Lett Appl Microbiol, 2005, 40(2), 92 - 8 A plate assay for simultaneous screening of polysaccharide- and protein-degrading micro-organisms; Ten LN et al.; Abstract l.n . ten, w.-t . im, m.-k . kim and s.-t . lee . 2004.Aims: To develop a plate assay for simultaneous screening of polysaccharide-degrading and protein-degrading micro-organisms . Methods and Results: A plate assay, based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of diversely coloured insoluble polysaccharides and dye-labelled collagen as chromogenic substrates, was developed . This method was successfully applied for isolating the diverse polysaccharide- and/or protein-degrading bacteria from soil and sludge samples . Selected strains were identified using 16S rDNA partial sequencing; most of them belong to the genera Bacillus, Cellulomonas and Cellulosimicrobium . Conclusions: This novel approach provides unique and valuable information for direct primary screening when the target of selection is micro-organisms exhibiting protein-degrading activity, polysaccharide-degrading activity or a specific combination of them . Significance and Impact of the Study: This plate assay is convenient and easy to perform, rapid, and more adaptable for screening of a large number of samples, compared with other existing methods in the literature. Pest Manag Sci, 2005 Feb, 61(2), 159 - 65 Identification of a chitinase-producing bacterium C4 and histopathologic study on locusts; Yong T et al.; In order to develop the potential of chitinase-producing micro-organisms as biocontrol agents for insect pests, five chitinase-producing bacterial strains (C1, C2, C3, C4 and C5) previously isolated from soil samples were chosen to infect grassland locusts . The data showed that the mortality rate of locusts fed with strain C4 was significantly higher than that of other groups, and its pathogenicity was confirmed by Koch's law . Midgut tissues of locusts infected with C4 were examined with a light microscope . Apparent histopathologic changes in midgut cells partly explained the pathogenesis of locusts . Therefore, strain C4 was considered to be a potential biocontrol agent . To determine the taxonomic position of C4, physiological and biochemical characteristics were determined and molecular identification was performed . The 16S rDNA gene of C4 was amplified, cloned and sequenced . Comparative sequence analysis demonstrated that C4 corresponded to the genera Sanguibacter, Oerskovia and Cellulomonas . On the basis of phenotypic characterization and sequence similarity analysis, strain C4 was more closely related to the genus Sanguibacter . This chitinase-producing strain C4, which closely corresponds to the species of the genus Sanguibacter and is pathogenic to locusts, is here reported for the first time . Copyright (c) 2004 Society of Chemical Industry. Biotechnol Lett, 2004 Sep, 26(17), 1329 - 33 Kinetic of improved production and carboxymethyl cellulose hydrolysis by an endo-glucanase from a derepressed mutant of Cellulomonas biazotea; Rajoka MI et al.; The maximum product yield of endo-glucanase (650 IU g(-1) substrate) from Cellulomonas biazotea mutant 51 Sm(r) was 1.5- to 2.5-fold more than was produced by the wild type cells and was twice that reported by previous researchers . Mutation substantially improved the enthalpy (DeltaH (*)) and entropy of activation (DeltaS (*)) for product formation, turnover number, specificity constant activation energy, free energies for transition state formation and substrate binding for CMC hydrolysis respectively. Biotechnol Prog, 2004 Sep-Oct, 20(5), 1479 - 89 Inexpensive and generic affinity purification of recombinant proteins using a family 2a CBM fusion tag; Rodriguez B et al.; The selective binding of the family 2a carbohydrate binding module (CBM2a) of xylanase 10A of the soil bacterium Cellulomonas fimi to a variety of cellulosic substrates is shown to provide a new, cost-effective affinity chromatography system for purification of recombinant protein . Genetic linkage of CBM2a to a target protein, in this case protein A from Staphylococcus aureus, results in a fusion protein that binds strongly to the particulate-cellulose resin Avicel PH101 and retains the biological activity of the fusion partner . Affinity purification of protein A-CBM2a from the supernatant of a recombinant E . coli JM101 culture results in a product purity of greater than 95% and a product concentration factor of 34 +/- 3 . Measured column parameters are combined with one-dimensional equations governing continuity and intraparticle diffusion to predict product breakthrough curves with good accuracy over the range of realistic operating conditions . Peak spreading within the column is controlled by intraparticle diffusion for CBM2a and by a combination of film mass transfer and intraparticle diffusion for the larger protein A-CBM2a fusion protein. J Biol Chem, 2004 Dec 24, 279(52), 54369 - 79 Epub 2004 Sep 27. The use of forced protein evolution to investigate and improve stability of family 10 xylanases . The production of Ca2+-independent stable xylanases; Andrews SR et al.; Metal ions such as calcium often play a key role in protein thermostability . The inclusion of metal ions in industrial processes is, however, problematic . Thus, the evolution of enzymes that display enhanced stability, which is not reliant on divalent metals, is an important biotechnological goal . Here we have used forced protein evolution to interrogate whether the stabilizing effect of calcium in an industrially relevant enzyme can be replaced with amino acid substitutions . Our study has focused on the GH10 xylanase CjXyn10A from Cellvibrio japonicus, which contains an extended calcium binding loop that confers proteinase resistance and thermostability . Three rounds of error-prone PCR and selection identified a treble mutant, D262N/A80T/R347C, which in the absence of calcium is more thermostable than wild type CjXyn10A bound to the divalent metal . D262N influences the properties of the calcium binding site, A80T fills a cavity in the enzyme, increasing the number of hydrogen bonds and van der Waals interactions, and the R347C mutation introduces a disulfide bond that decreases the free energy of the unfolded enzyme . A derivative of CjXyn10A (CfCjXyn10A) in which the calcium binding loop has been replaced with a much shorter loop from Cellulomonas fimi CfXyn10A was also subjected to forced protein evolution to select for thermostablizing mutations . Two amino acid substitutions within the introduced loop and the A80T mutation increased the thermostability of the enzyme . This study demonstrates how forced protein evolution can be used to introduce enhanced stability into industrially relevant enzymes while removing calcium as a major stability determinant. J Clin Microbiol, 2004 Jun, 42(6), 2495 - 500 Molecular epidemiologic evaluation of endocarditis due to Oerskovia turbata and CDC group A-3 associated with contaminated homograft valves; McNeil MM et al.; Oerskovia turbata is an unusual bacterial cause of endocarditis and septicemia in immunocompromised patients . In this study, we compared 12 isolates from a 1975 medical center cluster, 11 originally identified as O . turbata (four from the blood of a homograft aortic valve-associated endocarditis patient and seven from contaminated homograft valves) and one CDC group A-3 strain from the blood of a second endocarditis patient with fatal outcome, with eight control strains from unrelated locations . The control strains included type and reference strains of O . turbata, Cellulomonas hominis, and CDC group A-3 . The four blood isolates from the first patient and six of the valve isolates shared identical biochemical, antimicrobial susceptibility, and BglI ribotype patterns that differed from the second patient's isolate and control strains . The blood isolate from the second patient and the remaining valve isolate shared a phenotypic and genotypic profile and were phenotypically identical to, but epidemiologically different from, the CDC group A-3 reference strain with the strain-specific enzyme . Also, these isolates differed from the type strain and the other reference strains of C . hominis and O . turbata . Our results indicate that the four blood isolates from the first patient and six of the homograft valve isolates represent a single clone of O . turbata associated with endocarditis . Additionally, our results indicate that the blood isolate from the second patient and one of the homograft valve isolates differ from O . turbata and C . hominis and represent a unique clone of CDC group A-3 associated with fatal endocarditis. J Biol Chem, 2004 Jun 18, 279(25), 26619 - 26 Epub 2004 Apr 12. Structure and function of a family 10 beta-xylanase chimera of Streptomyces olivaceoviridis E-86 FXYN and Cellulomonas fimi Cex; Kaneko S et al.; The catalytic domain of xylanases belonging to glycoside hydrolase family 10 (GH10) can be divided into 22 modules (M1 to M22; Sato, Y., Niimura, Y., Yura, K., and Go, M . (1999) Gene (Amst.) 238, 93-101) . Inspection of the crystal structure of a GH10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) revealed that the catalytic domain of GH10 xylanases can be dissected into two parts, an N-terminal larger region and C-terminal smaller region, by the substrate binding cleft, corresponding to the module border between M14 and M15 . It has been suggested that the topology of the substrate binding clefts of GH10 xylanases are not conserved (Charnock, S . J., Spurway, T . D., Xie, H., Beylot, M . H., Virden, R., Warren, R . A . J., Hazlewood, G . P., and Gilbert, H . J . (1998) J . Biol . Chem . 273, 32187-32199) . To facilitate a greater understanding of the structure-function relationship of the substrate binding cleft of GH10 xylanases, a chimeric xylanase between SoXyn10A and Xyn10A from Cellulomonas fimi (CfXyn10A) was constructed, and the topology of the hybrid substrate binding cleft established . At the three-dimensional level, SoXyn10A and CfXyn10A appear to possess 5 subsites, with the amino acid residues comprising subsites -3 to +1 being well conserved, although the +2 subsites are quite different . Biochemical analyses of the chimeric enzyme along with SoXyn10A and CfXyn10A indicated that differences in the structure of subsite +2 influence bond cleavage frequencies and the catalytic efficiency of xylooligosaccharide hydrolysis . The hybrid enzyme constructed in this study displays fascinating biochemistry, with an interesting combination of properties from the parent enzymes, resulting in a low production of xylose. Biotechnol Lett, 2004 Feb, 26(4), 281 - 5 Kinetics of improved production and thermostability of an intracellular beta-glucosidase from a mutant-derivative of Cellulomonas biazotea; Rajoka MI et al.; The highest productivity (20 IU l(-1) h(-1)) of beta-glucosidase by a mutant of Cellulomonas biazotea was 2.5-fold more than that of the parent organism . The enzyme had a lower activation energy (57 kJ mol(-1)) than the native enzyme (68 kJ mol(-1)) . The enzyme from the mutant had enthalpy and entropy values for irreversible intactivation of 95.6 kJ mol(-1) and 60 J.mol(-1) K(-1) compared with 108 kJ mol(-1) and 86 J mol(-1) K(-1) for the native enzyme suggesting that the mutation had stabilized the enzyme. Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 533 - 6 Cellulomonas xylanilytica sp . nov., a cellulolytic and xylanolytic bacterium isolated from a decayed elm tree; Rivas R et al.; A Gram-positive, aerobic, non-motile bacterium was isolated from a decayed elm tree . Phylogenetic analysis based on 16S rDNA sequences revealed 99.0 % similarity to Cellulomonas humilata . Chemotaxonomic data that were determined for this isolate included cell-wall composition, fatty acid profiles and polar lipids; the results supported the placement of strain XIL11(T) in the genus Cellulomonas . The DNA G+C content was 73 mol% . The results of DNA-DNA hybridization with C . humilata ATCC 25174(T), in combination with chemotaxonomic and physiological data, demonstrated that isolate XIL11(T) should be classified as a novel Cellulomonas species . The name Cellulomonas xylanilytica sp . nov . is proposed, with strain XIL11(T) (=LMG 21723(T)=CECT 5729(T)) as the type strain. Environ Toxicol Chem, 2004 Feb, 23(2), 265 - 70 Microbial degradation of chlorobenzene under oxygen-limited conditions leads to accumulation of 3-chlorocatechol; Vogt C et al.; Five bacterial strains (Acidovorax facilis B517, Cellulomonas turbata B529, Pseudomonas veronii B547, Pseudomonas veronii B549, and Paenibacillus polymyxa B550) isolated on chlorobenzene as the sole source of carbon and energy were screened for the accumulation of the putative metabolic intermediate 3-chlorocatechol during growth on chlorobenzene under oxygen-limited conditions in the presence and absence of nitrate (1 mM) . 3-Chlorocatechol accumulated in the growth media of all five strains, but accumulation was significantly less in cultures of A . facilis B517 compared to the other four strains . The presence of nitrate did not influence the biological conversion pattern . However, biologically produced nitrite reacted with 3-chlorocatechol chemically, a reaction that masked the accumulation of 3-chlorocatechol . For P . veronii B549, a clear relationship between the presence of 3-chlorocatechol in the medium and low oxygen concentrations was demonstrated . The assumption is made that accumulation of 3-chlorocatechol is due to the low enzymatic turnover of the 3-chlorocatechol cleaving enzyme, catechol-1,2-dioxygenase, at low oxygen concentrations. Can J Microbiol, 2003 Nov, 49(11), 713 - 21 Influence of high salinities on the degradation of diesel fuel by bacterial consortia; Riis V et al.; Microbial communities from three Argentinean saline soils were extracted and tested for their ability to degrade diesel fuel in liquid culture at salinities between 0% and 25% . In each case, the degradation process was continuously monitored by measuring oxygen consumption . Two communities (CR1 and CR2) showed nearly equal degrees of degradation across a salinity range of 0%-10% (the former degrading about 63% of the diesel fuel and the latter about 70% after 53 and 80 d, respectively) . Furthermore, the degree of degradation was not significantly lower in the presence of 17.5% salt (58% and 65% degraded, respectively) . A third community (El Zorro) showed a maximum turnover at 5% salt (79% diesel fuel degraded) and significant degradation (66%) at a salinity of 10% . However, the degree of degradation by this community clearly dropped at 0% and 15% salt . None of the communities were able to degrade diesel fuel in the presence of 25% salt, but the living cell counts showed that components of the microbial population survived the long-term exposure . The surviving portion is obviously sufficient to allow substantial restoration of the original community, as verified by the BIOLOG method . Isolates of the CR1 community were identified as members of the genera Cellulomonas, Bacillus, Dietzia, and Halomonas . In light of our investigations, the bioremediation of contaminated saline soils should be quite possible if the salinity of the soil water is lower than 15% or if it is reduced below this limit by the addition of water. Appl Microbiol Biotechnol, 2004 Jun, 64(6), 794 - 9 Epub 2003 Dec 18. Effective extracellular trehalose production by Cellulosimicrobium cellulans; Seto A et al.; A bacterium isolated from a petal of Casa Blanca Lily (ST26 strain) produced a marked amount of extracellular trehalose (alpha- d-glucopyranosyl-{1,1}-alpha- d-glucopyranose) in culture medium containing glucose . 16S rDNA-based phylogeny showed that ST26 belongs to, or is related to, Cellulosimicrobium cellulans, a close relative of Cellulomonas spp . Various Cellulomonas strains obtained from culture collections also showed extracellular trehalose productivity, suggesting that trehalose production is a common property of this bacterial genus . ST26 accumulated trehalose in medium supplied with glucose but not with sucrose, glycerol or maltose . Effective extracellular trehalose production by ST26 was achieved by supplying 0.5-1% ammonium sulfate and 0.5-1% CaCO(3) . The addition of CaCO(3) adjusted the pH of the culture to around 5.0 . The optimized culture conditions yielded trehalose from glucose at a conversion rate of 61% . The addition of ammonium sulfate greatly reduced the dry cell weight of ST26 and intracellular content of trehalose, which suggests that the addition of ammonium sulfate makes ST26 cells leak trehalose into the medium . ST26 effectively propagated in minimal medium containing trehalose as a sole carbon source, which suggests that trehalose serves as a carbohydrate reserve of this organism. Protein Pept Lett, 2003 Dec, 10(6), 561 - 8 Kinetics and thermodynamics of the native and mutated extracellular endo-glucanases from Cellulomonas biazotea; Rajoka MI et al.; The mutation had dramatic effect on the kinetic and thermodynamic parameters inferring thermostability of endo-glucanase from Cellulomonas biazotea mutant 51 SM(r).The denaturation activation energies of native and mutated enzymes were 73.3 and 68.8 kJ/mol respectively . They showed compensation effect at 55 degrees C . Both enthalpy and entropy values of irreversible thermal inactivation for mutated enzyme were decreased suggesting that the mutation partly stabilized the enzyme. Curr Microbiol, 2003 Nov, 47(5), 359 - 63 Expression and characterization of the celcflB gene from Cellulomonas flavigena encoding an endo-beta-1,4-glucanase; Gutierrez-Nava A et al.; An endoglucanase gene called celcflB was isolated from a genomic library of C . flavigena . Its nucleotide sequence showed an ORF of 1725 bp with a GTG start codon . A promoter sequence was found inside the adjacent gene upstream from the start point of translation of celcflB gene . The gene celcflB was induced by sugarcane bagasse and repressed by glucose when C . flavigena was grown on these carbon sources, suggesting that celcflB gene is regulated at transcriptional level . The predicted amino acid sequence of the CelcflB protein presented a catalytic domain with a high homology to family 5 of glycosil hydrolases, and a cellulose binding domain identical to cellulose binding domains type II . The coding region of the celcflB gene was cloned into the expression vector pQE30 and the recombinant protein of 58 kDa presented endoglucanase activity towards carboxymethyl cellulose (CMC). Biotechniques, 2003 Nov, 35(5), 1032 - 8, 1041-2 Production of soluble and functional engineered antibodies in Escherichia coli improved by FkpA; Zhang Z et al.; Overproduction of genetically engineered antibodies, such as single-chain antibodies (scAbs) in Escherichia coli often results in insoluble and inactive products known as inclusion bodies . We now report that fusion or co-expression of FkpA, the E . coli periplasmic peptidyl-prolyl-isomerase with chaperone activity, substantially improves soluble and functional expression of scAbs . Anti-human bladder carcinoma scAb (PG) and anti-human CD3 x anti-human ovarian carcinoma-bispecific scAb (BH1) were fused with FkpA on the pTMF-based plasmid and expressed in E . coli . More than half of the amount of each expressed fusion protein FkpA-PG or FkpA-BH1 was soluble . In addition, the fusion protein cellulose-binding domain from Cellulomonas fimi (CBD)-PG and anti-human CD3 x anti-human CD28 x anti-human ovarian carcinoma-trispecific scAb (TRI) fused to the pelB (a signal peptide from pectate lysase B of a Bacillus sp.) signal sequence were co-expressed with FkpA under the control of the T7 promoter . A substantial portion of the co-expressed CBD-PG or TRI was soluble . Furthermore, PG, BH1, and TRI were biologically active as judged by ELISA and in vitro cytotoxicity assay . These results suggest that overexpression of FkpA should be useful in expressing heterologous proteins in E . coli. Arch Microbiol, 2003 Dec, 180(6), 434 - 43 Epub 2003 Oct 28. Biochemical and genetic characterization of ChiA, the major enzyme component for the solubilization of chitin by Cellulomonas uda; Reguera G et al.; Cellulomonas uda efficiently solubilized chitinous substrates with a simple chitinase system composed of an endochitinase, designated ChiA, which hydrolyzed insoluble substrates into long-chain chitooligosaccharides, and an as yet uncharacterized exochitinase activity . ChiA, isolated from culture supernatant fluids, was found to be a glycosylated endochitinase with an apparent molecular mass of approximately 70 kDa and pI of 8.5 . The gene encoding ChiA was cloned in Escherichia coli and sequenced, revealing an open reading frame of 1,716 bp encoding a 571-amino-acid protein with a predicted molecular mass of 59.2 kDa . The region upstream of chiA included a conserved -35 hexamer flanked by two direct repeats analogous to those found in many Streptomyces chitinase promoters, and thought to function as binding sequences for regulatory proteins . Analysis of the deduced amino acid sequence showed a modular protein consisting of a signal peptide at its N terminus, a family 2 carbohydrate-binding module (CBM2) that was closely related to the substrate-binding domains of glycosyl hydrolases from distantly related bacteria, and a family 18 glycosyl hydrolase catalytic module related to Streptomyces chitinases . In contrast to the fibronectin type III domains of Streptomyces chitinases, the linker region between modules in ChiA consisted of a long proline- and threonine-rich module, thought to contribute to the glycosylation and flexibility of the mature protein. Nucleosides Nucleotides Nucleic Acids, 2003 May-Aug, 22(5-8), 1695 - 8 Interactions of trimeric purine nucleoside phosphorylases with ground state analogues--calorimetric and fluorimetric studies; Wielgus-Kutrowska B et al.; Binding enthalpies, dissociation constants and stoichiometry of binding for interaction of trimeric calf spleen and Cellulomonas sp . purine nucleoside phosphorylases with their ground state analogues (substrates and inhibitors) were studied by calorimetric and spectrofluorimetric methods . Data for all ligands, with possible exception of hypoxanthine, are consistent with three identical non-interacting binding sites. Biotechnol Adv, 1997, 15(2), 315 - 31 The glucanases of Cellulomonas; Chaudhary P et al.; Cellulomonas is a unique bacterium possessing not only the capacity to degrade various carbohydrates, such as starch, xylan and cellulose, but crystalline cellulose as well . It has developed a complex battery of glucanases to deal with substrates possessing such extensive microheterogeneities . Some of these enzymes are multifunctional, as well as cross inducible, possessing a multi-domain structure; these enzymes are thought to have arisen by the shuffling of these domains . Intergeneric hybrids have been constructed between Cellulomonas and Zymomonas so as to enhance the industrial potential of this organism . This review examines the unique features of this microorganism and evaluates its key role in the conversion of complex wastes to useful products, by virtue of its unusual attributes. Gene, 2003 Sep 18, 314, 191 - 9 Frameshift mutation events in beta-glucosidases; Rojas A et al.; Compensated frameshift mutation is a modification of the reading frame of a gene that takes place by way of various molecular events . It appears to be a widespread event that is only observed when homologous amino acid and nucleodotide sequences are compared . To identify these mutation events, the sequence analysis rationale was based on the search for short regions that would have much lower degrees of conservation in protein, but not in DNA, in well-conserved beta-glucosidase families . We have restricted our study to a seed set of sequences of O-glycoside hydrolase families 1 and 3 . We found compensated frameshift mutation in the family of 1 beta-glucosidases for the Erwinia herbicola, Cellulomonas fimi, and (non-cyanogenic) Trifolium repens gene sequences, and in the family of 3 beta-glucosidases for the Clostridium thermocellum and Clostridium stercorarium gene sequences . By computational treatment, the observed mutation events in the gene frameshifting sub-sequence have been neutralised . Each nucleotide insertion must be eliminated and each nucleotide deletion must be substituted by the symbol N (any nucleotide) . When the frameshifting fragments of the amino acid sequences were substituted by the computationally neutralised subsequences, the beta-glucosidase alignments were improved . We also discuss the structural implications of the compensated frameshift mutations events. Biotechnol Lett, 2003 Jun, 25(12), 945 - 8 Kinetics of beta-glucosidase production by Saccharomyces cerevisiae recombinants harboring heterologous bgl genes; Rajoka MI et al.; The maximum productivity of beta-glucosidase by Saccharomyces cerevisiae recombinants under the control of GALI promoter was 100 IU l(-1) h(-1) . The highest productivity of beta-glucosidase by a S . cerevisiae recombinant was 16-fold more than that supported by Cellulomonas biazotea . The recombinants also co-produced ethanol from cellobiose: maximum product yield and productivity were 0.5 and 1.1 g ethanol g(-1) cellobiose and g ethanol l(-1) h(-1), respectively. Biochemistry, 2003 Jun 17, 42(23), 7195 - 204 Mechanism, mutagenesis, and chemical rescue of a beta-mannosidase from cellulomonas fimi; Zechel DL et al.; The chemical mechanism of a retaining beta-mannosidase from Cellulomonas fimi has been characterized through steady-state kinetic analyses with a range of substrates, coupled with chemical rescue studies on both the wild-type enzyme and mutants in which active site carboxyl groups have been replaced . Studies with a series of aryl beta-mannosides of vastly different reactivities (pK(a)(lg) = 4-10) allowed kinetic isolation of the glycosylation and deglycosylation steps . Substrate inhibition was observed for all but the least reactive of these substrates . Bronsted analysis of k(cat) revealed a downward breaking plot (beta(lg) = -0.54 +/- 0.05) that is consistent with a change in rate-determining step (glycosylation to deglycosylation), and this was confirmed by partitioning studies with ethylene glycol . The pH dependence of k(cat)/K(m) follows an apparent single ionization of a group of pK(a) = 7.65 that must be protonated for catalysis . The tentative assignment of E429 as the acid-base catalyst of Man2A on the basis of sequence alignments with other family 2 glycosidases was confirmed by the increased turnover rate observed for the mutant E429A in the presence of azide and fluoride, leading to the production of beta-mannosyl azide and beta-mannosyl fluoride, respectively . A pH-dependent chemical rescue of E429A activity is also observed with citrate . Substantial oxocarbenium ion character at the transition state was demonstrated by the alpha-deuterium kinetic isotope effect for Man2A E429A of alpha-D(V) = 1.12 +/- 0.01 . Surprisingly, this isotope effect was substantially greater in the presence of azide (alpha-D(V) = 1.166 +/- 0.009) . Likely involvement of acid/base catalysis was revealed by the pH dependence of k(cat) for Man2A E429A, which follows a bell-shaped profile described by pK(a) values of 6.1 and 8.4, substantially different from that of the wild-type enzyme . The glycosidic bond cleaving activity of Man2A E519A and E519S nucleophile mutants is restored with azide and fluoride and appears to correlate with the corresponding "glycosynthase" activities . The contribution of the substrate 2-hydroxyl to stabilization of the Man2A glycosylation transition state (DeltaDeltaG() = 5.1 kcal mol(-1)) was probed using a 2-deoxymannose substrate . This value, surprisingly, is comparable to that found from equivalent studies with beta-glucosidases despite the geometric differences at C-2 and the importance of hydrogen bonding at that position . Modes of stabilizing the mannosidase transition state are discussed. J Mol Microbiol Biotechnol, 2003, 5(1), 29 - 36 O-glycosylation of a recombinant carbohydrate-binding module mutant secreted by Pichia pastoris; Boraston AB et al.; Carbohydrate-binding modules (CBMs; previously called cellulose-binding domains) make excellent fusion partners for the immobilization or purification of polypeptides . However, their use in eukaryotic hosts has been limited by glycosylation, which interferes with the ability of the CBM to bind to cellulose . We have engineered the C-terminal carbohydrate-binding module from Cellulomonas fimi xylanase 10A such that it lacks N-glycosylation sites . This variant, called CBM2aNgly-, was produced and secreted by the methylotrophic yeast Pichia pastoris and found to be O-glycosylated . The O-linked glycans were composed entirely of mannose in a ratio of 1 mol of mannose to 4 mol of protein . The overall distribution of mannose on the O-glycosylated CBM mutant ranged from 1 to 9 mannose residues with the oligosaccharide sizes ranging from Man(1) to Man(4) . MALDI-TOF (all matrix-assisted-laser-desorption time of flight) mass spectrometry (MS) was used to map the O-glycosylation to three regions of the polypeptide, each region having a maximum of 4 mannose residues attached to each . Glycans chemically released from CBM2aNgly- and analyzed by fluorophore-assisted carbohydrate electrophoresis were found to contain alpha-1,2-, alpha-1,3-, and alpha-1,6-linkages . Significantly, the O-glycosylation did not influence binding, making CBM2aNgly- a suitable fusion partner for polypeptides produced in P . pastoris and other eukaryotic hosts . Eur J Biochem, 2003 Feb, 270(4), 771 - 8 Purification and cDNA cloning of a cellulase from abalone Haliotis discus hannai; Suzuki K et al.; A cellulase {endo-beta-1,4-D-glucanase (EC 3.2.1.4)} was isolated from the hepatopancreas of abalone Haliotis discus hannai by successive chromatographies on TOYOPEARL CM-650M, hydroxyapatite and Sephacryl S-200 HR . The molecular mass of the cellulase was estimated to be 66 000 Da by SDS/PAGE, thus the enzyme was named HdEG66 . The hydrolytic activity of HdEG66 toward carboxymethylcellulose showed optimal temperature and pH at 38 degrees C and 6.3, respectively . cDNAs encoding HdEG66 were amplified by the polymerase chain reaction from an abalone hepatopancreas cDNA library with primers synthesized on the basis of partial amino-acid sequences of HdEG66 . By overlapping the nucleotide sequences of the cDNAs, a sequence of 1898 bp in total was determined . The coding region of 1785 bp located at nucleotide position 56-1840 gave an amino-acid sequence of 594 residues including the initiation methionine . The N-terminal region of 14 residues in the deduced sequence was regarded as the signal peptide as it was absent in HdEG66 protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins . Thus, matured HdEG66 was thought to consist of 579 residues . The C-terminal region of 453 residues in HdEG66, i.e . approximately the C-terminal three quarters of the protein, showed 42-44% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)-cellulases from arthropods and Thermomonospora fusca . While the N-terminal first quarter of HdEG66 showed 27% identity to the carbohydrate-binding module (CBM) of a Cellulomonas fimi cellulase, CenA . Thus, the HdEG66 was regarded as the GHF9-cellulase possessing a family II CBM in the N-terminal region . By genomic PCR using specific primers to the 3'-terminal coding sequences of HdEG66-cDNA, a DNA of 2186 bp including three introns was amplified . This strongly suggests that the origin of HdEG66 is not from symbiotic bacteria but abalone itself. Wei Sheng Wu Xue Bao, 2001 Dec, 41(6), 741 - 4 {Study on the Apriona germari(Hope) larvae's intestinal bacterial flora}; He Z et al.; Intestinal flora of 47 Apriona germari(Hope) larvae, collected from fields, had been isolated and identified . The results showed that the predominant bacteria were Staphylococcus . Its viable count was 7.63 +/- 0.21, and the detection rate was 100% . Meanwhile, a strain of cellulose-utilizing bacterium was isolated from the fore-midgut fluid of A . germari larvae with the cellulose-congo red agar medium . The bacterium was tentatively identified as Cellulomonas . The detection rate of the cellulolytic bacterium was 23.40%, and the count was 3.84 +/- 0.54 approximately . Its contribution to the borer's cellulose digestion needs further investigations. Appl Microbiol Biotechnol, 2002 Oct, 60(1-2), 192 - 9 Epub 2002 Aug 22. Dissimilatory reduction of Cr(VI), Fe(III), and U(VI) by Cellulomonas isolates; Sani RK et al.; The reduction of Cr(VI), Fe(III), and U(VI) was studied using three recently isolated environmental Cellulomonas sp . (WS01, WS18, and ES5) and a known Cellulomonas strain ( Cellulomonas flavigena ATCC 482) under anaerobic, non-growth conditions . In all cases, these cultures were observed to reduce Cr(VI), Fe(III), and U(VI) . In 100 h, with lactate as electron donor, the Cellulomonas isolates (500 mg/l total cell protein) reduced nitrilotriacetic acid chelated Fe(III) {Fe(III)-NTA} from 5 mM to less than 2.2 mM, Cr(VI) from 0.2 mM to less than 0.001 mM, and U(VI) from 0.2 mM to less than 0.12 mM . All Cellulomonas isolates also reduced Cr(VI), Fe(III), and U(VI) in the absence of lactate, while no metal reduction was observed in either the cell-free or heat-killed cell controls . This is the first report of Cellulomonas sp . reducing Fe(III) and U(VI) . Further, this is the first report of Cellulomonas spp . coupling the oxidation of lactate, or other unknown electron donors in the absence of lactate, to the reduction of Cr(VI), Fe(III), and U(VI). Appl Microbiol Biotechnol, 2002 Oct, 60(1-2), 88 - 93 Epub 2002 Aug 08. Screening of facultative anaerobic bacteria utilizing D-xylose for xylitol production; Rangaswamy S et al.; Seventeen cultures belonging to three genera of facultative bacteria ( Serratia, Cellulomonas, and Corynebacterium) were screened for the production of xylitol, a sugar alcohol used as a sweetener in the pharmaceutical and food industries . The bacterial strains that utilized D-xylose for growth were investigated for xylitol production . A chromogenic assay of both solid and liquid cultures showed that ten of the 17 bacteria screened could grow on D-xylose and produce detectable quantities of xylitol during 24-96 h of fermentation . Among the screened cultures, Corynebacterium sp . B-4247 produced the highest amount of xylitol . In addition, the ten bacterial cultures that initially produced xylitol were studied for the effect of the environmental factors, such as temperature, concentration of D-xylose and aeration, on xylitol production. FEMS Microbiol Lett, 2002 Sep 10, 214(2), 205 - 9 Cellulomonas flavigena: characterization of an endo-1,4-xylanase tightly induced by sugarcane bagasse; Mayorga-Reyes L et al.; Xylanases, an important group of enzymes for biomass degradation in the industry, are commonly found forming complex multienzyme systems . As a preliminary step to the construction of efficient xylanase producers using genetic engineering, we have characterized a gene encoding an endo-beta-1,4 xylanase (xyncflA) from Cellulomonas flavigena . The xylanase activity and the xyncflA synthesis were higher when C . flavigena was grown on sugarcane bagasse . In this substrate, both activity and transcript increased with approximately the same rate during the culture period . When C . flavigena grew on glucose, low signal of mRNA was observed, suggesting that the xyncflA gene is regulated at the transcriptional level. J Int Med Res, 2002 Jul-Aug, 30(4), 422 - 7 Coryneform bacteria isolated from blood cultures and their antibiotic susceptibilities; Balci I et al.; We aimed to determine the types of corynebacteria isolated from the blood of patients at Gaziantep University Hospital, Turkey, and their antibiotic susceptibilities . Between February 1999 and June 2001, 3530 blood samples were cultured, of which 915 were found to be positive, and these were further investigated in the bacteriology laboratory . Among positive blood cultures, coryneform bacteria were identified in 31 (3.4%) isolates . Of these, 16 (51.6%) were Corynebacterium jeikeium, six (19.4%) were Corynebacterium striatum, four (12.9%) were Corynebacterium amycolatum, two (6.5%) were Cellulomonas species, two (6.5%) were Corynebacterium afermentans and one isolate (3.2%) was Corynebacterium propinquum . Antibiotic susceptibility tests showed that C . jeikeium was resistant to various antibiotics, whereas all isolates were susceptible to vancomycin and teicoplanin . This study illustrates the importance of taking coryneform bacteria into consideration when culturing blood samples . The need to identify the species and determine its antibiotic sensitivity is emphasized. Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1185 - 92 Cellulosimicrobium variabile sp . nov., a cellulolytic bacterium from the hindgut of the termite Mastotermes darwiniensis; Bakalidou A et al.; A novel cellulolytic and xylanolytic bacterium, strain MX5T, was isolated from the hindgut contents of the Australian termite Mastotermes darwiniensis (Froggatt) . The isolate was a facultative anaerobe and had a Gram-positive cell-wall profile . The rod-shaped bacterium formed irregular coryneform and coccoid cells during growth . Phylogenetic analysis of the 16S rDNA provided evidence that the organism was closely related to the as-yet undescribed cellulolytic strain SR272 and the non-validly described species 'Cellulomonas pachnodae' as well as Promicromonospora citrea and Promicromonospora sukumoe . Strain MX5T was assigned to the genus Cellulosimicrobium on the basis of phylogenetic and chemotaxonomic criteria . The murein of strain MX5T contained the diamino acid lysine . N-Glycolylmuramic acid, mycolic acids and hydroxy fatty acids were absent . The major neutral sugar in the cell wall was galactose and the major quinone was menaquinone MK-9(H4) . The predominant fatty acids were ai-C15:0, i-C15:0, i-C16:0 and C16:0 . The G+C content of the DNA was in a range 70-72 mol% . On the basis of 16S rDNA sequence similarities and chemotaxonomic features, MX5T was clearly different from Cellulosimicrobium cellulans and other validly described species within this phylogenetic group . For this reason, a novel species is described, for which the name Cellulosimicrobium variabile sp . nov . is proposed. Int J Syst Evol Microbiol, 2002 Jul, 52(Pt 4), 1105 - 11 Re-evaluation of the status of the genus Oerskovia, reclassification of Promicromonospora enterophila (Jáger et al . 1983) as Oerskovia enterophila comb . nov . and description of Oerskovia jenensis sp . nov . and Oerskovia paurometabola sp . nov; Stackebrandt E et al.; Phylogenetic analysis of Promicromonospora enterophila indicates that this taxon clusters with Cellulomonas species, adjacent to Cellulomonas turbata (basonym Oerskovia turbata) . 16S rDNA analysis, DNA-DNA reassociation, riboprinting, peptidoglycan analysis and determination of phenotypic properties of various strains of P . enterophila and C turbata reveal that they form a cluster that can be distinguished unambiguously from other Cellulomonas species by morphology, amino acid composition of the cell wall and 16S rDNA signatures . As a result of thispolyphasic study, it appears taxonomically reasonable to re-establish the genus Oerskovia for C turbata and to reclassify P . enterophila as Oerskovia enterophila comb . nov.; two novel species, Oerskovia jenensis sp . nov . (type strain DSM 46000T = CIP 100330T) and Oerskovia paurometabola sp . nov . (type strain DSM 14281T = LMG 20385T), are also proposed. J Clin Microbiol, 2002 Jul, 40(7), 2681 - 5 Catheter-related Microbacterium bacteremia identified by 16S rRNA gene sequencing; Lau SK et al.; We describe the application of 16S rRNA gene sequencing in defining two cases of catheter-related Microbacterium bacteremia . In the first case, a gram-positive bacillus was isolated from both the blood culture and central catheter tip of a 39-year-old woman with chronic myeloid leukemia . The API Coryne system identified the isolate as 98.9% Aureobacterium or Corynebacterium aquaticum . In the second case, a gram-positive bacillus was recovered from five sets of blood cultures from both central catheter and percutaneous venipuncture of a 5-year-old girl with acute myeloid leukemia . The isolate was identified by the API Coryne system as 99.7% Cellulomonas or Microbacterium species . Further phenotypic tests failed to identify the two isolates . 16S rRNA gene sequencing showed 99.4% similarity between the first isolate and Microbacterium oxydans and 98.7% similarity between the second isolate and Microbacterium trichotecenolyticum, indicating that both isolates were Microbacterium species . Microbacterium infections are rarely reported in the literature . Although the central venous catheter was previously proposed to be a source of bacteremia, the first case in this report represents the first culture-documented case of catheter-related Microbacterium bacteremia. J Mol Biol, 2002 Jun 21, 319(5), 1143 - 56 Differential oligosaccharide recognition by evolutionarily-related beta-1,4 and beta-1,3 glucan-binding modules; Boraston AB et al.; Enzymes active on complex carbohydrate polymers frequently have modular structures in which a catalytic domain is appended to one or more carbohydrate-binding modules (CBMs) . Although CBMs have been classified into a number of families based upon sequence, many closely related CBMs are specific for different polysaccharides . In order to provide a structural rationale for the recognition of different polysaccharides by CBMs displaying a conserved fold, we have studied the thermodynamics of binding and three-dimensional structures of the related family 4 CBMs from Cellulomonas fimi Cel9B and Thermotoga maritima Lam16A in complex with their ligands, beta-1,4 and beta-1,3 linked gluco-oligosaccharides, respectively . These two CBMs use a structurally conserved constellation of aromatic and polar amino acid side-chains that interact with sugars in two of the five binding subsites . Differences in the length and conformation of loops in non-conserved regions create binding-site topographies that complement the known solution conformations of their respective ligands . Thermodynamics interpreted in the light of structural information highlights the differential role of water in the interaction of these CBMs with their respective oligosaccharide ligands . (c) 2002 Elsevier Science Ltd. Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 875 - 81 Georgenia muralis gen . nov., sp . nov., a novel actinobacterium isolated from a medieval wall painting; Altenburger P et al.; Two bacterial strains, designated 1A-C(T) and 3A-1, were studied and, using these results and previously published data, taxonomically classified . Cells of the strains exhibited a rod-coccus cycle . The peptidoglycan determined for 1A-C(T) was of type A4alpha with lysine as the diagnostic cell-wall diamino acid and an interpeptide bridge of L-Lys <-- L-Glu . The menaquinone systems of the two strains contained MK-8(H4) (82-94%) and MK-7(H4) (3-11%) . The polar lipid profiles consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannoside, two unidentified phospholipids and an unidentified glycolipid . The fatty acid profiles contained predominantly ai-C15:0 and significant amounts of i-C14:0 and i-C15:1 fatty acids . Genomic fingerprints clearly distinguished strains 1A-C(T) and 3A-1 from each other . DNA-DNA relatedness between the two strains (92%) demonstrated that they are members of a single species . Analyses of the 16S rDNA sequences of strains IA-C(T) and 3A-1, which were almost identical (99.6% sequence similarity), and comparison with corresponding sequences demonstrated that they represent a novel lineage within the suborder Micrococcineae, most closely related to species of the genera Beutenbergia, Bogoriella and Cellulomonas (94.7-95.7% sequence similarity) . The results demonstrate that the two strains are members of a single new genus and a single novel species . Thus, the name Georgenia muralis gen . nov., sp . nov . is proposed . The type strain is strain 1A-C(T) (= DSM 14418T = CCM 4963T) . Another strain of the species is strain 3A-1 (= DSM 14419 = CCM 4964). Biochim Biophys Acta, 2002 Jun 3, 1597(2), 320 - 34 Purine nucleoside phosphorylase from Cellulomonas sp.: physicochemical properties and binding of substrates determined by ligand-dependent enhancement of enzyme intrinsic fluorescence, and by protective effects of ligands on thermal inactivation of the enzyme; Wielgus-Kutrowska B et al.; Purine nucleoside phosphorylase (PNP) from Cellulomonas sp., homotrimeric in the crystalline state, is also a trimer in solution . Other features of the enzyme are typical for "low molecular mass" PNPs, except for its unusual stability at pH 11 . Purine bases, alpha-D-ribose-1-phosphate (R1P) and phosphate enhance the intrinsic fluorescence of Cellulomonas PNP, and hence form binary complexes and induce conformational changes of the protein that alter the microenvironment of tryptophan residue(s) . The effect due to guanine (Gua) binding is much higher than those caused by other ligands, suggesting that the enzyme preferentially binds a fluorescent, most probably rare tautomeric anionic form of Gua, further shown by comparison of emission properties of the PNP/Gua complex with that of Gua anion and its N-methyl derivatives . Guanosine (Guo) and inosine (Ino) at 100 microM concentration show little and no effect, respectively, on enzyme intrinsic fluorescence, but their protective effect against thermal inactivation of the enzyme points to their forming weak binary complexes with PNP . Binding of Gua, hypoxanthine (Hx) and R1P to the trimeric enzyme is described by one dissociation constant, K(d)=0.46 microM for Gua, 3.0 microM for Hx, and 60 microM for R1P . The binding stoichiometry for Gua (and probably Hx) is three ligand molecules per enzyme trimer . Effects of phosphate on the enzyme intrinsic fluorescence are due not only to binding, but also to an increase in ionic strength, as shown by titration with KCl . When corrected for effects of ionic strength, titration data with phosphate are most consistent with one dissociation constant, K(d)=270 microM, but existence of a very weak binding site with K(d)>50 mM could not be unequivocally ruled out . Binding of Gua to the PNP/phosphate binary complex is weaker (K(d)=1.7 microM) than to the free enzyme (K(d)=0.46 microM), suggesting that phosphate helps release the purine base in the catalytic process of phosphorolysis.The results indicate that nonlinear kinetic plots of initial velocity, typical for PNPs, including Cellulomonas PNP, are not, as generally assumed, due to cooperative interaction between monomers forming the trimer, but to a more complex kinetic mechanism than hitherto considered. Appl Environ Microbiol, 2002 May, 68(5), 2198 - 208 Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants; Zinniel DK et al.; Endophytic bacteria reside within plant hosts without causing disease symptoms . In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species . We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates . In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log(10) CFU/g (fresh weight) . More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes . In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log(10) CFU/plant or higher . Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log(10) CFU/g (fresh weight) . Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log(10) CFU/g (fresh weight) up to 78 days postinoculation . Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses . This study defines for the first time the endophytic nature of Microbacterium testaceum . These microorganisms may be useful for biocontrol and other applications. Appl Biochem Biotechnol, 2001 Spring, 91-93, 575 - 92 Do cellulose binding domains increase substrate accessibility? Esteghlalian AR, Srivastava V, Gilkes NR, Kilburn DG, Warren RA, Saddle JN. This article provides an overview of various theories proposed during the past five decades to describe the enzymatic hydrolysis of cellulose highlighting the major shifts that these theories have undergone . It also describes the effect of the cellulose-binding domain (CBD) of an exoglucanase/xylanase from bacterium Cellulomonas fimi on the enzymatic hydrolysis of Avicel . Pretreatment of Avicel with CBDCex at 4 and 37 degrees C as well as simultaneous addition of CBDCex to the hydrolytic enzyme (Celluclast, Novo, Nordisk) reduced the initial rate of hydrolysis owing to irreversible binding of CBD proteins to the substrate's binding sites . Nonetheless, near complete hydrolysis was achieved even in the presence of CBDCex . Protease treatment of both pure and CBDCex-treated Avicel reduced the substrates' hydrolyzability, perhaps owing to proteolysis of the hydrolyzing enzyme (Celluclast) by the residual Proteinase K remaining in the substrate . Better protocols for complete removal of CBD proteins from the substrate need to be developed to investigate the effect of CBD adsorption on cellulose digestibility. Appl Microbiol Biotechnol, 2002 Mar, 58(4), 435 - 8 Epub 2002 Jan 19. Draw-fill batch culture mode for production of xylanases by Cellulomonas flavigena on sugar cane bagasse; Vega-Estrada J et al.; Draw-fill culture was evaluated as a method for xylanase production by Cellulomonas flavigena on sugar cane bagasse . Specific xylanase activity and volumetric xylanase activities were measured by harvesting 50%, 55%, 60% and 70% of fermented broth at the end of each subculture . Maximum specific (64 IU mg(-1) protein) and volumetric (166 IU ml(-1)) xylanase activities were obtained by harvesting 50-55% of broth . Values were 3.4 and 3.8 times greater than those obtained in batch cultures carried out under the same conditions . Enzyme productivity of 4.2 IU ml(-1) h(-1) was significantly greater than that obtained in continuous cultures (2.4 IU ml(-1) h(-1)) (P<0.05). Mol Cells, 2002 Feb 28, 13(1), 28 - 34 Cloning of the cel8Y gene from Pectobacterium chrysanthemi PY35 and its comparison to cel genes of soft-rot Pectobacterium; Cho SJ et al.; The phytopathogenic Pectobacterium chrysanthemi (Pch) PY35 secretes multiple isozymes of plant cell wall degrading enzyme cellulases . We cloned a second cel gene that encodes cellulase in Pch PY35 . The inserted 2 kb fragment was subcloned in order to geneate pPY710 (cel8Y) . The structural organization of the cel8Y gene consists of an open reading frame (ORF) of 999 bp that encodes 332 amino acid residues with a signal peptide of 23 amino acids . The predicted amino acid sequence of Cel8Y was very similar to that of Cellulomonas uda, but completely different from that of the Cel5Z of Pch PY35 . It belonged to the glycoside hydrolase family 8, based on amino acid sequence similarities in contrast to Cel5Z of Pch PY35, which was confirmed as family 5 . Cel8Y was not closely related to the known cellulases of Pectobacterium . It had the conserved region of the glycoside hydrolase family 8, ASDGDVLIAWALLKAGNKW . The apparent molecular mass of the Cel8Y protein was calculated to be approximately 34 kDa by a carboxymethylcellulosesodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE) . The Cel8Y had a calculated pl of 6.49 . It was optimally active at pH 7 with an approximate optimal temperature around 40 degrees C . The cellulase activity of Cel8Y was lower than that of Cel5Z. J Mol Biol, 2002 Feb 8, 316(1), 89 - 100 5-Methylthiopentose: a new substituent on lipoarabinomannan in Mycobacterium tuberculosis; Treumann A et al.; We have identified and characterised in several strains of Mycobacterium tuberculosis a new 5-methylthiopentose substituent on lipoarabinomannan (LAM) . The 5-methylthiopentose was initially observed in heteronuclear (1)H-(13)C-NMR spectra of intact, (13)C-enriched LAM . Oligosaccharides carrying this substituent were released from (13)C-enriched LAM and from unlabelled LAM using an endo-arabinanase from Cellulomonas gellida . The presence of the methylthio group in these oligosaccharides was established using NMR, high-resolution Fourier-transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry using a Q-TOF mass spectrometer . The 5-methylthiopentose is linked to a terminal mannose in the cap structures of these oligosaccharides as evidenced by tandem mass spectrometry and by NMR . We suggest interference with the signal transduction mechanisms of infected macrophages as a possible function for this newly discovered LAM substituent . Can J Microbiol, 2001 Dec, 47(12), 1063 - 7 Mapping of genes encoding glycoside hydrolases on the chromosome of Cellulomonas fimi; Stoll D; Cellulomonas fimi genomic DNA was digested with HpaI, MunI, HindIII, and NsiI, producing fragments ranging in size from 20 to 1400 kbp that were resolved by pulsed field gel electrophoresis . Genetic and physical linkages were determined by Southern blotting and were used to construct a genome map . Cellulomonas fimi has a single circular chromosome of approx . 4000 kbp . Except for two closely linked genes, cbh6A and cel5A, the genes known to encode glycoside hydrolases are scattered widely on the chromosome. FEMS Microbiol Lett, 2001 Nov 13, 204(2), 367 - 74 Chitin degradation by cellulolytic anaerobes and facultative aerobes from soils and sediments; Reguera G et al.; Species of strictly and facultatively anaerobic cellulolytic bacteria from soils and sediments were examined for the ability to degrade chitin . Of 22 species studied, 16 degraded insoluble chitin . Cellulomonas uda, which was selected for a comparative study of its cellulase and chitinase enzyme systems, produced different enzyme systems for the degradation of cellulose and chitin and different patterns of regulation of production of the two enzyme systems were observed . Moreover, C . uda utilized chitin as a source of nitrogen for the degradation of cellulose . In natural environments, the ability to use chitin as a nitrogen source may confer on cellulolytic microorganisms, such as C . uda, a selective advantage over other cellulolytic microbes. Protein Expr Purif, 2001 Oct, 23(1), 159 - 66 Purification and characterization of a major secretory cellobiase, Cba2, from Cellulomonas biazotea; Lau AT et al.; A novel cellobiase (Cba2) was purified from the culture supernatant of Cellulomonas biazotea and characterized . Cba2 appeared to be a major secretory cellobiase in C . biazotea as its enzymatic activity was estimated to represent over 40% of the total extracellular beta-glucosidase activity . The enzyme was purified over 260-fold subsequent to ammonium sulfate precipitation, gel-filtration chromatography, anion-exchange chromatography, and reversed-phase high-performance liquid chromatography . Cba2 was shown by SDS-PAGE to have a large molecular mass of 109 kDa, which makes it one of the largest secretory cellobiases characterized . Its homogeneity was confirmed by N-terminal amino acid sequencing . The K(m) and V(max) values were 0.025 mM and 0.0048 mM min(-1), respectively, for the Cba2 hydrolysis of p-nitrophenyl-beta-d-glucopyranoside, and 0.73 mM and 0.00033 mM min(-1), respectively, for the hydrolysis of cellobiose (at 37 degrees C and pH 7.0) . The purified enzyme has a pH optimum of 4.8 and the optimum temperature for activity is 70 degrees C . In view of the secretory nature of Cba2 and the fact that it is a major component of secretory cellobiases of C . biazotea, it is potentially important in the enzymatic degradation of cellulose, and its availability as a recombinant protein may facilitate the studies of its biotechnological applications . Biochem J, 2001 Sep 1, 358(Pt 2), 423 - 30 Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis; Boraston AB et al.; When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated . The glycans are of the high-mannose type, ranging in size from GlcNAc(2)Man(8) to GlcNAc(2)Man(14) . The N-linked glycans block the binding of CBM2a to cellulose . Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (K(a)) for the binding of CBM2a to bacterial microcrystalline cellulose approx . 10-fold, whereas glycans on Asn-87 destroy binding . The K(a) of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P . pastoris and is approx . half that of wild-type CBM2a produced by E . coli. Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1007 - 10 Reclassification of Cellulomonas cellulans (Stackebrandt and Keddie 1986) as Cellulosimicrobium cellulans gen . nov., comb . nov; Schumann P et al.; Phylogenetic analysis of 16S rDNA provides evidence that Cellulomonas cellulans branches outside the phylogenetic confines of the genus Cellulomonas . The distinct phylogenetic and chemotaxonomic status of Cellulomonas cellulans as a phylogenetic neighbour of the genus Promicromonospora, justifies the description of a new genus for which the name Cellulosimicrobium gen . nov . with the type species Cellulosimicrobium cellulans comb . nov . is proposed. Biochemistry, 2001 May 15, 40(19), 5700 - 7 Role of hydrogen bonding in the interaction between a xylan binding module and xylan; Xie H et al.; NMR studies of the internal family 2b carbohydrate binding module (CBM2b-1) of Cellulomonas fimi xylanase 11A have identified six polar residues and two aromatic residues that interact with its target ligand, xylan . To investigate the importance of the various interactions, free energy and enthalpy changes have been measured for the binding of xylan to native and mutant forms of CBM2b-1 . The data show that the two aromatic residues, Trp 259 and Trp 291, play a critical role in the binding, and similarly that mutants N264A and T316A have no affinity for the xylose polymer . Interestingly, mutations E257A, Q288A, N292A, E257A/Q288A, E257A/N292A, and E257A/N292A/Q288A do not significantly diminish the affinity of CBM2b-1 for the xylose polymers, but do influence the thermodynamics driving the protein-carbohydrate interactions . These thermodynamic parameters have been interpreted in light of a fresh understanding of enthalpy-entropy compensation and show the following . (1) For proteins whose ligands are bound on an exposed surface, hydrogen bonding confers little specificity or affinity . It also displays little cooperativity . Most specificity and affinity derive from binding between the face of sugar rings and aromatic rings . (2) Loss of hydrogen bonding interactions leads to a redistribution of the remaining bonding interactions such that the entropic mobility of the ligand is maximized, at the expense (if necessary) of enthalpically favorable bonds . (3) Changes in entropy and enthalpy in the binding between polysaccharide and a range of mutants can be interpreted by considering changes in binding and flexibility, without any need to consider solvent reorganization. Bioresour Technol, 2001 Jul, 78(3), 285 - 91 Regulation of cellulases and xylanases from a derepressed mutant of Cellulomonas flavigena growing on sugar-cane bagasse in continuous culture; Ponce-Noyola T et al.; When the wild type Cellulomonas flavigena was grown on glycerol, xylose or cellobiose, it produced basal levels of carboxymethyl-cellulase (CMCase), filter-paperase (FPase) and xylanase activities . By comparison, a catabolic derepressed mutant strain of the same organism produced markedly higher levels of these enzymes when grown on the same carbon sources . Sugar-cane bagasse induced both the wild type and the mutant strain to produce three- to eight-time higher levels of FPase and xylanase than was observed with xylose or cellobiose . Continuous culture was used to determine the minimal cellobiose or glucose concentrations that repress the enzyme synthesis in both strains . 2.5 g l(-1) glucose repressed FPase and xylanases from wild type, while 1.6 times more glucose was needed to repress the same activities in the PN-120 strain . In the same way, twofold more cellobiose was needed to reduce by 75% the CMCase and xylanase activities in the mutant compared to the wild type . The FPase in the presence of 4 g l(-1) cellobiose did not change in the same strain . Therefore, its derepressed and feedback resistant characters of PN-120 mutant are evident . On the other hand, isoelectrofocused crude extracts of mutant and wild strains induced by sugar-cane bagasse, did not show differences in protein patterns, however, the Schiffs staining was more intense in the PN-120 than in the wild strain . These results point out that the mutational treatment did not apparently change the extracellular proteins from mutant PN-120 and this could affect their regulation sites, since derepressed and feed-back resistant enzymes may be produced. Biochemistry, 2001 Feb 27, 40(8), 2468 - 77 Evidence for synergy between family 2b carbohydrate binding modules in Cellulomonas fimi xylanase 11A; Bolam DN et al.; Glycoside hydrolases often contain multiple copies of noncatalytic carbohydrate binding modules (CBMs) from the same or different families . Currently, the functional importance of this complex molecular architecture is unclear . To investigate the role of multiple CBMs in plant cell wall hydrolases, we have determined the polysaccharide binding properties of wild type and various derivatives of Cellulomonas fimi xylanase 11A (Cf Xyn11A) . This protein, which binds to both cellulose and xylan, contains two family 2b CBMs that exhibit 70% sequence identity, one internal (CBM2b-1), which has previously been shown to bind specifically to xylan and the other at the C-terminus (CBM2b-2) . Biochemical characterization of CBM2b-2 showed that the module bound to insoluble and soluble oat spelt xylan and xylohexaose with K(a) values of 5.6 x 10(4), 1.2 x 10(4), and 4.8 x 10(3) M(-1), respectively, but exhibited extremely weak affinity for cellohexaose (<10(2) M(-1)), and its interaction with insoluble cellulose was too weak to quantify . The CBM did not interact with soluble forms of other plant cell wall polysaccharides . The three-dimensional structure of CBM2b-2 was determined by NMR spectroscopy . The module has a twisted "beta-sandwich" architecture, and the two surface exposed tryptophans, Trp 570 and Trp 602, which are in a perpendicular orientation with each other, were shown to be essential for ligand binding . In addition, changing Arg 573 to glycine altered the polysaccharide binding specificity of the module from xylan to cellulose . These data demonstrate that the biochemical properties and tertiary structure of CBM2b-2 and CBM2b-1 are extremely similar . When CBM2b-1 and CBM2b-2 were incorporated into a single polypeptide chain, either in the full-length enzyme or an artificial construct comprising both CBM2bs covalently joined via a flexible linker, there was an approximate 18-20-fold increase in the affinity of the protein for soluble and insoluble xylan, as compared to the individual modules, and a measurable interaction with insoluble acid-swollen cellulose, although the K(a) (approximately 6.0 x 10(4) M(-1)) was still much lower than for insoluble xylan (K(a) = approximately 1.0 x 10(6) M(-1)) . These data demonstrate that the two family 2b CBMs of Cf Xyn11A act in synergy to bind acid swollen cellulose and xylan . We propose that the increased affinity of glycoside hydrolases for polysaccharides, through the synergistic interactions of CBMs, provides an explanation for the duplication of CBMs from the same family in some prokaryotic cellulases and xylanases. Microbiol Res, 2001 Mar, 155(4), 301 - 7 Vitamin requirements of hydrocarbon-utilizing soil bacteria; Radwan SS et al.; The numbers of oil-utilizing bacteria in several samples of clean and oil-polluted soils counted on vitamin-containing media were severalfold higher than the numbers counted on vitamin-free media . Colonies that grew on a medium containing a vitamin mixture were tested for growth on the same medium lacking any vitamins . More than 90% of the total colonies failed to grow . The remaining 10% grew, yet their growth was enhanced, when vitamins were added . The predominant oil-utilizing bacteria in one of the test desert soil samples were various strains of Cellulomonas flavigena and Rhodococcus erythropolis . Minor organisms belonged to the genera Pseudomonas, Bacillus and Arthrobacter . Two vitamin-requiring biovars of C . flavigena and R . erythropolis were selected for further study . Their growth on n-octadecane and phenanthrene as sole sources of carbon and energy as well as their potential for hydrocarbon consumption were enhanced by added vitamins, e.g . folic acid, pyridoxine, vitamin B12, biotin and others . In a field experiment, it was confirmed that vitamin fertilization of an oil-polluted sand sample enhanced the biodegradation of constituent hydrocarbons of that sample. Protein Expr Purif, 2001 Apr, 21(3), 417 - 23 A family 2a carbohydrate-binding module suitable as an affinity tag for proteins produced in Pichia pastoris; Boraston AB et al.; The family 2a carbohydrate-binding module (CBM), Cel5ACBM2a, from the C-terminus of Cel5A from Cellulomonas fimi, and Xyn10ACBM2a, the family 2a CBM from the C-terminus of Xyn10A from C . fimi, were compared as fusion partners for proteins produced in the methylotrophic yeast Pichia pastoris . Gene fusions of murine stem-cell factor (SCF) with both CBMs were expressed in P . pastoris . The secreted SCF-Xyn10ACBM2a polypeptides were highly glycosylated and bound poorly to cellulose . In contrast, fusion of SCF to Cel5ACBM2a, which lacks potential N-linked glycosylation sites, resulted in the production of polypeptides which bound tightly to cellulose . Cloning and expression of these CBM2a in P . pastoris without a fusion partner confirmed that N-linked glycosylation at several sites was responsible for the poor cellulose binding . The nonglycosylated CBMs produced in E . coli had very similar cellulose-binding properties . Protein Eng, 2000 Dec, 13(12), 873 - 9 Module shuffling of a family F/10 xylanase: replacement of modules M4 and M5 of the FXYN of Streptomyces olivaceoviridis E-86 with those of the Cex of Cellulomonas fimi; Kaneko S et al.; To facilitate an understanding of structure-function relationships, chimeric xylanases were constructed by module shuffling between the catalytic domains of the FXYN from Streptomyces olivaceoviridis E-86 and the Cex from Cellulomonas fimi . In the family F/10 xylanases, the modules M4 and M5 relate to substrate binding so that modules M4 and M5 of the FXYN were replaced with those of the Cex and the chimeric enzymes denoted FCF-C4, FCF-C5 and FCF-C4,5 were constructed . The k(cat) value of FCF-C5 for p-nitrophenyl-beta-D-cellobioside was similar to that of the FXYN (2.2 s(-1)); however, the k(cat) value of FCF-C4 for p-nitrophenyl-beta-D-cellobioside was significantly higher (7.0 s(-1)) . The loss of the hydrogen bond between E46 and S22 or the presence of the I49W mutation would be expected to change the position of Q88, which plays a pivotal role in discriminating between glucose and xylose, resulting in the increased k(cat) value observed for FCF-C4 acting on p-nitrophenyl-beta-D-cellobioside since module M4 directly interacts with Q88 . To investigate the synergistic effects of the different modules, module M10 of the FCF-C4 chimera was replaced with that of the Cex . The effects of replacement of module M4 and M10 were almost additive with regard to the K:(m) and k(cat) values. Protein Eng, 2000 Nov, 13(11), 801 - 9 Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues; McLean BW et al.; The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose . It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose . The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues . Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72) . Other residues on the binding face with hydrogen bonding potential were substituted with alanine . Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72 . Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity . Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface. J Bacteriol, 2001 Mar, 183(5), 1552 - 9 Properties and mutation analysis of the CelK cellulose-binding domain from the Clostridium thermocellum cellulosome; Kataeva IA et al.; The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBD(CelK)) was expressed in Escherichia coli and purified . It binds to acid-swollen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with capacities of 16.03 and 3.95 micromol/g of cellulose and relative affinities (K(r)) of 2.33 and 9.87 liters/g, respectively . The CBD(CelK) is the first representative of family IV CBDs to exhibit an affinity for BMCC . The CBD(CelK) also binds to the soluble polysaccharides lichenin, glucomannan, and barley beta-glucan, which are substrates for CelK . It does not bind to xylan, galactomannan, and carboxymethyl cellulose . The CBD(CelK) contains 1 mol of calcium per mol . The CBD(CelK) has three thiol groups and one disulfide, reduction of which results in total loss of cellulose-binding ability . To reveal amino acid residues important for biological function of the domain and to investigate the role of calcium in the CBD(CelK) four highly conserved aromatic residues (Trp(56), Trp(94), Tyr(111), and Tyr(136)) and Asp(192) were mutated into alanines, giving the mutants W56A, W94A, Y111A, Y136A, and D192A . In addition 14 N-terminal amino acids were deleted, giving the CBD-N(CelK) . The CBD-N(CelK) and D192A retained binding parameters close to that of the intact CBD(CelK), W56A and W94A totally lost the ability to bind to cellulose, Y136A bound to both ASC and BMCC but with significantly reduced binding capacity and K(r) and Y111A bound weakly to ASC and did not bind to BMCC . Mutations of the aromatic residues in the CBD(CelK) led to structural changes revealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation . Calcium content was drastically decreased in D192A . The results suggest that Asp192 is in the calcium-binding site of the CBD(CelK) and that calcium does not affect binding to cellulose . The 14 amino acids from the N terminus of the CBD(CelK) are not important for binding . Tyr136, corresponding to Cellulomonas fimi CenC CBD(N1) Y85, located near the binding cleft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBD(CelK) correctly folded. FEMS Microbiol Ecol, 2001 Jan, 34(3), 229 - 241 Enumeration and characterization of cellulolytic bacteria from refuse of a landfill; Pourcher A et al.; Enumeration and phenotypic characterization of aerobic cellulolytic bacteria were performed on fresh, 1 year old and 5 years old refuse samples of a French landfill site . Numbers of cellulolytic bacteria ranged from 1.1x10(6) to 2.3x10(8) c.f.u . (g dry wt.)(-1) and were lower in 5 years old refuse samples . A numerical analysis of phenotypic data based on 80 biochemical tests and performed on 321 Gram-positive isolates from refuse, revealed a high phenotypic diversity of cellulolytic bacteria which were distributed into 21 clusters . Based on the phenotypic analysis and the sequencing of 16S rDNA of five representative strains of major clusters, the predominant cellulolytic groups could be assigned to the family of Bacillaceae and to the genera Cellulomonas, Microbacterium and Lactobacillus . Furthermore, chemical parameters such as pH, carbohydrates and volatile solid contents influenced the composition of the cellulolytic bacterial groups which were reduced essentially to the family of Bacillaceae in the oldest refuse samples. Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 420 - 6 Cloning and characterization of thermostable endoglucanase (Cel8Y) from the hyperthermophilic Aquifex aeolicus VF5; Kim JO et al.; Aquifex aeolicus is the hyperthermophilic bacterium known, with growth-temperature maxima near 95 degrees C . The cel8Y gene, encoding a thermostable endoglucanase (Cel8Y) from Aquifex aeolicus VF5, was cloned into a vector for expression and expressed in Escherichia coli XL1-Blue . A clone of 1.7 kb fragment containing endoglucanase activity, designated pKYCY100, was sequenced and found to contain an ORF of 978 bp encoding a protein of 325 amino acid residues, with a calculated molecular mass of 38,831 Da . This endoglucanase was designated cel8Y gene . The endoglucanase has an 18-amino-acid signal peptide but not cellulose-binding domain . The endoglucanase of A . aeolicus VF5 had significant amino acid sequence similarities with endoglucanases from glycosyl hydrolase family 8 . The predicted amino acid sequence of the Cel8Y protein was similar to that of CMCase of Cellulomonas uda, BcsC of Escherichia coli, CelY of Erwinia chrysanthemi, and CMCase of Acetobacter xylinum . The molecular mass of Cel8Y was calculated to be 36,750 Da, which is consistent with the value obtained from result of CMC-SDS-PAGE of the purified enzyme . Cel8Y was thermostable, exhibiting maximal activity at 80 degrees C and pH optima of 7.0 and with half-lives of 2 h at 100 degrees C, 4 h at 90 degrees C . Acta Crystallogr D Biol Crystallogr, 2000 Dec, 56 Pt 12, 1560 - 8 Structure of a family IIIa scaffoldin CBD from the cellulosome of Clostridium cellulolyticum at 2.2 A resolution; Shimon LJ et al.; The crystal structure of the family IIIa cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit (CipC) of Clostridium cellulolyticum has been determined . The structure reveals a nine-stranded jelly-roll topology which exhibits distinctive structural elements consistent with family III CBDs that bind crystalline cellulose . These include a well conserved calcium-binding site, a putative cellulose-binding surface and a conserved shallow groove of unknown function . The CipC CBD structure is very similar to the previously elucidated family IIIa CBD from the CipA scaffoldin of C . thermocellum, with some minor differences . The CipC CBD structure was also compared with other previously described CBD structures from families IIIc and IV derived from the endoglucanases of Thermomonospora fusca and Cellulomonas fimi, respectively . The possible functional consequences of structural similarities and differences in the shallow groove and cellulose-binding faces among various CBD families and subfamilies are discussed. Biochem J, 2000 Nov 1, 351 Pt 3, 833 - 8 Identification of Glu-519 as the catalytic nucleophile in beta-mannosidase 2A from Cellulomonas fimi; Stoll D et al.; Incubation of the beta-mannosidase Man2A from Cellulomonas fimi with 2-deoxy-2-fluoro-beta-D-mannosyl fluoride (2FMan beta F) resulted in time-dependent inactivation of the enzyme (inactivation rate constant k(i)=0.57 min(-1), dissociation constant for the inactivator K(i)=0.41 mM) through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-mannosyl-beta-mannosidase 2A (2FMan-Man2A) enzyme intermediate, as observed by electrospray ionization mass spectrometry . The stoichiometry of inactivation was 1:1 . Removal of excess inactivator and regeneration of active enzyme by transglycosylation of the covalently attached inhibitor to gentiobiose {Glc beta(1-6)Glc} demonstrated that the covalent intermediate was catalytically competent . Comparison by MS of the peptic digests of 2FMan-Man2A with peptic digests of native Man2A revealed a peptide of m/z 1520 that was unique to 2FMan-Man2A, and one of m/z 1036.5 that was unique to a Man2A peptide . Their sequences, determined by collision-induced fragmentation, were CSEFGFQGPPTW and FGFQGPPTW, corresponding to residues 517-528 and 520-528 of Man2A respectively . The difference in mass of 483.5 between the two peptides equals the sum of the masses of the tripeptide CSE plus that of 2-fluoromannose . It was concluded that in 2FMan-Man2A, the 2-fluoromannose esterified to Glu-519 blocks hydrolysis of the Glu-519-Phe-520 peptide bond, and that Glu-519 is the catalytic nucleophile in this enzyme . This residue is conserved in all members of family 2 of the glycosyl hydrolases . This represents the first ever labelling and identification of an active-site nucleophile in a beta-mannosidase. Biochem J, 2000 Nov 1, 351 Pt 3, 649 - 59 Role of non-covalent enzyme-substrate interactions in the reaction catalysed by cellobiose phosphorylase from Cellulomonas uda; Nidetzky B et al.; Steady-state kinetic studies of the enzymic glucosyl transfer to and from phosphate catalysed by cellobiose phosphorylase from Cellulomonas uda have shown that this enzyme operates by a ternary-complex kinetic mechanism in which beta-cellobiose binds before phosphate, and beta-D-glucose and alpha-D-glucopyranosyl phosphate are released in that order . alpha-D-Glucopyranosyl fluoride (but not beta-D-glucopyranosyl fluoride) serves as alternative glucosyl donor for beta-cellobiose synthesis with a specificity constant that is one-ninth that of the corresponding enzymic reaction with alpha-D-glucopyranosyl phosphate (approximately 20000 M(-1).s(-1) at 30 degrees C) . The kinetic parameters for a complete series of deoxy and deoxyfluoro analogues of D-glucose have been determined and the data yield estimates of the net strengths of hydrogen-bonding interactions with the non-reacting hydroxy groups of D-glucose at the transition state (0.8-4.0 kcal/mol, where 1 cal identical with 4.184 J) and enable the prediction of the polarities of these hydrogen bonds . Each hydroxy group functions as donor of a hydrogen for bonding to probably a charged (at 3-OH) or neutral (at 2-OH and 6-OH) acceptor group on the enzyme . The equatorial 1-OH is essential for enzyme activity . Derivatives of D-glucose in which the 1-OH or the reacting 4-OH were replaced by hydrogen or fluorine have been tested as inhibitors to measure their affinities for the sugar-binding subsite +1 (numbered from the bond-cleaving/forming site) . The data show that hydrogen-bonding interactions between the 1-OH and 4-OH and charged groups on the enzyme stabilize the ground-state ternary complex of the enzymic synthesis of beta-cellobiose by 2.3 and 0.4 kcal/mol, respectively, and assist the precise positioning of beta-D-glucose for catalysis. Int J Food Microbiol, 2000 Sep 25, 60(2-3), 251 - 60 Microbial diversity during maturation and natural processing of coffee cherries of Coffea arabica in Brazil; Silv CF et al.; The magnitude and diversity of the microbial population associated with dry (natural) processing of coffee (Coffea arabica) has been assessed during a 2-year period on 15 different farms in the Sul de Minas region of Brazil . Peptone water-washed samples were taken of maturing cherries on trees (cherries, raisins and dried cherries) and from ground fermentations . The microbial load varied from 3 x 10(4) to 2.2 x 10(9) cfu/cherry with a median value of 1.6 x 10(7) cfu/cherry . The microbial load increased after heavy rainfall on cherries that were drying on the ground . At all stages, bacteria were usually the most abundant group, followed by filamentous fungi and finally yeasts . Counts of bacteria, yeasts and fungi varied considerably between farms and at different stages of maturation and processing and no consistent pattern could be seen . Yeasts showed an increase during the fermentation process . Median counts were not significantly different for fungi, yeasts and bacteria between the 2 years although Gram-negative bacteria dominated in the wet year and Gram-positive bacteria dominated in the dry year . Of a total of 754 isolates, 626 were identified to at least genus level comprising 44 genera and 64 different species . The 164 isolates of Gram-negative bacteria included 17 genera and 26 species, the most common of which were members of the genera Aeromonas, Pseudomonas, Enterobacter and Serratia . Of 191 isolates of Gram-positive bacteria, 23 were spore-forming and included six Bacillus species, and 118 were non-spore-formers of which over half were Cellulomonas with lesser numbers of Arthrobacter, Microbacterium, Brochothrix, Dermabacter and Lactobacillus . Of the 107 yeast isolates, 90 were identified into 12 genera and 24 different species and almost all were fermentative . The most common genera, in decreasing frequency, were Pichia, Candida, Arxula and Saccharomycopsis . There were many rarely described yeasts including Pichia lynferdii and Arxula adeninivorans . Almost all 292 fungal isolates were identified to genus level and 52 were identified to species level . Cladosporium, Fusarium and Penicillium each comprised about one third of the isolates and were found on all farms . Only 3% of the isolates were Aspergillus . Beauvaria, Monilia, Rhizoctonia and Arthrobotrys species were also occasionally found . The microbial flora is much more varied and complex than found in wet fermentations . The genera and species identified include members known to have all types of pectinase and cellulase activities. Biochemistry, 2000 Sep 26, 39(38), 11553 - 63 Detailed structural analysis of glycosidase/inhibitor interactions: complexes of Cex from Cellulomonas fimi with xylobiose-derived aza-sugars; Notenboom V et al.; Detailed insights into the mode of binding of a series of tight-binding aza-sugar glycosidase inhibitors of two fundamentally different classes are described through X-ray crystallographic studies of complexes with the retaining family 10 xylanase Cex from Cellulomonas fimi . Complexes with xylobiose-derived aza-sugar inhibitors of the substituted "amidine" class (xylobio-imidazole, K(i) = 150 nM; xylobio-lactam oxime, K(i) = 370 nM) reveal lateral interaction of the "glycosidic" nitrogen with the acid/base catalyst (Glu127) and hydrogen bonding of the sugar 2-hydroxyl with the catalytic nucleophile (Glu233), as expected . Tight binding of xylobio-isofagomine (K(i) = 130 nM) appears to be a consequence of strong interactions of the ring nitrogen with the catalytic nucleophile while, surprisingly, no direct protein contacts are made with the ring nitrogen of the xylobio-deoxynojirimycin analogue (K(i) = 5800 nM) . Instead the nitrogen interacts with two ordered water molecules, thereby accounting for its relatively weaker binding, though it still binds some 1200-fold more tightly than does xylobiose, presumably as a consequence of electrostatic interactions at the active site . Dramatically weaker binding of these same inhibitors to the family 11 xylanase Bcx from Bacillus circulans (K(i) from 0.5 to 1.5 mM) is rationalized for the substituted amidines on the basis that this enzyme utilizes a syn protonation trajectory and likely hydrolyzes via a (2,5)B boat transition state . Weaker binding of the deoxynojirimycin and isofagomine analogues likely reflects the energetic penalty for distortion of these analogues to a (2,5)B conformation, possibly coupled with destabilizing interactions with Tyr69, a conserved, catalytically essential active site residue. Proc Natl Acad Sci U S A, 2000 Sep 12, 97(19), 10342 - 7 Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose; Carrard G et al.; The cohesin-dockerin interaction in Clostridium thermocellum cellulosome mediates the tight binding of cellulolytic enzymes to the cellulosome-integrating protein CipA . Here, this interaction was used to study the effect of different cellulose-binding domains (CBDs) on the enzymatic activity of C . thermocellum endoglucanase CelD (1,4-beta-d endoglucanase, EC) toward various cellulosic substrates . The seventh cohesin domain of CipA was fused to CBDs originating from the Trichoderma reesei cellobiohydrolases I and II (CBD(CBH1) and CBD(CBH2)) (1,4-beta-d glucan-cellobiohydrolase, EC), from the Cellulomonas fimi xylanase/exoglucanase Cex (CBD(Cex)) (beta-1,4-d glucanase, EC), and from C . thermocellum CipA (CBD(CipA)) . The CBD-cohesin hybrids interacted with the dockerin domain of CelD, leading to the formation of CelD-CBD complexes . Each of the CBDs increased the fraction of cellulose accessible to hydrolysis by CelD in the order CBD(CBH1) < CBD(CBH2) approximately CBD(Cex) < CBD(CipA) . In all cases, the extent of hydrolysis was limited by the disappearance of sites accessible to CelD . Addition of a batch of fresh cellulose after completion of the reaction resulted in a new burst of activity, proving the reversible binding of the intact complexes despite the apparent binding irreversibility of some CBDs . Furthermore, burst of activity also was observed upon adding new batches of CelD-CBD complexes that contained a CBD differing from the first one . This complementation between different CBDs suggests that the sites made available for hydrolysis by each of the CBDs are at least partially nonoverlapping . The only exception was CBD(CipA), whose sites appeared to overlap all of the other sites. J Biol Chem, 2000 Jul 28, 275(30), 23020 - 6 Substrate specificity in glycoside hydrolase family 10 . Structural and kinetic analysis of the Streptomyces lividans xylanase 10A; Ducros V et al.; Endoxylanases are a group of enzymes that hydrolyze the beta-1, 4-linked xylose backbone of xylans . They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11 . The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.6 A resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z . S . (1994) J . Biol . Chem . 269, 20811-20814) . Here, we report the native structure of Xyl10A refined at a resolution of 1.2 A, which reveals many features such as the rare occurrence of a discretely disordered disulfide bond between residues Cys-168 and Cys-201 . In order to investigate substrate binding and specificity in glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the covalent cellobiosyl enzyme intermediates of Xyl10A were trapped through the use of appropriate 2-fluoroglycosides . The alpha-linked intermediate with the nucleophile, Glu-236, is in a (4)C(1) chair conformation as previously observed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsan, C., Warren, R . A . J., Withers, S . G., and Rose, D . R . (1998) Biochemistry 37, 4751-4758) . The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 subsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10. Biochemistry, 2000 Aug 1, 39(30), 8844 - 52 Binding site analysis of cellulose binding domain CBD(N1) from endoglucanse C of Cellulomonas fimi by site-directed mutagenesis; Kormos J et al.; Endoglucanase C (CenC), a beta1,4 glucanase from the soil bacterium Cellulomonas fimi, binds to amorphous cellulose via two homologous cellulose binding domains, termed CBD(N1) and CBD(N2) . In this work, the contributions of 10 amino acids within the binding cleft of CBD(N1) were evaluated by single site-directed mutations to alanine residues . Each isolated domain containing a single mutation was analyzed for binding to an insoluble amorphous preparation of cellulose, phosphoric acid swollen Avicel (PASA), and to a soluble glucopyranoside polymer, barley beta-glucan . The effect of any given mutation on CBD binding was similar for both substrates, suggesting that the mechanism of binding to soluble and insoluble substrates is the same . Tyrosines 19 and 85 were essential for tight binding by CBD(N1) as their replacement by alanine results in affinity decrements of approximately 100-fold on PASA, barley beta-glucan, and soluble cellooligosaccharides . The tertiary structures of unbound Y19A and Y85A were assessed by heteronuclear single quantum coherence (HSQC) spectroscopy . These studies indicated that the structures of both mutants were perturbed but that all perturbations are very near to the site of mutation. Enzyme Microb Technol, 2000 Aug 1, 27(3-5), 325 - 329 Effects of agitation level on the adsorption, desorption, and activities on cotton fabrics of full length and core domains of EGV (Humicola insolens) and CenA (Cellulomonas fimi); Azevedo H et al.; The activities (at pH 7 and 50 degrees C) of purified EGV (Humicola insolens) and CenA (Cellulomonas fimi) were determined on cotton fabrics at high and low levels of mechanical agitation . Similar activity measurements were also made by using the core domains of these cellulases . Activity experiments suggested that the presence of cellulose binding domains (CBDs) is not essential for cellulase performance in the textile processes, where high levels of mechanical agitation are applied . The binding reversibilities of these cellulases and their cores were studied by dilution of the treatment liquor after equilibrium adsorption . EGV showed low percentage of adsorption under both levels of agitation . It was observed that the adsorption/desorption processes of cellulases are enhanced by higher mechanical agitation levels and that the binding of cellulase with CBD of family I (EGV) is more reversible than that of CBD of the cellulase of family II (CenA). Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1279 - 85 Description of Bogoriellaceae fam . nov., Dermacoccaceae fam . nov., Rarobacteraceae fam . nov . and Sanguibacteraceae fam . nov . and emendation of some families of the suborder Micrococcineae; Stackebrandt E et al.; The hierarchic taxonomic framework described recently for the phylogenetic structure of the suborder Micrococcineae, class Actinobacteria, on the basis of 16S rDNA sequences and signature nucleotides was modified and extended . With the recent addition of novel taxa into the suborder, the phylogenetic coherence of some families was disrupted, leading to the emergence of novel lineages that, as judged by the depth of their branching points, were equivalent to those of described families . Bogoriellaceae fam . nov., Dermacoccaceae fam . nov., Rarobacteraceae fam . nov . and Sanguibacteraceae fam . nov . are proposed for these lineages . As a consequence of the restructuring process, some families have had to be emended, i.e . Dermatophilaceae, Cellulomonadaceae and Intrasporangiaceae. Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 993 - 6 Cellulomonas persica sp . nov . and Cellulomonas iranensis sp . nov., mesophilic cellulose-degrading bacteria isolated from forest soils; Elberson MA et al.; Two newly described species of mesophilic, cellulose-degrading, aerobic bacteria were isolated from forest humus soils along the southern border of the Caspian Sea . Cellulomonas persica and Cellulomonas iranensis are proposed as new specific epithets based on comparative sequence analyses of 16S rDNA, DNA-DNA hybridization and phenotypic characteristics . Formal species descriptions are provided. J Biol Chem, 2000 Jul 28, 275(30), 23027 - 33 Substrate specificity in glycoside hydrolase family 10 . Tyrosine 87 and leucine 314 play a pivotal role in discriminating between glucose and xylose binding in the proximal active site of Pseudomonas cellulosa xylanase 10A; Andrews SR et al.; The Pseudomonas family 10 xylanase, Xyl10A, hydrolyzes beta1, 4-linked xylans but exhibits very low activity against aryl-beta-cellobiosides . The family 10 enzyme, Cex, from Cellulomonas fimi, hydrolyzes aryl-beta-cellobiosides more efficiently than does Xyl10A, and the movements of two residues in the -1 and -2 subsites are implicated in this relaxed substrate specificity (Notenboom, V., Birsan, C., Warren, R . A . J., Withers, S . G., and Rose, D . R . (1998) Biochemistry 37, 4751-4758) . The three-dimensional structure of Xyl10A suggests that Tyr-87 reduces the affinity of the enzyme for glucose-derived substrates by steric hindrance with the C6-OH in the -2 subsite of the enzyme . Furthermore, Leu-314 impedes the movement of Trp-313 that is necessary to accommodate glucose-derived substrates in the -1 subsite . We have evaluated the catalytic activities of the mutants Y87A, Y87F, L314A, L314A/Y87F, and W313A of Xyl10A . Mutations to Tyr-87 increased and decreased the catalytic efficiency against 4-nitrophenyl-beta-cellobioside and 4-nitrophenyl-beta-xylobioside, respectively . The L314A mutation caused a 200-fold decrease in 4-nitrophenyl-beta-xylobioside activity but did not significantly reduce 4-nitrophenyl-beta-cellobioside hydrolysis . The mutation L314A/Y87A gave a 6500-fold improvement in the hydrolysis of glucose-derived substrates compared with xylose-derived equivalents . These data show that substantial improvements in the ability of Xyl10A to accommodate the C6-OH of glucose-derived substrates are achieved when steric hindrance is removed. Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 661 - 3 Reclassification of Actinomyces humiferus (Gledhill and Casida) as Cellulomonas humilata nom . corrig., comb . nov; Collins MD et al.; The placement of Actinomyces humiferus within the genus Actinomyces has always been controversial . A humiferus differs from typical members of the genus both phenotypically and in possessing a relatively high DNA G + C content . Comparative 16S rRNA gene sequencing has shown that A . humiferus is related only distantly to other species of the genus Actinomyces and is, in fact, a member of the genus Cellulomonas . On the basis of phylogenetic evidence, it is proposed that A . humiferus be reclassified in the genus Cellulomonas as Cellulomonas humilata nom . corrig., comb . nov. Biochemistry, 2000 Mar 14, 39(10), 2445 - 58 Structure and binding specificity of the second N-terminal cellulose-binding domain from Cellulomonas fimi endoglucanase C; Brun E et al.; The 1,4-beta-glucanase CenC from Cellulomonas fimi contains two cellulose-binding domains, CBD(N1) and CBD(N2), arranged in tandem at its N-terminus . These homologous CBDs are distinct in their selectivity for binding amorphous and not crystalline cellulose . Multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine the tertiary structure of CBD(N2) in the presence of saturating amounts of cellopentaose . A total of 1996 experimental restraints were used to calculate an ensemble of 21 final structures for the protein . CBD(Nu2) is composed of 11 beta-strands, folded into two antiparallel beta-sheets, with a topology of a jellyroll beta-sandwich . On the basis of patterns of chemical shift perturbations accompanying the addition of cellooligosaccharides, as well as the observation of intermolecular protein-sugar NOE interactions, the cellulose-binding site of CBD(N2) was identified as a cleft that lies across one face of the beta-sandwich . The thermodynamic basis for the binding of cellooligosaccharides was investigated using isothermal titration calorimetry and NMR spectroscopy . Binding is enthalpically driven and consistent with a structural model involving hydrogen bonding between the equatorial hydroxyls of the glucopyranosyl rings and polar amino acid side chains lining the CBD(N2) cleft . Affinity electrophoresis was used to determine that CBD(N2) also binds soluble beta-1,4-linked polymers of glucose, including hydroxyethylcellulose and beta-1,3-1,4-glucans . This study complements a previous analysis of CBD(N1) {Johnson, P . E., Joshi, M . D., Tomme, P., Kilburn, D . G., and McIntosh, L . P . (1996) Biochemistry 35, 14381-14394} and demonstrates that the homologous CBDs from CenC share very similar structures and sugar binding properties. Z Naturforsch {C}, 1999 Dec, 54(12), 1055 - 67 Synthesis of 6-aryloxy- and 6-arylalkoxy-2-chloropurines and their interactions with purine nucleoside phosphorylase from Escherichia coli; Bzowska A et al.; The phase transfer method was applied to perform the nucleophilic substitution of 2,6-dichloropurines by modified arylalkyl alcohol or phenols . Since under these conditions only the 6-halogen is exchanged, this method gives 2-chloro-6-aryloxy- and 2-chloro-6-arylalkoxy-purines . 2-Chloro-6-benzylthiopurine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl bromide . The stereoisomers of 2-chloro-6-(1-phenyl-1-ethoxy)purine were obtained from R- and S-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropurine . All derivatives were tested for inhibition with purified hexameric E . coli purine nucleoside phosphorylase (PNP) . For analogues showing IC50 < 10 microM, the type of inhibition and inhibition constants were determined . In all cases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, Kiu, was found to be several-fold lower than the competitive inhibition constant, Kic . This effect seems to be due to the 6-aryloxy- or 6-arylalkoxy substituent, because a natural PNP substrate adenine, as well as 2-chloroadenine, show mixed type inhibition with almost the same inhibition constants Kiu and Kic . The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzyloxy-2-chloro-, 2-chloro-6-(2-phenyl-1-ethoxy), 2-chloro-6-(3-phenyl-1-propoxy)- and 2-chloro-6-ethoxypurines (Kiu = 0.4, 0.6, 1.4, 1.4 and 2.2 microM, respectively) . The R-stereoisomer of 2-chloro-6-(1-pheny-1-ethoxy)purine has Kiu = 2.0 microM, whereas inhibition of its S counterpart is rather weak (IC50 > 12 microM) . More rigid (e.g . phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the purine base gave less potent inhibitors (IC50 = 26, 56 and > 100 microM, respectively) . The derivatives are selective inhibitors of hexameric "high-molecular mass" PNPs because no inhibitory activity vs . trimeric Cellulomonas sp . PNP was detected . By establishing the ligand-dependent stabilization pattern of the E . coli PNP it was shown that the new derivatives, similarly as the natural purine bases, are able to form a dead-end ternary complex with the enzyme and orthophosphate . It was also shown that the derivatives are substrates in the reverse synthetic direction catalyzed by E . coli PNP. FEMS Microbiol Lett, 2000 Feb 15, 183(2), 265 - 9 Mannanase Man26A from Cellulomonas fimi has a mannan-binding module; Stoll D et al.; A modular mannanase (Man26A) from the bacterium Cellulomonas fimi contains a mannan-binding module (Man26Abm) that binds to soluble but not to insoluble mannans . Man26Abm does not bind to cellulose, chitin or xylan . The K(d) for binding of Man26Abm to locust bean gum (LBG) is approximately 0.2 microM . Man26A is the first mannanase reported to contain a mannan-binding module. FEMS Microbiol Ecol, 2000 Jan 1, 31(1), 53 - 60 Phylogenetic and phenotypic diversity of 4-chlorobenzoate-degrading bacteria isolated from soils; Yi H et al.; Twenty numerically dominant 4-chlorobenzoate (4-CBA)-degrading bacteria were isolated from agricultural soils . The isolates were able to utilize 4-CBA as a sole source of carbon and energy . A total of 65% of the isolates was identified to the species level by fatty acid methyl ester (FAME) analysis, and the isolates were strains of Micrococcus, Pseudomonas, Oerskovia, Cellulomonas, and Arthrobacter species . The chromosomal DNA patterns of the isolates obtained by polymerase chain reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences were distinct from each other . Most of the isolates grew rapidly in 4-CBA medium, but their substrate utilization capabilities were generally restricted . Plasmid DNAs were detected from 55% of the isolates, and one strain, HR7, was shown to have self-transmissible, 4-CBA degradative plasmids . 4-CBA degradative enzymes were inducible by the presence of 4-CBA and most of the isolates appeared to mineralize it through 4-hydroxybenzoate rather than 4-chlorocatechol. J Mol Biol, 1999 Dec 17, 294(5), 1239 - 55 Crystal structure of the purine nucleoside phosphorylase (PNP) from Cellulomonas sp . and its implication for the mechanism of trimeric PNPs; Tebbe J et al.; The three-dimensional structure of the trimeric purine nucleoside phosphorylase (PNP) from Cellulomonas sp . has been determined by X-ray crystallography . The binary complex of the enzyme with orthophosphate was crystallized in the orthorhombic space group P212121 with unit cell dimensions a=64.1 A, b=108.9 A, c=119.3 A and an enzymatically active trimer in the asymmetric unit . X-ray data were collected at 4 degrees C using synchrotron radiation (EMBL/DESY, Hamburg) . The structure was solved by molecular replacement, with the calf spleen PNP structure as a model, and refined at 2.2 A resolution . The ternary "dead-end" complex of the enzyme with orthophosphate and 8-iodoguanine was obtained by soaking crystals of the binary orthophosphate complex with the very weak substrate 8-iodoguanosine . Data were collected at 100 K with CuKalpha radiation, and the three-dimensional structure refined at 2.4 A resolution . Although the sequence of the Cellulomonas PNP shares only 33 % identity with the calf spleen enzyme, and almost no identity with the hexameric Escherichia coli PNP, all three enzymes have many common structural features, viz . the nine-stranded central beta-sheet, the positions of the active centres, and the geometrical arrangement of the ligands in the active centres . Some similarities of the surrounding helices also prevail . In Cellulomonas PNP, each of the three active centres per trimer is occupied by orthophosphate, and by orthophosphate and base, respectively, and small structural differences between monomers A, B and C are observed . This supports cooperativity between subunits (non-identity of binding sites) rather than existence of more than one binding site per monomer, as previously suggested for binding of phosphate by mammalian PNPs . The phosphate binding site is located between two conserved beta- and gamma-turns and consists of Ser46, Arg103, His105, Gly135 and Ser223, and one or two water molecules . The guanine base is recognized by a zig-zag pattern of possible hydrogen bonds, as follows: guanine N-1...Glu204 O(epsilon1)...guanine NH2...Glu204 O(epsilon2) . The exocyclic O6 of the base is bridged via a water molecule to Asn246 N(delta), which accounts for the inhibitory, but lack of substrate, activity of adenosine . An alternative molecular mechanism for catalysis by trimeric PNPs is proposed, in which the key catalytic role is played by Glu204 (Glu201 in the calf and human enzymes), while Asn246 (Asn243 in the mammalian enzymes) supports binding of 6-oxopurines rather than catalysis . This mechanism, in contrast to that previously suggested, is consistent with the excellent substrate properties of N-7 substituted nucleosides, the specificity of trimeric PNPs versus 6-oxopurine nucleosides and the reported kinetic properties of Glu201/Ala and Asn243/Ala point variants of human PNP . FEBS Lett, 1999 Oct 22, 460(1), 61 - 6 An investigation of the nature and function of module 10 in a family F/10 xylanase FXYN of Streptomyces olivaceoviridis E-86 by module shuffling with the Cex of Cellulomonas fimi and by site-directed mutagenesis; Kaneko S et al.; Although the amino acid homology in the catalytic domain of FXYN xylanase from Streptomyces olivaceoviridis E-86 and Cex xylanase from Cellulomonas fimi is only 50%, an active chimeric enzyme was obtained by replacing module 10 in FXYN with module 10 from Cex . In the family F/10 xylanases, module 10 is an important region as it includes an acid/base catalyst and a substrate binding residue . In FXYN, module 10 consists of 15 amino acid residues, while in Cex it consists of 14 amino acid residues . The Km and kcat values of the chimeric xylanase FCF-C10 for PNP-xylobioside (PNP-X2) were 10-fold less than those for FXYN . CD spectral data indicated that the structure of the chimeric enzyme was similar to that of FXYN . Based on the comparison of the amino acid sequences of FXYN and Cex in module 10, we constructed four mutants of FXYN . When D133 or S135 of FXYN was deleted, the kinetic properties were not changed from those of FXYN . By deletion of both D133 and S135, the Km value for PNP-X2 decreased from the 2.0 mM of FXYN to 0.6 mM and the kcat value decreased from the 20 s(-1) of FXYN to 8.7 s(-1) . Insertion of Q140 into the doubly deleted mutant further reduced the Km value to 0.3 mM and the kcat value to 3.8 s(-1) . These values are close to those for the chimeric enzyme FCF-C10 . These results indicate that module 10 itself is able to accommodate changes in the sequence position of amino acids which are critical for enzyme function . Since changes of the spatial position of these amino acids would be expected to result in enzyme inactivation, module 10 must have some flexibility in its tertiary structure . The structure of module 10 itself also affects the substrate specificity of the enzyme. Gene, 1999 Sep 30, 238(1), 93 - 101 Module-intron correlation and intron sliding in family F/10 xylanase genes; Sato Y et al.; Xylanases are classified into two families, numbered F/10 and G/11 according to the similarity of amino acid sequences of their catalytic domain (Henrissat, B., Bairoch, A., 1993 . New families in the classification of glycosyl hydrolases based on amino acid sequence similarities . Biochem . J . 293, 781-788) . Three-dimensional structure of the catalytic domain of the family F/10 xylanase was reported (White, A., Withers, S.G., Gilkes, N.R., Rose, D.R., 1994 . Crystal structure of the catalytic domain of the beta-1,4-glycanase Cex from Cellulomonas fimi . Biochemistry 33, 12546-12552) . The domain was decomposed into 22 modules by centripetal profiles (Go, M., Nosaka, M., 1987 . Protein architecture and the origin of introns . Cold Spring Harbor Symp . Quant . Biol . 52, 915-924; Noguti, T., Sakakibara, H., Go, M., 1993 . Localization of hydrogen-bonds within modules in barnase . Proteins 16, 357-363) . A module is a contiguous polypeptide segment of amino acid residues having a compact conformation within a globular domain . Collected 31 intron sites of the family F/10 xylanase genes from fungus were found to be correlated to module boundaries with considerable statistical force (p values <0.001) . The relationship between the intron locations and protein structures provides supporting evidence for the ancient origin of introns, because such a relationship cannot be expected by random insertion of introns into eukaryotic genes, but it rather suggests pre-existence of introns in the ancestral genes of prokaryotes and eukaryotes . A phylogenetic tree of the fungal and bacterial xylanase sequences made two clusters; one includes both the bacterial and fungal genes, but the other consists of only fungal genes . The mixed cluster of bacterial genes without introns and the fungal genes with introns further supports the ancient origin of introns . Comparison of the conserved base sequences of introns indicates that sliding of a splice site occurred in Aspergillus kawachii gene by one base from the ancestral position . Substrate-binding sites of xylanase are localized on eight modules, and introns are found at both termini of six out of these functional modules . This result suggests that introns might play a functional role in shuffling the exons encoding the substrate-binding modules. Appl Microbiol Biotechnol, 1999 Aug, 52(2), 232 - 9 A beta-1,4-endoglucanase-encoding gene from Cellulomonas pachnodae; Cazemier AE et al.; A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity . Five endoglucanase-positive clones were isolated that carried identical DNA fragments . The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B) . The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50-55 degrees C . The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter . Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished . However, the regions in the Cel6A amino acid sequence at the positions 262-319 and 448-473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding. Appl Environ Microbiol, 1999 Sep, 65(9), 4099 - 107 Molecular and biochemical characterization of two xylanase-encoding genes from Cellulomonas pachnodae; Cazemier AE et al.; Two xylanase-encoding genes, named xyn11A and xyn10B, were isolated from a genomic library of Cellulomonas pachnodae by expression in Escherichia coli . The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da . Different domains could be identified in the Xyn11A protein on the basis of homology searches . Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence . Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains . The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da . Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified . It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains . Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified . A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology . C . pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid . Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the two Cellulomonas species were identified. J Microbiol Methods, 1999 Aug, 37(2), 101 - 9 Detection of a whitening fluorescent agent as an indicator of white paper biodegradation: a new approach to study the kinetics of cellulose hydrolysis by mixed cultures; Bichet-Hebe I et al.; A simple and reliable method to estimate paper degradation by cellulolytic bacteria is described . This method is based on the detection in the culture medium of a fluorescent whitening agent (FWA) added to white paper during the manufacturing process . Preliminary results using a Cellulomonas strain cultivated in a liquid medium containing FWA, indicated that this component is non-toxic at a final concentration of 0.01 per thousand (v/v) and that the fluorescence decreased during the first 24 h of incubation, i.e . during exponential growth phase, suggesting an adsorption of FWA on bacterial cells . Consequently, all experiments have been performed with a liquid medium containing FWA (0.01 per thousand v/v) and white paper (8.0 g/l) as cellulose source . Mixed bacterial populations (MBPs) were prepared from refuse samples . These MBPs, which mainly consisted of bacterial rod cells, were used as inocula and fluorescence was measured after 30 h of incubation, i.e . after the stationary phase was reached . A high linear correlation (R(2) = 0.979) was found between the percentages of degraded paper (%P) deduced from residual paper weight and the fluorescence values (F) of the culture medium and the following equation between %P and F was determined: %P = 8.71x10(-5) x F . An additional experiment using a second MBP showed a strong correlation (R(2) = 0.990) between the measured %P and the %P estimated from F values, confirming the reproducibility of the method . Moreover, the time course of paper degradation by five replicate flasks from a unique MBP was set up . Paper degradation was detected 3 to 5 days after the beginning of the stationary phase . The average degradation rate between the 7th and the 11th day of incubation was 11.4% per day . Rates of paper degradation ranged from 31 to 60% after 10 days and from 77 to 88% after 3 weeks of incubation, depending on the inoculum. Structure Fold Des, 1999 Jul 15, 7(7), 853 - 64 A family IIb xylan-binding domain has a similar secondary structure to a homologous family IIa cellulose-binding domain but different ligand specificity; Simpson PJ et al.; BACKGROUND: Many enzymes that digest polysaccharides contain separate polysaccharide-binding domains . Structures have been previously determined for a number of cellulose-binding domains (CBDs) from cellulases . RESULTS: The family IIb xylan-binding domain 1 (XBD1) from Cellulomonas fimi xylanase D is shown to bind xylan but not cellulose . Its structure is similar to that of the homologous family IIa