Elucidating the effects of laccase-modifying compounds treatments on bast and core fibers in flax pulp

Laccases in combination with various chemical compounds have been tested with a view to obtain environmental friendly, high‐value paper products from unbleached flax pulp, which is currently being assessed as a raw material for biotechnological innovation. With the aim of better understanding the effects of violuric acid (VA) and p‐coumaric acid (PCA) on flax pulp, changes in the chemical composition of the two major fiber types it contains were assessed. Following classification, the initial pulp was split into two fractions according to fiber size, namely: bast (long) fibers and core (short) fibers. Fiber size was found to significantly influence the properties of pulp and it response to various laccase treatments. The laccase‐PCA treatment substantially increased kappa number (KN) and color in both fiber fractions, which suggests grafting of the phenolic compound onto fibers. On the other hand, the laccase‐VA treatment produced long fibers with a low lignin content (KN = 1.3) and a high brightness (5% points higher than for the control fraction), which testifies to its bleaching efficiency. Both biotreatments produced long fibers containing highly crystalline cellulose and caused HexA removal from global and short fibers. On the other hand, the laccase treatments caused no morphological changes in the fibers, the integrity of which was largely preserved. As shown here, laccase acts as polymerization agent with PCA and as delignification agent with VA; also, the two enzymes systems act differently on bast and core fibers. Biotechnol. Bioeng. 2012;109: 225–233. © 2011 Wiley Periodicals, Inc.     

我国麻类资源的多样性及其保护利用对策

在“七五”、“八五”、“九五”研究的基础上,本文论述了我国麻类资源的种类、数量与分布以及保护利用现状等方面的问题,并提出了多样性的可持续发展和利用对策,为今后我国麻类资源的收集、保存、鉴定、创新和利用提供依据。     

Polysaccharides, tightly bound to cellulose in cell wall of flax bast fibre: Isolation and identification

Part of matrix polymers of flax bast fibre cell wall is tightly bound to cellulose and can not be extracted by conventional methods. To analyze these polymers, the residue, remaining after cell wall treatment with chelators and alkali, was dissolved in solution of lithium chloride in N,N-dimethylacetamide. Cellulose was precipitated by water and completely degraded by cellulase, giving the possibility to separate matrix polysaccharides, which remained in polymeric form. The obtained polymers were fractionated by gel permeation chromatography and characterized by monosaccharide analysis, staining with LM5 antibody and Yariv reagent, ¹H and ¹³C NMR. The total yield of the polysaccharides that are tightly bound to cellulose in flax fibre, was 4.6%. The major fractions (molecular mass 100–400 kDa) were composed of galactose, accompanied by two other significant monomers, GalA and Rha, with the ratio 1.1–1.4. Composition and structure of the cellulose bound galactan permit to consider it as fragment of the high-molecular mass (2000 kDa) galactan, synthesized by the developing fibres, while forming the secondary cell wall of gelatinous type.     

麻类脱胶高效菌株果胶裂解酶基因克隆与表达

【目的】克隆麻类脱胶高效菌株Dickeya sp.DCE-01的果胶裂解酶基因并进行原核表达,对表达产物进行纯化和酶学性质研究。【方法】根据该菌株全基因组序列预测的果胶裂解酶基因Q59419设计引物,PCR扩增后将该基因连接到pEASY-E1和pACYCDuet-1载体上,导入E.coli BL21(DE3)进行表达。选择酶活力高的阳性克隆子进行大量诱导表达后,采用超滤和Sephadex G-100凝胶层析两步法纯化出果胶裂解酶,研究其酶学性质。【结果】克隆到果胶裂解酶基因pel(GenBank登录号:JX964997),其序列全长1 128 bp,编码375个氨基酸。pACYCDuet-1-pel-BL表达胞外果胶裂解酶活力最高,发酵液粗酶活达298.8 IU/mL。其最适反应温度为50°C,最适pH为9.0;保温1 h,酶活稳定温度≤45°C,稳定pH为9.0?10.0。酶催化作用依赖于Ca2+,其最适作用浓度为2 mmol/L;Zn2+、Ca2+和NH4+促进酶活力,Fe3+和Pb2+严重抑制酶活力;聚半乳糖醛酸钠为该酶的最适底物。【结论】从麻类脱胶高效菌株中发掘到碱性果胶裂解酶基因,其表达产物在生物质加工过程中具有重要工业化应用前景。     

Variability in the composition of tissue-specific galactan from flax fibers

Tissue-specific galactan of sclerenchyma fibers, with cell walls of the gelatinous type, was examined in flax plants (Linum usitatissimum L.) of 23 various genotypes. The content and average degree of polymerization of side chains of galactan were estimated before its deposition into the cell wall. The variability of the analyzed parameters of tissue-specific galactan from flax fibers was high; within the same genotype, the scope of paratypic variability between replicates and years of research was comparable to variability between different genotypes. The average length of side chains in the studied samples ranged from 5 to 41 galactose residues. The average degrees of polymerization of galactan side chains in flax fibers was found to be discrete, which could be explained by block assemblage of the polymer in the Golgi apparatus.     

Raman imaging to investigate ultrastructure and composition of plant cell walls: distribution of lignin and cellulose in black spruce wood (Picea mariana)

A detailed understanding of the structural organization of the cell wall of vascular plants is important from both the perspectives of plant biology and chemistry and of commercial utilization. A state-of-the-art 633-nm laser-based confocal Raman microscope was used to determine the distribution of cell wall components in the cross section of black spruce wood in situ. Chemical information from morphologically distinct cell wall regions was obtained and Raman images of lignin and cellulose spatial distribution were generated. While cell corner (CC) lignin concentration was the highest on average, lignin concentration in compound middle lamella (CmL) was not significantly different from that in secondary wall (S2 and S2–S3). Images generated using the 1,650 cm⁻¹ band showed that coniferaldehyde and coniferyl alcohol distribution followed that of lignin and no particular cell wall layer/region was therefore enriched in the ethylenic residue. In contrast, cellulose distribution showed the opposite pattern—low concentration in CC and CmL and high in S2 regions. Nevertheless, cellulose concentration varied significantly in some areas, and concentrations of both lignin and cellulose were high in other areas. Though intensity maps of lignin and cellulose distributions are currently interpreted solely in terms of concentration differences, the effect of orientation needs to be carefully considered to reveal the organization of the wood cell wall.The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin. This article was written and prepared by U.S. Government employees on official time, and it is therefore in the public domain and not subject to copyright. The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service.     

Cu/Zn superoxide dismutases in developing cotton fibers: evidence for an extracellular form

Hydrogen peroxide and other reactive oxygen species are important signaling molecules in diverse physiological processes. Previously, we discovered superoxide dismutase (SOD) activity in extracellular protein preparations from fiber-bearing cotton (Gossypium hirsutum L.) seeds. We show here, based on immunoreactivity, that the enzyme is a Cu/Zn-SOD (CSD). Immunogold localization shows that CSD localizes to secondary cell walls of developing cotton fibers. Five cotton CSD cDNAs were cloned from cotton fiber and classified into three subfamilies (Group 1: GhCSD1; Group 2: GhCSD2a and GhCSD2b; Group 3: GhCSD3 and GhCSD3s). Members of Group 1 and 2 are expressed throughout fiber development, but predominant during the elongation stage. Group 3 CSDs are also expressed throughout fiber development, but transiently increase in abundance at the transition period between cell elongation and secondary cell wall synthesis. Each of the three GhCSDs also has distinct patterns of expression in tissues other than fiber. Overexpression of cotton CSDs fused to green fluorescent protein in transgenic Arabidopsis demonstrated that GhCSD1 localizes to the cytosol, GhCSD2a localizes to plastids, and GhCSD3 is translocated to the cell wall. Subcellular fractionation of proteins from transgenic Arabidopsis seedlings confirmed that only c-myc epitope-tagged GhCSD3 co-purifies with cell wall proteins. Extracellular CSDs have been suggested to be involved in lignin formation in secondary cell walls of other plants. Since cotton fibers are not lignified, we suggest that extracellular CSDs may be involved in other plant cell wall growth and development processes.     

Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers

Summary.  Methods for cryogenic fixation, freeze substitution, and embedding were developed to preserve the cellular structure and protein localization of secondary-wall-stage cotton (Gossypium hirsutum L.) fibers accurately for the first time. Perturbation by specimen handling was minimized by freezing fibers still attached to a seed fragment within 2 min after removal of seeds from a boll still attached to the plant. These methods revealed native ultrastructure, including numerous active Golgi bodies, multivesicular bodies, and proplastids. Immunolocalization in the context of accurate structure was accomplished after freeze substitution in acetone only. Quantitation of immunolabeling identified sucrose synthase both near the cortical microtubules and plasma membrane and in a proximal exoplasmic zone about 0.2 μm thick. Immunolabeling also showed that callose (β-1,3-glucan) was codistributed with sucrose synthase within this exoplasmic zone. Similar results were obtained from cultured cotton fibers. The distribution of sucrose synthase is consistent with its having a dual role in cellulose and callose synthesis in secondary-wall-stage cotton fibers. Received August 19, 2002; accepted November 12, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131, U.S.A. E-mail: candace.haigler@ttu.edu     

A potential role for sinapyl p-coumarate as a radical transfer mechanism in grass lignin formation

Grass lignins are differentiated from other lignin types by containing relatively large amounts of p-coumaric acid (pCA) acylating the C-9 position of lignin subunits. In the case of a mature corn (Zea mays L.) stems, pCA constitutes 15–18% of a dioxane soluble enzyme lignin. The major portion of the pCA is specifically attached to syringyl residues. Studies with isolated corn wall peroxidases show that pCA readily undergoes radical coupling in the presence of hydrogen peroxide, whereas sinapyl alcohol radical coupling proceeds more slowly. Analysis of corn wall peroxidases did not reveal specific enzymes that would lead to the preferred incorporation of sinapyl alcohol as seen in other plants. The addition of ethyl ferulate, methyl p-coumarate, or sinapyl p-coumarate conjugates to a reaction mixture containing peroxidase, sinapyl alcohol, and hydrogen peroxide stimulated the rate of sinapyl alcohol radical coupling by 10–20-fold. Based on spectral analysis it appears that pCA and ferulate radicals form rapidly, but the radical is readily transferred to sinapyl alcohol. The newly formed sinapyl alcohol radicals undergo coupling and cross-coupling reactions. However, sinapyl alcohol radicals do not cross-couple with pCA radicals. As long as hydrogen peroxide is limiting pCA remains uncoupled. Ferulates have similar reaction patterns in terms of radical transfer though they appear to cross-couple in the reaction mixture more readily then pCA. The role of pCA may be to internally provide a radical transfer mechanism for optimizing radical coupling of sinapyl alcohol into the growing lignin polymer. Attachment of some pCA to sinapyl alcohol ensures localization of the radical transfer mechanism in areas where sinapyl alcohol is being incorporated into lignin.     

Label-free in situ imaging of lignification in the cell wall of low lignin transgenic Populus trichocarpa

Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulose and lignin distribution in wood cell walls. Lignin reduction in wood can be achieved by, for example, transgenic suppression of a monolignol biosynthesis gene encoding 4-coumarate-CoA ligase (4CL). Here, we use confocal Raman microscopy to compare lignification in wild type and lignin-reduced 4CL transgenic Populus trichocarpa stem wood with spatial resolution that is sub-μm. Analyzing the lignin Raman bands in the spectral region between 1,600 and 1,700 cm⁻¹, differences in lignin signal intensity and localization are mapped in situ. Transgenic reduction of lignin is particularly pronounced in the S2 wall layer of fibers, suggesting that such transgenic approach may help overcome cell wall recalcitrance to wood saccharification. Spatial heterogeneity in the lignin composition, in particular with regard to ethylenic residues, is observed in both samples. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of hypergravity conditions on elongation growth and lignin formation in the inflorescence stem of Arabidopsis thaliana

The effects of hypergravity on elongation growth and lignin deposition in secondary cell walls of the Arabidopsis thaliana (L.) Heynh. inflorescence stem were examined in plants grown for 3 days after exposure to hypergravity in the direction from shoot to root at 300 g for 24 h. The content of acetylbromide-extractable lignins in a secondary cell wall fraction prepared by enzyme digestion of inflorescence stem segments removing primary cell wall components was significantly increased by the hypergravity stimulus. Xylem vessels, particularly in a region closer to the base of the inflorescence stem, increased in number. Gadolinium chloride at 0.1 mM, a blocker of mechanoreceptors, partially suppressed the effect of hypergravity on lignin deposition in the secondary cell wall fraction. These results suggest that mechanoreceptors are responsible for hypergravity-induced lignin deposition in secondary cell walls in A. thaliana inflorescence stems.     

Another evidence for inhibitory effect of auxin in adventitious bud formation of decapitated flax (Linum usitatissimum L.) seedlings

Adventitious buds were formed on the hypocotyls of decapitated flax seedlings. Scanning electron and light microscopic examinations of hypocotyls showed that epidermal cells divided to produce meristematic spots from which several leaf primordia were formed. Between leaf primordia and the original vascular tissues of hypocotyls, new xylem cells were formed which connected them. About 10, 30 and 60% of adventitious buds were formed on upper, middle and basal parts of hypocotyls of decapitated seedlings, respectively. Removal of apical meristem together with longer hypocotyl zero to four cm long below the apical meristem) induced higher percentage of adventitious bud formation in the remaining hypocotyl. When the entire hypocotyl was cut into 16 segments (0.25 cm each) and these segments were cultured on MS medium containing 3% sucrose and 0.8% agar, adventitious buds were mainly formed in the lowest five segments. These results suggested that there was a gradient of inhibitory factor(s) from apical to basal part of hypocotyl with respect to adventitious bud formation. Auxin transport inhibitors, morphactin and TIBA induced adventitious bud formation on intact seedlings by suppressing the basipetal movement of auxin.     

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.     

Biochemical origin and refractory properties of humic acid extracted from maize plants: the contribution of lignin

The soil organic carbon (SOC) pool is the largest terrestrial reservoir of carbon and plant residues play an important role in its maintenance. Up to 70–80% of SOC in arable soil is composed of humic substances (HS). In these soils post-harvested residues, left in arable soil after harvesting the crops, are the basic source of humus. Previous research indicated that maize plants residue contain a humic acid (HA) fraction possessing recalcitrant compounds that contributed to soil-HA fraction. This study presents updated results obtained using Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS) to provide an indication of the contribution of the lignin to the soil HA. Results obtained indicated that the HAs from maize plants were mainly composed of lignin-derived moieties that were likely derived from the partial hydrolysis of p-coumaric and ferulic acid that are linked to lignin, polysaccharides or other biopolymers of the cell wall. Lignin composing the HAs derived from plants and incubated in soil was substantially preserved. Nevertheless the modification of the syringyl/guaiacyl ratio and the oxidation of the side-chains of lignin, suggested a turnover of lignin-derived molecules in soil-HA fraction. This fact indicated an involvement of the alkali insoluble fraction of maize plant residue (humin) in the soil-HA formation, up-dating our previous knowledge.     

The mediation of veratryl alcohol in oxidations promoted by lignin peroxidase: the lifetime of veratryl alcohol radical cation

The kinetics of decay of veratryl alcohol radical cation, generated by cerium(IV) ammonium nitrate induced oxidation of veratryl alcohol, have been followed spectrophotometrically in a stopped-flow apparatus. In acidic aqueous acetonitrile the radical cation was found to decay by a first-order process, due to deprotonation from the alpha-carbon leading to an alpha-hydroxybenzyl radical with the rate constant of 17.1+/-0.5 s(-1). This value is in full agreement with those obtained by pulse radiolysis studies but much lower than the value (1.2x10(3) s(-1)) indirectly determined by EPR experiments. The implications of these results with respect to the possible role of veratryl alcohol as a mediator in the oxidative biodegradation of lignin catalysed by lignin peroxidase are discussed.     

Biocatalysis in ionic liquids for lignin valorization: Opportunities and recent developments

Lignin holds tremendous potential as a renewable feedstock for upgrading to a number of high-value chemicals and products that are derived from the petroleum industry at present. Since lignin makes up a significant fraction of lignocellulosic biomass, co-utilization of lignin in addition to cellulose and hemicelluloses is vital to the economic viability of cellulosic biorefineries. The recalcitrant nature of lignin, originated from the molecule's compositional and structural heterogeneity, however, poses great challenges toward effective and selective lignin depolymerization and valorization. Ionic liquid (IL) is a powerful solvent that has demonstrated high efficiency in fractionating lignocellulosic biomass into sugar streams and a lignin stream of reduced molecular weight. Compared to thermochemical methods, biological lignin deconstruction takes place at mild temperature and pressure while product selectivity can be potentially improved via the specificity of biocatalysts (lignin degrading enzymes, LDEs). This review focuses on a lignin valorization strategy by harnessing the biomass fractionating capabilities of ILs and the substrate and product selectivity of LDEs. Recent advances in elucidating enzyme-IL interactions as well as strategies for improving enzyme activity in IL are discussed, with specific emphases on biocompatible ILs, thermostable and IL-tolerant enzymes, enzyme immobilization, and surface charge engineering. Also reviewed is the protein engineering toolsets (directed evolution and rational design) to improve the biocatalysts' activity, stability and product selectivity in IL systems. The alliance between IL and LDEs offers a great opportunity for developing a biocatalytic route for lignin valorization.     

ESR evidence for sequential divalent cation binding in higher plant cell walls

Electron spin resonance linewidth measurements have been made on intact cell walls exchanged with various combinations of Mn²⁺ and Ca²⁺. These experiments were performed to find the Mn²⁺ nearest-neighbor distance and thereby determine whether carboxylate-Mn²⁺ complexes potentiate ion association at adjacent sites on cell wall polyuronides. Our results show that as the fraction of available binding sites occupied by Mn²⁺ increased from 2% to 27%, the nearest-neighbor distance parameter decreased only from 14 to 11 Å. These distances are close to polyuronide interanionic spacings. The small change in the distance parameter with concentration is evidence for sequential rather than random binding. Competitive ion-exchange with Ca²⁺ was found to reduce the Mn²⁺ spin-spin line broadening at similar total bound Mn²⁺ concentrations. This is expected only if Ca²⁺ competes at adjacent sites. The data presented offer strong support for the hypothesis that carboxylate groups near already occupied sites have a greater affinity for divalent cations than other sites along the polyuronide main chain.