Prebleaching of kraft pulp with full-length and truncated forms of a thermostable modular xylanase from Rhodothermus marinus

Full-length and truncated forms of a modular thermostable xylanase (EC 3.2.1.8., glycoside hydrolase family 10) were used in bleaching sequences of hardwood and softwood kraft pulps. Enzymatic treatment led to brightness gains of all pulps but the result depended on the pulp source. The presence of the additional domains in the full-length enzyme (including carbohydrate-binding modules) did not improve the bleaching process. No significant change in viscosity was seen after enzyme treatments indicating an unaffected pulp fibre length.     

Novel architecture of family-9 glycoside hydrolases identified in cellulosomal enzymes of Acetivibrio cellulolyticus and Clostridium thermocellum

We have sequenced a new gene, cel9B, encoding a family-9 cellulase from a cellulosome-producing bacterium, Acetivibrio cellulolyticus. The gene includes a signal peptide, a family-9 glycoside hydrolases (GH9) catalytic module, two family-3 carbohydrate-binding modules (CBM3c-CBM3b tandem dyad) and a C-terminal dockerin module. An identical modular arrangement exists in two putative GH9 genes from the draft sequence of the Clostridium thermocellum genome. The three homologous CBM3b modules from A. cellulolyticus and C. thermocellum were overexpressed, but, surprisingly, none bound cellulosic substrates. The results raise fundamental questions concerning the possible role(s) of the newly described CBMs. Phylogenetic analysis and preliminary site-directed mutagenesis studies suggest that the catalytic module and the CBM3 dyad are distinctive in their sequences and are proposed to constitute a new GH9 architectural theme.     

Additional Carbohydrate-Binding Modules Enhance the Insoluble Substrate-Hydrolytic Activity of β-1,3-Glucanase from Alkaliphilic Nocardiopsis sp. F96

β-1,3-Glucanase (BglF) from Nocardiopsis sp. F96 is composed of only a catalytic domain. To improve the enzymatic properties of BglF, we attempted to construct chimeric enzymes consisting of BglF and some carbohydrate-binding modules, such as the C-terminal additional domain (CAD) and the N-terminal additional domain (NAD) of β-1,3-glucanase H from Bacillus circulans IAM1165 and the chitin-binding domain (ChBD) of chitinase from alkaliphilic Bacillus sp. J813. CAD-fused BglF (BglF-CAD), NAD-fused BglF (NAD-BglF), both NAD- and CAD-fused BglF (NAD-BglF-CAD) and ChBD-fused BglF (BglF-ChBD) were constructed and characterized. The addition of CAD caused increases in binding abilities and hydrolytic activities toward insoluble β-1,3-glucans. As well as BglF-CAD, the binding ability and hydrolytic activity of BglF-ChBD toward pachyman were also increased. The hydrolytic activity of BglF-CAD at pH 9–10 was higher than that of BglF. The relative activities of BglF-CAD and BglF-ChBD at around 50–70 °C were higher than that of BglF.     

Anti-tumor Effect Induced by the DEPC-treated MH134 Cells in C3H Mouse

The mechanism of association of pectin by calcium ions was studied to elucidate the gelling process. The molecular weight and size were determined by light scattering measurements on samples of pectin demethylated in gradation (ELM-pectin) by pectinesterase from Aspergillus japonicus, acid demethylated pectin (CLM-pectin), and sodium polygalacturonic acid (PGA). The molecular size of ELM-pectin which was prepared from identical materials increased quantitatively as demethylation progressed. The molecular size of CLM-pectin and PGA was larger than ELM-pectin even though the methoxyl content was similar. This probably resulted from differences in molecular structure. When Ca²⁺ was added to ELM-pectin, as demethylation progressed, molecular weight increased due to cross-linking induced by Ca^{2 +} ; however, the increase was small, when Ca²⁺ was added to CLM-pectin, molecular weight increased greatly; however, the molecular size was small, and a slight contraction of molecular was caused by cross-linking, Ca²⁺ addition to PGA resulted in enhancement of phenomena observed with CLM-pectin.     

Catalytic properties of the bifunctional soybean beta-glucan-binding protein, a member of family 81 glycoside hydrolases

The beta-glucan-binding protein (GBP) of soybean (Glycine max L.) has been shown to contain two different activities. As part of the plasma membrane-localized pathogen receptor complex, it binds a microbial cell wall elicitor, triggering the activation of defence responses. Additionally, the GBP is able to hydrolyze beta-1,3-glucans, as present in the cell walls of potential pathogens. The substrate specificity, the mode of action, and the stereochemistry of the catalysis have been elucidated. This defines for the first time the inverting mode of the catalytic mechanism of glycoside hydrolases belonging to family 81.     

Structure,Dynamics, and Specificity of Endoglucanase D from Clostridium cellulovorans

The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the β-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising an N-terminal catalytic domain and a C-terminal carbohydrate-binding module, which is attached via a flexible linker. Here, we present the 2.1-Å-resolution crystal structures of full-length EngD with and without cellotriose bound, solution small-angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active-site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases, the active-site cleft of EngD is partially enclosed by Trp162 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products, suggesting that Trp162 plays a direct role in substrate binding.     

The first structure of a glycoside hydrolase family 61 member, Cel61B from Hypocrea jecorina, at 1.6 A resolution

The glycoside hydrolase (GH) family 61 is a long-recognized, but still recondite, class of proteins, with little known about the activity, mechanism or function of its more than 70 members. The best-studied GH family 61 member, Cel61A of the filamentous fungus Hypocrea jecorina, is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. We present here the first structure for this family, that of Cel61B from H. jecorina. The best-quality crystals were formed in the presence of nickel, and the crystal structure was solved to 1.6 Å resolution using a single-wavelength anomalous dispersion method with nickel as the source of anomalous scatter. Cel61B lacks a carbohydrate-binding module and is a single-domain protein that folds into a twisted β-sandwich. A structure-aided sequence alignment of all GH family 61 proteins identified a highly conserved group of residues on the surface of Cel61B. Within this patch of mostly polar amino acids was a site occupied by the intramolecular nickel hexacoordinately bound in the solved structure. In the Cel61B structure, there is no easily identifiable carbohydrate-binding cleft or pocket or catalytic center of the types normally seen in GHs. A structural comparison search showed that the known structure most similar to Cel61B is that of CBP21 from the Gram-negative soil bacterium Serratia marcescens, a member of the carbohydrate-binding module family 33 proteins. A polar surface patch highly conserved in that structural family has been identified in CBP21 and shown to be involved in chitin binding and in the protein's enhancement of chitinase activities. The analysis of the Cel61B structure is discussed in light of our continuing research to better understand the activities and function of GH family 61.     

Preparation of Triacylglycerol Molecular Species by Interesterification Using Endocellular Lipase in n-Hexane

The interesterification of triacylglycerol with fatty acid was done to prepare triacylglycerol molecular species. Optimum operating conditions for the interesterification using a 1,3-positional specific endocellular lipase from Rhizopus japonicus NR400 in a batch system were investigated. The reaction was done at 40°C for 5 hr in the following system: Trioleoylglycerol-palmitic acid = 1:3.5 (mol/mol), 10 ml n-hexane/g trioleoylglycerol, and 2500 units of enzyme/g trioleoylglycerol. Under these conditions, the content of palmitoyl groups in 1,3-positions of triacylglycerol was about 60 mol%. Additional interesterification (2-cycle reaction) using palmitic acid and the novel triacylglycerol prepared by one-step interesterification (1-cycle reaction) resulted in a preparation of highly pure 1,3-dipalmitoyl-2-oleoylglycerol.     

Physical association of the catalytic and helper modules of a family-9 glycoside hydrolase is essential for activity

Clostridium thermocellum cellulase 9I (Cel9I) is a non-cellulosomal tri-modular enzyme, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b). The presence of CBM3c was previously shown to be essential for activity, however the mechanism by which it functions is unclear. We expressed the three recombinant modules independently in Escherichia coli and examined their interactions. Non-denaturing gel electrophoresis, isothermal titration calorimetry, and affinity purification of the GH9-CBM3c complex revealed a specific non-covalent binding interaction between the GH9 module and CBM3c. Their physical association was shown to recover 60-70% of the intact Cel9I endoglucanase activity.

Structured summary:

MINT-6946626:Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)MINT-6946649:Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by molecular sieving (MI:0071)MINT-6946687:Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by isothermal titration calorimetry (MI:0065)MINT-6946706:Cel9I (uniprotkb:Q02934) binds (MI:0407) to Cel9I (uniprotkb:Q02934) by pull down (MI:0096)     

Recognition of cellooligosaccharides by a family 28 carbohydrate-binding module

The crystal structures of a carbohydrate-binding module (CBM) family 28 domain of endoglucanase Cel5A from Clostridium josui have been determined in ligand-free and complex forms with cellobiose, cellotetraose, and cellopentaose as the first complex structures of this family. In the cleft of a β-sandwich fold, the ligands are recognized by stacking interactions and hydrogen bonds. Conformations of the bound cellooligosaccharides are similar to those in crystals and solution but clearly different from the cellulose structure. Interestingly, the glucan chain bound on CBM28 is in the opposite direction of that bound to CBM17, although these families share significant structural similarity.     

Specific recognition of saturated and 4,5-unsaturated hexuronate sugars by a periplasmic binding protein involved in pectin catabolism

The process of pectin depolymerization by pectate lyases and glycoside hydrolases produced by pectinolytic organisms, particularly the phytopathogens from the genus Erwinia, is reasonably well understood. Indeed each extracellular and intracellular catabolic stage has been identified using either genetic, bioinformatic or biochemical approaches. Nevertheless, the molecular details of many of these stages remain unknown. In particular, the mechanism and ligand binding profiles for the transport of pectin degradation products between cellular compartments remain entirely uninvestigated. Here we present the structure of TogB, a 45.7 kDa periplasmic binding protein from Yersinia enterocolitica. This protein is a component of the TogMNAB ABC transporter involved in the periplasmic transport of oligogalacturonides. In addition to the unliganded complex (at 2.2 A), we have also determined the structures of TogB in complex with digalacturonic acid (at 2.2 A), trigalacturonic acid (at 1.8 A) and 4,5-unsaturated digalacutronic acid (at 2.3 A). The molecular determinants of oligogalacturonide binding include a novel salt-bridge between the non-reducing sugar uronate group, selectivity for the unsaturated ligand, and the overall sugar configuration. Complementing this are UV difference and isothermal titration calorimetry experiments that highlight the thermodynamic basis of ligand specificity. The ligand binding profiles of the TogMNAB transporter complex nicely complement pectate lyase-mediated pectin degradation, which is a significant component of pectin depolymerization reactions.     

Domain coupling in a multimodular cellobiohydrolase CbhA from Clostridium thermocellum

Cellobiohydrolase A (CbhA) from Clostridium thermocellum is composed of an N-terminal carbohydrate-binding domain 4 (CBD4), an immunoglobulin-like domain (Ig), a glycoside hydrolase 9 (GH9), X1(1) and X1(2) domains, a CBD3, and a dockerin domain. All domains, except the Ig, bind Ca2+. The following constructs were made: X1(2), X1(1)X1(2), CBD3, X1(1)X1(2)-CBD3, Ig, GH9, Ig-GH9, Ig-GH9-X1(1)X1(2), and Ig-GH9-X1(1)X1(2)-CBD3. Interactions between domains in (1) buffer, (2) with Ca2+, or (3) ethylenediaminetetraacetic acid (EDTA) were studied by differential scanning calorimetry. Thermal unfoldings of all constructs were irreversible. Calcium increased T(d) and cooperativity of unfolding. Multi-domain constructs exhibited more cooperative unfolding in buffer and in the presence of EDTA than did individual domains. They denatured by mechanism simpler than expected from their modular architecture. The results indicate that domain coupling in thermophilic proteins constitutes a significant stabilizing factor.     

The proteins involved in sucrose synthesis in the marine cyanobacterium Synechococcus sp. PCC 7002 are encoded by two genes transcribed from a gene cluster

It has been reported that higher plants and cyanobacteria synthesize sucrose (Suc) by a similar sequential action of sucrose-phosphate synthase (SPS) and sucrose-phosphate phosphatase (SPP). In the genome of the marine unicellular cyanobacterium Synechococcus sp. PCC 7002 there is a sequence that was not annotated as a putative SPP encoding gene (sppA), although the sequence was available. In this study, we functionally characterize the sppA gene of that strain and demonstrate that it is cotranscribed with spsA, the SPS encoding gene. This is the first report on the coordination of Suc synthesis gene expression in an oxygenic-photosynthetic organism.     

A novel petal up-regulated PhXTH7 promoter analysis in Petunia hybrida by using bioluminescence reporter gene

Flower opening is an important phenomenon in plant that indicates the readiness of the flower for pollination leading to petal expansion and pigmentation. This phenomenon has great impact on crop yield, which makes researches of its mechanism attractive for both plant physiology study and agriculture. Gene promoters directing the expression in petal during the petal cell wall modification and expansion when flower opens could be a convenient tool to analyze or monitor gene expression targeting this event. However, there are no reports of isolated gene promoters that can direct gene expression in petal or petal limb during the rapid cell wall dynamics when the flower opens. Xyloglucan endotransglucosylase/hydrolase 7 (XTH7), a cell wall modifying enzyme, was reported having up-regulated gene expression in the petal of Arabidopsis thaliana and Petunia hybrida. In this study, we fused a 1,904 bp length P. hybrida XTH7 promoter with a gene encoding a bright bioluminescent protein (Green enhanced Nano-lantern) to report gene expression and observed petal up-regulated bioluminescence activity by means of a consumer-grade camera. More importantly, this novel promoter demonstrated up-regulated activity in the petal limb of P. hybrida matured flower during flower opening. P. hybrida XTH7 promoter would be a useful tool for flowering study, especially for petal expansion research during flower opening.     

家蚕 Fhx/P25 基因的一种新的转录模式分析研究

家蚕 Fhx/P25 蛋白是丝素蛋白的主要成分之一,过去报道只在家蚕后部丝腺特异的转录表达 . 通过对大规模的家蚕 EST 序列分析发现, Fhx/P25 基因不仅在家蚕后部丝腺高效转录,而且在家蚕幼虫五龄第三天的卵巢组织及其他组织也有转录;分析还发现 Fhx/P25 基因在丝腺和卵巢组织中有不同的转录起始位点,在卵巢组织中的转录起始位点比在丝腺中的至少要提前 115 bp 左右 . 用 RT-PCR 和 FQ-PCR 进一步验证,以上分析结果均正确 . 分析还发现 Fhx/P25mRNA 存在选择性拼接 . 以上结果表明 Fhx/P25 基因并不是组织特异转录基因,它的转录表达存在复杂的调控机制,可能还有其他功能 .