Identification and partial purification of an oxidase from lignifying xylem of Leyland cypress (X Cupressocyparis leylandii)

 Oxidase activity was exclusively present in lignifying cells of developing xylem of Leyland cypress. The oxidase was enriched in 200 mM CaCl₂ extracts of crude cell walls and seems to be ionically associated with the cell walls. Oxidase activity was selected and concentrated using affinity chromatography on Concanavalin-A Sepharose which suggests that it is a high-mannose type glycoprotein. A subsequent purification step using gel permeation chromatography on Sephadex GF-150 partially separated the oxidase activity from peroxidase activity. An oxidase band of apparent Mr 92 kD capable of oxidising N, N, N′, N′ - tetramethyl phenylene diamine/α-naphthol was identified after non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 92 kD oxidase band was enriched in the oxidase-rich fraction and absent from the peroxidase-rich fraction from the gel permeation step. In addition, the 92 kD oxidase band could be differentiated from peroxidase bands because it was not intensified by the addition of hydrogen peroxide. The partially purified oxidase effectively oxidised and polymerised coniferyl alcohol to form insoluble material that yielded a Fourier transform infra-red spectrum similar to dehydrogenation polymers of coniferyl alcohol. This coniferyl alcohol oxidase appears to be specific to lignifying xylem cells and may participate in lignin deposition but further studies are required to fully define this oxidase and its possible homology with other oxidases identified in the lignifying xylem of different species of trees. Received: 20 May 1997 / Accepted: 7 August 1997     

Chemical composition of Casparian strips isolated from Clivia miniata Reg. roots: evidence for lignin

Endodermal cell walls and xylem vessels were isolated enzymatically from Clivia miniata Reg. roots. Transmission-electron-microscopic investigation of cross-sections of intact C. miniata roots and scanning-electron-microscopic investigation of isolated endodermal cell walls indicated that the root endodermis of C. miniata is essentially in its primary state of development. Isolated Casparian strips and xylem vessels were subjected to two different degradation methods usually applied to prove the existence of lignin, namely, cupric oxide oxidation and thioacidolysis. The reaction products obtained were typical aromatic derivatives of the natural lignin precursors coniferyl and sinapyl alcohols, and, in traces, of p-coumaryl alcohol, indicating the occurrence of lignin in the polymers from both Casparian strips and xylem vessels. The qualitative chemical compositions of the polymers from the two sources were similar, whereas the quantitative compositions were different, indicating that the molecular structure of the lignin polymer in the Casparian strips was different from that in the xylem vessels. Thus, for the first time, direct chemical evidence has been obtained that Casparian strips of C. miniata roots contain lignin as a major cell wall polymer.The author is indebted to Prof. Dr. G. Krohne (Zentrale Abteilung für Elektronenmikroskopie, Universität Würzburg, Germany) and to Prof. Dr. R. Guggenheim (Labor für Rasterelektronenmikroskopie, Universität Basel, Schweiz) for offering the opportunity for transmission-electron-microscopic and low-temperature scanning-electron-microscopic investigations, respectively. Financial support by the Deutsche Forschungsgemeinschaft is gratefully acknowledged.     

Characterization of polyphenols in cell walls of cultured Populus trichocarpa tissues

The amount and composition of cell wall-bound polyphenol (lignin) in cultured Populus trichocarpa tissues which formed numerous xylem elements (xylogenic) or no xylem (non-xylogenic) were compared. Polyphenol accounted for ca 15% of the dry wt of the cell wall and did not differ significantly in amount in xylogenic and non-xylogenic tissues. The syringic acid derivatives, 3,4.5-trimethoxybenzoic acid, was identified as one of the oxidation products of methylated cell walls and was recovered in similar amounts irrespective of xylem formation. In contrast, lignin from xylogenic cultures contained more p-coumaryl alcohol derivatives and less coniferyl alcohol derivatives than lignin from non-xylogenic cultures. In this respect the lignin composition of xylogenic tissues closely resembled that from stems.     

Identification and partial characterization of a coniferyl alcohol oxidase from lignifying xylem of Sitka spruce (Picea sitchensis)

Oxidase activity in the developing xylem of branches of Sitka spruce [Picea sitchensis] (Bong) Carr. was expressed in synchrony with the deposition of lignin. The activity was closely associated with the cell wall but it could be extracted by elution with salt solutions such as 1 M NaCl or CaCl₂. A number of different oxidase isoforms with isoelectric points in the range 8–5 were present in these cell wall extracts. These enzymes displayed a marked preference for the oxidation of coniferyl alcohol and efficiently initiated polymerization of coniferyl alcohol into insoluble, lignin-like polymers. They also had a substrate preference and profile of sensitivity to inhibitors that was dissimilar to those reported for classical catechol oxidase or laccase-type polyphenol oxidases. A novel procedure that combines extraction and affinity chromatography on Concanavalin-A to select high-mannose-type glycoproteins provided oxidase activity at higher purity and yield than previously used methods. A single band of oxidase activity (apparent M_r approx. 84 kDa) which was capable of oxidizing α-naphthol/N,N,N′N′-tetramethyl p-phenylene diamine in the absence of added hydrogen peroxide was detected in these cell wall extracts using non-denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The addition of hydrogen peroxide did not intensify the staining of this band but it confirmed the presence of a true peroxidase band of apparent M_r approx. 40 kDa. The properties of this coniferyl alcohol oxidase are different from those of laccase-type polyphenol oxidases (EC 1.10.3.2) previously implicated in lignin deposition in tree species, and their possible roles in this process are discussed. Received: 9 January 1997 / Accepted: 14 March 1997     

Purification and characterization of a novel laccase from the ascomycete Trichoderma atroviride: Application on bioremediation of phenolic compounds

The extracellular laccase produced by the ascomycete Trichoderma atroviride was purified and characterized and its ability to transform phenolic compounds was determined. The purified laccase had activity towards typical substrates of laccases including 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), dimethoxyphenol (2,6-DMP), syringaldazine and hydroquinone. The enzyme was a monomeric protein with an apparent molecular mass of 80 kDa and an isoelectric point of 3.5. The pH optima for the oxidation of ABTS and 2,6-DMP were 3 and 5, respectively, and the optimum temperature was 50 °C with 2,6-DMP. The laccase was stable at slightly acidic pH (4 and 5). It retained 80% of its activity after 4 h incubation at 40 °C. Under standard assay conditions, K_m values of the enzyme were 2.5 and 1.6 mM towards ABTS and 2,6-DMP, respectively. This enzyme was able to oxidize aromatic compounds present in industrial and agricultural wastewater, as catechol and o-cresol, although the transformation of chlorinated phenols required the presence of ABTS as mediator.     

Laccase activities of Penicillium chrysogenum in relation to lignin degradation

 An extracellular laccase capable of oxidizing ABTS (the diammonium salt of 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid) was detected in ligninolytic cultures of Penicillium chrysogenum. By contrast, no lignin peroxidase, manganese-dependent peroxidase or aryl-alcohol oxidase was detected at any time during culturing. Both ABTS laccase activity and mineralization of dehydrogenative polymerizate of coniferyl alcohol were regulated by the C/N ratio in the medium and partially inhibited in the presence of thioglycolic acid, suggesting that both events are associated. In the presence of several known laccase inducers neither ABTS laccase activity nor mineralization rates were enhanced. However, a new laccase was detected in P. chrysogenum, able to oxidize 2,6-dimethoxyphenol but not involved in lignin mineralization. Studies with the known ligninolytic basidiomycete Trametes villosa suggest that lignin degradation by this fungus also involves the action of laccase. Received: 6 July 1995/Received revision: 28 October 1995/Accepted: 6 November 1995     

Peroxidase and not laccase is the enzyme responsible for cell wall lignification in the secondary thickening of xylem vessels inLupinus

Summary The post-exponential growth phase of lupin (Lupinus albus cv. Multolupa) hypocotyls is characterized by a strong deposition of lignins in the primary and secondary walls of the xylem vessels. Coinciding with this phenomenon, there is a clearly peroxidatic activity in both the primary cell walls and the outer-most layers of the secondary thickening of the xylem vessels, as demonstrated by 3,3-diaminobenzidine cytochemistry. This activity was completely inhibited by KCN and the removal of H₂O₂ and was not due to laccase since this enzyme shows an almost total inability to oxidize 3,3-diaminobenzidine both in the presence and in the absence of H₂O₂. The absence of laccase-like activities in cell walls of vascular cells was supported by the fact that cell wall proteins from vascular cells were only capable of oxidizing 3,3-diaminobenzidine and coniferyl alcohol in the presence of H₂O₂. These results support the idea of an exclusive role of peroxidase (and exclude any role for laccase) in lignin formation in the secondary thickening of xylem vessels inLupinus.     

Characterization of basic <Emphasis Type="Italic">p</Emphasis>-coumaryl and coniferyl alcohol oxidizing peroxidases from a lignin-forming <Emphasis Type="Italic">Picea abies</Emphasis> suspension culture

A Norway spruce (Picea abies) tissue culture line that produces extracellular lignin into the culture medium has been used as a model system to study the enzymes involved in lignin polymerization. We report here the purification of two highly basic culture medium peroxidases, PAPX4 and PAPX5, and isolation of the corresponding cDNAs. Both isoforms had high affinity to monolignols with apparent K_m values in μM range. PAPX4 favoured coniferyl alcohol with a six-fold higher catalytic efficiency (V_max/K_m) and PAPX5 p-coumaryl alcohol with a two-fold higher catalytic efficiency as compared to the other monolignol. Thus coniferyl and p-coumaryl alcohol could be preferentially oxidized by different peroxidase isoforms in this suspension culture, which may reflect a control mechanism for the incorporation of different monolignols into the cell wall. Dehydrogenation polymers produced by the isoforms were structurally similar. All differed from the released suspension culture lignin and milled wood lignin, in accordance with previous observations on the major effects that e.g. cell wall context, rate of monolignol feeding and other proteins have on polymerisation. Amino acid residues shown to be involved in monolignol binding in the lignification-related Arabidopsis ATPA2 peroxidase were nearly identical in PAPX4 and PAPX5. This similarity extended to other peroxidases involved in lignification, suggesting that a preferential structural organization of the substrate access channel for monolignol oxidation might exist in both angiosperms and gymnosperms.     

Oxidation of cinnamyl alcohols and aldehydes by a basic peroxidase from lignifying Zinnia elegans hypocotyls.

The xylem of 26-day old Zinnia elegans hypocotyls synthesizes lignins derived from coniferyl alcohol and sinapyl alcohol with a G/S ratio of 43/57 in the aryl-glycerol-beta-aryl ether core, as revealed by thioacidolysis. Thioacidolysis of Z. elegans lignins also reveals the presence of coniferyl aldehyde end groups linked by beta-0-4 bonds. Both coniferyl and sinapyl alcohols, as well as coniferyl and sinapyl aldehyde, are substrates of a xylem cell wall-located strongly basic peroxidase, which is capable of oxidizing them in the absence and in the presence of hydrogen peroxide. This peroxidase shows a particular affinity for cinnamyl aldehydes with kappa(M) values in the mu(M) range, and some specificity for syringyl-type phenols. The affinity of this strongly basic peroxidase for cinnamyl alcohols and aldehydes is similar to that shown by the preceding enzymes in the lignin biosynthetic pathway (microsomal 5-hydroxylases and cinnamyl alcohol dehydrogenase), which also use cinnamyl alcohols and aldehydes as substrates, indicating that the one-way highway of construction of the lignin macromolecule has no metabolic "potholes" in which the lignin building blocks might accumulate. This fact suggests a high degree of metabolic plasticity for this basic peroxidase, which has been widely conserved during the evolution of vascular plants, making it one of the driving forces in the evolution of plant lignin heterogeneity.     

Decomposition of 14C-labelled lignin and phenols by a Nocardia sp.

A Gram-positive bacterium which was isolated from a Finnish soil and identified as a Nocardia sp., was able to decompose lignin and to assimilate lignin degradation products as a carbon source. It could release ¹⁴CO₂ from ¹⁴C-labelled methoxyl groups, side chains or ring carbons of coniferyl alcohol dehydropolymers (DHP) and from specifically ¹⁴C-labelled lignin of plant material. Furthermore, it could release ¹⁴CO₂ from phenolcarboxylic and cinnamic acids and alcohols labelled in the OCH₃, COOH groups, side chain or aromatic ring carbons.Non-Common Abbreviations Used DHP dehydropolymers of coniferyl alcohol     

Regulation of Peroxidase-Dependent Oxidation of Phenolics by Ascorbic Acid: Different Effects of Ascorbic Acid on the Oxidation of Coniferyl Alcohol by the Apoplastic Soluble and Cell Wall-Bound Peroxidases from Epicotyls of Vigna angularis

Intercellular washing fluid (IWF) and washed cell walls obtainedfrom epicotyls of Vigna angularis catalyzed the oxidation ofconiferyl alcohol in the presence of hydrogen peroxide, indicatingthe presence of both soluble and bound peroxidases in the cellwalls. The products of oxidation of coniferyl alcohol were identicalin both cases. Ascorbic acid inhibited the oxidation of coniferylalcohol. The inhibition was due to the rapid reduction of anoxidized intermediate of coniferyl alcohol by ascorbic acid,with resultant regeneration of coniferyl alcohol. However, theinhibitory effects of ascorbic acid were different in the caseof IWF and cell walls. Ascorbic acid completely inhibited theoxidation of coniferyl alcohol by IWF peroxidase as long asascorbic acid was available, whereas the oxidation of coniferylalcohol by cell wall-bound peroxidase was competitively inhibitedby ascorbic acid. Ascorbic acid was present in cell walls andlignin was formed in cell walls during aging of stem. Basedon these results, a possible function for ascorbic acid in theregulation of oxidation of phenolics in cell walls is discussed. (Received March 19, 1993; Accepted May 24, 1993)     

Characterization of two laccases of loblolly pine (Pinus taeda) expressed in tobacco BY-2 cells

We previously showed that eight laccase genes (Lac 1–Lac 8) are preferentially expressed in differentiating xylem and are associated with lignification in loblolly pine (Pinus taeda) [Sato et al. (2001) J Plant Res 114:147–155]. In this study we generated transgenic tobacco suspension cell cultures that express the pine Lac 1 and Lac 2 proteins, and characterized the abilities of these proteins to oxidize monolignols. Lac 1 and Lac 2 enzymatic activities were detected only in the cell walls of transgenic tobacco cells, and could be extracted with high salt. The optimum pH for laccase activity with coniferyl alcohol as substrate was 5.0 for Lac 1 and between 5.0 and 6.0 for Lac 2. The activities of Lac 1 and Lac 2 increased as the concentration of CuSO₄ in the reaction mixtures increased in the range from 1 to 100 μM. Both enzymes were able to oxidize coniferyl alcohol and to produce dimers of coniferyl alcohol. These results are consistent with the hypothesis that Lac 1 and Lac 2 are involved in lignification in differentiating xylem of loblolly pine.     

The syringaldazine-oxidizing peroxidase PXP 3-4 from poplar xylem: cDNA isolation,characterization and expression

The cell wall polymer lignin is believed to be condensed by specific cell wall-localized oxidoreductases. In many plants species, including poplar, the peroxidase-directed oxidation of the lignin analogue syringaldazine (SYR) has been localized to cells that undergo secondary wall formation, a process that includes lignification. As a first step to analyse the corresponding peroxidases, we have isolated previously two anionic isoenzymes (PXP 3-4 and PXP 5) from poplar xylem (Populus trichocarpa), which use SYR as a substrate. Here, we demonstrate that these enzymes are responsible for the visualized SYR oxidation in the developing xylem. The cDNA that corresponds to PXP 3-4 was isolated and the deduced protein was found closely related to the other SYR-oxidizing peroxidase PXP 5 (ca. 98% of identity). PXP 3-4 was expressed in a baculovirus expression system yielding high levels of active peroxidase (3 mg/l medium). The heterologously produced protein showed characteristics similar to those of the corresponding protein from poplar xylem (enzymatic properties, isoelectric point, and migration in a native gel). PXP 3-4 was expressed in the stem and in the root xylem. The data demonstrate that PXP 3-4 (and/or PXP 5) are present in differentiating xylem, supporting a function in secondary cell wall formation.     

Cloning of sucrase genes from Streptococcus mutans in bacteriophage lambda

Abstract An extracellular peroxidase was purified by chromatofocusing column chromatography from the growth medium of ligninolytic cultures of the white-rot fungus Phanerochaete chrysosporium Burds BKM-1767. The enzyme was electrophoretically pure with an M _r of 45 000–47 000. It contained an easily dissociable heme, and required Mn²⁺ ions for activity. In the presence of hydrogen peroxide and Mn²⁺ it oxidized compounds such as vanillylacetone, 2,6-dimethyloxyphenol, curcumin, syringic acid, guaiacol, syringaldazine, divanillylacetone, and coniferyl alcohol. It did not oxidize veratryl alcohol. In reactions requiring Mn²⁺ and O₂, but not hydrogen peroxide, the enzyme oxidized glutathione, dithiothreitol, and NADPH with production of hydrogen peroxide. The hydrogen peroxide produced could be used as a co-substrate by ligninases such as those that oxidize veratryl alcohol, or by the peroxidase itself to oxidize lignin model compounds.     

Screening for lignin degrading bacteria by means of 14C-labelled lignins

Several Nocardia and Pseudomonas spp., as well as some unidentified bacteria, isolated from lake water containing high loads of waste lignin, were tested for their capacity to release ¹⁴CO₂ from specifically ¹⁴C-labelled dehydropolymer of coniferyl alcohol (DHP) or corn stalk lignins. The bacteria were selected according to their ability to degrade phenolic compounds. However, only some of them could release significant amounts of ¹⁴CO₂ from the labelled lignin. The tested Nocardia spp. were more active than the Pseudomonas spp. and the unidentified bacteria. The most active strains belonged to N. autotrophica. These strains released CO₂ significantly from the methoxyl group and transformed the other carbons from the phenylpropane skeleton of lignin also into CO₂. Other less demethylating strains also released little CO₂ from the other carbons of the lignin molecule. From corn stalk materials which were specifically labelled in the lignin part only small amounts of labelled CO₂ were released.Non-Common-Abbreviation Used DHP dehydropolymers of coniferyl alcohol     

Metabolite Profiling Reveals a Role for Atypical Cinnamyl Alcohol Dehydrogenase CAD1 in the Synthesis of Coniferyl Alcohol in Tobacco Xylem

In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes. Sequences of NtCAD1-1 and NtCAD1-7 were deposited in GenBank under accession numbers AY911854 and AY911855, respectively.     

Oxidase of the white rot fungus Panus tigrinus 8/18

Extracellular oxidase of the white rot fungus Panus tigrinus earlier reported as laccase)contains copper but has no absorption spectrum typical of ‘blue’ oxidases. Thioglycolate and sodium azide inhibit the activity of this enzyme at concentrations 2.5–3 orders lower than those needed for fungal laccases. The oxidase of P. tigrinus oxidizes syringaldazine, coniferyl alcohol, ABTS, syringic acid, diaminobenzidine, guaiacol, catechol and vanillylacetone with different efficiencies. Oxygen consumption and no hydrogen peroxide formation were detected during substrate oxidation by P. tigrinus oxidase. It is proposed that P. tigrinus oxidase is a new ligninolytic enzyme.     

Kinetics of Veratryl Alcohol Oxidation by Lignin Peroxidase and in situ Generated Hydrogen Peroxide in an Electrochemical Reactor

The cathodic reduction of oxygen to hydrogen peroxide, the current efficiency for the production of H₂O₂ and the oxidation of veratryl alcohol with an in situ generated hydrogen peroxide‐lignin peroxidase complex were studied in this paper. The complex was prepared by utilizing a novel preparation technique in an electrochemical reactor. The oxidation of veratryl alcohol (VA; 3,4‐dimethoxybenzyl alcohol) was carried out with or without lignin peroxidase under an electric field. The redox properties of veratryl alcohol on a carbon electrode in the presence of lignin peroxidase have been investigated using cyclic voltammetry. The kinetics of veratryl alcohol oxidation in an electrochemical reactor were compared to the oxidation when hydrogen peroxide was supplied externally. Further, the oxidation of veratryl alcohol by lignin peroxidase was optimized in terms of enzyme dosage, pH, and electrical potential. The novel electroenzymatic method was found to be effective using in situ generated hydrogen peroxide for the oxidation of veratryl alcohol by lignin peroxidase.     

Impact of different levels of cinnamyl alcohol dehydrogenase down-regulation on lignins of transgenic tobacco plants

The effects of cinnamyl alcohol dehydrogenase (CAD, EC.1.1.1.195) down-regulation on lignin profiles of plants were analysed in four selected transgenic lines of tobacco (Nicotiana tabacum L. cv. Samsun) exhibiting different levels of CAD activity (8–56% of the control). A significant decrease in thioacidolysis yields (i.e. yield of β-O-4 linked monomers) and in the ratio of syringyl to guaiacyl monomers (S/G) was observed for three transgenic lines and the most drastic reduction (up to 50%) was correlated with the lowest level of CAD activity. Higher lignin extractability by mild alkali treatment was confirmed, and, in addition to a tenfold increase in C₆-C₁ aldehydes, coniferyl aldehyde was detected by high-performance liquid chromatography in the alkali extracts from the xylem of transgenic plants. In-situ polymerisation of cinnamyl aldehydes in stem sections of untransformed tobacco gave a xylem cell wall coloration strikingly similar to the reddish-brown coloration of the xylem of antisense CAD-down-regulated plants. Overall, these data provide new arguments for the involvement of polymerised cinnamyl aldehydes in the formation of the red-coloured xylem of CAD-down-regulated plants. Received: 24 January 1997 / Accepted: 14 May 1997     

Comparative study of substrates of fungal laccase

Coriolus versicolor, Pycnoporus cinnabarinus and Pycnoporus coccineus were grown under conditions to produce extracellular laccase. Prior to estimating enzyme activity, culture fluids were pretreated with catalase to destroy hydrogen peroxide and hence minimize peroxidase activity which might interfere with laccase determinations. Similar trends in enzyme assay were shown when colour reagents contained either syringaldazine or 3-dimethylaminobenzoic acid plus 3-methyl-2-benzothiazolone hydrasone as laccase substrates. Use of 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as laccase substrate showed a different trend which was attributed to peroxidatic activity of the catalase using hydrogen peroxide generated by fungal oxidases. Peroxidatic activity was not observed with the other substrates.