Localization of Xyloglucan in the Macromolecular Complex Composed of Xyloglucan and Cellulose in Pea Stems

A macromolecular complex composed of xyloglucan and cellulosewas isolated from elongating regions of stems of etiolated pea(Pisum sativum L. var Alaska) seedlings and binding of a xyloglucan-specificantibody was examined after treatment of the complex with endo-1,4-ß-glucanaseor 24% KOH. The antibody bound to the complex but the extentof binding was reduced after treatment of the complex with endo-1,4-ß-glucanaseand was hardly detectable after treatment with 24% KOH. Themolecular weight of the xyloglucan that remained (5%) in theß-glucanase-treated complexes was less than 9,200.Pea xyloglucan was allowed to bind to enzymeand alkali-treatedcomplexes to generaly reconstituted complexes. The amount ofthe antibody that bound to each type of reconstituted complexwas similar but was much lower than that bound to the nativecomplex. Immunogold labeling indicated that most of the antigenwas widely distributed between microfibrils in the native complex,whereas the antigen appeared to be confined to the microfibrilsin the reconstituted complexes. These findings suggest thata part of each xyloglucan molecule is strongly associated withcellulose microfibrils while the rest is free of the microfibrilsin the native complex. ¹This work was supported in part by a grant from the YamadaScience Foundation.     

Enzymatic Degradation of GlycosaminogIycans

Abstract

Glycosarninoglycans (GAGs) play an intricate role in the extracellular matrix (ECM), not only as soluble components and polyelectrolytes, but also by specific interactions with growth factors and other transient components of the ECM. Modifications of GAG chains, such as isomerization, sulfation, and acetylation, generate the chemical specificity of GAGs. GAGS can be depolymerized enzymatically either by eliminative cleavage with lyases (EC 4.2.2.-) or by hydrolytic cleavage with hydrolases (EC 3.2.1.-). Often, these enzymes are specific for residues in the polysaccharide chain with certain modifications. As such, the enzymes can serve as tools for studying the physiological effect of residue modifications and as models at the molecular level of protein-GAG recognition. This review examines the structure of the substrates, the properties of enzymatic degradation, and the enzyme substrate-interactions at a molecular level. The primary structure of several GAGS is organized macro-scopicallyby segregation into alternating blocks of specific sulfation patterns and microscopicallyby formation of oligosaccharide sequences with specific binding functions. Among GAGs, considerable dermatan sulfate, heparin and heparan sulfate show conformational flexibility in solution. They elicit sequence-specific interactions with enzymes that degrade them, as well as with other proteins, however, the effect of conformational flexibility on protein-GAG interactions is not clear. Recent findings have established empirical rules of substrate specificity and elucidated molecular mechanisms of enzyme-substrate interactions for enzymes that degrade GAGs. Here we propose that local formation of polysaccharide secondary structure is determined by the immediate sequence environment within the GAG polymer, and that this secondary structure, in turn, governs the binding and catalytic interactions between proteins and GAGs.     

瘤胃微生物对纤维素的降解及其应用

瘤胃微生物主要包括细菌、真菌和原生动物。其中,瘤胃细菌和瘤胃真菌能分泌纤维素酶,对纤维素有较强的降解能力,主要介绍了瘤胃微生物对纤维素的降解作用及其广阔的应用前景。     

Water storage in the wood and xylem cavitation in 1-year-old twigs of Populus deltoides Bartr.

The possible role of water expelled from cavitated xylem conduits in the rehydration of water-stressed leaves has been studied in one-year-old twigs of populus deltoides Bartr. Twigs were dehydrated in air. At desired values of leaf water potential (Ψ_l) (between near full turgor and -1.62 MPa), twigs were placed in black plastic bags for 1–2h. Leaf water content was measured every 3–5 min before bagging and every 10 min in the dark. Hydraulic conductivity and xylem cavitation were measured both in the open and in the dark. Cavitation was monitored as ultrasound acoustic emissions (AE). A critical Ψ_l value of -0.96 MPa was found, at which AE increased significantly while the leaf water deficit decreased by gain of water. Since the twigs were no longer attached to roots, it was concluded that water expelled from cavitated xylem conduits was transported to the leaves, thus contributing to their rehydration. Xylem cavitation is discussed in terms of a ‘leaf water deficit buffer mechanism’, under not very severe water stress conditions.     

Roles of Cellulose and Xyloglucan in Determining the Mechanical Properties of Primary Plant Cell Walls

The primary cell walls of growing and fleshy plant tissue mostly share a common set of molecular components, cellulose, xyloglucan (XyG), and pectin, that are required for both inherent strength and the ability to respond to cell expansion during growth. To probe molecular mechanisms underlying material properties, cell walls and analog composites from Acetobacter xylinus have been measured under small deformation and uniaxial extension conditions as a function of molecular composition. Small deformation oscillatory rheology shows a common frequency response for homogenized native cell walls, their sequential extraction residues, and bacterial cellulose alone. This behavior is characteristic of structuring via entanglement of cellulosic rods and is more important than cross-linking with XyG in determining shear moduli. Compared with cellulose alone, composites with XyG have lower stiffness and greater extensibility in uniaxial tension, despite being highly cross-linked at the molecular level. It is proposed that this is due to domains of cross-linked cellulose behaving as mechanical elements, whereas cellulose alone behaves as a mat of individual fibrils. The implication from this work is that XyG/cellulose networks provide a balance of extensibility and strength required by primary cell walls, which is not achievable with cellulose alone.     

Degradation of Xyloglucan by Wall-Bound Enzymes from Soybean Tissue II: Degradation of the Fragment Heptasaccharide from Xyloglucan and the Characteristic Action Pattern of the {alpha}-D-Xylosidase in the Enzyme System

Hydrolysis of the fragment heptasaccharide (glucose : xylose= 4 : 3) from xyloglucan with an enzyme preparation from soybeancell wall produced a penta- and a trisaccharide. The resultsof fragmentation analysis of these oligosaccharides with Aspergillusoryzae ß-D-glucosidase indicate the following structuresfor the penta- and trisaccharide. The detection of these intermediate products suggested thatdegradation of the heptasaccharide took place by sequentialsplitting of the -D-xylosidic and ß-D-glucosidic linkages.A characteristic action pattern of the a-D-xylosidase in theenzyme preparation was found. ¹Present address: Department of Biology, McGill University,Montreal, Canada. ²Present address: Department of Botany, Iowa State University,Ames, Iowa 50010, U.S.A. (Received August 20, 1982; Accepted December 7, 1982)     

Degradation of the Pine Wood Structure in Ozonolytic Delignification

Russian Journal of Bioorganic Chemistry - Transformations of pine wood during exposure to ozone were studied. The content of lignin and cellulose in the cellulose-containing material (CCM) from...     

曾候乙墓穴木椁微生物降解对木材危害及防治措施研究

从曾候乙墓分离到的16株菌,其中有11株为芽孢杆菌属类,4株微杆菌属,1林为黄色杆菌属。将16株细菌分别侵染6种木材,结果证明细菌降解对6种木材均有不同的韧性下降,个别木材出现腐烂病灶。2号菌降解后对泡桐、枣树、马尾松、香樟、刺槐和桑树分别下降8％,12％,10％,5％,10％和13％。3,5,6,7,8,9和16号菌对上述6种木材也有不同程度影响。而4,11,12,13,14和15号菌对6种木材的韧性下降影响不明显。但其渗透性大大提高了,因此也会影响木材的使用年限。采用杀菌、杀虫的石油气,乙醇等有机溶济,防治微生物降解产物对木材的浸蚀,均有较好的防菌、防认腐效果。     

The Structural Basis for the Susceptibility of Gangliosides to Enzymatic Degradation

The conformational properties of GM2, GalNac-4(Neu5Ac-3) Gal-4Glc-1Cer have been compared to those of 6-GM2, in which the linkage between the GalNAc and Gal was altered from GalNac-4Gal- to GalNac-6Gal-, and to those of GD1a, Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-3)Gal-4Glc-1Cer, and GalNAc-GD1a.Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions.The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Ac-3Gal-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two core GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.     

盐胁迫对转AtNHX1基因杨树光合特性与叶绿体超微结构的影响

以欧美107杨(Populus×euramericana ‘Neva',Wt)和转拟南芥液泡膜Na~+/H~+逆向转运蛋白基因AtNHX1的欧美107杨新品系(Tr) 幼苗为材料,研究了高低度盐胁迫对两品系幼苗光合色素含量、光合参数和叶绿体超微结构的影响,以阐明转AtNHX1基因杨树的耐盐性与其光合作用及叶绿体结构之间的关系.结果表明:(1)盐处理后,两品系叶片叶绿素含量、类胡萝卜素含量、净光合速率、蒸腾速率和气孔导度均下降,且高盐度处理下降幅度更大;同等盐度处理下,Tr品系叶片叶绿素含量、净光合速率和气孔导度的下降幅度显著低于Wt品系,且在高盐度处理间差异更大;两品系杨树叶片P_n下降的原因在低盐处理时以气孔限制为主,而在高盐下则是气孔限制和非气孔限制共同作用的结果.(2)盐胁迫对T_r 品系叶片叶绿体超微结构的影响较轻,其在高盐下仍保持了较好的内部结构;盐胁迫Wt品系叶绿体则缩皱成球形,内部结构趋向简单,以至解体,脂质球显著增多.可见,盐胁迫导致杨树叶绿体结构破坏而引起叶绿体色素含量下降,最终降低其光合作用效率;同等盐度胁迫下,转AtNHX1基因品系叶片保持了较完整的叶绿体超微结构、更高的叶绿素含量,能维持较好的光合状态,从而表现出较高的耐盐能力.     

转UGPase基因喜树木材化学成分与生长速率研究

分析了田间栽培条件下2年生转UGPase基因喜树与对照株的木材化学成分与生长速率。结果表明,转UGPase基因喜树综纤维素含量达到78.87%,比对照株相比提高了2.23%;纤维素含量为36.34%,与对照株相比没有明显提高;木质素含量为15.05%,较对照株降低了1.75%;两者的灰分含量均较低且无显著差异;冷、热水抽提物含量为7.62%与10.17%,分别提高了2.04%与2.13%;1% NaOH抽提物含量为27.13%,提高了1.27%。因此,就综纤维素、木质素、灰分含量而言,转UGPase基因喜树为优质纸浆材,水抽提物和1% NaOH抽提物的含量略高,在纸浆生产中需加以重视。本文还对转UGPase基因喜树与对照株的株高、基径、生物量进行了动态监测,结果表明,从5月25日到11月10日的生长季中,其株高平均增加121 cm,对照株平均仅67.8 cm,株高生长速率提高了78.47%;基径平均增加1 792 cm,对照株平均仅0.532 8 cm,提高了236.37%;地上部分生物量的积累与对照相比提高了322.61%,即转入UGPase基因使喜树生长速率显著提高。因此,虽然转UGPase基因喜树的综纤维素和纤维素含量没有明显提高,但其生长速率快,生物量增长显著,间接提高了纤维素与喜树碱的产量。因此,转基因喜树较普通喜树更符合纸浆材速生、纤维素含量高和产量高、木质素含量低的基本要求,可在生产中进一步推广。     

Free Energy Diagram for the Heterogeneous Enzymatic Hydrolysis of Glycosidic Bonds in Cellulose

Kinetic and thermodynamic data have been analyzed according to transition state theory and a simplified reaction scheme for the enzymatic hydrolysis of insoluble cellulose. For the cellobiohydrolase Cel7A from Hypocrea jecorina (Trichoderma reesei), we were able to measure or collect relevant values for all stable and activated complexes defined by the reaction scheme and hence propose a free energy diagram for the full heterogeneous process. For other Cel7A enzymes, including variants with and without carbohydrate binding module (CBM), we obtained activation parameters for the association and dissociation of the enzyme-substrate complex. The results showed that the kinetics of enzyme-substrate association (i.e. formation of the Michaelis complex) was almost entirely entropy-controlled and that the activation entropy corresponded approximately to the loss of translational and rotational degrees of freedom of the dissolved enzyme. This implied that the transition state occurred early in the path where the enzyme has lost these degrees of freedom but not yet established extensive contact interactions in the binding tunnel. For dissociation, a similar analysis suggested that the transition state was late in the path where most enzyme-substrate contacts were broken. Activation enthalpies revealed that the rate of dissociation was far more temperature-sensitive than the rates of both association and the inner catalytic cycle. Comparisons of one- and two-domain variants showed that the CBM had no influence on the transition state for association but increased the free energy barrier for dissociation. Hence, the CBM appeared to promote the stability of the complex by delaying dissociation rather than accelerating association.     

Enzymatic saccharification of cellulose in membrane reactors

The kinetics of enzymatic saccharification of ball-milled sugar-cane bagasse, sorghum stubble and peanut shells was studied and their conversions compared. Particle size analyses were performed on the bagasse sample and pure cellulose (Solka-Floc). It was revealed that most of the size reduction of cellulose particle took place between 0 5% conversion. Means of using commercially available ultrafiltration units as continuous-flow membrane reactors to reduce glucose inhibition were tested and compared using Solka-Floc as substrate. It was pointed out that a low conversion CSTR placed between a ball-mill and a hollow-fibre cartridge could reduce the cost of pretreatment and prevent possible blockage of hollow fibres.     

Ion-mediated increase in the hydraulic conductivity of Laurel stems: role of pits and consequences for the impact of cavitation on water transport

Changes in hydraulic conductivity (K(h)) were measured in stems of Laurus nobilis L. during perfusion with KCl, NaCl or sucrose solutions. Ionic solutes induced marked increase of K(h) with respect to deionized water but sucrose had no effect. The kinetics of KCl-induced K(h) increase was measured together with changes in [K(+)] of the perfused solution. K(h) increases were paralleled by increases in the [K(+)](out)/[K(+)](in) ratio. Samples of different lengths or with increasing percentage loss of conductivity (PLC) due to xylem cavitation were tested, with the aim of increasing radial flow through intervessel pits. KCl solutions enhanced the K(h) of 12-cm-long samples with a concentration-dependent effect up to 100 mm KCl. DeltaK(h) increased from 3 to 30% in 1.5- and 12-cm-long samples, respectively and remained constant for longer samples. Increasing PLC induced an exponential increase in DeltaK(h). PLC measured with KCl solutions was significantly less than that measured with deionized water, suggesting that measurements of PLC can be affected by the composition of the perfused solution. Experiments support the hypothesis that the 'ionic effect' is mediated by physico-chemical changes of pectins of the pit membranes and raise the possibility that plants might alter the ionic composition of the xylem sap to alleviate the hydraulic impact of cavitation.     

Direct Tests of Enzymatic Heme Degradation by the Malaria Parasite Plasmodium falciparum

Malaria parasites generate vast quantities of heme during blood stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygenase (HO)-like protein from Plasmodium falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with PfHO''s lack of a heme-coordinating His residue and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood stage infection.     

Enzymatic digestion of alkaline‐sulfite pretreated sugar cane bagasse and its correlation with the chemical and structural changes occurring during the pretreatment step

Sugar cane bagasse is recalcitrant to enzymatic digestion, which hinders the efficient conversion of its polysaccharides into fermentable sugars. Alkaline‐sulfite pretreatment was used to overcome the sugar cane bagasse recalcitrance. Chemical and structural changes that occurred during the pretreatment were correlated with the efficiency of the enzymatic digestion of the polysaccharides. The first 30 min of pretreatment, which removed approximately half of the initial lignin and 30% of hemicellulose seemed responsible for a significant enhancement of the cellulose conversion level, which reached 64%. After the first 30 min of pretreatment, delignification increased slightly, and hemicellulose removal was not enhanced; however, acid groups continued to be introduced into the residual lignin. Water retention values were 145% to the untreated bagasse and 210% to the bagasse pretreated for 120 min and fiber widths increased from 10.4 to 30 μm, respectively. These changes were responsible for an additional increase in the efficiency of enzymatic hydrolysis of the cellulose, which reached 92% with the 120 min pretreated sample. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:890–895, 2013     

Xylem perfusion of tap root segments of Plantago maritima: the physiological significance of electrogenic xylem pumps

Abstract A method is described for perfusing xylem vessels in tap root segments of the halophyte P. maritima. Use of excised segments allowed recording of the trans-root potential (TRP) at both ends of a segment. It was shown that there can be a spatial variation of electrogenic ion pump activity along the xylem in one root segment. The pH of perfusion solutions, differing in buffering capacity, was adjusted by the root segment to pH 5.1–5.6 during How through the xylem. This pH range was similar to that of sap produced by root pressure. The K⁺ activity in the outflow solution (K⁺_out) was rather constant at 12–13 mol m^?l3 despite input K⁺ activities ranging from 8 to 20 mol m^?l3. Addition of fusicoccin (10^?l2 mol m^?l3) to the perfusion solution induced a strong acidification of the xylem sap, a decrease in K⁺_out and an increase in Na⁺_out. Inhibition of aerobic respiration through anoxia inhibited electrogenic proton pumping into the xylem and led to an increase in K⁺_out and a decrease in Na⁺_out. It is suggested that transport of K⁺ and Na⁺ to the shoot of the halophyte P. maritima is regulated in the tap root by means of ion exchange between xylem vessels and xylem parenchyma and that this exchange is energized by proton translocating ATPases.