Purification, biochemical characterization, and structure of recombinant endo-1,4-β-xylanase XylE

The gene xylE encoding endo-1,4-β-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding β-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ_1/2 of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K _m) and 75 μmol/min per mg (V _max) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 ? resolution. The 3D structure of an endo-1,4-β-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.     

Mutagenit?tsuntersuchungen mit 1,4-Dimethylsulfonoxy-1,4-Dimethylbutan anDrosophila melanogaster

Zusammenfassung Die orale Verabfolgung von Dimethyl-Myleran löste in den Männchen des Stammes Berlin wild vonDrosophila melanogaster etwa 6% geschlechtsgebundene Letalmutationen aus. Die beobachteten Mutationen wurden hauptsächlich in postmeiotischen Keimzellstadien induziert. Die Fertilität der behandelten Tiere wurde erheblich gesenkt.Dimethyl-Myleran wirkt unter vergleichbaren Bedingungen etwa dreimal stärker mutagen und deutlich stärker sterilisierend als Myleran. Die Sensibilität des Spermatogenesezyklus gegenüber der mutagenen Wirkung von Dimethyl-Myleran und Myleran ist gleich. Die vermutlichen Ursachen für die unterschiedliche biologische Wirksamkeit der beiden Verbindungen werden diskutiert.     

O-Succinyl-l-homoserine-based C4-chemical production: succinic acid,homoserine lactone, γ-butyrolactone, γ-butyrolactone derivatives,and 1,4-butanediol

There has been a significant global interest to produce bulk chemicals from renewable resources using engineered microorganisms. Large research programs have been launched by academia and industry towards this goal. Particularly, C4 chemicals such as succinic acid (SA) and 1,4-butanediol have been leading the path towards the commercialization of biobased technology with the effort of replacing chemical production. Here we present O-Succinyl-l-homoserine (SH) as a new, potentially important platform biochemical and demonstrate its central role as an intermediate in the production of SA, homoserine lactone (HSL), γ-butyrolactone (GBL) and its derivatives, and 1,4-butanediol (BDO). This technology encompasses (1) the genetic manipulation of Escherichia coli to produce SH with high productivity, (2) hydrolysis into SA and homoserine (HS) or homoserine lactone hydrochloride, and (3) chemical conversion of either HS or homoserine lactone HCL (HSL·HCl) into drop-in chemicals in polymer industry. This production strategy with environmental benefits is discussed in the perspective of targeting of fermented product and a process direction compared to petroleum-based chemical conversion, which may reduce the overall manufacturing cost.     

Expression change of β-1,4 galactosyltransferase I, V mRNAs and Galβ1,4GlcNAc group in rat sciatic nerve after crush

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of β-1,4-galactosylation is performed by a family of β-1,4-galactosyltransferases (β-1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. β-1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides. In the present study, Real-time PCR revealed that the β-1,4-GalT I and V mRNAs reached peaks at 2 w after sciatic nerve crush. In situ hybridization showed that at 1 d after sciatic nerve crush, the expression levels of β-1,4-GalT I and V mRNAs were strong at the crush site, and decreased gradually from crush site to the distal segments. In addition, combined in situ hybridization for β1,4-GalT I and V mRNAs and immunohistochemistry for S100 showed that β1,4-GalT I and V mRNAs were mainly located in Schwann cells. Lectin blot showed that the expression of Galβ1,4GlcNAc group increased at 6 h immediately, reached a peak at 12 h and remained elevated up to 4 w after sciatic nerve crush. In conclusion, β1,4-GalT I and V might play important roles in the regeneration of the injuried sciatic nerve, and upregulation of Galβ1,4GlcNAc group might be correlated with the process of the sciatic nerve injury.     

Absorption of the indigestible disaccharide, β-1,4-mannobiose, from coconut by the rat portal vein

The intestinal absorption of β-1,4-mannobiose by rats was investigated. Mannobiose was detected in the portal vein plasma by matrix-assisted laser desorption ionization/time of flight mass spectrometry after its administration to rats. The presence of mannobiose in the rat plasma was confirmed by an experiment using β-mannosidase. These results indicate that mannobiose was directly absorbed through the intestines even without being hydrolyzed.     

OsGLU1, A Putative Membrane-bound Endo-1,4-ß-D-glucanase from Rice, Affects Plant Internode Elongation

A dwarf mutant glu was identified from screening of T-DNA tagged rice population. Genetic analysis of the T1 generation of glu revealed that a segregation ratio of wild-type:dwarf phenotype was 3:1, suggesting that the mutated phenotype was controlled by a single recessive nuclear locus. The mutated gene OsGLU1, identified by Tail-PCR, encodes a putative membrane-bound endo-1,4-β-D-glucanase, which is highly conserved between mono- and dicotyledonous plants. Mutation of OsGLU1 resulted in a reduction in cell elongation, and a decrease in cellulose content but an increase in pectin content, suggesting that OsGLU1 affects the internode elongation and cell wall components of rice plants. Transgenic glu mutants harboring the OsGLU1 gene complemented the mutation and displayed the wild-type phenotype. In addition, OsGLU1 RNAi plants showed similar phenotype as the glu mutant has. These results indicate that OsGLU1 plays important roles in plant cell growth. Gibberellins and brassinosteroids induced OsGLU1 expression. In rice genome, endo-1,4-β-D-glucanases form a multiple gene family with 15 members, and each may have a distinct expression pattern in different organs. These results indicate that endo-1, 4-β-D-glucanases may play diverse roles in growth and developmental process of rice plants. Hua-Lin Zhou, Si-Jie He: These authors contributed equally to this work     

cDNA cloning and bacterial expression of an endo-β-1,4-mannanase, AkMan, from Aplysia kurodai

Previously we isolated an endo-β-1,4-mannanase (EC 3.2.1.78), AkMan, from the digestive fluid of a common sea hare Aplysia kurodai and demonstrated that this enzyme had a broad pH optimum spanning 4.0 to 7.5 and an appreciably high heat stability in this pH range (Zahura et al., Comp. Biochem. Physiol., B157, 137-148 (2010)). In the present study, we cloned the cDNA encoding AkMan and constructed a bacterial expression system for this enzyme to enrich information about the primary structure and the characteristic properties of this enzyme. cDNA fragments encoding AkMan were amplified by PCR followed by 5'- and 3'-RACE PCRs from the A. kurodai hepatopancreas cDNA using degenerated primers designed on the basis of partial amino-acid sequences of AkMan. The cDNA including entire translational region of AkMan consisted of 1392bp and encoded 369 amino-acid residues. The N-terminal region of 17 residues of the deduced sequence except for the initiation Met was regarded as the signal peptide of AkMan and the mature enzyme region was considered to comprise 351 residues with a calculated molecular mass of 39961.96Da. Comparison of the primary structure of AkMan with other β-1,4-mannanases indicated that AkMan belongs to the subfamily 10 of glycosyl-hydrolase-family-5 (GHF5). Phylogenetic analysis for the GHF5 β-1,4-mannanases indicated that AkMan together with other molluscan β-1,4-mannanases formed an independent clade of the subfamily 10 in the phylogenetic tree. The recombinant AkMan (recAkMan) was expressed with an Escherichia coli BL21(DE3)-pCold1 expression system as an N-terminal hexahistidine-tagged protein and purified by Ni-NTA affinity chromatography. The recAkMan showed the broad pH optimum in acidic pH range as did native AkMan; however, heat stability of recAkMan was considerably lower than that of native enzyme. This may indicate that the stability of AkMan is derived from an appropriate folding and/or some posttranslational modifications in Aplysia cells.     

Recombinant expression,characterization, and pulp prebleaching property of a Phanerochaete chrysosporium endo-β-1,4-mannanase

A Phanerochaete chrysosporium cDNA predicted to encode endo-1,4-β-d-mannanase, man5D, was cloned and expressed in Aspergillus niger. The coding region of the gene man5D was predicted to contain, in order from the N-terminal: a secretory signal peptide, cellulose-binding domain, linker region, and glycosyl hydrolase family 5 catalytic site. The enzyme was purified from culture filtrate of A. niger transformants that carried the recombinant man5D. Recombinant Man5D had an apparent molecular size of about 65 kDa by SDS-PAGE, and optimal activity at pH 4.0–6.0 and 60 °C. It was stable from pH 4.0 to 8.0 and up to 60 °C. The enzyme showed affinity for Avicel cellulose, suggesting that the predicted cellulose-binding domain is biologically functional. The specific activities of Man5D on mannan, galactomannan, and glucomannan at pH 5 and 60 °C ranged from 160 to 460 μmol/(min mg), with apparent K_m values from 0.54 to 2.3 mg/mL. Product analysis results indicated that Man5D catalyzes endo-cleavage, and appears to have substantial transglycosylase activity. When used to treat softwood kraft pulp, Man5D hydrolyzed mainly glucomannan and exhibited a positive effect as a prebleaching agent. Compared to a commercial prebleaching with xylanase, the prebleaching effect of Man5D was weaker but with reduced loss of fibre yield as determined by the release of solubilized sugars.     

α-1,4-Glucan lyase,a new class of starch/glycogen-degrading enzyme

Antibodies have been raised against an -1,4-glucan lyase purified from the red alga Gracilariopsis lemaneiformis (Bory) Dawson, Acleto et Foldvik. Localization of -1,4-glucan lyase in ultra-thin sections of the red alga was performed using immunogold/transmission electron microscopy. The enzyme was found exclusively in the stroma of the chloroplasts of the algal cells, not in the cell wall, cytosol or around the cytosolic starch granules. Partial amino-acid sequences of the algal lyase, with a total length of 100 amino-acid residues, were obtained. No sequence homology was found with proteins and peptides of known sequences.This work was supported by grants from the Swedish Council for Planning and Coordination of Research (FRN), Carl Tryggers Foundation, and Hierta-Retzius Fund. We thank Ms Katrin Österlund and Anette Axén for their expert technical assistance with this work.     

An α-glucoside of 1,4-dideoxy-1,4-imino-d-lyxitol with an eleven carbon side chain

The distribution of pyrrolidine-type iminosugars with a long-side chain appears to be restricted to the relatively unrelated plant families Moraceae, Campanulaceae, and Hyacinthaceae. In a search for glycosidase inhibitors in these plant families, we isolated the 1,4-dideoxy-1,4-imino-d-lyxitol (DIL) glucoside bearing the 1,2,11-trihydroxyundec-4-ene side chain at the C-1α position from the roots of Adenophora triphylla. This iminosugar was a powerful and selective inhibitor of coffee bean α-galactosidase, with an IC₅₀ value of 8 μM.     

Identification and characterization of the Sda β1,4,N-acetylgalactosaminyltransferase from pig large intestine

The high occurrence in large intestine epithelial cells from pig of a -N-acetylgalactosaminyltransferase with a substrate specificity very similar to that of the Sd^a 1,4-N-acetylgalactosaminyltransferase from other tissues is reported. The enzyme strictly recognized the NeuAc2,3Gal terminal sequence ofN- andO-linked oligosaccharides bound to glycoproteins. The transferase activity required Mn²⁺ and an optimum pH of 7.4. In contrast to the kidney Sd^a-enzyme from humans and other mammals, the microsomal fraction of pig colonic cells expressed a very high activity even in the absence of Triton X-100. A rapid procedure is presented for the large scale preparation of GalNAc1,4(NeuAc2,3)Gal1,4Glc from NeuAc2,3Gal1,4Glc. The biosynthesized tetrasaccharide was completely resistant to the action of neuraminidase fromVibrio cholerae, whereas about 60% ofN-acetylneuramic acid was cleaved by neuraminidase from Newcastle disease virus. HPLC separation of different compounds is reported.     

Distinct expression profiles of UDP-galactose: β-D-galactoside α1,4-galactosyltransferase and UDP-galactose: β-D-galactoside β1,4-galactosyltransferase in pigeon, ostrich and chicken

We previously identified two novel enzymes in pigeon, α1,4- and β1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Galα1-4Gal and Galβ1-4Gal sequences on glycoproteins, respectively. No such glycan structures and/or enzymes have been found in mammals, suggesting that the expression of these enzymes diverged during the course of vertebrate evolution. To compare their expression profiles among avian species, we first established a method for detecting the activities of these two GalTs based on the two-dimensional high pressure liquid chromatography mapping technique, using 2-aminopyridine-derivatized asialo-biantennary N-glycans as an acceptor substrate. When we analyzed the activities of GalTs in pigeon liver extracts in the presence of UDP-Gal, 13 different products containing Galα1-4Galβ1-4GlcNAc, Galβ1-4Galβ1-4GlcNAc and/or Galα1-4Galβ1-4Galβ1-4GlcNAc branches were identified. The newly formed glycosidic linkages of the enzymatic products were determined by nuclear magnetic resonance and methylation analysis, as well as by galactosidase digestions. The activities of both α1,4- and β1,4-GalTs were detected in various tissues in pigeon, although their relative activities were different in each tissue. In contrast, ostrich expressed β1,4-GalT, but not α1,4-GalT, in all tissues analyzed, whereas neither α1,4- nor β1,4-GalT activity was detected in chicken. These results indicate that α1,4- and β1,4-GalTs are expressed in a species-specific manner and are distributed throughout the entire body of pigeon or ostrich when the enzymes are present.     

Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50℃ and 60 ℃; for EG45 it was 50 ℃. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 ℃, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 ℃ for 24 h. However, less than 10% residual activity of EG45 was detected at 50 ℃. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan （from oat spelt） and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.     

Purification, characterization and modular organization of a cellulose-binding protein, CBP105, a processive β-1,4-endoglucanase from Cellulomonas flavigena

A cellulose-binding protein of 105 kDa (CBP105) from Cellulomonas flavigena was purified and its gene was cloned. CBP105 is a processive endoglucanase with maximum activity on carboxymethyl cellulose (CMC) at pH 7.5 and 60°C. Limited proteolysis suggested that CBP105 is composed of one catalytic domain (CD) and two carbohydrate-binding modules (CBM). The nucleotide sequence of the cbp105 gene (AY729806) indicates that CBP105 is a modular enzyme with a family 9 glycoside hydrolase CD linked to a family 3 CBM, two fibronectin III-like domains and a family 2 CBM. This structural organization may be responsible for CBP105 processive CMC degradation.     

Cytochemical localization of GalNAc and GalNAcβ1,4Galβ1,4 disaccharide in mouse zona pellucida

Carbohydrate residues contained in the zona pellucida play a key role in the process of sperm-egg interaction. In vitro fertilization experiments have shown that a specific monoclonal antibody against GalNAcş,4Galş,4 disaccharide inhibits fertilization in mice. In the present study, the ultrastructural cytochemical localization of GalNAc residues and the GalNAcş,4Galş,4 disaccharide was carried out in ovarian and postovulatory oocytes by using lectin-gold cytochemistry and immunocytochemistry. Plant lectins SBA and DBA showed an affinity for the entire zona pellucida matrix of ovarian oocytes throughout the follicular maturation; however, immunoreactivity for GalNAcş,4Galş,4 disaccharide was not detected in ovarian oocytes at the earliest stages of follicular development but was found to be associated with the inner region of the zona matrix at the trilaminar primary follicle stage. The Golgi apparatus, vesicular aggregates, and cortical granules of the oocyte were intensely labeled by SBA and DBA throughout follicular development. Immunoreactivity to GalNAcş,4Galş,4 disaccharide was first observed in the Golgi apparatus and vesicular aggregates in trilaminar primary follicles. No immunoreactivity was observed in the cortical granules. In postovulatory oocytes, results were similar to those observed in ovarian oocytes. Our results thus suggest that (1) GalNAcş,4Galş,4 disaccharide residues are present only in the inner region of the zona pellucida and, therefore, might be involved in sperm penetration through the zona pellucida, (2) the inner and outer regions of the zona pellucida contain different oligosaccharide chains, (3) the vesicular aggregates detected in the oocyte could represent an intermediate step in the secretory pathway of zona pellucida glycoproteins and might be involved in the formation of cortical granules.     

Production of hydrogen from α-1,4- and β-1,4-linked saccharides by marine hyperthermophilic Archaea

Nineteen hyperthermophilic heterotrophs from deep-sea hydrothermal vents, plus the control organism Pyrococcus furiosus, were examined for their ability to grow and produce H₂ on maltose, cellobiose, and peptides and for the presence of the genes encoding proteins that hydrolyze starch and cellulose. All of the strains grew on these disaccharides and peptides and converted maltose and peptides to H₂ even when elemental sulfur was present as a terminal electron acceptor. Half of the strains had at least one gene for an extracellular starch hydrolase, but only P. furiosus had a gene for an extracellular β-1,4-endoglucanase. P. furiosus was serially adapted for growth on CF11 cellulose and H₂ production, which is the first reported instance of hyperthermophilic growth on cellulose, with a doubling time of 64 min. Cell-specific H₂ production rates were 29 fmol, 37 fmol, and 54 fmol of H₂ produced cell⁻¹ doubling⁻¹ on α-1,4-linked sugars, β-1,4-linked sugars, and peptides, respectively. The highest total community H₂ production rate came from growth on starch (2.6 mM H₂ produced h⁻¹). Hyperthermophilic heterotrophs may serve as an important alternate source of H₂ for hydrogenotrophic microorganisms in low-H₂ hydrothermal environments, and some are candidates for H₂ bioenergy production in bioreactors.     

Pectin Biosynthesis: GALS1 in Arabidopsis thaliana Is a β-1,4-Galactan β-1,4-Galactosyltransferase

β-1,4-Galactans are abundant polysaccharides in plant cell walls, which are generally found as side chains of rhamnogalacturonan I. Rhamnogalacturonan I is a major component of pectin with a backbone of alternating rhamnose and galacturonic acid residues and side chains that include α-1,5-arabinans, β-1,4-galactans, and arabinogalactans. Many enzymes are required to synthesize pectin, but few have been identified. Pectin is most abundant in primary walls of expanding cells, but β-1,4-galactan is relatively abundant in secondary walls, especially in tension wood that forms in response to mechanical stress. We investigated enzymes in glycosyltransferase family GT92, which has three members in Arabidopsis thaliana, which we designated GALACTAN SYNTHASE1, (GALS1), GALS2 and GALS3. Loss-of-function mutants in the corresponding genes had a decreased β-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher β-1,4-galactan content. The plants did not have an obvious growth phenotype. Heterologously expressed and affinity-purified GALS1 could transfer Gal residues from UDP-Gal onto β-1,4-galactopentaose. GALS1 specifically formed β-1,4-galactosyl linkages and could add successive β-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as β-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties.     

ROOT GROWTH INHIBITING, a Rice Endo-1,4-β-d-Glucanase, Regulates Cell Wall Loosening and is Essential for Root Elongation

The molecular mechanism involved in cell wall dynamics has not been well clarified, although it is quite important for organ growth. We characterized a rice mutant, root growth inhibiting (rt), which is defective in root elongation. The rt mutant showed a severe defect in cell elongation at the root-elongating zone with additional collapse of epidermal and cortex cells at the root tip caused by the defect in the smooth exfoliation of root cap cells. Consistent with these phenotypes, expression of the RT gene, which encodes a member of the membrane-anchored endo-1,4-??-d-glucanase, was specifically localized in the root-elongating zone and at the junction between epidermal and root cap cells. The enzymatic analysis of root extracts from the wild-type and rt mutant indicated that RT hydrolyzes noncrystalline amorphous cellulose. The cellulose content was slightly increased but the crystallinity of cellulose was decreased in the rt root. In addition, the hemicellulose composition was different between wild-type and rt roots. The total extensibility was significantly lower in the rt root explants. Based on these results, we concluded that RT is involved in the disassembly of the cell wall for cell elongation in roots as well as for root cap exfoliation from the epidermal cell layer by hydrolyzing the noncrystalline amorphous cellulose fibers of cellulose microfibrils resulting in loosening of the hemicellulose and cellulose interaction.