Screening of wood-rotting fungi for kraft pulp bleaching by the Poly R decolorization test and biobleaching of hardwood kraft pulp byPhanerochaete crassa WD1694

From among 419 wood-rotting fungi 10 were selected by the Poly R decolorization test, and their ability to bleach hardwood kraft pulp was assayed. Of the 10 selected, 6 fungi (i.e.,Phanerochaete crassa WD1694 and F150;Pleurotus pulmonarius PSC-H, PSC-M, and PSC-T;and Pleurotus species A119) showed much higher bleaching ability thanPhanerochaete chrysosponum BKMF1767 orTrametes versicolor WD1670, both of which are well-known high ligninolytic fungi.P. crassa WD1694 had the highest bleaching ability among the selected strains, and it increased the pulp brightness from 28 to 54, with a corresponding decrease in kappa number from 16 to 6 after 10 days of cultivation and alkali extraction. MnP was a predominant ligninolytic enzyme ofP. crassa WD1694 during the biobleaching.This research was presented in part at the 46th Annual Meeting of the Japan Wood Research Society, Kumamoto, April 1996     

Glyoxal oxidase supplies hydrogen peroxide at hyphal tips and on hyphal wall to manganese peroxidase of white-rot fungus Phanerochaete crassa WD1694

Peroxidase activity staining localized at hyphal tips of white-rot fungus Phanerochaete crassa WD1694 that was cultivated in a shaken liquid culture containing unbleached kraft pulp was investigated. Manganese peroxidase was detected in culture solution, washing solution of mycelium, and mycelial extract. Glyoxal oxidase was detected only in mycelial extract and was not detected in culture solution. Addition of hydrogen peroxide generated peroxidase activity staining in the culture solution. Addition of catalase resulted in no staining in the culture of P. crassa WD1694, and the addition of methylglyoxal resulted in marked peroxidase activity staining at hyphal tips and on hyphal wall. In an optimized culture, glyoxal oxidase was produced in culture solution. Although the production of glyoxal oxidase and manganese peroxidase had a positive correlation, the secretion and the peak of glyoxal oxidase was observed 3 and 2 days later than those of manganese peroxidase. The N-terminal sequence of purified glyoxal oxidase had very high homology with that of P. chrysosporium. These results elucidated the hydrogen peroxide supply system in lignin biodegradation by white-rot fungi, i.e., while remaining on the hyphal cell wall, glyoxal oxidase provides hydrogen peroxide to manganese peroxidase that had diffused into the culture solution beforehand.     

Selective staining and visualization of hyphal sheath of a white-rot fungus <Emphasis Type="Italic">Phanerochaete crassa</Emphasis> WD1694 with phloxine B

The hyphal sheath is a morphological feature of many kinds of fungi. Although the fine structures of the sheath have been studied in detail by a number of electron microscopy techniques, the function and physiology of the hyphal sheath are not yet clarified. One reason for this is that the hyphal sheath is a colorless, mucilaginous, and delicate material so that it is not easily identified. We developed a simple method to visualize and identify the hyphal sheath of the white-rot fungus Phanerochaete crassa WD1694. The small mycelial pellets in shaken liquid cultures of P. crassa WD1694 were stained directly with phloxine B. Both the hyphae and the hyphal sheath that filled the gaps between each of the hyphae were visualized and observed by light microscopy. The stained hyphae were further studied by transmission electron microscopy, atomic force microscopy, and fl uorescence microscopy. Based on these observations, we confirmed that the staining of the hyphae was also due to the presence of the hyphal sheath that closely covered the fungal cell wall. These results clearly showed that the hyphal sheath was selectively stained with phloxine B and could be observed and identified by conventional light microscopy. Part of this report was presented at the 50th Lignin Symposium, Nagoya, October 2005     

Extracellular peroxidase activity at the hyphal tips of the white-rot fungus Phanerochaete crassa WD1694

The white-rot fungus Phanerochaete crassa WD1694 was cultivated and peroxidase activity staining was performed to determine the sites at which the extracellular peroxidase reaction actually occurs in vivo. Although the ligninolytic peroxidases were found in the culture filtrates, the culture medium did not show a color reaction. However, a particularly strong color reaction was observed on the hyphal tips. Visible spectra and absorbance of the staining were analyzed by microspectrophotometry, and the catalytic rates of the peroxidase reaction at the hyphal tips were calculated. The estimated catalytic rate of the peroxidase reaction at the hyphal tips peaked at 794 μM/min, expressed as the consumption rate of H₂O₂, on day 3 of the cultivation. Analysis of the extracellular enzyme eluted with 0.1% Tween 80 from the mycelium revealed that manganese peroxidase accounted for 89% of all the peroxidase activity measured. The results clearly showed the existence of the concentrated manganese peroxidase reaction around the hyphal tips of the organism.     

Isolation of cDNA and genomic fragments encoding the major manganese peroxidase isozyme from the white rot basidiomycetePleurotus ostreatus

We have isolated the cDNA and genomic sequences encoding the major isozyme of manganese peroxidase, MnP3, from the white rot basidiomycetePleurotus ostreatus strain IS1. The genemnp3 is interrupted by 10 introns and encodes a mature protein of 357 amino acid residues with a 26-amino-acid signal peptide. The amino acid residues known to be involved in peroxidase function and those that form the Mn-binding site in thePanerochaete chrysosporium MnP isozyme are conserved in MnP3. Comparison of the deduced primary structure of MnP3 with those of other peroxidases from various white rot fungi suggested that MnPs fromP. ostreatus andTrametes versicolor belong to a subgroup that is more similar to the lignin peroxidases than MnPs fromP. chrysosporium orCeriporiopsis subvermispora.     

Extracellular peroxidase reaction at hyphal tips of white-rot fungus <Emphasis Type="Italic">Phanerochaete crassa</Emphasis> WD1694 and in fungal slime

The distribution of an extracellular peroxidase reaction by white-rot fungus Phanerochaete crassa WD1694 was visualized by peroxidase activity staining. The extracellular peroxidase reaction occurred at the hyphal tips and in the fungal slime filling the gaps between the hyphae. We investigated whether the peroxidase reaction occurred from the hyphal tips or in the slime. The hyphal tips were observed by phase-contrast microscopy, which showed that slime did not exist around the hyphal tips. Time-course observation of hyphal tips showed that peroxidase staining became thick and intense at the tips that did not have fungal slime. Daily observation of the peroxidase staining revealed that the staining was first observed at the hyphal tips. Furthermore, strongly stained hyphae were observed in the stained slime. These results suggested that an active species that oxidizes a peroxidase substrate is first produced at the tips of the hyphae, and then occurs in the slime via diffusion when slime exists around the hyphae. Our results show that the extracellular peroxidase reaction that is important to lignin biodegradation by white-rot fungi occurs directly at the tips of the hyphae and in the slime. Part of this report was presented at the 50th Lignin Symposium, October 19–20, 2005, Nagoya, Japan     

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     

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     

猴头菌锰过氧化物酶1基因在构巢曲霉的异源转化与表达

为提高猴头菌菌株CB1锰过氧化物酶(MnP)基因的表达产量,采用PEG/CaCl2介导的原生质体转化方法,将携带有He-mnp1的重组质粒pLB01/He-mnp1转入到构巢曲霉尿嘧啶尿苷营养缺陷菌株TN02A7的原生质体中,获得了转化子菌株TN02A7-He-mnp1,并在乙醇脱氢酶启动子alcA(p)控制下实现了异源表达。将TN02A7-He-mnp1、TN02A7、构巢曲霉野生型菌株WJA01、猴头菌菌株CB1在相同的木质素环境下进行培养并检测MnP酶活性,结果表明:转化子菌株TN02A7-He-mnp1在0.05 g.L-1血红素的情况下、诱导96 h后酶活性最高为38.31 U.L-1,比不添加血红素的酶活力高8.64倍,但比猴头菌菌株CB1的酶活力低,而TN02A7与WJA01始终无MnP酶活性,说明基因He-mnp1已经成功地被转化到TN02A7-He-mnp1中,并在木质素环境下得到表达,血红素是重组MnP基因异源表达的限制性因素之一。本文为生产MnP和提高MnP产量提供了新的途径。     

Reduction of ferric chelate caused by various wood-rot fungi

The reduction of ferric chelate caused by various wood-rot fungi was analyzed. Ferric chelate reductive activity was detected in cell-free extracts of seven wood-rot fungi:Phanerochaete chrysosporium, P. sordida YK-624,Ganoderma sp. YK-505,Coriolus versicolor, Bjerkandera adusta, Tyromyces palustris, andGloeophyllum trabeum. These fungi produced NADPH- or NADH-dependent ferric chelate reductive enzymes (or both) of different molecular weight. In the liquid culture ofP. sordida YK-624 andC. versicolor, a positive correlation was observed between extracellular MnP activity and intracellular NADPH-dependent ferric chelate reductive activity.     

油桐尺蛾核型多角体病毒lef-8基因结构及系统进化分析

lef-8基因作为杆状病毒重要的早期表达基因,编码病毒RNA聚合酶的最大亚基。本文通过对油桐尺蛾核型多角体病毒lef-8基因进行测序,对其基因结构及其在杆状病毒中的系统进化进行了分析。发现lef-8基因在油桐尺蛾核型多角体病毒中编码区长2 634 bp,编码877个氨基酸。在5’-UTR区具有可能为其启动子及转录起始位点的信号序列,在3’-UTR区具有加尾信号序列,但这些序列的具体功能还有待进一步研究。其蛋白序列与常见的模式NPV同源性较低,但具有共同的签名序列。通过对杆状病毒lef-8基因的系统进化分析,发现颗粒体病毒和核型多角体病毒明显分为两支,而核型多角体病毒中又明显分为4个分支,其进化关系在很大程度上与其寄主的进化关系相关。     

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     

哈茨木霉天冬氨酸蛋白酶基因SA76的3＇RACE扩增和序列分析

由EST获得全长cDNA对于结构基因组学和功能基因组学都是至关重要的,cDNA末端快速扩增技术RACE是该领域中的重要研究方法.利用BD SMART RACE技术扩增编码分泌天冬氨酸蛋白酶SA76基因的3＇末端,将其与哈茨木霉cDNA文库中的SA76基因的EST序列进行序列拼接,获得2019bp的全长cDNA序列,其开放读码框长1593bp,5＇非编码区266bp,3＇非编码区201bp,编码530个氨基酸,有信号肽.哈茨木霉天冬氨酸蛋白酶基因与玉蜀黍赤霉、粗糙脉孢菌、球毛壳菌天冬氨酸蛋白酶基因的同源性分别为53%, 37%, 36%.利用BD SMART RACE技术首次从哈茨木霉中克隆天冬氨酸蛋白酶基因,为验证SA76基因的功能奠定基础,为进一步研究蛋白酶的作用机制及生物防治功能提供依据.     

3种木腐菌木质纤维素相关酶基因的TRAP标记体系优化与遗传变异初探

以黑龙江省东北林业大学帽儿山实验林场的3种典型木材腐朽菌为试验材料,采用TRAP分子标记的手段,针对5种木质素与纤维素相关酶基因LiP、MnP、Lac、CBHⅡ、CDH设计引物,初步分析这些编码基因的多态性。试验确定了3种木腐菌TRAP分子标记的反应体系和反应程序。经过对32对引物进行筛选试验,共确定11对引物,包括3对标记MnP编码基因的引物、3对标记Lac编码基因的引物、3对标记CBH编码基因的引物,以及2对标记CDH编码基因的引物。结果表明:3种木腐菌的TRAP-PCR标记共产生了265条条带,其中多态性条带206条,占总条带的77.74%,多态性最高为100%,最低为60%,证明了TRAP分子标记可以应用于木腐菌的遗传分析。     

Variations of wood basic density with tree age and social classes in the axial direction within Pinus massoniana stems in Southern China

Context

Reliable estimates of wood density (WD) within individual trees could maximize the value of Pinus massoniana for specific end-use.

Aim

We examined and quantified the axial patterns of WD in trees with different social status in the stands.

Methods

Wood disks were sampled at the bottom, breast height, and middle of each 1-m sections from 108 stems, harvesting from three social classes in subtropical forests. A mixed-effects model was designed to quantify axial variation.

Results

The WD at different height was significantly different from the whole-stem WD (WWD) except the relative height of 0.1. An overall decrease of 133.8 kg m^?3 in WD was found from stem base to top. WD was significantly influenced by relative heights, tree age, and social class. WD of each relative height in mature trees was significantly higher than that of younger trees. Tree social class can affect WD development in the axial direction at age classes 2 and 3. Combining the fixed plus random effects, the final model explained 91 % of the observed variation in WD.

Conclusion

The WD development patterns in the axial direction vary considerably among tree age, diameter at breast height, and social class. To distinguish and supply timber for specific end-uses, we should use the axial variation in disk WD (DWD) instead of WWD directly. The accurate predictions of WD provided by the model could be used to optimally classify logs into different product classes and maximize economic benefits. We can use DWD at the relative height of 0.1 instead of WWD of a single tree.     

美洲黑杨PdZFR基因的克隆和分析

根据美洲黑杨PdZFR部分cDNA序列,采用拟基因组文库步行法和RACE克隆了该基因全长cDNA及基因组DNA, 并对该基因的启动子、转录起始位点、内含子分布、剪切方式、编码的氨基酸序列和该基因在染色体上的分布位置进行了分析。组织或器官特异表达分析表明:该基因与植物发育相关。     

Variation analyses of growth and wood properties of <Emphasis Type="Italic">Larix olgensis</Emphasis> clones in China

The growth traits (tree height, diameter at breast height, and stem straightness degree) and wood properties [wood density (WD), fiber length, fiber width, ash content (AC), lignin content, cellulose content, hemicellulose content (HEC), and holocellulose content] of 208 26-year-old Larix olgensis clones were analyzed. Except for WD and AC, there were significant differences (p < 0.01) for all traits among clones. The phenotypic coefficient of variation and repeatability of all traits were 9.34–35.33% and 0.218–0.930, respectively. Tree height and diameter at breast height showed significant positive correlation; however, the correlation coefficients among growth characteristics and wood properties were mostly not significant. Ten clones (L70, L56, L82, L90, L59, L91, L61, L92, L86, and L64) were selected as excellent clones under a selection rate of 5%, using tree height, diameter at breast height, and stem straightness degree as evaluation indexes, providing genetic gains of 28.69, 17.96, and 0.67%, respectively. Ten clones (L88, L305, L59, L66, L253, L304, L277, L298, L248, and L293) were selected as excellent clones using wood properties as an evaluation index, with a selection rate of 5%, providing genetic gains in WD, fiber length, fiber width, cellulose content, and HEC of 4.14, 3.64, 9.28, 6.77, and 9.61%, respectively. This study provides a theoretical basis for selecting excellent L. olgensis clones.     

麻疯树磷酸烯酮式丙酮酸羧化酶em>pepc基因全长cDNA克隆及序列分析

应用RT-PCR和RACE法从麻疯树总RNA中分离出pepc基因全长cDNA,长度为3 142 bp,阅读框2 898 bp,编码965个氨基酸,在GenBank中登录,序列号为EU069413。它的氨基酸序列与蓖麻、陆地棉、橙、大豆、花生、烟草、油菜、拟南芥的氨基酸同源性分别为94.94%、92.46%、90.60%、90.50%、90.50%、88.33%、84.61%、82.44%。该基因编码了pepc基因家族中的pepc1,蛋白属于C₃型PEPC。推测了pepc编码蛋白分子量为110.6 kD,并对其稳定性、二级结构、疏水性等特性进行了分析,最后确定了该蛋白的功能位点和结构域。     

Biological characteristics of five wood-rotting fungi and wood-decaying ability to Betula platyphylla

In this paper, the growth rate of five wood-rotting fungi, i.e., Coriolus versicolor, Irpex lacteus, Fomes fomentarius, Piptoporus betulinus and Pholiota adiposa, in solid medium and their biomass in liquid culture medium were compared by measuring mycelium length and dry mass. The activity of three main ligninolytic enzymes in those fungi, namely LiP, MnP and Lac, were also tested by colorimetry. At the same time, these fungi were used to decay the wood samples from 300 natural trees of white birch, to study their wood-decaying ability by measuring wood mass loss. The result showed that the growth rate, biomass, ligninolytic enzyme activity, and wood-decaying ability of the fungi were incompletely correlated. The growth rates of C. versicolor and I. lacteus were faster than those of P. betulinus and F. fomentarius; P. adiposa was the slowest in growth. The biomass of P. betulinus was the highest; C. versicolor, I. lacteus and F. fomentarius were in the middle, and P. adiposa was the lowest. There existed LiP, MnP and Lac activities in all fungi except P. betulinus, and the enzyme activities induced by wood powder were all higher than those of the control. The Lac of I. lacteus and the LiP of F. fomentarius and P. adiposa were only expressed in wood powder medium; the longer the fungi were cultured, the higher activity the enzyme had. The decomposition ability of C. versicolor to wood samples was the highest, followed by F. fomentarius and P. betulinus; I. lacteus and P. adiposa were the lowest.     

毛竹PeAP2基因及其启动子的克隆与表达初步分析

APETALA2(AP2)基因在植物生长发育过程中发挥着重要作用.利用RT-PCR和RACE方法,从毛竹中克隆到1个AP2同源基因的全长cDNA序列,命名为PeAP2.序列分析表明:PeAP2基因全长1 750 bp,其中,5′端非编码区106bp,3′端非编码区174 bp,开放阅读框1 470 bp,编码1个489 aa的蛋白,该蛋白含有2个AP2结构域,属于AP2/EREBP家族的AP2亚家族.PeAP2蛋白与来自其它单子叶植物的AP2蛋白均有着较高同源性,其中,与二穗短柄草的AP2蛋白同源性最高,达74.85％.实时定量PCR分析显示:PeAP2基因在毛竹的根、茎、叶、鞘和节5种器官中均有表达,其中,叶片中的表达丰度最高,鞘中次之,而在根、茎、节中的表达丰度接近,均较低.利用hiTAIL-PCR方法克隆获得了PeAP2上游启动子区序列1 359 bp,分析显示其含有光、激素等多种信号应答相关的作用元件.