Studies on hypersaline-tolerant white-rot fungi III: biobleaching of unbleached kraft pulp by hypersaline-tolerant manganese peroxidase from a marine white rot isolate, <Emphasis Type="Italic">Phlebia </Emphasis>sp. MG-60

 A marine white rot isolate, Phlebia sp. MG-60, secreted lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase under different sea salt incubation conditions. Its MnP production was strongly enhanced by adding 3% sea salts, and the MnP showed high tolerance to sea salts and NaCl. The crude enzyme secreted at 3% sea salt concentration by Phlebia sp. MG-60, in which the main component was MnP (cMnP), was then used to bleach unbleached hardwood kraft pulp (UKP) in vitro. The pulp was brightened 11 points by 4 U of cMnP, and the kappa number was decreased 6 points when only 0.5 mM H₂O₂ was added continuously. When 0.5 mM H₂O₂ (1.22 mg H₂O₂ /g pulp) was added at the initial bleaching, the pulp brightness increased 6 points with a dosage of 4 U of cMnP. When crude MnPs were employed to bleach UKP with organic-free model white-water instead of the Milli Q water usually used, the pulp was brightened 10 and 13 points by 4 and 20 U of cMnP, respectively, and 5 and 6 points by 4 and 20 U of MnP, respectively, of Phanerochaete chrysosporium. Received: September 28, 2001 / Accepted: March 15, 2002 Correspondence to:R. Kondo     

Genomic gene encoding manganese peroxidase from a white-rot fungus Phanerochaete crassa WD1694

The gene encoding manganese peroxidase of a white-rot fungus Phanerochaete crassa WD1694 was cloned and sequenced. Four genomic clones were sequenced in which 3 clones were existed as alleles. The analysis of intron–exon structures divided the 4 clones into three subfamilies that corresponded to mnp2 and mnp3 of Phanerochaete chrysosporium, and a new subfamily possessing only five introns. The purified P. crassa WD1694 MnP consisted of 4 isozymes with same molecular weight, same N-terminal sequence, and different pI. N-terminal sequence of deduced protein of P. crassa mnpB3 gene was identical to those of 4 MnP isozymes; however, the other 3 mnp genes had different N-terminal sequence. The molecular weight of encoded mature protein of mnpB3 gene and purified MnP had a gap that could be difference between MnP proteins encoded by single gene. The results suggested that 4 MnP isozymes of P. crassa WD1694 arose from single gene.     

Polyethylene degradation by lignin-degrading fungi and manganese peroxidase

Degradation of high-molecular-weight polyethylene membrane by lignin-degrading fungi, IZU-154, Phanerochaete chrysosporium, and Trametes versicolor, was investigated under various nutritional conditions. IZU-154 showed the most significant polyethylene degradation among the three lignin-degrading fungi under nitrogen- or carbon-limited culture conditions. Furthermore, for T. versicolor and P. chrysosporium, the addition of Mn(II) into nitrogen- or carbon-limited culture medium enhanced polyethylene degradation. These results suggest that polyethylene degradation is related to ligninolytic activity of lignin-degrading fungi. Treatment of polyethylene membrane with partially purified manganese peroxidase (MnP) caused significant degradation in the presence of Tween 80, Mn(II), and Mn(III) chelator. This result demonstrates that MnP is the key enzyme in polyethylene degradation by lignin-degrading fungi.This study was presented in part at the 9th International Symposium on Wood and Pulping Chemistry, Montreal, Canada, June 9–12, 1997     

Biobleaching of unbleached and oxygen-bleached hardwood kraft pulp by culture filtrate containing manganese peroxidase and lignin peroxidase fromPhanerochaete chrysosporium

Unbleached and oxygen-bleached hardwood kraft pulp (UKP and OKP), respectively, were bleached with a culture filtrate containing manganese peroxidase (MnP) and lignin peroxidase (LiP) fromPhanerochaete chrysosporium Burds. The brightness increases of UKP upon biobleaching with the culture filtrate with and without MnSO₄ were the same. The brightness increase of OKP with MnSO₄ decreased to about half that seen without MnSO₄. Changes in the brightness of UKP and OKP by treatment with the culture filtrate were determined. The brightness increased sharply by about eight points during the first 3h. The 3-h treatment was repeated seven times. The brightness increased linearly with the bleaching of UKP. On bleaching of OKP, the brightness increased slowly and stopped at about 78%.Part of this report was presented at the 62nd Pulp and Paper Research Conference of the Japan Tappi, Tokyo, June 1995     

A comparison between decay patterns of the white‐rot fungus Pleurotus ostreatus in chestnut–leaved oak (Quercus castaneifolia) shows predominantly simultaneous attack both in vivo and in vitro

In this research, we examined decay patterns occurring in Quercus castaneifolia wood under natural conditions compared with controlled decay in vivo. Pleurotus ostreatus‐infected oak wood was obtained from the Sari forests in the north of Iran. The species causing decay was verified as P. ostreatus using rDNA‐ITS sequencing of pure cultures from infected sapwood. In addition to P. ostreatus, two wood‐inhabiting Ascomycota, Trichoderma harzianum and T. lixii, were present. Mass loss in oak sapwood samples exposed to P. ostreatus for 60 days was around 10 per cent. Samples were prepared from both naturally decayed wood and wood decayed under controlled conditions and examined using microscopy. P. ostreatus was found to produce a simultaneous white‐rot decay pattern in both conditions.     

Immunolocalization of an anionic peroxidase in differentiating poplar xylem

Peroxidases are the major candidate enzymes involved in dehydrogenative polymerization of monolignols. Peroxidases have the signal sequence at their N-terminus and this suggests that they are transported to extracellular spaces or developing cell walls. In this study, we focused on an anionic peroxidase isozyme encoded by prxA3a, which seems to be related to lignification. To investigate the localization of peroxidase in differentiating xylem cells of poplar (Populus sieboldii × Populus grandidentata), anti-PRX3 antibody was raised against the anionic peroxidase. Western blotting and peroxidase activity inhibition assay showed specificity of the antibody. Labeling by anti-PRX3 antibody was localized in vessels and fibers during the secondary wall formation and was observed along the plasma membrane beside the microtubules. The labeling was not seen in the cell wall, where localization of peroxidases was expected during lignification. The peroxidase isozyme, which is suggested to be involved in monolignol polymerization, is localized on the plasma membrane and its localization might be regulated by microtubules.     

Cladistic relationships among the Pleurotus ostreatus complex, the Pleurotus pulmonarius complex, and Pleurotus eryngii based on the mitochondrial small subunit ribosomal DNA sequence analysis

The cladistic analysis of the V4 domain sequences, performed by UPGMA, the neighbor-joining, and parsimony methods, revealed that the 19 Pleurotus strains tested in this study evolved along three lineages, each corresponding to a separate biological species: the Pleurotus ostreatus complex, the Pleurotus pulmonarius complex, and Pleurotus eryngii. Moreover, the cladistic positions of the 3 biological species show that the P. ostreatus complex and P. eryngii were derived from a common ancestor at a later stage of evolution, and that the common ancestor had diverged from the lineage of the P. pulmonarius complex during an earlier evolutionary event. The sequences of the 5 portion of the mt SSU rDNA among the strains of the P. ostreatus complex had 99.2%–99.6% homology. All test strains in the P. pulmonarius complex had completely identical sequences. The homology of the strain sequences between the P. ostreatus complex and the P. pulmonarius complex ranged from 96.0% to 96.3%. The sequence of the strain of P. eryngii showed 97.8%–98.3% and 96.5% homologies with those of the strains in the P. ostreatus and the P. pulmonarius complexes, respectively.     

杏鲍菇和乳白耙齿菌锰过氧化物酶活性规律的研究

采用LNAS培养基在4种不同培养液中,对白腐菌杏鲍菇和乳白耙齿菌在28℃下进行静止培养,得到了不同时间内的培养液,用紫外-可见分光光度计检测了在470 nm处对于2,6-DMP的氧化作用后光密度值的变化情况,作为锰过氧化物酶（MnP）产生和活性大小的依据,从而获得了杏鲍菇和乳白耙齿菌锰过氧化物酶与培养基的成分和酶作用底物的关系。结果表明,杏鲍菇和乳白耙齿菌均可产生MnP,对比4种成分不同的培养基酶活力测定结果,在培养液内添加酶的底物木屑和2,6-DMP后可提高MnP的分泌量。以前的研究认为：Mn2＋是MnP产生的必要因子。而本试验研究结果表明,Mn2＋并不是杏鲍菇和乳白耙齿菌产生MnP所必需的。论文为进一步利用这两种真菌产木质素降解酶,以及为进一步深入研究这两种真菌MnP对木质素及其它异生物质降解的作用机理提供基础的酶学研究。     

Isolation and characterization of a novel anionic peroxidase cDNA found in poplar (Populus nigra) suspension cultured cells

A cDNA clone fromPopulus nigra L. var.italica Koehne, denoted PCY2-6, coding for an anionic peroxidase has been isolated, cloned, and characterized. PCY2-6 is 1160 bp long; and its deduced product, PnC26, contains 343 amino acid residues. The mature protein has a calculated isoelectric point of 4.09. The protein contains two motifs typical of peroxidase and 10 potentialN-glycosylation sites. PnC26 is therefore classified as an anionic peroxidase. The mRNA of the PCY2-6 gene family was detected in immature and mature leaves and in two parts of current-year stems: the shoot tip and the older stem. The mRNA of PCY2-6 gene family was found to localize in the phloem and cortex of the current-year stems. We therefore conclude that expression of the PCY2-6 gene family is related to bark development.     

Karyotype analysis of interspecific fusants of basidiomycetes by pulsed-field gel electrophoresis

The purpose of this research was to analyze the karyotype of the interspecific fusants of twoPleurotus species. Auxotrophic mutants derived from the cultivated strain ofP. ostreatus andP. cornucopiae were used. Protoplasts were fused electrically, and the fusants were selected under auxotrophic complementation. Esterase isozyme analysis showed that several fusants had isozyme bands originating from both parental strains, and others had unilateral isozyme bands. The fusant that had expressed isozyme bands of both parental strains showed chromosomal DNA bands of both of the parental strains in pulsedfield gel electrophoresis analysis. Despite the above results, the chromosomal composition of the fusants obtained by the pulsed-field gel electrophoresis did not exhibit all of the bands of both fusion parents.     

A histological investigation of Oriental beech wood decayed by Pleurotus ostreatus and Trametes versicolor

Chemical, light and electron microscopic studies were carried out on wood of Oriental beech (Fagus orientalis Lipsky) decayed by the white‐rot fungi Pleurotus ostreatus and Trametes versicolor for 30, 60 and 120 days according to the modified European standard EN 113. Mass loss as well as lignin, cellulose and carbohydrate content were determined before and after fungal attack. There were no significant differences of wood mass loss and chemical composition between both fungi at the end of incubation. After each incubation period, small specimens were stained for microscopic studies. The micromorphology of fungal cell wall degradation was rather similar for both fungi. Both decreased the cell wall thickness to the same extent. The accumulation of hyphae as well as the rupture of cell walls was also similar. The occurrence of hyphae, cavities in the pits and vessel walls followed nearly the same patterns. The parenchyma cells were completely destroyed. Altogether, both fungi produced a simultaneous white rot in Oriental beech wood.     

Biodegradation of Hevea brasiliensis wood by Rigidoporus lignosus and Phellinus noxius1

In vitro wood slats degradation assays reveal that both the white root rot fungus R. lignosus and the brown root rot fungus P. noxius cause a white rot of wood. In vivo (infected tap roots) they cause the same type of decay. Nevertheless lignin determination show the rubber-tree ability to react against the parasite aggression by increased lignification of tissues.     

Comparative study on the durability of heat-treated White Birch (Betula papyrifera) subjected to the attack of brown and white rot fungi

Abstract

The effect of heat treatment on decay resistance of white birch was evaluated for different incubation periods ranging from 2 to 12 weeks using three species of brown rot and one species of white rot fungus. The results of weight loss tests showed that the white rot fungus, Trametes versicolor, effectively degraded the untreated wood (73.5%). While the degradation of untreated wood by brown rot fungi species, Gloephyllum trabeum (11.6%) and Conifora puteana (6.2%), was considerably less compared to T. versicolor, the third brown rot fungi studied, Poria placenta, caused an appreciable degradation of the same species (52.4%). The results clearly showed that the heat treatment reduced the effect of fungi attack on white birch. Increasing the heat treatment temperature from 195 to 215°C resulted in reduction of weight loss, consequently, reduction in fungal attack. As an example, the weight loss reductions due to T. versicolor, P. placenta, G. trabeum and C. puteana attack was 62.2%, 71.3%, 89.6% and 100%, respectively, compared to the weight loss of untreated wood when it is heat treated at 215°C. Thus, these results confirmed that the heat treatment increased the biological resistance of white birch.     

Introduction of mitochondrial DNA from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> into <Emphasis Type="Italic">Pleurotus pulmonarius</Emphasis> by interspecific protoplast fusion

Interspecific protoplast fusion between two monokaryotic strains (a methionine auxotrophic and chloramphenicol-resistant Pleurotus ostreatus strain and a wild-type strain of Pleurotus pulmonarius) was carried out to introduce mitochondrial DNA (mtDNA) from P. ostreatus into P. pulmonarius. Because mycelial colonies regenerated on minimum medium containing chloramphenicol only after the treatment of protoplast electrofusion, the regenerants were regarded as protoplast fusants. The fusants isolated from regenerated colonies were uninucleate, and their resistance of chloramphenicol seemed to be a stable trait. The fusants mated with P. pulmonarius but not with P. ostreatus, and showed almost identical random amplified polymorphic DNA (RAPD) patterns to that of the parental P. pulmonarius monokaryon. The sizes of mitochondrial genomes were estimated to be 81.5 kb for P. ostreatus monokaryon, 54.9 kb for P. pulmonarius monokaryon, and 84.5 or 86.0 kb for the fusants. BamHI and EcoRI digest patterns of the fusant mtDNAs were almost the same as the parental P. ostreatus monokaryon. From the above results, the fusants seemed to carry nuclei derived from the monokaryon of P. pulmonarius and mtDNA including the chloramphenicol-resistance gene from the P. ostreatus monokaryon, suggesting that the P. ostreatus mtDNA had been introduced into P. pulmonarius cells by interspecific protoplast fusion. Contribution No. 383 of the Tottori Mycological Institute     

Molecular identification of sex in <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> L. using isozyme and RAPD markers

In many dioecious plants, gender affects economic value, breeding schemes and opportunities for commercial harvests. Hippophae rhamnoides L. is a dioecious plant species in which female genotypes are commercially preferred over male genotypes. Its berries have rich medicinal, nutritional and pharmaceutical properties because of their large amounts of vitamins, essential oils, proteins, fatty acids, free amino acids and flavanoids. Primary limitation for breeding H. rhamnoides L. is its dioecious nature, since gender cannot be identified by traditional methods. Therefore, some reliable and quick methods need to be developed. This communication deals with the development of isozyme and RAPD markers for early sex identification in this dioecious tree. The isozyme analysis was conducted with four enzyme systems, viz. peroxidase, esterase, malate dehydrogenase and catalase. The peroxidase enzyme system produced a female specific sex marker, which successfully differentiated between the staminate and pistillate genotypes of H. rhamnoides L. Thirty five random decamer primers were used in our study and one male sex linked marker was identified. OPD-20 (5′-ACTTCGCCAC-3′) displayed a band at 911 bp that expressed polymorphism between male and female genotypes. The staminate and pistillate genotypes could be distinguished using RAPD marker OPD-20911. These results revealed the immense potential of peroxidase isozyme patterns and RAPD as genetic markers for sex identification in H. rhamnoides L.     

Structural changes of residual lignin in softwood kraft pulp treated with manganese peroxidase

Structural changes of residual lignin in unbleached softwood kraft pulp (SWKP) during manganese peroxidase (MnP) treatment were investigated to obtain some understanding of the biobleaching action of SWKP with MnP treatment. Alkaline-extracted lignin from darkened SWKP by MnP showed more intense color and contained moreo-quinone than that from control SWKP. However, no difference in the conjugated-carbonyl was observed between the lignins from MnP-treated and control SWKP. The nitrobenzene oxidation analysis revealed that oxidative condensation of non-condensed lignin in SWKP occurs during an early stage of MnP treatment. These observations were supported by the model experiment in which the lignin prepared from control SWKP was subjected to MnP treatments three times, and the changes of color and functional groups in the lignin were determined after each treatment. These results suggested that an increase ino-quinone and the condensation reaction of non-condensed lignin in SWKP are responsible for the characteristic darkening of SWKP during MnP treatment. It was also ascertained that darkened lignin was degraded and brightened by repeated MnP treatments.     

Mutagenesis, heterogeneous gene expression, and purification and amino acid substitution analyses of plant peroxidase, PrxA3a

We introduced mutations into prxA3a, a peroxidase gene of hybrid aspen, Populus kitakamiensis, to substitute the amino acid residues at the surface of the protein, and analyzed substrate specificities. PrxA3a and mutated enzymes heterogeneously gene expressed in Saccharomyces cerevisiae were purified by Ni affinity chromatography, hydrolysis of sugar chain (Endoglycosidase H_f) and gel filtration. The substrate specificities were altered by substituted amino acid residues. PrxA3a F77Y A165W acquired the substrate specificity to m-chlorophenol. PrxA3a F77Y and PrxA3a F77YA165W could polymerize sinapyl alcohol. In addition, PrxA3a A165W, F77Y, and F77YA165W improved cytochrome c oxidizing activity. These substituted amino acid residues should function as a catalytic site outside of the heme pocket.     

Environment-friendly short-term protection of palm wood against mould and rot fungi

Felled palm trunks are susceptible to fungi as long as their moisture content is above fibre saturation. During this period, palm wood has to be protected against mould and rot fungi. The study was aimed at testing environment-friendly organic acids for their protecting efficiency. Small samples of date palm (Phoenix dactylifera) and oil palm (Elaeis guineensis) wood were treated with weak organic acids and subsequently infected by moulds and wood-decay fungi. Short dipping of the samples in solutions of 5% acetic acid and propionic acid, respectively, protected all samples for two months from colonization by Aspergillus niger, Penicillium sp., Cladosporium sp. and by a natural infection. Boric acid (4%) used in practice for protection was ineffective. Decay tests with the white-rot fungus Pleurotus ostreatus, the brown-rot species Coniophora puteana and the soft-rot fungus Chaetomium globosum showed that both acids prevented most samples from fungal colonization for three weeks and reduced the decay considerably during two months.