Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of<Emphasis Type="Italic"> PHGPX</Emphasis> and<Emphasis Type="Italic"> URE2</Emphasis> genes in aluminum resistance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (Al) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 , phgpx2 , and phgpx3), were tested for their sensitivity to Al. The triple mutant, phgpx1 /2/3, was more sensitive to Al (55% reduction in growth at 300 M Al) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to Al resistance. The hypersensitivity of phgpx3 to Al was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to Al in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3 and phgpx1/2/3 mutants were sensitive to oxidative stress (exposure to H₂O₂ or diamide). Lipid peroxidation was also increased in the phgpx1/2/3 mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1 and gtt2), glutathione biosynthesis (gsh1 and gsh2), glutathione reductase (glr1) and a glutathione transporter (opt1) did not show hypersensitivity to Al relative to the parental strain BY4741. Interestingly, a strain deleted for URE2, a gene which encodes a prion precursor with homology to GSTs, also showed hypersensitivity to Al. The hypersensitivity of the ure2 mutant could be overcome by complementation with URE2. Expression of URE2 in the parental strain increased approximately 2-fold in response to exposure to 100 M Al. Intracellular oxidation levels in the ure2 mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H₂O₂) increase compared to BY4741; however, the ure2 mutant showed no change in lipid peroxidation compared to the control. The phgpx1/2/3 and ure2 mutants both showed increased accumulation of Al. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in Al toxicity/resistance in S. cerevisiae.Communicated by D.Y. Thomas     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the genus <Emphasis Type="Italic">Clivia</Emphasis>

We have developed a protocol for the in vitro propagation of the genus Clivia. Shoots were regenerated when fragments of the peduncle-pedicel junction (PP junction) from young inflorescences were used as explants. The optimal media for PP junction were Murashige and Skoog (MS)-based medium containing 10 M of 6-benzyladenine (BA) and 10 M of 2,4-dichlorophenoxyacetic acid (2,4-D) or MS supplemented with 5 M BA, 10 M -naphthaleneacetic acid (NAA), 250 mg l^-1 glutamine and 500 mg l^–1 casein hydrolysate and their usage depended on the breeding lines. Multiplication from initiations and in vitro seedlings was the best when the explants were cut longitudinally through the meristem and placed on MS plus 44 M BA. Plantlets were transferred on to hormone -free MS medium with charcoal for rooting.     

Antifibrotic effects of <Emphasis Type="Italic">Salvia miltiorrhiza </Emphasis>on dimethylnitrosamine-intoxicated rats

Excessive oxidative stress is implicated in hepatic fibrogenesis. Extracts of Salvia miltiorrhiza (Sm) have been shown to protect cells against oxidative stress. In this study we investigated the in vitro and in vivo effects of Sm on hepatic fibrosis. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with transforming growth factor-1 (TGF-1). The inhibitory effects of Sm (50~400 g/ml) on TGF-1-induced -smooth muscle actin (-SMA) secretion and the mRNA expressions of fibrosis-related genes, including -SMA, connective tissue growth factor (CTGF), and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed. Fibrosis was induced by dimethylnitrosamine (DMN) administration in rats. DMN-treated rats were randomly assigned to 1 of 4 groups: saline, Sm (20 mg/kg), Sm (100 mg/kg), or silymarin (100 mg/kg), each given by gavage twice daily for 5 weeks starting from the onset of DMN administration. Sm (200 and 400 g/ml) significantly inhibited TGF-1-stimulated -SMA secretion and the mRNA expressions of -SMA, CTGF, and TIMP-1 in HSC-T6 cells. Fibrosis scores of livers from DMN-treated rats with either a low (1.8 ± 0.2) or high (1.8 ± 0.1) dose of Sm, or silymarin (1.4 ± 0.2) were significantly reduced in comparison with DMN-treated rats receiving saline (3.1 ± 0.1). Hepatic collagen contents were also significantly reduced by either Sm or silymarin treatment. The mRNA expression levels of -SMA, TGF-1, and procollagen I were all attenuated in Sm- and silymarin-treated rats. Moreover, levels of plasma aspartate transaminase activities were reduced by Sm and silymarin treatment. In conclusion, our results show that Sm exerted antifibrotic effects in both HSC-T6 cells and in rats with DMN-induced fibrosis.     

A novel lectin (morniga M) from mulberry (<Emphasis Type="Italic">Morus nigra</Emphasis>) bark recognizes oligomannosyl residues in N-Glycans

Morniga M is a jacalin-related and mannose-specific lectin isolated from the bark of the mulberry (Morus nigra). In order to understand the function and application of this novel lectin, the binding property of Morniga M was studied in detail using an enzyme-linked lectinosorbent assay and lectin-glycan inhibition assay with extended glycan/ligand collection. From the results, it was found that the di-, tri-, and oligomannosyl structural units of N-glycans such as those of the bovine ₁-acid glycoprotein (gp) and lactoferrin were the most active gps, but not the O-glycans or polysaccharides including mannan from yeast. The binding affinity of Morniga M for ligands can be ranked in decreasing order as follows: gps carrying multiple N-glycans with oligomannosyl residues >> N-glycopeptide with a single trimannosyl core > Tri-Man oligomer [Man1 6(Man 1 3) Man], Penta-Man oligomer [Man1 6(Man1 3)Man1 6(Man1 3) Man] Man 1 2, 3 or 6 Man > Man > GlcNAc, Glc >> L-Fuc, Gal, GalNAc (inactive), demonstrating the unique specificity of this lectin that may not only assist in our understanding of cell surface carbohydrate ligand-lectin recognition, but also provide informative guidelines for the application of this structural probe in biotechnological and clinical regimens, especially in the detection and purification of N-linked glycans.     

Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in<Emphasis Type="Italic"> Xenopus laevis</Emphasis> oocytes

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken 7, chicken 42 and the Drosophila melanogaster/chicken hybrid receptors SAD/2 and ALS/2. No agonist action of NTX (0.1–100 M) was observed on chicken 7, chicken 42 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (7) or heteromeric (42) nicotinic AChRs. Block by NTX of the chicken 7, chicken 42 and the SAD/2 and ALS/2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.     

A Motility Revertant of the <Emphasis Type="Italic">ndvB</Emphasis> Mutant of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

A motility revertant of a Bradyrhizobium japonicum ndvB mutant was isolated and characterized. The ndvB mutants of B. japonicum have been reported to be osmotically sensitive, as well as defective in motility, periplasmic cyclic -(13), (16)-D-glucan synthesis, and symbiosis with soybean. The motility revertant was restored for osmotic tolerance but not for cyclic -glucan production or effective symbiosis. These results support our hypothesis that cyclic -glucans have an important role in symbiosis—the suppression of a plant defense response—in addition to their role in periplasmic osmoprotection.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Plant regeneration from hairy-root cultures transformed by infection with<Emphasis Type="Italic"> Agrobacterium rhizogenes</Emphasis> in<Emphasis Type="Italic"> Catharanthus roseus</Emphasis>

Hypocotyl explants of Catharanthus roseus produced hairy roots when cultured on Murashige and Skoog (MS) basal medium after infection by Agrobacterium rhizogenes. Explants gave rise to adventitious shoots at a frequency of up to 80% when cultured on MS medium supplemented with 31.1 M 6-benzyladenine and 5.4 M -naphthaleneacetic acid. There was a significant difference in the frequency of adventitious shoot formation for each hairy-root line derived from a different cultivar. Plants derived from hairy roots exhibited prolific rooting and had shortened internodes. Approximately half of the plants had wrinkled leaves and an abundant root mass with extensive lateral branching, but otherwise appeared morphologically normal. Plants with hairy roots that were derived from the cultivar Cooler Apricot developed flowers with petals that were white in the proximal region, whereas the wild-type flower petals are red. PCR and Southern blot analyses revealed that plants derived from hairy roots retained the Ri TL-DNA.Abbreviations BA 6-Benzyladenine - MS Murashige and Skoog medium - NAA -Naphthaleneacetic acid - SH Schenk and Hildebrandt mediumCommunicated by I.S. Chung     

Glycoglycerolipid from the Membranes of <Emphasis Type="Italic">Acholeplasma laidlawii</Emphasis> Binds to Human Immunodeficiency Virus-1 (HIV-1) and Accelerates Its Entry into Cells

We have reported previously that glycoglycerolipids derived from the membranes of Acholeplasma laidlawii, 3-O-[2-O-(-D-glucopyranosyl)-6-O-acyl--glucopyranosyl]-1,2-di-O-acyl-sn-glycerols (GAGDGs) bind to human cell lines. In addition, the GAGDGs were found to augment HIV-1 infection in human cell lines. Here we show that GAGDG binds to HIV-1 and facilitates the entry of HIV-1 into cells. The binding ability of GAGDG to HIV-1 was blocked by anti-GAGDG serum. Binding assay with synthetic GAGDGs and related compounds showed that the presence of branching form of acyl chains at the C14 or C16 position, glucose, and the acyl chain binding to the glucose were critical for efficient binding. GAGDG efficiently augmented the entry of HIV-1 into cells in a single-cycle replication assay. These results indicate that GAGDG of A. laidlawii membranes participates in the facilitation of HIV-1 infection.     

The moss<Emphasis Type="Italic"> Physcomitrella patens</Emphasis> releases a tetracyclic diterpene

The presence of the tetracyclic diterpene 16-hydroxykaurane (16-hydroxy-ent-kaurane, C₂₀H₃₄O, CAS 5524–17–4) was detected in sterile cell cultures of the moss Physcomitrella patens (Hedw.) B.S.G. using gas chromatography and mass spectrometry. 16-hydroxykaurane was found to be a major lipid compound in P. patens, with an estimated intracellular concentration of up to 0.84 mmol/l and an extracellular concentration of up to 9.3 µmol/l. The overall content of 16-hydroxykaurane (in milligrams) produced per culture reached 0.37-fold that of chlorophyll a+b. In agar cultures with low air exchange, 16-hydroxykaurane forms needle-like crystals on tissue and on the inner surface of the culture vessels, indicating that it is being released into the atmosphere. Solid phase microextraction confirmed the air-bound release of 16-hydroxykaurane. To our knowledge this is the first report on the release of a plant-derived tetracyclic diterpene into the air.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. ReskiThis work is dedicated to the 65th birthday of Prof. Heinz Hahn.     

The karyology of the cyprinid genera <Emphasis Type="Italic">Scardinius</Emphasis> and <Emphasis Type="Italic">Rutilus</Emphasis> in southern Europe

The chromosomes of eight species of Rutilus and Scardinius, mostly endemic to the Italian and the Balkan peninsula, were analyzed by conventional and other banding techniques. Parallel analyses were conducted also on two leuciscine species, Alburnus albidus, for which we provide the first karyological analysis, and Leuciscus cephalus. All species examined displayed the same karyotype (2n=50 chromosomes, 8 metacentric+13 submetacentric+4 subtelo/acrocentric pairs) with nucleolus organizer regions (NORs) on the ends of the shorter arms of a medium-sized submetacentric pair. In contrast, interspecific variation was observed in the distribution of constitutive heterochromatin. The variation observed in this genomic material proved to be systematically and phylogenetically informative. Indeed, a peritelomeric C-band on the first telocentric pair characterizes species of Rutilus and Scardinius. In both genera heterochromatin differentiation appears to be directed to a centromere–telomere direction, particularly evident along the metacentric elements of their karyotypes.An erratum to this article can be found at     

Regeneration of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) by a cyclic organogenic system

In a five-step procedure, plants were regenerated from meristematic tissue initiated from nodal tissue in four pea cultivars (Espace, Classic, Solara, and Puget). In step 1, stem tissue with one node (1-cm size) was subcultured on medium containing thidiazuron. As a result multiple shoots were produced, appearing normal or swollen at their bases. The multiple shoots were subcultured in the same medium, resulting in the formation of a green hyperhydric tissue in the swollen bases of the multiple shoots, which is fully covered with small buds [bud-containing tissue (BCT)]. In step 2, BCT fragments were isolated and subcultured in the same medium and, as a result, they were able to reproduce themselves in a cyclic fashion. In step 3, subculture of BCT on medium supplemented with a combination of gibberelic acid, 6-benzyladenine and -naphthalene acetic acid (NAA), resulted in the formation of shoots, which were rooted in step 4 on medium supplemented with 0.5 mg/l NAA, indole-3-acetic acid (IAA) or indole-3-butyric acid. In step 5, in vitro plants were transferred to the greenhouse for acclimatisation and further development. The four varieties tested were all able to produce meristematic tissue, suggesting that its production is genotype independent.     

Callus induction and <Emphasis Type="Italic">de novo</Emphasis> regeneration from callus in Guar (<Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>)

Guar (Cyamopsis tetragonoloba L. Taub) is a drought tolerant and multipurpose grain legume cash crop grown primarily under rainfed conditions in several countries. The effect of various growth regulators and their combinations on a variety of explants, namely the embryo, cotyledons, cotyledonary nodes, shoot tip and hypocotyle, has been studied and an efficient system for callus induction and regeneration from callus has been developed. It was established that Murashige and Skoogs culture medium containing 2,4-dichlorophenoxyacetic acid (10.0M) in combination with 6-benzylaminopurine (5.0M) with embryo or cotyledon explants is most suitable for induction of green and friable morphogenic callus, with a range of 82.5–95% of cultured explants responding to callus induction. Efficient de novo shoot regeneration was achieved by culturing the callus obtained on this medium on Murashige and Skoogs medium containing 1-naphthlenacetic acid (13.0M) in combination with 6-benzylaminopurine (5.0M) with a range of 82.1–88.4% of callus clumps producing 20–25 shoots. In vitro rooting of cultured shoots was obtained on half-salt concentration of Murashige and Skoogs culture medium supplied with indole-3-butyric acid (5.0M) on which 82–90% of cultured shoots produced healthy roots. The in vitro regenerated plants were grown to pod setting and subsequent maturity under greenhouse conditions.     

Molecular cloning and characterization of a laccase gene from the basidiomycete<Emphasis Type="Italic"> Fome lignosus</Emphasis> and expression in<Emphasis Type="Italic"> Pichia pastoris</Emphasis>

A cDNA encoding for a laccase was isolated from the white-rot fungus Fome lignosus by RT-PCR. It contained an open reading frame of 1,557 bp. The deduced mature protein consisted of 497 amino acids and was preceded by a signal peptide of 21 amino acids. The genomic DNA of the laccase, containing 11 introns, was cloned by PCR. The cDNA was cloned into the vectors pGAPZA and pGAPZA, and expressed in the Pichia pastoris GS115. Laccase-secreting transformants were selected by their ability to oxidize the substrate 22-azinobis-(3-ethylbenzthiaoline-6-sufonic acid) (ABTS). The laccase activity obtained with the native signal peptide was found to be fivefold higher than that obtained with the -factor secretion signal peptide. The presence of 0.4 mM copper was necessary for optimal activity of the enzyme. The highest activity value reached 9.03 U ml^–1, and the optimal secreting time was 2~3 days at 20°C. The crude laccase was stable in a pH range from 6.0 to 10.0 and at temperatures lower than 30°C in pH4.5 for 24 h. The molecular mass of the enzyme was estimated to be 66.5 kDa by SDS-PAGE. The optimum pH and temperature were 2.4 and 55°C. The K_m and V_max values for ABTS were 177 M and 23.54 mol min^–1 respectively. The extent of glycosylation of the purified enzyme was 58.6%.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Heterologous expression of metabotropic glutamate receptor subtype 1 in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The upstream region of the isocitrate lyase gene (UPR-ICL) from the n-alkane-utilizing yeast Candida tropicalis serves as a useful promoter of gene expression in the yeast Saccharomyces cerevisiae. The production of rat metabotropic glutamate receptor 1 (mGluR1), which belongs to the G-protein-coupled receptor (GPCR) family, was tested under the control of UPR-ICL. Expression of mGluR1 was found in recombinant clones and enhanced by replacing the signal sequence of mGluR1 with the corresponding region of the -factor receptor (Ste2), which is a GPCR found in S. cerevisiae. Moreover, the membrane fraction from a recombinant clone associated with Vesl-1S/Homer-1a protein binds the mGluR1 in rat cerebellum. These results suggest that the UPR-ICL-controlled gene expression system is useful for heterologous GPCRs in S. cerevisiae.     

Organization of genes required for gellan polysaccharide biosynthesis in<Emphasis Type="Italic"> Sphingomonas elodea</Emphasis> ATCC 31461

Sphingomonas elodea ATCC 31461 produces gellan, a capsular polysaccharide that is useful as a gelling agent for food and microbiological media. Complementation of nonmucoid S. elodea mutants with a gene library resulted in identification of genes essential for gellan biosynthesis. A cluster of 18 genes spanning 21 kb was isolated. These 18 genes are homologous to genes for synthesis of sphingan polysaccharide S-88 from Sphingomonas sp. ATCC 31554, with predicted amino acid identities varying from 61% to 98%. Both polysaccharides have the same tetrasaccharide repeat unit, comprised of [4)--l-rhamnose-(13)--d-glucose-(14)--d-glucuronic acid-(14)--d-glucose-(1]. Polysaccharide S-88, however, has mannose or rhamnose in the fourth position and has a rhamnosyl side chain, while gellan has no sugar side chain but is modified by glyceryl and acetyl substituents. Genes for synthesis of the precursor dTDP-l-rhamnose were highly conserved. The least conserved genes in this cluster encode putative glycosyl transferases III and IV and a gene of unknown function, gelF. Three genes (gelI, gelM, and gelN) affected the amount and rheology of gellan produced. Four additional genes present in the S-88 sphingan biosynthetic gene cluster did not have homologs in the gene cluster for gellan biosynthesis. Three of these gene homologs, gelR, gelS, and gelG, were found in an operon unlinked to the main gellan biosynthetic gene cluster. In a third region, a gene possibly involved in positive regulation of gellan biosynthesis was identified.     

Purification and characterization of an<Emphasis Type="Italic"> N</Emphasis>-acetylglucosaminidase produced by a<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> strain which controls<Emphasis Type="Italic"> Crinipellis perniciosa</Emphasis>

Isolate 1051 of Trichoderma harzianum, a mycoparasitic fungus, was found to impair development of the phytopathogen, Crinipellis perniciosa, in the field. This Trichoderma strain growing in liquid medium containing chitin produced substantial amounts of chitinases. The N-acetylglucosaminidase present in the culture-supernatant was purified to homogeneity by gel filtration and hydrophobic interaction chromatography, as demonstrated by SDS-PAGE analysis. The enzyme had a molecular mass of 36 kDa and hydrolyzed the synthetic substrate -nitrophenyl-N-acetylglucosaminide (NGlcNAc) with Michaelis–Menten kinetics. Maximal activities were determined at pH 4.0 and a temperature range of 50–60°C. K _m and V _max values for NGlcNAc hydrolysis were 8.06 moles ml^–1 and 3.36 moles ml^–1 min^–1, respectively, at pH 6.0 and 37°C. The enzyme was very sensitive to Fe³⁺, Mn²⁺ and Co²⁺ ions, but less sensitive to Zn²⁺, Al³⁺, Cu²⁺ and Ca²⁺. Glucose at a final concentration of 1 mM inhibited 65% of the original activity of the purified enzyme. Determination of the product (reducing sugar) of hydrolysis of C. perniciosa mycelium and scanning electron microscopic analysis revealed that the N-acetylglucosaminidase hydrolyses the C. perniciosa cell wall.     

Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from<Emphasis Type="Italic"> Bifidobacterium adolescentis</Emphasis> DSM20083

Clones of a genomic library of Bifidobacterium adolescentis were grown in minimal medium with sucrose as sole carbon source. An enzymatic fructose dehydrogenase assay was used to identify sucrose-degrading enzymes. Plasmids were isolated from the positive colonies and sequence analysis revealed that two types of insert were present, which only differed with respect to their orientation in the plasmid. An open reading frame of 1,515 nucleotides with high homology for sucrose phosphorylases was detected on these inserts. The gene was designated SucP and encoded a protein of 56,189 Da. SucP was heterologously expressed in Escherichia coli, purified, and characterized. The molecular mass of SucP was 58 kDa, as estimated by SDS-PAGE, while 129 kDa was found with gel permeation, suggesting that the native enzyme was a dimer. The enzyme showed high activity towards sucrose and a lower extent towards -glucose-1-phosphate. The transglucosylation properties were investigated using a broad range of monomeric sugars as acceptor substrate for the recombinant enzyme, while -glucose-1-phosphate served as donor. d- and l-arabinose, d- and l-arabitol, and xylitol showed the highest production of transglucosylation products. The investigated disaccharides and trisaccharides were not suitable as acceptors. The structure of the transglucosylation product obtained with d-arabinose as acceptor was elucidated by NMR. The structure of the synthesized non-reducing dimer was -Glcp(11)-Araf.