- bound ca bohyd a es educed, bu did no elimina e his binding (Figu e 2). In con as , he binding o he soluble cul u e medium pe oxidases o he polyme s was limi ed. The highes appa en a ini y was o calcium algina e, which bound 5 ±0.5% o he soluble pe oxidase ac i i y (Figu e 2). Only basic pe oxidases om he cul u e medium bound o lignin as shown in he IEF gel s ained o pe oxidase ac i i y (Figu e 3). The abili y o T. hi su a laccase, M. albomyces laccase and HRP o bind o he DHP p oduced by HRP was also measu ed. Unde he expe imen al condi ions 8.0 ±1.1% o he T. hi su a laccase ac i i y and 3.8 ±3.1% o ha o M. albomyces we e bound o he polyme . No signi ican binding o HPR could be de ec ed (da a no shown). DISCUSSION In his wo k we ha e in es iga ed he ole o oxida i e enzymes, isola ed om No way sp uce, on lignin s uc u e wi h he help o DHPs p oduced om coni e yl alcohol. The esul s show ha di e en enzymes, o a ying enzyme ac i i y amoun s unde o he wise iden ical condi ions, p oduce phenolic polyme s wi h di e en p opo ions o he lignin subs uc u es. Lignin polyme g ows when an unpai ed elec on in he polyme adical o ms a co alen bond wi h an unpai ed elec on in he monolignol adical. Thus, all enzymes ha can p oduce a polyme om monolignols mus also oxidize he polyme ic subs a e (di ec ly o indi ec ly). Howe e , only high oxida ion a e o he polyme will p oduce in amolecula c oss-links, such as he 5-5 bond ha occu s in he 5-5-O-4 subs uc u e ypical o coni e wood (Ka hunen e al., 1995; Kukkola e al., 2003). This sugges s ha he ailu e o mos in i o-polyme s o FIGURE 2 | Binding o he soluble cul u e medium p o eins (A), and he ex ac ed, lignin-bound p o eins (B) o lignin-like and anionic polysaccha ide polyme s. EL, ex acellula lignin; Deglycos. EL, ex acellula lignin a e he glycosyl hyd olase ea men ; DHP, dehyd ogena ion polyme om coni e yl alcohol made wi h HRP; MWL, milled wood lignin; Ca-PG, Ca-polygalac u ona e; Ca-ALG, Ca-algina e. Ligh g ay, unbound ac ion; da k g ay, bound ac ion. mimic na u al lignins esul s om an insu icien oxida ion o he g owing polyme . The p esen s udy shows ha he No way sp uce cell suspension cul u e has a subse o pe oxidases, which can bind o lignin-like and pec ic polyme s (Figu es 2,3), and which can oxidize polyme ic lignin (Table 3). This se o ex acellula lignin-ex ac ed pe oxidases, as well as T. hi su a laccase ha also has a conside able polyme -oxidizing abili y (Table 2), p oduced DHPs in a high yield (>50%) and wi h a high p opo ion o β-O-4 subs uc u es (Table 3). This is conside ed as a signa u e o end-wise polyme iza ion (Sa kanen, 1971), equi ing oxida ion o he polyme o p oceed. Enzymes such as HRP o M. albomyces laccase wi h lowe polyme -oxidizing abili y (Table 2), when dosed a he same le el, p oduced a condensed polyme wi h less β-O-4 and a high p opo ion o β-5 linkages (Table 3). In e es ingly, DHP p oduced wi h he ex acellula lignin-ex ac ed enzymes (a 200 nka ) was close o na u al sp uce lignin (MWL), mo e simila han any o he DHPs F on ie s in Plan Science | www. on ie sin.o g 8Oc obe 2016 | Volume 7 | A icle 1523 Wa inowski e al. Polyme Oxida ion De e mines Lignin S uc u e FIGURE 3 | No way sp uce cul u e medium p o eins sepa a ed in an isoelec ic ocusing gel and s ained o pe oxidase ac i i y. (1) P o eins unbound o MWL, (2) P o eins bound o MWL. Numbe s beside he IEF gel indica e he de e mined pH g adien in he gel. p oduced wi h o he enzymes es ed. The main con ibu o o his simila i y was, in addi ion o he β-O-4 con en , he ela i ely high con en o 5-5-O-4 bonds (Table 3). Wi h a mo e spa ing amoun o he lignin-ex ac ed enzymes (5 nka ) we ob ained a low polyme yield and a bond dis ibu ion simila o ha in DHP p oduced wi h HRP a a 200-nka dosage (Table 3). This obse a ion led o a sugges ion ha he key ac o in he syn hesis o na u al-like lignin is no he polyme -oxidizing abili y pe se, bu ins ead, he a io o polyme o monome oxida ion occu ing in he ac ual eac ion. One way o inc ease polyme oxida ion is o ha e enzymes ha a e specialized in his unc ion; howe e , also an inc ease in he enzyme ac i i y should ha e he same e ec since i leads o a apid consump ion o he monome s. The e o e, also HRP should be able o p oduce a na u al-like lignin i su icien amoun o he ac i i y was applied. Indeed, expe imen s wi h 5000 nka and 20,000 nka o HRP did p oduce a p ecipi a e in a high yield (58%, Table 3). Con a y o mos o he DHPs assayed, bu simila o some epe i ions o he expe imen wi h 200 nka o he ex acellula lignin-ex ac ed enzymes, hese polyme s could no be made soluble o he NMR analysis o he ac ual bond dis ibu ions, which is a possible indica o o c osslinking o he polyme . Inc easing he oxida i e enzyma ic ac i i y o p oduce mo e na u al-like lignins is essen ially he same bu an ex ended e ec , which is known om compa isons o “zu op ” and “zulau ” polyme s, whe e “zu op ” polyme s ha e a highe molecula mass and a s uc u e ha is mo e simila o ha in na u al lignins (F eudenbe g, 1956). Zu op e e s o g adual eeding o he subs a es in o he eac ion essel, i.e., inc easing he enzyme o subs a e a io (somewha simula ing lignin biosyn hesis), while in a zulau eac ion all subs a es a e added in he beginning o he eac ion. Sy jänen and B unow (2000) u ilized he same p inciple in an elegan expe imen al se up, whe e coni e yl alcohol was p o ided e y slowly o a HRP- con aining solu ion h ough a dialysis memb ane, p oducing a na i e-like polyme wi h inc eased p opo ion o 5-5-O-4 bonds. Méchin e al. (2007) ha e also es ed how pe oxidase ac i i y a ec s he DHP s uc u e, concluding howe e ha o zu op polyme s he s uc u e was mo e na u al-like when lowe enzyme ac i i ies we e used. This seemingly con adic o y esul is a leas pa ly due o a di e en measu e o “na u al- like”, which o Méchin e al. (2007) was he abundance o β-O-4 bonds. In he p esen s udy, we used a ios o all ou bond ypes, wi h 5-5-O-4 ha ing a high weigh . As s a ed abo e, his bond ype o ms only when he g owing polyme is oxidized e icien ly. In ac , high edox po en ial o he guaiacyl g oups p e en s hei c oss-coupling un il he concen a ion o he mo e eac i e coni e yl alcohol monome s is su icien ly low and app oaches ze o (B unow e al., 1998). In xylem cells he concen a ion o monolignols app oaches ze o a e p og ammed cell dea h o ligni ying cells. Indeed, in sp uce his is accompanied by an inc ease in 5-5-O-4 s uc u es in he S3 laye o he seconda y cell wall (Kukkola e al., 2003). Ou esul s a e in line wi h he iew ha lignin polyme iza ion is a s ochas ic p ocess whe e he p obabili ies o a ious bond ypes a e de e mined by he concen a ion a ios, esonance s uc u es and oxida ion po en ials o he adicals in ol ed (Adle , 1977; G abbe e al., 1996; Sy jänen and B unow, 1998, 2000; Sasaki e al., 2004, 2008). As he la e wo a e essen ially cons an , he concen a ion a io o polyme o monome adicals eme ges as a p incipal way how lignin s uc u e is de e mined. Oxida i e enzymes di e in hei abili y o accep la ge subs a es. In popla , one o he sec e ed, cell wall-bound pe oxidases showed inc eased polyme -oxidizing ac i i y ha ela ed o y osine esidues ha elay elec ons o he su ace o he enzyme (Sasaki e al., 2008; Shige o e al., 2012), a simila solu ion as in ungal enzymes ha oxida i ely deg ade lignin F on ie s in Plan Science | www. on ie sin.o g 9Oc obe 2016 | Volume 7 | A icle 1523