Characterization of an extracellular lignin peroxidase of the lignocellulolytic actinomycete Streptomyces viridosporus

Previously we reported production of an extracellular lignin-inducible peroxidase by Streptomyces viridosporus (M. Ramachandra, D.L. Crawford, and A.L. Pometto III, Appl. Environ. Microbiol. 53:2754-2760, 1987). This peroxidase was shown to oxidize 3,4-dihydroxyphenylalanine, 2,4-dichlorophenol, homoprotocatechuic acid, caffeic acid, and N,N,N',N'-tetramethylphenylenediamine and was found in higher than normal levels in strains enhanced for lignocellulose degradation. In the present study, we used a pure extracellular enzyme preparation with high peroxidase isoform P3 activity to oxidize lignin substructure model compounds of both the 1,2-diaryl propane and arylglycerol-beta-aryl ether types and containing C alpha-carbonyl and C alpha-hydroxyl groups. The reactions were monitored by gas chromatography-mass spectrometry and high-pressure liquid chromatography techniques. In the presence, but not the absence, of hydrogen peroxide, the enzyme preparation catalyzed C alpha-C beta bond cleavage in the side chains of the diaryl ethers 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propan-1-one (II) and the diaryl ethane 1-(4-methoxyphenyl)-2-(phenyl)ethan-1-one (III). Rapid hydrogen peroxide consumption was observed when the enzyme preparation was added to either milled corn lignin or lignocellulose. Additional characterizations showed that this enzyme is a heme protein (Soret band, 408 nm) and a major component of the ligninolytic system of S. viridosporus T7A. This is the first report of a lignin peroxidase in a bacterium. We have designated this new lignin peroxidase as ALiP-P3.     

Characterization of an extracellular lignin peroxidase of the lignocellulolytic actinomycete Streptomyces viridosporus.

Previously we reported production of an extracellular lignin-inducible peroxidase by Streptomyces viridosporus (M. Ramachandra, D.L. Crawford, and A.L. Pometto III, Appl. Environ. Microbiol. 53:2754-2760, 1987). This peroxidase was shown to oxidize 3,4-dihydroxyphenylalanine, 2,4-dichlorophenol, homoprotocatechuic acid, caffeic acid, and N,N,N',N'-tetramethylphenylenediamine and was found in higher than normal levels in strains enhanced for lignocellulose degradation. In the present study, we used a pure extracellular enzyme preparation with high peroxidase isoform P3 activity to oxidize lignin substructure model compounds of both the 1,2-diaryl propane and arylglycerol-beta-aryl ether types and containing C alpha-carbonyl and C alpha-hydroxyl groups. The reactions were monitored by gas chromatography-mass spectrometry and high-pressure liquid chromatography techniques. In the presence, but not the absence, of hydrogen peroxide, the enzyme preparation catalyzed C alpha-C beta bond cleavage in the side chains of the diaryl ethers 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propan-1-one (II) and the diaryl ethane 1-(4-methoxyphenyl)-2-(phenyl)ethan-1-one (III). Rapid hydrogen peroxide consumption was observed when the enzyme preparation was added to either milled corn lignin or lignocellulose. Additional characterizations showed that this enzyme is a heme protein (Soret band, 408 nm) and a major component of the ligninolytic system of S. viridosporus T7A. This is the first report of a lignin peroxidase in a bacterium. We have designated this new lignin peroxidase as ALiP-P3.     

Characterization of two lignin peroxidase clones from Phanerochaete chrysosporium

Two cDNA clones encoding lignin peroxidase isozymes from Phanerochaete chrysosporium have been isolated and characterized. One of the clones, lambda ML-4, encodes isozyme H8 as does the previously reported clone lambda ML-1 [Tien, M. and Tu, C.-P.D. Nature 326 (1987) 520-523; 328, 742]. Our data are consistent with lambda ML-1 and lambda ML-4 being allelic variants. The other clone, lambda ML-5, encodes a homologous isozyme. We have also isolated the genomic clone corresponding to lambda ML-4 cDNA. Conserved residues thought to be essential for peroxidase function were identified in the predicted amino acid sequences of both cDNA clones. Northern blot analyses indicate that these isozymes are expressed during secondary metabolism, appearing on day 4 of growth and increasing on days 5 and 6.     

Characterization of a lignin peroxidase gene from the white-rot fungus Trametes versicolor.

A genomic library of the white-rot fungus Trametes versicolor has been constructed and a gene coding for a lignin peroxidase has been isolated and sequenced. The gene, which contains 6 introns, encodes a protein of 346 amino acid residues, preceded by a tentative 26-residue signal peptide. The deduced amino-terminal sequence agrees with the amino-terminal end of a lignin peroxidase isozyme previously isolated from carbon-limited cultures of T versicolor.     

Expression in yeast of secreted lignin peroxidase with improved 2,4-dichlorophenol degradability by DNA shuffling

Lignin peroxidase (LiP) from Phanerochaete chrysosporium was shown to mineralize a variety of recalcitrant aromatic compounds and oxidize a number of polycyclic aromatic and phenolic compounds. The major problem of the wild type LiP is that it can be inactivated by excess H(2)O(2) and high concentrations of aromatic compounds. We applied a directed evolution technique coupled with a rapid colorimetric screening method to obtain mutant genes with improved H(2)O(2) stability and polychlorinated phenol degradability, and they were successfully expressed as the secretive LiPs in recombinant Saccharomyces cerevisiae. The resulting variants showed approximately 1.6-fold improved 2,4-dichlorophenol (2,4-DCP) degradation activity and stability against H(2)O(2) compared with the parent strain. The kinetic properties of the variants toward 2,4-DCP and H(2)O(2) were also increased compared with the wild type for all three mutants studied. Amino acid sequence analysis indicated that the greatest number of amino acid substitutions was located near the surface or Ca(2+) binding sites of the enzyme.     

Oxidation of a tetrameric nonphenolic lignin model compound by lignin peroxidase

The present study maps the active site of lignin peroxidase in respect to substrate size using either fungal or recombinant wild type, as well as mutated, recombinant lignin peroxidases. A nonphenolic tetrameric lignin model was synthesized that contains beta-O-4 linkages. The fungal and recombinant wild type lignin peroxidase both oxidized the tetrameric model forming four products. The four products were identified by mass spectral analyses and compared with synthetic standards. They were identified as tetrameric, trimeric, dimeric, and monomeric carbonyl compounds. All four of these products were also formed from single turnover experiments. This indicates that lignin peroxidase is able to attack any of the C(alpha)-C(beta) linkages in the tetrameric compound and that the substrate-binding site is well exposed. Mutation of the recombinant lignin peroxidase (isozyme H8) in the heme access channel, which is relatively restricted and was previously proposed to be the veratryl alcohol-binding site (E146S), had little effect on the oxidation of the tetramer. In contrast, mutation of a Trp residue (W171S) in the alternate proposed substrate-binding site completely inhibited the oxidation of the tetrameric model. These results are consistent with lignin peroxidase having an exposed active site capable of directly interacting with the lignin polymer without the advent of low molecular weight mediators.     

Characterization of a new lignin peroxidase gene (GLG6) from Phanerochaete chrysosporium

Sequence analysis of a new lignin peroxidase (LIP) gene, GLG6, from P. chrysosporium showed that it encodes a mature LIP protein with a predicted Mr of 36,454. A 28 aa signal peptide precedes the mature protein. The coding region of GLG6 is interrupted by nine introns ranging in size from 50-57 bp. GLG6 encoded-LIP has 72%, 88% and 82% homology, respectively, to the LIP isozymes H2, H3, and H10. Comparison of the N-terminal sequence of GLG6-encoded LIP to that of the LIP proteins H2, H8, H10 also showed that the former is relatively less related to the H2 protein than it is to the H8 and H10 proteins. Expression of GLG6, similar to the other LIP genes, occurs only during secondary metabolism.     

Characterization of catechol derivative removal by lignin peroxidase in aqueous mixture

The use of lignin peroxidase (LIP) as an alternative method for the removal of four catechols (1,2-dihydroxybenzene): catechol (CAT), 4-chlorocatechol (4-CC), 4,5-dichlorocatechol (4,5-DCC) and 4-methylcatechol (4-MC) typical pollutants in wastewater derived from oil and paper industries, was evaluated. The removal of 2 mM catecholic substrates by 1 μM LIP after 1 h was in the following order: 4,5-DCC (95%) > 4-CC(90%) > CAT(55%) > 4-MC(43%). Except for 4-MC, all reactions were accompanied by the formation of insoluble products, leading to LIP precipitation. LIP was exposed to soluble or insoluble product-dependent inactivation, depending on the substrates tested, immediately at the start of the reactions. Despite immediate enzyme inactivation, removal of catecholic substrates continued, resulting in oligomeric product formation. Major oxidation products analyzed were compatible with dimeric, trimeric and tetrameric structures. Ether linkages and a benzoquinone structure were detected in two purified oligochlorocatechols.Catechol derivatives removal initiated by LIP, seems to be different for each catecholic substrate in terms of substrate consumption and transformation, and of enzyme activity.     

Phenolics, lignin content and peroxidase activity in Picea omorika lines

We analyzed low molecular mass phenolics, lignin content and both soluble and cell wall bound peroxidase activity in the needles of three Picea omorika (Pancic) Purkyne lines grown in the generative seed orchard. The highest values of the total phenol content as well as of catechine, caffeic acid, coniferyl alcohol, isoferulic acid and lignin concentration were detected in B5 line (“semidichotomy” line). The soluble guaiacol peroxidase activity was the highest in A3 line (line “borealis”). The highest activity of cell wall bound peroxidases was measured in B5 line, and it was in correlation with lignin content.     

Description of a versatile peroxidase involved in the natural degradation of lignin that has both manganese peroxidase and lignin peroxidase substrate interaction sites

Two major peroxidases are secreted by the fungus Pleurotus eryngii in lignocellulose cultures. One is similar to Phanerochaete chrysosporium manganese-dependent peroxidase. The second protein (PS1), although catalyzing the oxidation of Mn2+ to Mn3+ by H2O2, differs from the above enzymes by its manganese-independent activity enabling it to oxidize substituted phenols and synthetic dyes, as well as the lignin peroxidase (LiP) substrate veratryl alcohol. This is by a mechanism similar to that reported for LiP, as evidenced by p-dimethoxybenzene oxidation yielding benzoquinone. The apparent kinetic constants showed high activity on Mn2+, but methoxyhydroquinone was the natural substrate with the highest enzyme affinity (this and other phenolic substrates are not efficiently oxidized by the P. chrysosporium peroxidases). A three-dimensional model was built using crystal models from four fungal peroxidase as templates. The model suggests high structural affinity of this versatile peroxidase with LiP but shows a putative Mn2+ binding site near the internal heme propionate, involving Glu36, Glu40, and Asp181. A specific substrate interaction site for Mn2+ is supported by kinetic data showing noncompetitive inhibition with other peroxidase substrates. Moreover, residues reported as involved in LiP interaction with veratryl alcohol and other aromatic substrates are present in peroxidase PS1 such as His82 at the heme-channel opening, which is remarkably similar to that of P. chrysosporium LiP, and Trp170 at the protein surface. These residues could be involved in two different hypothetical long range electron transfer pathways from substrate (His82-Ala83-Asn84-His47-heme and Trp170-Leu171-heme) similar to those postulated for LiP.     

粉刺侧孢霉产木质素过氧化物酶酶活性与pH值的关系

研究了粉刺侧孢霉 (Phanerochaetechrysosporium)在以微晶纤维素为C源时 ,木素过氧化酶与 pH值的关系 .试验采用 37～ 39°C下液体振荡培养 ,藜芦醇为产酶诱导剂 ,测试不同起始 pH值所得木素过氧化物酶活性 .结果表明 ,起始 pH值 6.0所得酶活最高达 0 .1 2 6U·ml- 1 ,说明以微晶纤维素代替葡萄糖为C源 ,其最佳 pH值相对较高 .菌丝球直径与酶活呈正相关 .     

Effect of copper on peroxidase activity and lignin content in Raphanus sativus

Copper (Cu²⁺) significantly inhibits the growth of radish (Raphanus sativus) seedlings at the concentration of 1 μM. As far as the relationship between the growth of radish roots and peroxidase (POD) activity is concerned, the reduction of radish roots is correlated with the induction of cationic and anionic PODs. The data show that the increase of cationic PODs (pI 8.6 and pI 9.3) and anionic PODs (pI 5.1 and pI 3.5) activities was correlated with the rise in lignin content in Cu-treated tissues. In our investigation, among the radish root PODs, the cationic pI 8.6 POD isozyme displayed a high affinity (K_m of 57.9 μM) for syringaldazine and the similar value of catalytic efficiency jointly with the anionic pI 5.1 POD, 0.14 and 0.12 μM^–1 s^–1, respectively. The results suggest that the increase of cationic POD (pI 8.6) induced by Cu treatment can be a good candidate for lignification in radish roots.     

Substrate specificity of lignin peroxidase and a S168W variant of manganese peroxidase

Lignin peroxidase (LiP) and manganese peroxidase (MnP) are structurally similar heme-containing enzymes secreted by white-rot fungi. Unlike MnP, which is only specific for Mn(2+), LiP has broad substrate specificity, but it is not known if this versatility is due to multiple substrate-binding sites. We report here that a S168W variant of MnP from Phanerochaete chrysosporium not only retained full Mn(2+) oxidase activity, but also, unlike native or recombinant MnP, oxidized a multitude of LiP substrates, including small molecule and polymeric substrates. The kinetics of oxidation of most nonpolymeric substrates by the MnP variant and LiP were similar. The stoichiometries for veratryl alcohol oxidation by these two enzymes were identical. Some readily oxidizable substrates, such as guaiacol and ferrocyanide, were oxidized by MnP S168W and LiP both specifically and nonspecifically while recombinant MnP oxidized these substrates only nonspecifically. The functional similarities between this MnP variant and LiP provide evidence for the broad substrate specificity of a single oxidation site near the surface tryptophan.     

Genetic factors influencing lignin peroxidase activity in Phanerochaete chrysosporium ME446

Haploid recombinant progeny of Phanerochaete chrysosporium ME446, genome compositions of which had been defined by RFLP-mapping, vary in their idiophasic behaviour. This allowed us to formulate a model of the sequence of idiophasic activities. One component of this variation, the amount of lignin peroxidase activity, is independent of the allele distributions of the lignin peroxidase gene clusters, but correlates with the allele distribution of another locus. This locus appears to control the spread of the lignin peroxidase-active state within the idiophasic mycelial mat and may be the mating-type locus. The successful determination of linkage relied on analysis of chromosome intervals rather than linkage to single markers; this approach should be generally useful for analysing quantitative characters by RFLP mapping.     

Impact of pellet size on growth and lignin peroxidase activity of Phanerochaete chrysosporium

We investigated the influence of pellet size on the growth and lignin peroxidase (LiP) productivity of Phanerochaete chrysosporium. Different pellet sizes were obtained by varying the vessel diameter under constant shaking conditions. Under these varying conditions the pellet size was in the range of 2–18 mm, while the number of pellets in a single vessel varied from around 1,200 in the Erlenmeyer flask to around 6 in the narrowest vessel. A correlation between the final pellet size and the shear rate was obtained, demonstrating that the pellet size is mainly affected by hydrodynamics. The growth of large pellets was described by a cubic growth model. Despite different pellet sizes, LiP activity appeared in all vessels, but the onset of LiP activity showed a delay based upon the pellet size, while maximal LiP activities varied by only 15%, being around 850 U/l.     

A critical study of lignin peroxidase activity assay by veratryl alcohol oxidation

Summary The effects of various parameters on Phanerochaete chrysosporium lignin peroxidase activity as obtained in ligninase assay based on the oxidation of veratryl alcohol were investigated. Marked differences in the ligninase activity were observed when the temperature and pH were varied within the ranges of 23 to 37°C and 2.5 to 4.0, respectively, reported to have been used by various research groups. Further, both veratryl alcohol, and hydrogen peroxide concentration had a significant effect on ligninase activity.     

Molecular analysis of a Bjerkandera adusta lignin peroxidase gene

Summary A cDNA clone, LPO-1, encoding a major lignin peroxidase from the basidiomycete Bjerkandera adusta was isolated and characterized. The nucleotide sequence of LPO-1 predicts a mature protein consisting of 349 amino acids with a molecular weight of 37,225 preceded by a signal peptide of 23 amino acid residues. We have also cloned and sequenced the gene encoding lignin peroxidase from B. adusta. Comparison of these sequences reveals a lignin peroxidase gene structure consisting of 1,116 bp of protein-encoding DNA that is interrupted by four intervening sequences. The putative eukaryotic regulatory sequence, a TATA box, is present at position — 75 relative to the translational initiation codon. Amino acid sequence homology between the coding regions of LPO-1 and of the lignin peroxidase cDNA clone ML-1 from Phanerochaete chrysosporium is 61%. Offprint requests to: M. Kuwahara     

Characterization of a class alpha glutathione-S-transferase with glutathione peroxidase activity in human liver microsomes

A 25.5kDa class alpha glutathione S-transferase (GST) designated as microsomal Ya-GST or M-GSTA has been purified to electrophoretic homogeneity from human liver microsomes. Limited proteolysis, gel filtration chromatography followed by EDTA, and alkaline Na(2)CO(3) treatments of microsomes indicate that the M-GSTA is intrinsic to the microsomes. Western immunoblot analysis revealed that human liver M-GSTA and the previously reported 17-kDa microsomal GST (FEBS Lett. 315 (1993) 77) did not have immunological cross reactivity. The enzyme showed conjugation activity towards substrates like 1-chloro-2,4-nitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 4-hydroxy-2-nonenal (4-HNE), a genotoxic alpha,beta-unsaturated aldehyde product of lipid peroxidation. In addition, the M-GSTA exhibited significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides as well as phosphatidylcholine hydroperoxide, but not with H(2)O(2). C-terminal amino acid sequence analysis revealed a high homology with the human liver cytosolic GST-A1 and A3 isozymes. Western immunoblot analyses of the microsomes prepared from human hepatoblastoma (HepG2) showed that the expression of this M-GSTA was induced upon treatment with such prooxidants as H(2)O(2), suggesting that it may play an important role in the protection of cellular membranes from peroxidative damage.