Characterization of a recombinant l-rhamnose isomerase from Bacillus subtilis and its application on production of l-lyxose and l-mannose

The gene of an l-rhamnose isomerase (RhaA) from Bacillus subtilis was cloned to the pET28a(+) and then expressed in the E. coli ER2566. The expressed enzyme was purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzyme existed as a 194 kDa tetramer and the maximal activity was observed at pH 8.0 and 60°C. The RhaA displayed activity for l-rhamnose, l-lyxose, l-mannose, d-allose, d-gulose, d-ribose, and l-talose, among all aldopentoses and aldohexoses and it showed enzyme activity for l-form monosaccharides such as l-rhamnose, l-lyxose, l-mannose, and l-talose. The catalytic efficiency (k _cat/K _m) of the recombinant enzyme for l-rhamnose, l-lyxose, and l-mannose were 7,460, 1,013, and 258 M/sec. When l-xylulose 100 g/L and l-fructose 100 g/L were used as substrates, the optimum concentrations of RpiB were determined with 6 and 15 U/mL, respectively. The l-lyxose 40 g/L was produced from l-xylulose 100 g/L by the enzyme during 60 min, while l-mannose 25 g/L was produced from l-fructose 100 g/L for 80 min. The results suggest that RhaA from B. subtilis is a potential producer of l-form monosaccharides.     

Chromosome counts of some Cuban Angiosperms

Chromosome numbers are reported for 14 species collected in Cuba. The first chromosome records are reported forAlbizzia cubana Britton etWilson (2n=26),Atkinsia cubensis (Britton etWilson)Howard (2n=26),Caesalpinia violacea (Mill.)Standl. (2n=24),Colubrina ferruginosa Brongn. (2n=24). Chromosome numbers of the following species were confirmed:Albizzia lebbeck (L.)Benth. (2n=26),Canavalia maritima (Aubl.)Thouars (2n=22),Casuarina equisetifolia Forst. (2n=18),Cedrela mexicana M.J. Roem. (2n=56),Delonix regia (Bojer)Raf. (2n=28),Guazuma tomentosa H.B.K. (2n=16),Lysiloma bahamense Benth. (2n=26),L. latisiliqua (L.)Benth. (2n=26),Samanea saman (Jacq.)Merrill (2n=26),Thespesia populnea (L.)Soland (2n=26).     

l-Arabinose/d-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of l-arabinose to l-arabonate with Saccharomyces cerevisiae

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for l-arabonate production from l-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a d-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a l-arabinose/d-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP⁺ but uses also NAD⁺ as a cofactor, and showed highest catalytic efficiency (k _cat/K _m) towards l-arabinose, d-galactose and d-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of l-arabinose, and the stable oxidation product detected is l-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear l-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for l-arabinose uptake, resulted in production of 18 g of l-arabonate per litre, at a rate of 248 mg of l-arabonate per litre per hour, with 86 % of the provided l-arabinose converted to l-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for l-arabonate production in yeast.     

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C₆₅H₉₃N₁₇O₃₂, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.     

Notes on some African hepatic genera 1–5

The present study deals with five genera of hepatics in Africa, Isotachis Mitt., Anastrophyllum (Spruce) Steph., Tritomaria Schiffn. ex Loeske, Gymnocoleopsis (Schust.) Schust. and Lophozia (Dum.) Dum. All African populations of the genus Isotachis Mitt. are considered to be one species, I. aubertii (Schwaegr.) Mitt. Four species of Anastrophyllum (Spruce) Steph. (s.l.), A. auritum (Lehm.) Steph., A. piligerum (Nees) Spruce, A. subcomplicatum (Lehm. et Lindenb.) Steph. and A. minutum (Schreb.) Schust., and two species of Tritomaria Schiffn. et Loeske, T. camerunensis S. Arnell and T. exsecta (Schrad.) Schiffn. ex Loeske occur in Africa. Gymmocoleopsis multiflora (Steph.) Schust. represents a genus and species hitherto unreported for the African flora. Finally, five Lophozia (Dum.) Dum. species, L. argentina (Steph.) Schust., L. capensis S. Arnell, L. decolorans (Limpr.) Steph., L. hedbergii S. Arnell and L. tristaniana (S. Arnell) Váňa, are reported from central and southern Africa; two of these (L. argentina (Steph.) Schust. and L. decolorans (Limpr.) Steph.) represent the first reports from Africa.     

Annotated chromosome numbers of selected asiaticPotentilla species

Chromosome numbers are reported for 19 collections representing 16 AsiaticPotentilla taxa. The first chromosome records are reported forP. desertorum Bunge var.arnavatensis Wolf (2n=28),P. festiva Soják (2n=28),P. griffithii Hook f. subsp.beauvaisii (Cardot) Soják (2n=42),P. micropetala D. Don subsp.byssitecta (Soják) Měsí?ek etSoják (2n=14),P. mollissima Lehm. (2n=28),P. moorcroftii Wall. exLehm. (2n=42),P. multicaulis Bunge (2n=14),P. [x]omissa Soják (2n=35, 56, 70) andP. stanjukoviczii Ovcz. exKoczk. (2n=14). Counts differing from those previously recorded are given forP. algida Soják (2n=56) andP. flagellaris Willd. exSchlecht. (2n=42). Chromosome numbers of the following species were confirmed:P. [x]agrimonioides Bieb. (2n=42),P. chinensis Ser. in DC. (2n=14),P. fragarioides L. (2n=14),P. lineata Trev. (2n=14) andP. sericea L. (2n=28). Taxonomy is briefly discussed. A new combinationP. micropetala D. Don subsp.byssitecta (Soják) Měsí?ek etSoják stat. nov. is proposed.     

Improved Protease Stability of the Antimicrobial Peptide Pin2 Substituted with d-Amino Acids

Cationic antimicrobial peptides (AMPs) have attracted a great interest as novel class of antibiotics that might help in the treatment of infectious diseases caused by pathogenic bacteria. However, some AMPs with high antimicrobial activities are also highly hemolytic and subject to proteolytic degradation from human and bacterial proteases that limit their pharmaceutical uses. In this work a d-diastereomer of Pandinin 2, d-Pin2, was constructed to observe if it maintained antimicrobial activity in the same range as the parental one, but with the purpose of reducing its hemolytic activity to human erythrocytes and improving its ability to resist proteolytic cleavage. Although, the hydrophobic and secondary structure characteristics of l- and d-Pin2 were to some extent similar, an important reduction in d-Pin2 hemolytic activity (30–40 %) was achieved compared to that of l-Pin2 over human erythrocytes. Furthermore, d-Pin2 had an antimicrobial activity with a MIC value of 12.5 μM towards Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and two strains of Pseudomonas aeruginosa in agar diffusion assays, but it was half less potent than that of l-Pin2. Nevertheless, the antimicrobial activity of d-Pin2 was equally effective as that of l-Pin2 in microdilution assays. Yet, when d- and l-Pin2 were incubated with trypsin, elastase and whole human serum, only d-Pin2 kept its antimicrobial activity towards all bacteria, but in diluted human serum, l- and d-Pin2 maintained similar peptide stability. Finally, when l- and d-Pin2 were incubated with proteases from P. aeruginosa DFU3 culture, a clinical isolated strain, d-Pin2 kept its antibiotic activity while l-Pin2 was not effective.     

Chromosome numbers in selectedHieracium species in the Krkonoše Mts. (the West Sudeten)

Chromosome numbers are given of 15 species of the genusHieracium L. s. str., representing seven species groups (in the sense of Flora Europaea, roughly corresponding to Zahn's “species principales”) from the Krkono?e Mts., N. Bohemia and SW Poland. For the first time chromosome numbers are reported forH. melanocephalum Tausch (2n=27),H. tubulosum Tausch (2n=36),H. schustleri Zlatník (2n=36),H. fritzei F. Schultz (2n=27),H. rohlenae Zlatník (2n=27),H. nigrescens Willd. (2n=36),H. decipiens Tausch (2n=36),H. atrellum Juxip inSchischkin etBobrov (2n=27),H. subnigrescens (Fries exNorrlin)Dahlst. (2n=36),H. sudeticum Sternberg (2n=36),H. pedunculare Tausch (2n=36),H. glandulosodentatum Uechtr. (2n=36),H. wimmeri Uechtr. (2n=27). InHieracium alpinum L. s. str. the number 2n=27 has been confirmed. The results show a high proportion of tetraploid taxa; no diploids have been found.     

Probing the specificity of gamma-glutamylamine cyclotransferase: an enzyme involved in the metabolism of transglutaminase-catalyzed protein crosslinks

γ-Glutamylamine cyclotransferase (gGACT) catalyzes the intramolecular cyclization of a variety of l-γ-glutamylamines producing 5-oxo-l-proline and free amines. Its substrate specificity implicates it in the downstream metabolism of transglutaminase products, and is distinct from that of γ-glutamyl cyclotransferase which acts on l-γ-glutamyl amino acids. To elucidate the mechanism by which gGACT distinguishes between l-γ-glutamylamine and amino acid substrates, the specificity of the rabbit kidney enzyme for the amide region of substrates was probed through the kinetic analysis of a series of l-γ-glutamylamines. The isodipeptide N ^?-(l-γ-glutamyl)-l-lysine 1 was used as a reference. The kinetic constants of the l-γ-glutamyl derivative of n-butylamine 7, were nearly identical to those of 1. Introduction of a methyl or carboxylate group on the carbon adjacent to the side-chain amide nitrogen in l-γ-glutamylamine substrates resulted in a dramatic decrease in substrate properties for gGACT thus providing an explanation of why gGACT does not act on l-γ-glutamyl amino acids except for l-γ-glutamylglycine. Placement of substituents on carbons further removed from the side-chain amide nitrogen in l-γ-glutamylamines restored activity for gGACT, and l-γ-glutamylneohexylamine 19 had a higher specificity constant (k _cat /K _m) than 1. gGACT did not exhibit any stereospecificity in the amide region of l-γ-glutamylamine substrates. In addition, analogues (26–30) with heteroatom substitutions for the γ methylene position of the l-γ-glutamyl moiety were examined. Several thiocarbamoyl derivatives of l-cysteine (28–30) were excellent substrates for gGACT.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

Chromosome counts and chromosome morphology of some selected species

In the years 1976–1981 we studied chromosome counts and karyotypic formulae of the following 29 species of plants from 41 localities (of these 6 from Bohemia, 32 from Moravia, 3 from Slovakia):Batrachium baudotii (Godron) F. W. Schultz,Chenopodium rubrum L.,C. polyspermum L.,C. murale L.,C. ficifolium Sm.,C. opulifolium Schrader ex DC. inLam. et DC.,C. strictum Roth [subsp.strictum, subsp.glaucophyllum (Aellen)Aellen inJust etAellen, subsp.striatiforme Uotila],Arenaria grandiflora L.,Illecebrum verticillatum L.,Spergula morisonii Boreau inDuchartre,Spergularia marginata (DC. inLam. et DC.)Kittel S. marina (L.)Griseb.,S. rubra (L.) J. etC. Presl,Silene conica L.,Sisymbrium loeselii L.,S. volgense Bieb. exE. Fourn.,S. orientale L. [subsp. orientale, subsp.macroloma (A. Pomel)Dvo?ák],S. officinale (L.)Scop.,Descurainia sophia (L.)Webb exPrantl inEngler etPrantl,Nasturtium officinale R. Br. inAiton,Barbarea arcuata (Opiz inPresl J. et C.)Reichenb.,Lunaria annua L.,Soldanella montana Willd.,S. carpatica Vierh. inUrban etGraebner,Lotus tenuis Waldst. etKit. exWilld.,L. uliginosus Schkuhr,Trigonella monspeliaca L.,Geranium sibiricum L.,Lactuca tatarica (L.)C. A. Meyer.     

Studien über dieJungermannioideae (Hepaticae) 6.Jungermannia Subg.Solenostoma: Europäische und nordamerikanische Arten

Dersechste Beitrag aus der Serie der vorbereiteten Studien über die SubfamilieJungermannioideae (Jungermanniaceae, Hepaticae) enthält die taxonomische Bearbeitung des Subg.Solenostoma (Mitt.)Amak. (GattungJungermannia L. emend.Dum.) in Europa (inkl. Kaukasus und Kleinasien) und Nordamerika. Im genannten Gebiet kommen 10 Arten vor, u. zw.J. caespiticia Lindenb.,J. caucasica Váňa,J. confertissima Nees,J. gracillima Sm.,J. handelii (Schiffn.)Amak.,J. jenseniana Grolle,J. lignicola (Schiffn.)Grolle,J. pyriflora Steph.,J. rubra Gott. exUnd. undJ. sphaerocarpa Hook. Die ArtJ. confertissima Nees (bisher alsSolenostoma levieri (Steph.)Steph. bezeichnet) gehört zu der SektionDesmorhiza Amak., alle anderen Arten gehören zu der SektionSolenostoma.     

Ein Beitrag zur Nomenklatur sphinctozoider Schwämme (Porifera)

The sphinctozoid sponge generaFania Senowbari-Daryan 1990 andSpica Termier &Termier 1977 are preoccupied.Fania is replaced byFanthalamia nom. nov. andSpica by the younger synonymFistulispongia Termier &Termier 1977. The invalid subfamily name FaniinaeSenowbari-Daryan 1990 is replaced by Fanthalamiinae n. subfam. The invalid family and subfamily names SpicidaeTermier &Termier 1977 and SpicinaeSenowbari-Daryan 1990 respectively are replaced by FistulispongiidaeTermier atTermier 1977 and FistulispongiinaeSenowbari-Daryan 1990. The generaWaagenium de Laubenfels 1957 andCatubria Merla 1931 were previously overlooked.Waagenium DeLaubenfels 1957 is a younger synonym ofColospongia Laube 1865. The position ofCatubria Merla 1931 is uncertain. Most probablyCatubria is an alga.     

Hybridization studies inSilene subgen.Petrocoptis (Caryophyllaceae)

Silene subgenusPetrocoptis comprises sexual diploid taxa and it is restricted to calcareous cliffs in the Iberian peninsula. Artificial crosses involvingSilene pyrenaica (Bergeret)Mayol etRosselló (≡Petrocoptis pyrenaica (Bergeret)Walp.),Silene laxipruinosa Mayol etRosselló andS. montserratii subsp.crassifolia (Rouy)Mayol etRosselló (≡P. crassifolia Rouy) were attempted to assess the extent of barriers developed within the subgenusPetrocoptis. Usually, intraspecific crosses involving allopatric populations were successful, suggesting that geographically isolated populations are not genetically isolated. Cross-compatibility was noted among the polymorphicS. pyrenaica, which developed fertile F₁ hybrids. All other interspecific crosses failed due to cross- or seed-incompatibility. Crossing results agree with available evidence supporting the merging of segregates ofS. pyrenaica within a single taxon.     

Selected chromosome counts of the Czechoslovak flora III

Chromosome numbers compared with as yet published data are given for the following 12 Phanerogams (both native species and aliens) from Czechoslovakia:Ambrosia trifida L.,Cardamine chelidonia L.,Dephne cneorum L.,Epipactis albensis Nováková etRydlo,Linum flavum L.subsp flavum, Lunaria rediviva L.,Nepeta grandiflora M.BIEB.,Reseda luteola L.,Thlaspi montanum L.,Tithymalus salicifolius (Host)Klotzsch etGarcke,Tithymalus virgultosus (Klokov) Holub andVerbascum speciosum Schrad. subsp.speciosum. The chromosome number 2n=40 is presented for the first time in autogamousEpipactis albensis Nováková etRydlo. New chromosome numbers were found inCardamine chelidonia L. (2n=32) and inTithymalus salicifolius (Host) Klotzch etGarcke (2n=40). Known but less frequent cytotypes are reported inLinum flavum L. subsp.flavum (2n=28) and inVerbascum speciosum Schrad. subsp.speciosum (2n=30).     

New chromosome numbers inTaraxacum with reference to SAT-chromosomes

Chromosome numbers of eight agamospermousTaraxacum microspecies are reported for the first time:T hypanicum Tzvel. [sect.Dioszegia (Heuff.) Heuff.,T. neosivaschicum Tzvel. [sect.Palustria (Lindb, fil.)Dahlst.],T. pseudohoppeanum Kirschner et?těpánek (sect.Erythrocarpa Dahlst.),T. schroeterianum H.-M. (sect.Rhodocarpa Van Soest),T. marmottae C.E. Sonck,T. lambinonii Van Soest [sect.Erythrosperma (Lindb. fil.)Dahlst.],T. zermattense Dahlst, andT. magnoobliquum Van Soest (taxa of uncertain classification). All these polyploid microspecies have satellite chromosomes in their karyotypes. Advanced karyological characters were found in sect.Dioszegia, hitherto regarded as a primitive section ofTaraxacum: triploidy in agamospermousT. hypanicum Tzvel. and satellite chromosomes as in this taxon, as in two diploid sexualsT. serotinum (W. et K.)Poiret andT. haussknechtii Uechtr. The results presented are compared to the literature data.     

Bemerkungen zu einigen balkanischenOxytropis-Arten

The present paper deals with the following three mountainOxytropis species:Oxytropis urumovii Jáv. inUrumov, an endemic species of the Pirin (Bulgaria);O. dinarica (Murb.) Wettst. distributed predominantly in Yugoslavia, andO. campestris which is common in the East Alps, but very rare in the Balkans. Three subspecies ofO. dinarica were distinguished: subsp.dinarica, subsp.weberi Chrtek etChrtková, and subsp.velebitica Chrtek etChrtková. In addition, a new combinationO. halleri Bunge exKoch subsp.korabensis (Kümmerle etJáv.)Chrtek etChrtková is given.     

LignicolousHyphomycetes from Czechoslovakia 1.Brachysporium

Four species of the genusBrachysporium Sacc. emend.Mason etHughes are treated in this paper from Czechoslovakia:B. nigrum (Link)Hughes,B. obovatum (Berk.)Sacc. and two new species,B. abietinum Hol.-Jech. andB. brevius Hol.-Jech.