Role of veratryl alcohol in lignin degradation by Phanerochaete chrysosporium

Several aromatic compounds increased initial lignin degradation rates in cultures of Phanerochaete chrysosporium. This activation was connected to increased H₂O₂ production and glucose oxidation rates. Veratryl alcohol, a natural secondary metabolite of P. chrysosporium, also activated the lignin-degrading system. In the presence of added veratryl alcohol the ligninolytic system appeared 6–8 h earlier than in reference cultures. This effect was only seen when lignin was added after the primary growth was completed because lignin itself also caused earlier appearance of the degradative system. In cultures which received no added lignin or veratryl alcohol the ligninolytic activity only appeared once the alcohol started to accumulate. The degradation patterns of veratryl alcohol and lignin were similar. The activity levels of lignin degradation and glucose oxidation could be regulated by veratryl alcohol concentration. It is suggested that either veratryl alcohol itself or a metabolite derived from it is actually responsible for the low levels of ligninolytic activity in glucose grown cultures.     

Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism,growth, and protein synthesis of Pseudomonas halodurans and Alteromonas luteo-violaceus during sulfate limitation

Sulfate concentration in the growth medium exerted a strong influence on the sulfur content of protein in two marine bacteria, Pseudomonas halodurans and Alteromonasluteo-violaceus, but the distribution of sulfur in major biochemical fractions was not affected. 90% of the total cellular sulfur was contained in low molecular weight organic compounds and protein; inorganic sulfate was not an important component. The sulfur content of isolated protein and total cellular sulfur increased in proportion to the external sulfate concentration for both bacteria, reaching a maximum at about 100–250 M. The growth rate of P. halodurans only was dependent on the sulfate concentration.Sulfur starvation of cells labeled to equilibrium with ³⁵S-sulfate resulted in a rapid decrease in low molecular weight organic S with a concommitant increase in alcohol soluble (P. halodurans) or residue protein (A. luteo-violaceus). Although cell division was prevented, total protein increased in both bacteria, resulting in synthesis of sulfur-deficient protein. This effect was most pronounced in P. halodurans.Addition of ³⁵S-sulfate to sulfur-starved A. luteo-violaceus further demonstrated that sulfur metabolism was restricted primarily to the synthesis and utilization of sulfurcontaining protein precursors. The low molecular weight organic S pool was replenished rapidly, and the pool size per cell reached twice the normal value before cell division resumed. Incorporation into protein was very rapid.Abbreviations L.M.W. low molecular weight - TCA trichloroacetic acid     

>Decolourisation and depolymerisation of solubilised low-rank coal by the white-rot basidiomycete Phanerochaete chrysosporium

Peroxidases secreted by the white-rot basidiomycete Phanerochaete chrysosporium can oxidise a wide range of recalcitrant compounds including lignin and aromatic xenobiotics. Since low-rank coals such as brown coal and lignite retain structural features of the parent lignin, we investigated the possibility that P. chrysosporium is capable of acting on a brown coal, with the production of useful low-molecular-mass compounds. In nitrogen-limiting liquid medium containing 0.03% solubilised Morwell brown coal, P. chrysosporium was found to convert about 85% of the coal after 16 days incubation to a form not recoverable by alkali-washing and acid-precipitation. The modal molecular mass of the residual coal macromolecules was reduced from the initial 65kDa to 32 kDa. Extensive bleaching of the coal coincided with the presence of extracellular lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP), although both LiP and MnP activity were lower in cultures containing coal. These reductions are accounted for by interference with the enzyme assays by solubilised coal and by binding of MnP to precipitated coal. LiP was about eight times more sensitive than MnP to inhibition by solubilised coal. In nitrogen-sufficient medium containing solubilised coal, neither coal modification nor LiP activity were observed, suggesting that LiP is an essential component of the bleaching process.     

Purification and characterization of lignin peroxidases from <Emphasis Type="Italic">Penicillium decumbens</Emphasis> P6

Summary Peroxidases are essential enzymes in biodegradation of lignin and lignite which have been investigated intensively in the white-rot fungi. This is the first report of purification and characterization of lignin peroxidase from Penicillium sp. P6 as lignite degradation fungus. The results indicated that the lignin peroxidase of Penicillium decumbens P6 had physical and chemical properties and a N-terminal amino acid sequence different from the lignin peroxidases of white-rot fungi. The lignin peroxidase was isolated from a liquid culture of P. decumbens P6. This enzyme had a molecular weight of 46.3 KDa in SDS-PAGE and exhibited greater activity, temperature stability and wider pH range than those previously reported. The isolation procedure involved (NH₄)₂SO₄ precipitation, ion-exchange chromatography on DEAE-cellulose and CM-cellulose, gel filtration on Sephadex G-100, and non-denaturing, discontinuous polyacrylamide gel electrophoresis. The K _m and V _max values of this enzyme using veratryl alcohol as substrate were 0.565 mmol L ⁻¹ and 0.088 mmol (mg protein) ⁻¹ min ⁻¹ respectively. The optimum pH of P6 lignin peroxidase was 4.0, and 70.6 of the relative activity was remained at pH 9.0. The optimum temperature of the enzyme was 45 °C.     

Biodegradation of kraft lignin by a newly isolated bacterial strain, <Emphasis Type="Italic">Aneurinibacillus aneurinilyticus</Emphasis> from the sludge of a pulp paper mill

A kraft lignin-degrading bacterium (ITRC S ₇ ) was isolated from sludge of pulp and paper mill and characterized as Aneurinibacillus aneurinilyticus by biochemical tests and 16SrRNA gene sequencing. The bacterium did not utilize kraft lignin (KL) as the sole source of carbon and energy. However, this strain reduced the color (58%) and lignin content (43%) from kraft lignin-mineral salt medium when supplemented with glucose at pH 7.6 and 30°C after 6 days. The degradation on addition of glucose in culture medium is clear evidence of co-metabolism of KL by A. aneurinilyticus. The analysis of lignin degradation products by GC-MS in ethyl acetate extract from an A. aneurinilyticus-inoculated sample revealed the formation of low molecular weight aromatic compounds such as guaiacol, acetoguaiacone, gallic acid and ferulic acid, indicating that the bacterium can oxidize of the sinapylic (G units) and coniferylic (S units) alcohol units which are the basic moieties that build the hardwood lignin structure. The low molecular weight aromatic compounds identified in extracts of the inoculated sample favors the idea of biochemical modification of the KL to a single aromatic unit.     

Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures

Lignin and lignans share monolignols as common precursors and are both potentially involved in plant defence against pathogens. In this study, we investigated the effects of fungal elicitors on lignin and lignan metabolism in flax (Linum usitatissimum) cell suspensions. Cell suspension cultures of flax were treated with elicitor preparations made from mycelium extracts of Botrytis cinerea, Phoma exigua and Fusarium oxysporum F ssp lini. Elicitors induced a rapid stimulation of the monolignol pathway, as confirmed by the increase in PAL (phenylalanine ammonia-lyase, EC 4.1.3.5), CCR (cinnamoyl-CoA reductase EC 1.2.1.44) and CAD (cinnamyl alcohol dehydrogenase EC 1.1.1.195) gene expression and PAL activity. At the same time, CCR activity only increased significantly in F. oxysporum-treated cells 24 h post elicitation. On the other hand, CAD activity measured for coniferyl alcohol formation was transiently decreased but a substrate-specific activation of CAD activity was observed in F. oxysporum-treated cells when using sinapyl alcohol as substrate. The accumulation of monolignol-derived products varied according to the elicitor used. B. cinerea or P. exigua-elicited cell cultures were characterised by a reinforcement of the cell wall by a deposit of 8-O-4′-linked non-condensed lignin structures and phenolic monomers, while at the same time no stimulation of 8-8′-linked lignan or 8-5′-linked phenylcoumaran lignan accumulation was observed. Additionally, elicitation of cell cultures with F. oxysporum extracts even triggered a strong incorporation of monolignols in the non condensed labile ether-linked lignin fraction concomitantly with a decrease in lignan and phenylcoumaran lignan accumulation. Several hypotheses are proposed to explain the putative role of these compounds in the defence response of flax cells against pathogens. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. C. Hano and M. Addi contributed equally to this work.     

Fungal elicitor-mediated responses in pine cell cultures

A tissue culture system has been developed to examine phenylpropanoid metabolism induced in pine tissues by an ectomycorrhizal symbiont. An elicitor preparation from the ectomycorrhizal fungus Thelephora terrestris Fr. induced enhanced phenolic metabolism in suspension cultured cells of Pinus banksiana Lamb., as indicated by tissue lignification and accumulation of specific methanol-extractable compounds in the cells. Induction of lignification was observed as early as 12 h after elicitation. The activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the entry-point enzyme into phenylpropanoid metabolism, also increased within the same time-frame in elicited cells. Significant increases in PAL activity were evident by 6 h after elicitation, and, by 12 h after elicitation, PAL activity in elicited cells was ten times greater than that in the corresponding controls. Lignification of the elicited tissue was also accompanied by an increase in the activity of other enzymes associated with lignin synthesis, including caffeic acid O-methyl transferase (EC 2.1.1.46), hydroxycinnamate:CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-), coniferin glucosidase (EC 3.2.1.21) and peroxidase (EC 1.11.1.7). The increase in total peroxidase activity was associated with a change in the pattern of soluble peroxidase isoforms. The pine cell culture-ectomycorrhizal elicitor system provides a good model for molecular analysis of the process of lignification in an economically important softwood species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4CL hydroxycinnamate:Coenzyme A ligase (EC 6.2.1.12) - CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.-) - COMT S-adenosyl-l-methionine:caffeate O-methyl transferase (EC 2.1.1.46) - HPLC high-pressure liquid chromatography - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TGA thioglycolic acid To whom correspondence should be addressedFinancial assistance for this work was provided by the Natural Sciences and Engineering Research Council of Canada.     

Identification of low molecular weight aromatic compounds by gas chromatography–mass spectrometry (GC–MS) from kraft lignin degradation by three Bacillus sp.

Three bacterial strains identified as Paenibacillus sp., Aneurinibacillus aneurinilyticus and Bacillus sp. have been shown to decolourise kraft lignin in 6 days of incubation. The release of low molecular weight aromatic compounds by these bacterial strains during degradation of kraft lignin was analysed by GC–MS analysis. The total ion chromatograph (TIC) of ethyl acetate extract from kraft lignin sample inoculated by Paenibacillus sp. was similar to control except some minor changes in the chromatographic profile indicating incapability of this bacterium to modify kraft lignin. On the other hand the TIC of ethyl acetate extract from kraft lignin inoculated by A. aneurinilyticus and Bacillus sp. caused formation of several aromatic lignin-related compound that were not present in the extract of control. The compounds identified in extract of the sample degraded by A. aneurinilyticus were guaiacol, acetoguiacone, gallic acid and ferulic acid while t-cinnamic acid, 3,4,5 trimethoxy benzaldehyde, and ferulic acid by Bacillus sp. indicating oxidization of coniferylic (G units) and sinapylic (S units) alcohol of lignin polymer. Differences between the identified compounds from different bacterial treatment were strain-specific. Among the identified aromatic compounds, ferulic acid and 3,4,5-trimethoxy benzaldehyde could be useful to the industry of preservatives, aromas and perfumes.     

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.     

Biodegradation of kraft lignin by a bacterial strain Comamonas sp. B-9 isolated from eroded bamboo slips

Aims: The aim was to obtain evidences for lignin degradation by unicellular bacterium Comamonas sp. B‐9. Methods and Results: Comamonas sp. B‐9 was inoculated into kraft lignin‐mineral salt medium (KL‐MSM) at pH 7·0 and 30°C for 7 days of incubation. The bacterial growth, chemical oxygen demand (COD) reduction, secretion of ligninolytic enzymes and productions of low‐molecular‐weight compounds revealed that Comamonas sp. B‐9 was able to degrade kraft lignin (KL). COD in KL‐MSM reduced by 32% after 7 days of incubation. The maximum activities of manganese peroxidase (MnP) of 2903·2 U l^?1 and laccase (Lac) of 1250 U l^?1 were observed at 4th and 6th day, respectively. The low‐molecular‐weight compounds such as ethanediol, 3, 5‐dimethyl‐benzaldehyde and phenethyl alcohol were formed in the degradation of KL by Comamonas sp. B‐9 based on GC‐MS analysis. Conclusions: This study confirmed that Comamonas sp. B‐9 could utilize KL as a sole carbon source and degrade KL to low‐molecular‐weight compounds. Significance and Impact of the Study: Comamonas sp. B‐9 may be useful in the utilization and bioconversion of lignin and lignin‐derived aromatic compounds in biotechnological applications. Meanwhile, using Comamonas sp. B‐9 in treatment of wastewater in pulp and paper industry is a meaningful work.     

A Phanerochaete chrysosporium mutant defective in lignin degradation as well as several other secondary metabolic functions

A pleiotropic mutant of Phanerochaete chrysosporium 104-2 lacking phenol oxidase and unable to form fruit bodies and a revertant strain 424-2 were isolated after UV mutagenesis. Strains 104-2 and 424-2 had no apparent dysfunction in primary metabolism with glucose as a carbon source. Unlike the wild type strain and strain 424-2, strain 104-2 was unable to evolve ¹⁴CO₂ from ¹⁴C ring, side chain and 3-O-¹⁴C-methoxy labeled lignin. In addition, strain 104-2 was unable to evolve ¹⁴CO₂ from a variety of lignin model compounds including ¹⁴C-4-methoxy labeled veratrylglycerol--guaiacyl (V) ether, -¹⁴C-guaiacylglycerol--guaiacyl ether (VI), as well as 1-(¹⁴C-4-methoxy, 3-methoxyphenyl)1,2 propene (III) and 1-(¹⁴C-4-methoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IV). The addition of peroxidase/H₂O₂ to cultures of strain 104-2 did not alter its capacity to degrade the labeled lignins. A variety of unlabeled lignin model compounds previously shown to be degraded by the wild type organism including -aryl ether dimers and diaryl propane dimers were also not degraded by the mutant 104-2. The revertant strain 424-2 regained the capacity to degrade these compounds. The substrates described are degraded by oxygen requiring system(s) expressed during the secondary phase of growth, suggesting this pleiotropic mutant is possibly defective in the onset of postprimary metabolism. The inability of the mutant to produce the secondary metabolite veratryl alcohol and to elaborate enzymes in the veratryl alcohol biosynthetic pathway supports this hypothesis.Abbreviations GLC gas liquid chromatography - TMSi trimethylsilyl - MS mass spectrometry - LDS lignin degrading system     

Purification,characterization, and coal depolymerizing activity of lignin peroxidase from <Emphasis Type="Italic">Gloeophyllum sepiarium</Emphasis> MTCC-1170

Lignin peroxidase from the liquid culture filtrate of Gloeophyllum sepiarium MTCC-1170 has been purified to homogeneity. The molecular weight of the purified enzyme was 42 kDa as determined by SDS-PAGE. The K _m values were 54 and 76 μM for veratryl alcohol and H₂O₂, respectively. The pH and temperature optima were 2.5 and 25°C, respectively. Depolymerization of coal by the fungal strain has been demonstrated using humic acid as a model of coal. Depolymerization of humic acid by the purified lignin peroxidase has been shown by the decrease in absorbance at 450 nm and increase in absorbance at 360 nm in presence of H₂O₂. Depolymerization of humic acid by the purified enzyme has also been demonstrated by the decrease in the viscosity with time of the reaction solution containing humic acid, H₂O₂, and the purified lignin peroxidase. The influence of NaCl and NaN₃ and inhibitory effects of various metal chelating agents on the lignin peroxidase activity were studied.     

Elicitor-mediated induction of enzymes of lignin biosynthesis and formation of lignin-like material in a cell suspension culture of spruce (Picea abies)

Incubations of photomixotrophic suspension culture cells of spruce (Picea abies) (L.) (Karst) with an autoclaved cell wall preparation of Rhizosphaera kalkhoffii as elicitor led to a rapid increase of the activity of a number of enzymes involved in lignin biosynthesis. l-phenylalanine ammonia-lyase (EC 4.3.1.5) was induced about 10-fold, feruloyl-Coenzyme A reductase (ED 1.2.1.44) 4-fold, cinnamyl alcohol dehydrogenase (NADP⁺) (EC 1.1.1.195) 2-fold and peroxidase (EC 1.11.1.7) about 1.5-fold. The induction of the enzymes, with the exception of the peroxidase, was transient, showing maximal activity within 3 days after elicitation. Extracellular peroxidase activity, determined in the culture medium, rapidly decreased on initiation of elicitation.Concomitant with the increase of activity of the enzymes of lignin synthesis was a rapid clouding of the culture medium. Phloroglucinol-HCl staining revealed the presence of lignin-like material in the medium and also in the cells. The IR-spectrum of this material was identical with the IR-spectrum of authentic spruce lignin.Abbreviations PAL l-phenylalanine ammonia-lyase - FCR feruloyl-Coenzyme A reductase - CAD cinnamyl alcohol dehydrogenase - POD peroxidase     

Modification of Water-Soluble Coal-Derived Products by Dibenzothiophene-Degrading Microorganisms

To study mechanisms by which microorganisms oxidize thiophenic sulfur in coal, we tested bacterial cultures for the ability to degrade dibenzothiophene (DBT), DBT-5-oxide, and DBT-sulfone and to modify water-soluble coal products derived from Illinois no. 6 and Ugljevik coals. In yeast extract medium, the majority of selected isolates degraded DBT and accumulated DBT-5-oxide in culture fluids; all but one of the cultures degraded DBT-5-oxide, and none of them degraded DBT-sulfone. Elemental analysis data indicated that the microbial cultures were able to decrease the amount of sulfur in soluble coal products derived from Illinois no. 6 and Ugljevik coals. However, these data suggested that microbially mediated sulfur removal from soluble Ugljevik coal occurred by nonspecific mechanisms. That is, extensive degradation of the carbon structure was concurrent with the loss of sulfur. This conclusion was supported by X-ray photoelectron spectroscopic data which indicated that the reduced sulfur forms in the soluble Ugljevik coal product was not oxidized by microbial treatment.     

Regulation of anthocyanin and lignin synthesis in Petunia hybrida cell suspensions

In Petunia hybrida cv. Violet 30 cell suspensions the phenylpropanoid pathway can be induced to produce lignin and anthocyanins. Orthovanadate addition leads to lignin accumulation, subculturing the cells using small inoculum sizes (<2 g fresh weight l^-1) gives rise to both anthocyanin and lignin production. Orthovanadate has a negative effect on cell growth. By replacing the medium, one day after orthovanadate addition, by medium without elicitor, we were able to restore growth without disturbing the lignin accumulation. The activity of phenylalanine ammonia-lyase (PAL) increased immediately after orthovanadate addition; this increase stopped upon medium replacement without affecting the lignin production. Reduction of the NAA concentration from 2 mg l^-1 to 0.1 mg l^-1, subsequent to the elicitation by orthovanadate or dilution stress, gave rise to a further increase in the production of lignin and anthocyanins respectively. Decreasing the NAA concentration without a prior elicitation, didn't have any effect on either PAL activity or product formation.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BSA bovine serum albumine - FW fresh weight - NAA naphthaleneacetic acid - PAL phenylalanine ammonia-lyase - PPP phenyl propanoid pathway     

Colonization and degradation of oxidized bituminous and lignite coals by fungi

Summary APenicillium sp. previously shown to grow on lignite coals degraded an air-oxidized bituminous coal (Illinois #6) to a material that was more than 80% soluble in 0.5 N NaOH. Scanning electron microscopy of the oxidized Illinois #6 revealed colonization of the surface by thePenicillium sp., production of conidia, and erosion of the coal surface. The average molecular weight (MW) of Illinois #6 degraded by the fungus and base-solubilized was approximately 1000 Da. The average MW for base-solubilized Illinois #6 that was not exposed to the fungus was 6000 Da, suggesting solubilizing mechanisms other than base catalysis. A spectrophotometric assay to quantify the microbial conversion of biosolubilized coal was developed. Standard curves were constructed based on the absorbance at 450 nm of different quantities of microbe-solubilized coal. An acid precipitation step was necessary to remove medium and/or microbial metabolites from solubilized coal to prevent overestimation of the extent of coal biosolubilization. Furthermore, the absorption spectra for different coal products varied, necessitating construction of standard curves for individual coals.     

Influence of methyl jasmonate on podophyllotoxin and 6-methoxypodophyllotoxin accumulation in Linum album cell suspension cultures

The accumulation of podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (6MPTOX) was enhanced about twofold in the suspension culture of Linum album line 2-5 aH following the addition of methyl jasmonate (MeJas) to the cultivation medium, reaching 7.69±1.45 mg/g dry weight and 1.11±0.09 mg/g dry weight, respectively. There was no increase in 6MPTOX accumulation following the addition of MeJas to suspension cells of L. album line X4SF, whereas PTOX accumulation was enhanced about tenfold to 0.49±0.10 mg/g dry weight. Phenylalanine ammonia-lyase activity increased immediately after the addition of MeJas to a cell suspension culture of line X4SF, reaching a maximum between 4 h and 1 day after elicitation, while cinnamyl alcohol dehydrogenase activity and the lignin content of the cells were not affected.     

Bioalteration of Kraft pine lignin byPhanerochaete chrysosporium

Bioalteration of the organic-soluble ether-insoluble fraction of Kraft pine lignin (KL-O) was studied. Various types of inocula ofPhanerochaeta chrysosporium were compared using a standing mode of cultivation under nitrogen limitation. Pellet inoculated and mycelial cultures required a period of about 10 days to become ligninolytically active. When spores were used as inoculum the bioalteration of lignin commenced earlier but the rate was considerably less. The total decrease in absorbance measured amounted to 61% within 20 days after addition of lignin to active mycelial cultures. This corresponded to a reduction of 50% in Klason lignin. Further reduction was possible only when the free lignin was extracted from the culture, purified and mixed with new active cells. Elemental analysis, Klason lignin content, absorptivity coefficient, molecular weight distribution and the presence of saccharides and proteins for free and cell recovered lignin were compared with KL-O. Microscopic observation showed the formation of new out-growths in the form of short hyphae appearing concurrently with ligninolytic activity.Abbreviations GPC Gel Permeation Chromatography - TLC Thin Layer Chromatography - rpm revolutions per minute - vvm volume per volume per minute     

Activity of ligninase peroxidase from Acinetobacter anitratus and the degradation of Pinus pinaster lignin

The ligninase activity of Acinetobacter anitratus cells and enzyme extracted from that bacterium was measured with a model substrate (benzyl alcohol) and two lignin substrates: Pinus pinaster lignin precipitated from kraft black liquor and treated black liquor. The activity of this intracellular enzyme was confirmed on benzyl alcohol, showing a sigmoid Michaelis curve for constant H₂O₂ concentration. Enzymatic activity on treated black liquor leads to the production of low molecular weight compounds and to partial polymerisation of lignin. The evolution of the molecular weight distribution of the original sample with time was followed by high pressure liquid chromatography.     

Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation

Cell wall-bound peroxidase (EC 1.11.1.7) isoenzymes (P1-P5) from cells of Zinnia elegans L. that were differentiating into tracheary elements were separated and characterized to obtain information about the relationships between these isoenzymes and the biosynthesis of lignin. Fractionation of Zinnia cells by centrifugation in solutions of Percoll revealed that P1, P2, and P5 were present in differentiated tracheary elements. These peroxidase isoenzymes were separated by several column-chromatographic steps. During hydrophobic chromatography on Phenyl Superose, P5 activity was separated into activities P5A and P5B. Enzymatically pure preparations of P1, P3, P5A, and P5B were finally obtained and used for the characterization of each isoenzyme. The optimum pH was 5.5–6.0 for P1, 5.0–7.5 for P3, 5.0 for P5A, and 4.0 for P5B. Each of the isoenzymes oxidized coniferyl alcohol efficiently, whereas p-coumaryl alcohol and sinapyl alcohol were poor substrates for all the isoenzymes. An absolute requirement for Ca²⁺ ions was demonstrated for P3. Based on these results, possible roles of peroxidase isoenzymes in the formation of lignin during the differentiation of tracheary elements are discussed.Abbreviations DAB diaminobenzidine - GTA equal proportions of 3,3-dimethylglutaric acid, tris(hydroxymethyl)aminomethane, and 2-amino-2-methyl-1,3-propanediol - TE tracheary element The authors are very grateful to Professor M. Tanahashi of Gifu University for providing hydroxycinnamyl alcohols. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan to H.F.