Biochemistry, 1996 Oct 8, 35(40), 13165 - 72 Mechanistic consequences of mutation of active site carboxylates in a retaining beta-1,4-glycanase from Cellulomonas fimi; MacLeod AM et al.; The exoglucanase/xylanase Cex from Cellulomonas fimi is a retaining glycosidase which functions via a two-step mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme intermediate . The roles of three conserved active site carboxylic acids in this enzyme have been probed by detailed kinetic analysis of mutants modified at these three positions . Elimination of the catalytic nucleophile (E233A) results in an essentially inactive enzyme, consistent with the important role of this residue . However addition of small anions such as azide or formate restores activity, but as an inverting enzyme since the product formed under these conditions is the alpha-glycosyl azide . Shortening of the catalytic nucleophile (E233D) reduces the rates of both formation and hydrolysis of the glycosyl-enzyme intermediate some 3000-4000-fold . Elimination of the acid/base catalyst (E127A) yields a mutant for which the deglycosylation step is slowed some 200-300-fold as a consequence of removal of general base catalysis, but with little effect on the transition state structure at the anomeric center . Effects on the glycosylation step due to removal of the acid catalyst depend on the aglycon leaving group ability, with minimal effects on substrates requiring no general acid catalysis but large (> 10(5)-fold) effects on substrates with poor leaving groups . The Bronsted beta 1g value for hydrolysis of aryl cellobiosides was much larger (beta 1g approximately -1) for the mutant than for the wild-type enzyme (beta 1g = -0.3), consistent with removal of protonic assistance . The pH-dependence was also significantly perturbed . Mutation of a third conserved active site carboxylic acid (E123A) resulted in rate reductions of up to 1500-fold on poorer substrates, which could be largely restored by addition of azide, but without the formation of glycosyl azide products . These results suggest a simple strategy for the identification of the key active site nucleophile and acid/base catalyst residues in glycosidases without resort to active site labeling. Int J Syst Bacteriol, 1996 Oct, 46(4), 1078 - 82 Reassessment of the phylogenetic position of the bacterium associated with Whipple's disease and determination of the 16S-23S ribosomal intergenic spacer sequence; Maiwald M et al.; Whipple's disease is a rare chronic illness associated with an unculturable bacterium that is constantly present in affected tissues . This bacterium was previously characterized at the molecular level by PCR and sequencing of the 16S rRNA gene . On the basis of 1,321 nucleotides of the sequence of its gene coding for 16S rRNA (16S rDNA), a phylogenetic relationship to the actinomycetes was established . In this study, we determined an almost complete 16S rDNA sequence (1,495 nucleotides), the 16S-23S ribosomal intergenic spacer sequence, and 200 nucleotides of the 23S rRNA gene . The 16S rDNA sequence was compared with the large number of actinomycete sequences that have been added to the database since the original study . Phylogenetic analysis revealed a branching position as the deepest branch of the cluster comprising the actinomycetes with group B peptidoglycan between this group and the family Cellulomonadaceae . This provides additional information on the phylogenetic position of this bacterium and some clues as to its characteristics . The spacer region between the 16S and 23S rRNA genes is 294 nucleotides long and does not contain tRNA genes . As has been shown in other instances, the increased variability of the ribosomal intergenic spacer compared with the 16S rRNA gene makes it a potential target for use in the differentiation of strains of the bacterium associated with Whipple's disease. J Bacteriol, 1996 Sep, 178(17), 5153 - 8 A methyl-accepting protein involved in multiple-sugar chemotaxis by Cellulomonas gelida; Hsing W et al.; Tethered-cell and capillary assays indicated that L-methionine is required by Cellulomonas gelida for its normal cell motility pattern and chemotaxis and that S-adenosylmethionine is involved in sugar chemotaxis by this cellulolytic bacterium . In addition, in vivo methylation assays showed that several proteins were methylated in the absence of protein synthesis . The incorporated methyl groups were alkali sensitive . Of special interest was the observation that the methylation level of a 51,000-Mr protein increased two- to fivefold upon addition of various sugar attractants and decreased after the removal of the attractants . The increase was less pronounced in mutants defective in sugar chemotaxis and appeared to be specifically involved with sugar chemotaxis . Furthermore, cell fractionation and in vitro methylation assays demonstrated that the 51,000-Mr protein is located in the cytoplasmic membrane . These results suggest that a specific methyl-accepting chemotaxis protein is involved in multiple-sugar chemotaxis by C gelida . During chemotaxis, the changes of methylesterase activity in C gelida cells were similar to those in Escherichia coli RP437 cells, as determined by a continuous-flow assay for methanol evolution . Thus, the mechanism of methyl-accepting chemotaxis protein-mediated chemotaxis of the gram-positive C . gelida appears to be similar to that of the gram-negative E . coli rather than to that of other gram-positive bacteria, such as Bacillus subtilis. J Bacteriol, 1996 Aug, 178(15), 4751 - 7 Nucleotide sequence of a beta-1,3-glucanase isoenzyme IIA gene of Oerskovia xanthineolytica LL G109 (Cellulomonas cellulans) and initial characterization of the recombinant enzyme expressed in Bacillus subtilis; Ferrer P et al.; The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined . Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium . The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity . It can be included in the glycosyl hydrolase family 16 . Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca . 4.0). J Bacteriol, 1996 Jul, 178(14), 4216 - 23 Characterization of CenC, an enzyme from Cellulomonas fimi with both endo- and exoglucanase activities; Tomme P et al.; The cenC gene, encoding beta-1,4-glucanase C (CenC) from Cellulomonas fimi, was overexpressed in Escherichia coli with a tac-based expression vector . The resulting polypeptide, with an apparent molecular mass of 130 kDa, was purified from the cell extracts by affinity chromatography on cellulose followed by anion-exchange chromatography . N-terminal sequence analysis showed the enzyme to be properly processed . Mature CenC was optimally active at pH 5.0 and 45 degrees C . The enzyme was extremely active on soluble, fluorophoric, and chromophoric glycosides (4-methylumbelliferyl beta-glycosides, 2'-chloro-4'-nitrophenyl-beta-D-cellobioside, and 2'-chloro-4'-nitrophenyl-lactoside) and efficiently hydrolyzed carboxymethyl cellulose, barley beta-glucan, lichenan, and, to a lesser extent, glucomannan . CenC also hydrolyzed acid-swollen cellulose, Avicel, and bacterial microcrystalline cellulose . However, degradation of the latter was slow compared with its degradation by CenB, another C . fimi cellulose belonging to the same enzyme family . CenC acted with inversion of configuration at the anomeric carbon, in accordance with its classification as a family 9 member . The enzyme released mainly cellobiose from soluble cellodextrins and insoluble cellulose . Attack appeared to be from the reducing chain ends . Analysis of carboxymethyl cellulose hydrolysis suggests that CenC is semiprocessive enzyme with both endo- and exoglucanase activities. Biosci Biotechnol Biochem, 1996 Jun, 60(6), 957 - 61 Degradation of beta 1-->6 galactofuranoside linkages in the polysaccharide of Fusarium sp . M7-1 by endo-beta-galactofuranosidase from Bacillus sp; Iwahara S et al.; A polysaccharide, in which the main part of the side chains were depleted, was prepared from the acidic polysaccharides of Fusarium sp . M7-1 by digestion with lyase of Cellulomonas sp . and mild acid treatment . This polysaccharide was degraded into several fragments, neutral oligosaccharides, neutral polysaccharide, and acidic polysaccharide, by an enzyme, endo-beta-galactofuranosidase, produced by Bacillus sp . The main components of the oligosaccharides were isolated and identified as Gal{sequence: see text} The molecular mass of the neutral polysaccharide fragment was estimated to be about 6000 Da by gel filtration chromatography . The polysaccharide fragment consisted of an alpha 1-->6 linked mannan main chain to which various sugars, namely Glc, Man, and Rha were attached through alpha 1-->3 (or 2) linkages . The molecular mass of the acidic polysaccharide fragment was estimated to be about 6000 Da from the amounts of the reducing terminal galactose . The chemical structures of the oligosaccharides derived from the acidic polysaccharide fragment by mild acid hydrolysis were identified as reported structural units {Iwahara et al., J . Biochem., 112, 355-359 (1992)} . The structure of the mild-acid-resistant part of the acidic polysaccharide fragment was assumed to be a polyuronide to which various sugars such as Glc, Man, and GlcNac are attached as the side chains . The linkage modes of each sugar are not clear. Biochem J, 1996 Apr 15, 315 ( Pt 2), 467 - 72 Substrate specificity of endoglucanase A from Cellulomonas fimi: fundamental differences between endoglucanases and exoglucanases from family 6; Damude HG et al.; Values of kcat . and Km for the hydrolysis of cellotetraose, cellotriose, beta-cellobiosyl fluoride and various beta-aryl cellobiosides by endoglucanase A (CenA) from Cellulomonas fimi indicate that specific binding interactions between the reducing-end glucose residues of cellotetraose and cellotriose and the enzyme at the transition state provide enormous stabilization, endowing glucose with the "effective leaving group ability' of 2,4-dinitrophenol . As has been seen with several other inverting glycosidases, CenA hydrolyses the "wrong' anomer of its glycosyl fluoride substrate, alpha-cellobiosyl fluoride, according to non-Michaelian kinetics . This indicates that CenA carries out this hydrolysis by a mechanism involving binding of two substrate molecules in the active site (Hehre, Brewer and Genghof (1979) J . Biol . Chem . 254, 5942-5950} in contrast with that reported for cellobiohydrolase II, another family-6 enzyme {Konstantinidis, Marsden and Sinnott (1993) Biochem . J . 291, 833-838} . The pH profiles for wild-type CenA indicate that kcat . for CenA depends on the presence of both a protonated group and a deprotonated group for full activity, consistent with the presence of an acid and a base catalyst at the active site . By contrast, the profile for the Asp252Ala mutant of CenA shows a dependence only on a base-catalytic group, thereby confirming the role of Asp-252 as an acid catalyst . These results show that hydrolysis by CenA occurs by a typical inverting mechanism involving both acid and base catalysis, as first proposed by Koshland . It also suggests that endoglucanases from family 6 may function by fundamentally different mechanisms for exoglucanases in this family. Anal Biochem, 1996 Feb 15, 234(2), 119 - 25 A mass spectrometry-based approach for probing enzyme active sites: identification of Glu 127 in Cellulomonas fimi exoglycanase as the residue modified by N-bromoacetyl cellobiosylamine; Tull D et al.; We have identified the residue in Cellulomonas fimi exoglycanase modified by N-bromoacetyl cellobiosylamine as Glu 127 using a new combination of experimental approaches . The enzyme was quantitatively inhibited with the affinity label N-bromoacetyl cellobiosylamine and cleaved with pepsin . The N-acetyl cellobiosylamine-modified peptide was identified by comparative peptide mapping of the digests derived from labeled and unlabeled proteins by reverse-phase high-performance liquid chromatography connected online to an electrospray ionization mass spectrometer . The modified residue in the labeled peptide was determined by using a novel protein sequencing chemistry which is based on monitoring the amino acid derivatives released by stepwise peptide degradation using electrospray ionization mass spectrometry . Tandem mass spectrometry was used for further structural characterization of the cleaved residue . We show that the residue modified by N-bromoacetyl cellobiosylamine is Glu 127 . This residue has been identified previously as the acid-base catalyst by using a combination of mutagenic and kinetic analyses . Our results therefore demonstrate the usefulness of this type of affinity label in identifying important catalytic residues in glycosidases and suggest that this new experimental approach can be applied generally to any labeled protein in which the mass of the label is known and thus represents an alternative approach to the current methods used to identify labeled residues within proteins. Biosci Biotechnol Biochem, 1995 Dec, 59(12), 2263 - 7 High cell density cultivation and high recombinant protein production of Escherichia coli strain expressing uricase; Nakagawa S et al.; Uricase from Cellulomonas flavigena SK-4 is an industrially useful enzyme for commercial formulations of hair coloring . The uricase production by recombinant Escherichia coli strain with a high cell density cultivation technique was described . Of three kinds of media, synthetic media with the feeding of a high concentration of glucose solution were suitable for high cell density cultivation . As for feeding, both biomass concentration and uricase productivity were increased by about two (61.2 g dry cell weight (DCW)/liter) and three times (1037 U/ml broth), respectively, in 24 h by continuous supply . In the case of feeding by a DO-stat method, however, cell concentration was comparable to continuous glucose supply but uricase activity was reduced . By supplying pure oxygen to compensate for oxygen limitation during cultivation, the highest values of 77.4g DCW/liter and 1113 U/ml broth of the uricase activity were achieved with the total cultivation time of 15 h. Biochem J, 1995 Oct 1, 311 ( Pt 1), 67 - 74 Cellobiohydrolase B, a second exo-cellobiohydrolase from the cellulolytic bacterium Cellulomonas fimi; Shen H et al.; The gene cbhB from the cellulolytic bacterium Cellulomonas fimi encodes a polypeptide of 1090 amino acids . Cellobiohydrolase B (CbhB) is 1037 amino acids long, with a calculated molecular mass of 109765 Da . The enzyme comprises five domains: an N-terminal catalytic domain of 643 amino acids, three fibronectin type III repeats of 97 amino acids each, and a C-terminal cellulose-binding domain of 104 amino acids . The catalytic domain belongs to family 48 of glycosyl hydrolases . CbhB has a very low activity on CM-cellulose . Viscometric analysis of CM-cellulose hydrolysis indicates that the enzyme is an exoglucanase . Cellobiose is the major product of hydrolysis of cellulose . In common with two other exoglycanases from C . fimi, CbhB has low but detectable endoglucanase activity . CbhB is the second exo-cellobiohydrolase found in C . fimi . Therefore, the cellulase system of C . fimi resembles those of fungi in comprising multiple endoglucanases and cellobiohydrolases. Carbohydr Res, 1995 Sep 8, 274, 137 - 53 Syntheses and testing of substrates and mechanism-based inactivators for xylanases; Ziser L et al.; The syntheses of the 2,5- and 3,4-dinitrophenyl beta-xylobiosides by two separate routes are described, as well as the syntheses of the 2,4-dinitrophenyl beta-glycosides of 2-chloro-2-deoxy-xylobiose and 2-deoxy-2-fluoro-xylobiose . Both the 3,4- and 2,5-dinitrophenyl beta-xylobiosides proved to be good substrates for the Bacillus subtilis xylanase, with kcat/Km values of 1.0 and 34.4 mM-1 s-1, respectively . Excellent time-dependent inactivation of the exoxylanase/glucanase from Cellulomonas fimi was provided by 2,4-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside, according to inactivation parameters of ki = 0.057 min-1 and Ki = 0.0035 mM. J Bacteriol, 1995 Aug, 177(15), 4356 - 63 Comparison of a fungal (family I) and bacterial (family II) cellulose-binding domain; Tomme P et al.; A family II cellulose-binding domain (CBD) of an exoglucanase/xylanase (Cex) from the bacterium Cellulomonas fimi was replaced with the family I CBD of cellobiohydrolase I (CbhI) from the fungus Trichoderma reesei . Expression of the hybrid gene in Escherichia coli yielded up to 50 mg of the hybrid protein, CexCBDCbhI, per liter of culture supernatant . The hybrid was purified to homogeneity by affinity chromatography on cellulose . The relative association constants (Kr) for the binding of Cex, CexCBDCbhI, the catalytic domain of Cex (p33), and CbhI to bacterial microcrystalline cellulose (BMCC) were 14.9, 7.8, 0.8, and 10.6 liters g-1, respectively . Cex and CexCBDCbhI had similar substrate specificities and similar activities on crystalline and amorphous cellulose . Both released predominantly cellobiose and cellotriose from amorphous cellulose . CexCBDCbhI was two to three times less active than Cex on BMCC, but significantly more active than Cex on soluble cellulose and on xylan . Unlike Cex, the hybrid protein neither bound to alpha-chitin nor released small particles from dewaxed cotton fibers. Biochemistry, 1995 May 30, 34(21), 6993 - 7009 Solution structure of a cellulose-binding domain from Cellulomonas fimi by nuclear magnetic resonance spectroscopy; Xu GY et al.; Multidimensional, multinuclear nuclear magnetic resonance spectroscopy combined with dynamical simulated annealing has been used to determine the structure of a 110 amino acid cellulose-binding domain (CBD) from Cex, a beta-1,4-glycanase from the bacterium Cellulomonas fimi (CBDcex) . An experimental data set comprising 1795 interproton NOE-derived restraints, 50 phi, 34 chi 1, and 106 hydrogen bond restraints was used to calculate 20 final structures . The calculated structures have an average root-mean-square (rms) deviation about the mean structure of 0.41 A for backbone atoms and 0.67 A for all heavy atoms when fitted over the secondary structural elements . Chromatography, ultracentrifugation, and 15N NMR relaxation experiments demonstrate that CBDcex is a dimer in solution . While attempts to measure NOEs across the dimer interface were unsuccessful, a computational strategy was employed to generate dimer structures consistent with the derived data set . The results from the dimer calculations indicate that, while the monomer topologies produced in the context of the dimer can be variable, the relative positioning of secondary structural elements and side chains present in the monomer are restored upon dimer formation . CBDcex forms an extensive beta-sheet structure with a beta-barrel fold . Titration with cellohexaose, {beta-D-glucopyranosyl-(1,4)}5-D-glucose, establishes that Trp 54 and 72 participate in cellulose binding . Analysis of the structure shows that these residues are adjacent in space and exposed to solvent . Together with other proximate hydrophilic residues, these residues form a carbohydrate-binding cleft, which appears to be a feature common to all CBDs of the same family. Gene, 1995 May 26, 158(1), 23 - 9 The gene encoding the beta-1,4-endoglucanase (CelA) from Myxococcus xanthus: evidence for independent acquisition by horizontal transfer of binding and catalytic domains from actinomycetes; Quillet L et al.; The celA gene encoding a beta-1,4 endoglucanase (CelA) from Myxococcus xanthus has been cloned in Escherichia coli and sequenced . The C-terminal region of CelA displayed a high level of similarity with the catalytic domain of several Egl belonging to the glycosyl hydrolases family 6 (CenA from Cellulomonas fimi, CelA from Microbispora bispora, E2 from Thermonospora fusca, CasA from Streptomyces KSM9 and CelA1 from Streptomyces halstedii) and less similarity to the cellobiohydrolases of the fungi Trichoderma reesei and Agaricus bisporus . Using PCR amplification we found in another myxobacterium, Stigmatella aurantiaca, a part of a glycosyl hydrolase belonging to the same family . The N-terminal part of CelA displayed significant similarities with the cellulose-binding domain of other cellulases belonging to a rare subset of family II, such as the avicelase I from Streptomyces reticuli, both tandem repeats N1 and N2 of the cellulase CenC from Cellulomonas fimi, and the N-terminal part of the Egl E1 from Thermonospora fusca . Analyses of the multiple alignments and reconstruction of phylogenetic trees strongly suggest that both domains of CelA were acquired by independent horizontal transfers between Gram+ soil bacteria and scavenging myxobacteria followed by domain shuffling. Bioseparation, 1995 Apr, 5(2), 95 - 104 Purification of human interleukin-2 using the cellulose-binding domain of a prokaryotic cellulase; Ong E et al.; Engineering gene fusions which introduce an affinity tag linked to the target polypeptide by a specific protease cleavage site is widely used to facilitate recombinant protein purification . A fusion protein CBDAPT-IL-2, comprised of the cellulose-binding domain (CBD) and Pro-Thr (PT) rich linker of the Cellulomonas fimi endo-beta-1,4-glucanase A (CenA) and a factor Xa cleavage sequence (IleGluGlyArg) fused to the N terminus of human interleukin-2, was produced in Escherichia coli, Streptomyces lividans and mammalian COS cells . CBDAPT-IL-2, secreted from S . lividans or COS cells or recovered from the insoluble fraction of E . coli, could be purified by adsorption on cellulose . The intact fusion protein adsorbed to cellulose was hydrolyzed in situ with factor Xa to release active interleukin-2. Biochem J, 1995 Apr 1, 307 ( Pt 1), 191 - 5 A modular xylanase containing a novel non-catalytic xylan-specific binding domain; Black GW et al.; Xylanase D (XYLD) from Cellulomonas fimi contains a C-terminal cellulose-binding domain (CBD) and an internal domain that exhibits 65% sequence identity with the C-terminal CBD . Full-length XYLD binds to both cellulose and xylan . Deletion of the C-terminal CBD from XYLD abolishes the capacity of the enzyme to bind to cellulose, although the truncated xylanase retains its xylan-binding properties . A derivative of XYLD lacking both the C-terminal CBD and the internal CBD homologue did not bind to either cellulose or xylan . A fusion protein consisting of the XYLD internal CBD homologue linked to the C-terminus of glutathione S-transferase (GST) bound to xylan, but not to cellulose, while GST bound to neither of the polysaccharides . The Km and specific activity of full-length XYLD and truncated derivatives of the enzyme lacking the C-terminal CBD (XYLDcbd), and both the CBD and the internal CBD homologue (XYLDcd), were determined with soluble and insoluble xylan as the substrates . The data showed that the specific activities of the three enzymes were similar for both substrates, as were the Km values for soluble substrate . However, the Km values of XYLD and XYLDcbd for insoluble xylan were significantly lower than the Km of XYLDcd . Overall, these data indicate that the internal CBD homologue in XYLD constitutes a discrete xylan-binding domain which influences the affinity of the enzyme for insoluble xylan but does not directly affect the catalytic activity of the xylanase . The rationale for the evolution of this domain is discussed. J Biol Chem, 1995 Mar 3, 270(9), 4383 - 6 Enhancement of the endo-beta-1,4-glucanase activity of an exocellobiohydrolase by deletion of a surface loop; Meinke A et al.; In the commonly accepted mechanism for enzymatic hydrolysis of cellulose, endo-beta-1,4-glucanases randomly cleave glucosidic bonds within glucan polymers, providing sites for attack by exo-cellobiohydrolases (EC 3.2.1.91) . It has been proposed that hydrolysis by Trichoderma reesei cellobiohydrolase II is restricted to the ends of cellulose polymers because two surface loops cover its active site to form a tunnel . In a closely related endoglucanase, E2 from Thermomonospora fusca, access to the substrate appears to be relatively unhindered because the carboxyl-proximal loop is shortened, and the amino-proximal loop is displaced . The hypothesis was examined by deletion of a region in Cellulomonas fimi cellobiohydrolase A corresponding to part of the carboxyl-proximal loop of T . reesei cellobiohydrolase II . The mutation enhanced the endoglucanase activity of the enzyme on soluble O-(carboxymethyl)cellulose and altered its activities on 2',4'-dinitrophenyl-beta-D-cellobioside, insoluble cellulose, and cellotetraose. Biochemistry, 1995 Feb 21, 34(7), 2220 - 4 Site-directed mutation of the putative catalytic residues of endoglucanase CenA from Cellulomonas fimi; Damude HG et al.; The catalytic domains of beta-1,4-glucanases can be grouped into families of related amino acid sequences . The endoglucanase CenA from Cellulomonas fimi is a member of family B . All enzymes from this family are believed to hydrolyze beta-1,4-glucosidic bonds using a general acid-base catalytic mechanism resulting in inversion of anomeric configuration at the scissile bond . Three-dimensional structures for two cellulases from family B have been determined by X-ray crystallographic analysis . These structures show that there are four Asp residues which are in a position to function as acid catalyst, base catalyst, and/or transition state stabilizers . These aspartates are conserved in all members of family B . The roles of Asp216, Asp252, Asp287, and Asp392, the corresponding amino acids in CenA, were determined . These aspartates have been systematically replaced with alanine and glutamate via site-directed mutagenesis, and the resulting effect on activity, substrate specificity, and overall structure has been determined . Changes in overall structure were monitored using circular dichroism spectroscopy, and no significant differences between the wild-type and mutant proteins were found . Active site structure was also found to be intact as all proteins bound to a cellobiose affinity column . The Michaelis-Menten parameters of the enzyme were determined on 2,4-dinitrophenyl cellobioside as well as (carboxymethyl)-cellulose and phosphoric acid-swollen cellulose . Initial characterization of mutant proteins indicates that Asp252 and Asp392 are the acid and base catalysts, respectively, in CenA . Residue Asp287 appears to aid Asp252 in acid catalysis, and Asp216 is not absolutely required for catalysis. Lett Appl Microbiol, 1995 Feb, 20(2), 69 - 75 Phenotypic and phylogenetic characterization of some unknown coryneform bacteria isolated from bovine blood and milk: description of Sanguibacter gen.nov; Fernandez-Garayzabal JF et al.; 16S rRNA gene sequencing studies were performed on some Gram-positive coryneform bacteria of unknown taxonomic position isolated from blood and milk of healthy cows . Comparative sequence analysis demonstrated that the milk isolates corresponded to Oerskovia xanthineolytica whereas those from blood consisted of two distinct, albeit highly related species, within the family Cellulomonadaceae . Based on the phylogenetic and phenotypic distinctiveness of the blood isolates, it is proposed that they be classified in a new genus Sanguibacter. Zentralbl Bakteriol, 1995 Jan, 282(1), 13 - 7 Detection of sialidase activity in Oerskovia (Cellulomonas) turbata; Muller HE; The enzyme N-acylneuraminate glycohydrolase, E.C . 3.2.1.18 (sialidase or neuraminidase) was detected in all five strains of Oerskovia (Cellulomonas) turbata and in a further strain isolated from clinical material . The detection of sialidase was performed by different methods, i.e . colorimetric determination of liberated chromogen, immunoelectrophoresis and paper chromatography . The Oerskovia turbata sialidase is able to cleave different linkages between N-acylneuraminic acid and the carbohydrate chains of oligosaccharides or polysaccharides and of glycoproteins, i.e . the 2-->3, 2-->6, and 2-->8 linkages . Finally, the ecologic and pathogenic role of neuraminidase is discussed. Ann N Y Acad Sci, 1994 Nov 30, 745, 372 - 82 The expression of recombinant proteins on the external surface of Escherichia coli . Biotechnological applications; Francisco JA et al.; The expression of recombinant proteins on the external surface of Gram-negative bacteria is expected to open the way for a number of significant biotechnological applications, including the development of live bacterial vaccines, the production of whole cell adsorbents, the preparation of whole cell catalysts, and the display and selection of peptide and antibody libraries . We have developed a fusion protein system for the production of active recombinant proteins on the surface of Escherichia coli . Using this system we have expressed beta-lactamase, the Cellulomonas fimi exoglucanase Cex as well as its cellulose binding domain, and an antidigoxin single chain Fv antibody fragment on the cell surface . Recently we have begun to explore some of the potential applications for cell-surface expression. Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11383 - 7 C1-Cx revisited: intramolecular synergism in a cellulase; Din N et al.; Endoglucanase A (CenA) from the bacterium Cellulomonas fimi is composed of a catalytic domain and a nonhydrolytic cellulose-binding domain that can function independently . The individual domains interact synergistically in the disruption and hydrolysis of cellulose fibers . This intramolecular synergism is distinct from the well-known intermolecular synergism between individual cellulases . The catalytic domain corresponds to the hydrolytic Cx system and the cellulose-binding domain corresponds to the nonhydrolytic C1 system postulated by Reese et al . {Reese, E . T., Sui, R . G . H . & Levinson, H . S . (1950) J . Bacteriol . 59, 485-497} to be required for the hydrolysis of cellulose. Biochemistry, 1994 Oct 25, 33(42), 12546 - 52 Crystal structure of the catalytic domain of the beta-1,4-glycanase cex from Cellulomonas fimi; White A et al.; beta-1,4-Glycanases, principally cellulases and xylanases, are responsible for the hydrolysis of plant biomass . The bifunctional beta-1,4-xylanase/glucanase Cex from the bacterium Cellulomonas fimi, one of a large family of cellulases/xylanases, depolymerizes oligosaccharides and releases a disaccharide unit from the substrate nonreducing end . Hydrolysis occurs with net retention of the anomeric configuration of the sugar through a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate . The active site nucleophile, Glu233, has been unambiguously identified by trapping of such an intermediate {Tull et al . (1991) J . Biol . Chem . 266, 15621-15625} and the acid/base catalyst, Glu127, by detailed kinetic analysis of mutants {MacLeod et al . (1994) Biochemistry 33, 6371-6376} . However, little is known about the enzyme's overall folding and its active site architecture . We report here the high-resolution crystal structure of the catalytic domain of Cex . The atomic structure refinement results in a model that includes 2400 protein atoms and 45 water molecules, with an R-factor of 0.217 for data extending to 1.8-A resolution . The protein forms an eight-parallel-stranded alpha/beta-barrel, which is a novel folding pattern for a microbial beta-glycanase . The active site, inferred from the location of Glu233, Glu127, and other conserved residues, is an open cleft on the carboxy-terminal end of the alpha/beta-barrel . An extensive hydrogen-bonding network stabilizes the ionization states of the key residues; in particular, the Asp235-His205-Glu233 hydrogen-bonding network may play a role in modulating the ionization state of Glu233 and in controlling local charge balance during the reaction. Biochem J, 1994 Sep 1, 302 ( Pt 2), 463 - 9 Changes in the molecular-size distribution of insoluble celluloses by the action of recombinant Cellulomonas fimi cellulases; Kleman-Leyer KM et al.; Specific patterns of attacks of cotton, bacterial cellulose and bacterial microcrystalline cellulose (BMCC) by recombinant cellulases of Cellulomonas fimi were investigated . Molecular-size distributions of the celluloses were determined by high-performance size-exclusion chromatography . Chromatography of cotton and bacterial celluloses revealed single major peaks centered over progressively lower molecular-mass positions during attack by endoglucanase CenA . In advanced stages, a second peak appeared at very low average size (approx . 11 glucosyl units); ultimate weight losses were approximately 30% . The isolated catalytic domain of CenA, p30, gave results very similar to those with complete CenA . CenA did not effectively depolymerize or solubilize BMCC significantly . Molecular-size distributions of cotton and bacterial cellulose incubated with endoglucanases CenB or CenD exhibited one major peak regardless of incubation time; low-molecular-mass fragments did not accumulate . Weight losses were 40 and 35% respectively . The single peak shifted to lower-molecular-mass positions as incubation continued, but high-molecular-mass material persisted . CenB and CenD readily attacked and solubilized BMCC (approx . 70%) . We conclude that CenA attacks cellulose by preferentially cleaving completely through the cellulose microfibrils at the amorphous sites, and much more slowly by degrading the crystalline surfaces . Conversely, CenB and CenD cleave the amorphous regions much less efficiently while vigorously degrading the surfaces of the crystalline regions of the microfibrils. Bioorg Med Chem, 1994 Jun, 2(6), 429 - 32 Enzymatic asymmetric synthesis of alpha-methyl arylalkylamines and alpha-methyl arylalkylalcohols by arylalkyl acylamidases; Ogawa J et al.; With the novel microbial enzyme, 'arylalkyl acylamidase', optically active alpha-methyl arylalkylamines and alpha-methyl arylalkylalcohols have been obtained through enantioselective hydrolysis of their racemic amides and esters . (S)-Enantiomers of 1-methylbenzylamine, 1-methyl-3-phenylpropylamine and 1-methyl-3-phenylpropanol of high optical purity (> 94% e.e.) were synthesized with the cells of Nocardia erythropolis IAM 1440 or Cellulomonas fimi AKU 671 . (R)-Enantiomer of 1-methyl-3-phenylpropylamine and (S)-enantiomer of 1-methyl-2-phenylpropanol of high optical purity (> 95% e.e.) were synthesized with the crude preparation of arylalkyl acylamidase of Pseudomonas putida Sc2 AKU 881. Biochemistry, 1994 May 24, 33(20), 6363 - 70 Mechanisms of cellulases and xylanases: a detailed kinetic study of the exo-beta-1,4-glycanase from Cellulomonas fimi; Tull D et al.; The exoglucanase/xylanase from Cellulomonas fimi (Cex) has been subjected to a detailed kinetic investigation with a range of aryl beta-D-glycoside substrates . This enzyme hydrolyzes its substrates with net retention of anomeric configuration, and thus it presumably follows a double-displacement mechanism . Values of kcat are found to be invariant with pH whereas kcat/Km is dependent upon two ionizations of pKa = 4.1 and 7.7 . The substrate preference of the enzyme increases in the order glucosides < cellobiosides < xylobiosides, and kinetic studies with a range of aryl glucosides and cellobiosides have allowed construction of Broensted relationships for these substrate types . A strong dependence of both kcat (beta 1g = -1) and kcat/Km (beta 1g = -1) upon leaving group ability is observed for the glucosides, indicating that formation of the intermediate is rate-limiting . For the cellobiosides a biphasic, concave downward plot is seenj for kcat, indicating a change in rate-determining step across the series . Pre-steady-state kinetic experiments allowed construction of linear Broensted plots of log k2 and log (k2/Kd) for the cellobiosides of modest (beta 1g = -0.3) slope . These results are consistent with a double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and hydrolyzed via oxocarbonium ion-like transition states . Secondary deuterium kinetic isotope effects and inactivation experiments provide further insight into transition-state structures and, in concert with beta 1g values, reveal that the presence of the distal sugar moiety in cellobiosides results in a less highly charged transition state.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 May 24, 33(20), 6371 - 6 The acid/base catalyst in the exoglucanase/xylanase from Cellulomonas fimi is glutamic acid 127: evidence from detailed kinetic studies of mutants; MacLeod AM et al.; The exoglucanase/xylanase Cex from Cellulomonas fimi hydrolyzes beta-1,4-glycosidic bonds with net retention of anomeric configuration, releasing the disaccharides beta-cellobiose or beta-xylobiose . It uses a double-displacement mechanism involving a glycosyl-enzyme intermediate which is formed and hydrolyzed with general acid/base catalytic assistance . Glu127 was proposed as the acid/base catalyst on the basis of sequence alignments, and mutants at this position were constructed in which the glutamic acid is replaced by alanine or glycine . The following kinetic analysis provides firm support for the assignment of Glu127 as the acid/base catalyst and suggests a more general strategy for identification of this residue in other glycosidases . Substrates which do not require protonic assistance for initial bond cleavage exhibit kcat/Km values similar to those of wild-type enzyme, whereas substrates which do require assistance have kcat/Km values over 6000-fold smaller . Thus rate constants for glycosylation are affected to different degrees by this substitution, depending upon their need for acid catalysis . The deglycosylation rate constant is decreased 200-fold by such substitution, due to the removal of general base catalytic assistance . In the presence of sodium azide a new product, beta-cellobiosyl azide, is formed with these mutants whereas only cellobiose is formed with wild-type enzyme or the Glu127Asp mutant under similar conditions . Addition of azide results in very significant increases in kcat values, ranging from 8-fold for 4''-nitrophenyl cellobioside to over 200-fold for 2'',4''-dinitrophenyl cellobioside, whereas kcat/Km values for these substrates remain essentially constant . No effects on rate upon azide addition are seen with substrates containing aglycons of poor leaving group ability.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Microbiol, 1994 May, 12(3), 413 - 22 Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi is a beta-1,4-exocellobiohydrolase analogous to Trichoderma reesei CBH II; Meinke A et al.; The gene cbhA from the cellulolytic bacterium Cellulomonas fimi encodes a protein of 872 amino acids designated cellobiohydrolase A (CbhA) . Mature CbhA contains 832 amino acid residues and has a predicted molecular mass of 85,349 Da . It is composed of five domains: an N-terminal catalytic domain, three repeated sequences of 95 amino acids, and a C-terminal cellulose-binding domain typical of other C . fimi glycanases . The structure and enzymatic activities of the CbhA catalytic domain are closely related to those of CBH II, an exocellobiohydrolase in the glycosyl hydrolase family B from the fungus Trichoderma reesei . CbhA is the first such enzyme to be characterized in bacteria . The data support the proposal that extended loops around the active site distinguish exohydrolases from endohydrolases in this enzyme family. Glycobiology, 1994 Apr, 4(2), 165 - 73 Enzymatic evidence for the presence of a critical terminal hexa-arabinoside in the cell walls of Mycobacterium tuberculosis; McNeil MR et al.; A species of Cellulomonas was isolated from soil by enrichment culture and shown to secrete enzymes capable of degrading mycobacterial cell wall arabinogalactan, both the insoluble peptidoglycan-bound and base-solubilized forms . The major degradation product was purified and characterized as a hexa-arabinofuranoside, {beta-D-Araf-(1-->2)-alpha-Araf-(1-->}2-->3,5-alpha-D-Ara f-(1-->5)-D-Araf . The non-reducing ends of this unit are the sites of mycolic acid attachment and, as they also appear in lipoarabinomannan (LAM), the point of mannose capping in some mycobacteria . Thus, elaboration of the structure of this focal hexasaccharide is critical to our understanding of much of the physiology and pathogenesis of mycobacteria . The extracellular enzymes of Cellulomonas sp . also released the disaccharide, alpha-D-Araf-(1-->5)-D-Araf, from internal linear regions of arabinan and, surprisingly, convert the linear galactan backbone into cyclic oligosaccharides of the structure {-->5-D-Galf-(1-->6)-beta-D-Galf-(1-->}n where n is 2, 3 or 4 . Thus, the preparation contains Schardinger-like enzyme activity . This group of enzymes are powerful tools for the dissection of the mycolylarabino-galactan-peptidoglycan (mAGP) complex of mycobacteria towards understanding its role in drug resistance, disease processes and mycobacterial physiology. Biochem Biophys Res Commun, 1994 Mar 30, 199(3), 1223 - 8 Stereochemical course of hydrolysis catalysed by Cellulomonas fimi CenE, a member of a new family of beta-1,4-glucanases; Shen H et al.; The gene for a previously identified, extracellular, 120 kDa cellulose-binding protein (Cbp120) was isolated from a Cellulomonas fimi genomic library and expressed in Escherichia coli . Qualitative analysis of CM-cellulose hydrolysis shows that Cbp120 is an endo-beta-1,4-glucanase . Cbp120, now renamed CenE, catalyzes hydrolysis of cellohexaose with inversion of anomeric carbon configuration, characteristic of a single displacement reaction . Partial sequencing of its gene shows that CenE has significant sequence similarity with the catalytic domains of five enzymes from cellulolytic bacteria . It is proposed that the six enzymes form a new family of beta-1,4-glucanases . CenE is the first enzyme from this family to be characterized stereochemically. Mol Microbiol, 1994 Feb, 11(4), 747 - 55 The cellulose-binding domain of endoglucanase A (CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding; Din N et al.; Cellulomonas fimi endo-beta-1,4-glucanase A (CenA) contains a discrete N-terminal cellulose-binding domain (CBDCenA) . Related CBDs occur in at least 16 bacterial glycanases and are characterized by four highly conserved Trp residues, two of which correspond to W14 and W68 of CBDCenA . The adsorption of CBDCenA to crystalline cellulose was compared with that of two Trp mutants (W14A and W68A) . The affinities of the mutant CBDs for cellulose were reduced by approximately 50- and 30-fold, respectively, relative to the wild type . Physical measurements indicated that the mutant CBDs fold normally . Fluorescence data indicated that W14 and W68 were exposed on the CBD, consistent with their participation in binding to cellobiosyl residues on the cellulose surface. J Bacteriol, 1994 Feb, 176(4), 999 - 1008 Streptomyces lividans glycosylates the linker region of a beta-1,4-glycanase from Cellulomonas fimi; Ong E et al.; The beta-1,4-glycanase Cex of the gram-positive bacterium Cellulomonas fimi is a glycoprotein comprising a C-terminal cellulose-binding domain connected to an N-terminal catalytic domain by a linker containing only prolyl and threonyl (PT) residues . Cex is also glycosylated by Streptomyces lividans . The glycosylation of Cex produced in both C . fimi and S . lividans protects the enzyme from proteolysis . When the gene fragments encoding the cellulose-binding domain of Cex (CBDCex), the PT linker plus CBDCex (PT-CBDCex), and the catalytic domain plus CBDCex of Cex were expressed in S . lividans, only PT-CBDCex was glycosylated . Therefore, all the glycans must be O linked because only the PT linker was glycosylated . A glycosylated form and a nonglycosylated form of PT-CBDCex were produced by S . lividans . The glycosylated form of PT-CBDCex was heterogeneous; its average carbohydrate content was approximately 10 mol of D-mannose equivalents per mol of protein, but the glycans contained from 4 to 12 alpha-D-mannosyl and alpha-D-galactosyl residues . Glycosylated Cex from S . lividans was also heterogeneous . The presence of glycans on PT-CBDCex increased its affinity for bacterial microcrystalline cellulose . The location of glycosylation only on the linker region of Cex correlates with the properties conferred on the enzyme by the glycans. Mol Microbiol, 1994 Jan, 11(2), 375 - 82 Evidence for a general role for high-affinity non-catalytic cellulose binding domains in microbial plant cell wall hydrolases; Millward-Sadler SJ et al.; Cellulases expressed by Cellulomonas fimi consist of a catalytic domain and a discrete non-catalytic cellulose-binding domain (CBD) . To establish whether CBDs are common features of plant cell-wall hydrolases from C . fimi, the molecular architecture of xylanase D (XYLD) from this bacterium was investigated . The gene encoding XYLD, designated xynD, consisted of an open reading frame of 1936 bp encoding a protein of M(r) 68,000 . The deduced primary sequence of XYLD was confirmed by the size (64 kDa) and N-terminal sequence of the purified recombinant xylanase . Biochemical analysis of the purified enzyme revealed that XYLD is an endoacting xylanase which displays no detectable activity against polysaccharides other than xylan . The predicted primary structure of XYLD comprised an N-terminal signal peptide followed by a 190-residue domain that exhibited significant homology to Family-G xylanases . Truncated derivatives of xynD, encoding the N-terminal 193 amino acids of mature XYLD directed the synthesis of a functional xylanase, confirming that the 190-residue N-terminal sequence constitutes the catalytic domain . The remainder of the enzyme consisted of two approximately 90-residue domains, which exhibited extensive homology with each other, and limited sequence identity with CBDs from other polysaccharide hydrolases . Between the two putative CBDs is a 197-amino-acid sequence that exhibits substantial homology with Rhizobium NodB proteins . The four discrete domains in XYLD were separated by either threonine/proline-or novel glycine-rich linker regions . Although full-length XYLD adsorbed to cellulose, truncated derivatives of the enzyme lacking the C-terminal CBD hydrolysed xylan but did not bind to cellulose.(ABSTRACT TRUNCATED AT 250 WORDS) Protein Eng, 1994 Jan, 7(1), 117 - 23 An internal cellulose-binding domain mediates adsorption of an engineered bifunctional xylanase/cellulase; Tomme P et al.; A chimeric xylanase/endoglucanase (XynCenA) with an internal cellulose-binding domain was constructed by fusing the Bacillus subtilis xyn gene fragment to the 5'-end of the Cellulomonas fimi cenA . A polyhistidine-encoding sequence was also fused to the 5'-end of the xyn gene . The gene fusion was overexpressed in Escherichia coli and the fusion polypeptide purified from the cell extracts using the polyhistidine tail . The hybrid protein behaved like the parental endoglucanase or xylanase when assayed on a number of soluble and insoluble cellulosic substrates or xylans . The presence of two distinct active sites and the internal cellulose-binding domain did not significantly affect the hydrolysis of any of these substrates . However, the fusion protein exhibited a strong affinity for both microcrystalline cellulose (Avicel) and regenerated chitin . Like the parental endoglucanase, bound XynCenA could not be eluted from these polysaccharides with either low or high salt buffer or distilled water . More stringent conditions, such as 1% SDS or 8 M guanidinium hydrochloride, fully desorbed the protein . The fusion protein did not adsorb significantly to insoluble xylan. Folia Microbiol (Praha), 1994, 39(4), 251 - 4 Cloning of endoglucanase genes from Cellulomonas biazotea into E . coli and S . cerevisiae using shuttle vector YEp24; Parvez S et al.; We constructed a SmaI genomic library of Cellulomonas biazotea DNA in E . coli and in the S . cerevisiae shuttle vector, YEP 24 . Three clone were identified that conferred the ability for E . coli or S . cerevisiae transformants to produce carboxymethylcellulase (CMCase) . Cells transformed with these clones were compared with one another and with nontransformed cells for hyper-production of CMCase . In vivo and in vitro studies indicated that the CMCase genes were fully expressed and the enzyme activity was located extracellularly . The optimum pH and temperature for the CMCase thus cloned were pH 7 and 50 degrees C, respectively, as was the case for the donor. Int J Biol Macromol, 1993 Dec, 15(6), 347 - 51 Visualization of the adsorption of a bacterial endo-beta-1,4-glucanase and its isolated cellulose-binding domain to crystalline cellulose; Gilkes NR et al.; Endo-beta-1,4-glucanase A (CenA), a cellulase from the bacterium Cellulomonas fimi, is composed of two domains: a catalytic domain and a cellulose-binding domain . Adsorption of CenA and its isolated cellulose-binding domain (CBD.PTCenA) to Valonia cellulose microcrystals was examined by transmission electron microscopy using an antibody sandwich technique (CenA/CBD.PTCenA-alpha CenA IgG-protein A-gold conjugate) . Adsorption of both CenA and CBD.PTCenA occurred along the lengths of the microcrystals, with an apparent preference for certain crystal faces or edges . CenA or CBD.PTCenA, but not the isolated catalytic domain, were shown to prevent the flocculation of microcrystalline bacterial cellulose . The cellulose-binding domain may assist crystalline cellulose hydrolysis in vitro by promoting substrate dispersion. Biotechnology (N Y), 1993 Dec, 11(13), 1570 - 3 A bifunctional affinity linker to couple antibodies to cellulose; Ramirez C et al.; We have constructed a fusion protein between the staphylococcal A protein and the cellulose binding domain of an exoglucanase (Cex) from Cellulomonas fimi that can be directly immobilized on cellulose while retaining its capacity to bind immunoglobulin G molecules . The cellulose domain provides binding that does not interfere with the biological activity of the fusion partner, does not involve hazardous chemicals and the matrix does not need to be chemically activated which reduces its cost . We have tested some of the possible applications of the fusion protein and show that it can be used in immunoassays, affinity chromatography and immunoprecipitations. Carbohydr Res, 1993 Oct 18, 249(1), 77 - 90 Syntheses of 2-deoxy-2-fluoro mono- and oligo-saccharide glycosides from glycals and evaluation as glycosidase inhibitors; McCarter JD et al.; Several fluorinated oligosaccharides, including 2-deoxy-2-fluoro derivatives of cellobiose, maltose, and maltotriose were synthesized by the action of fluorine or acetyl hypofluorite on the corresponding glycal peracetates . Temperature effects on the stereoselectivities of these reactions were examined . Addition of acetyl hypofluorite to several 2-substituted glycals in the gluco or galacto series gave 2,2-disubstituted arabino- or lyxo-hexose derivatives; 3,4,6-tri-O-acetyl-2-fluoro-D-glucal or the analogous galactal yielded 2-deoxy-2,2-difluoro arabino- or lyxo-hexose peracetates, whereas 2-acetoxy-3,4,6-tri-O-acetyl-D-glucal or the analogous galactal gave 2(R)-2-acetoxy-2-fluoro-arabino- or lyxo-hexose peracetates, respectively . 2-Acetamido-3,4,6-tri-O-acetyl-D-glucal gave 2(R)-2-acetamido-2-acetoxy-3,4,6-tri-O-acetyl-alpha-D-arabino-hexopyrano syl fluoride . 2,4-Dinitrophenyl 2-deoxy-2-fluoro-beta-cellobioside was an inactivator of the exoglucanase from Cellulomonas fimi while 2-deoxy-2-fluoro-alpha-maltosyl and alpha-maltotriosyl fluorides were slow substrates of human pancreatic alpha-amylase and rabbit muscle glycogen debranching enzyme, respectively. Glycobiology, 1993 Oct, 3(5), 497 - 506 Structural definition of the non-reducing termini of mannose-capped LAM from Mycobacterium tuberculosis through selective enzymatic degradation and fast atom bombardment-mass spectrometry; Chatterjee D et al.; The application of extracellular arabinases from a Cellulomonas sp . and fast atom bombardment-mass spectrometry (FAB-MS) provided new insight into the structure of lipoarabinomannan (LAM) of Mycobacterium tuberculosis, a key molecule in the pathogenesis and physiology of the tubercle bacillus . Previously, the non-reducing arabinan ends of LAM from the virulent (Erdman) strain of M . tuberculosis were shown to be 'capped' by short (alpha 1-->2)-linked mannopyranose (Manp)-containing oligosaccharides, a product called ManLAM . The structural relationship between these Manp units and the underlying arabinofuranose (Araf)-containing arabinan was examined by digesting ManLAM from M.tuberculosis Erdman with the Cellulomonas enzyme, resolving fragments by various means and subjecting the derivatized oligoglycosylalditols to FAB-MS . The sequences Manp2Araf4, Manp3Araf4 and Manp1-6Araf6 were recognized as the major terminal motifs . Upon complete structural definition, all of the Ara6-containing products were shown to be based on a 3,5-linked branched Araf unit, whereas those containing Ara4 were linear . Minor non-mannosylated terminal arrangements containing Ara4-6, branched, linear and cyclical, were also recognized . In addition, the mannan 'core' of ManLAM was isolated from enzyme digests and shown to contain segments of the phosphatidylinositol anchor and a 'stub' of the arabinan side-chain in the form of a 'linker' alpha-Araf-(1-->5)-Araf unit attached to C-2, apparently of the penultimate 2,6-linked Manp residue . The structural unravelling of this complex molecule further substantiates the case for structural and biological similarities to the enterobacterial lipopolysaccharides/lipoglycans and other important 'capped' lipooligomers such as the lipooligosaccharides of Neisseria species and the lipophosphoglycan of Leishmania promastigotes. Appl Environ Microbiol, 1993 Sep, 59(9), 3032 - 43 DNA sequences and expression in Streptomyces lividans of an exoglucanase gene and an endoglucanase gene from Thermomonospora fusca; Jung ED et al.; Two genes encoding cellulases E1 and E4 from Thermomonospora fusca have been cloned in Escherichia coli, and their DNA sequences have been determined . Both genes were introduced into Streptomyces lividans, and the enzymes were purified from the culture supernatants of transformants . E1 and E4 were expressed 18- and 4-fold higher, respectively, in S . lividans than in E . coli . Thin-layer chromatography of digestion products showed that E1 digests cellotriose, cellotetraose, and cellopentaose to cellobiose and a trace of glucose . E4 is poor at degrading cellotriose and cleaves cellopentaose to cellotetraose and glucose or cellotriose and cellobiose . It readily cleaves cellotetraose to cellobiose . E1 shows 59% identity to Cellulomonas fumi CenC in a 689-amino-acid overlap, and E4 shows 80% identity to the N terminus of C . fimi CenB in a 441-amino-acid overlap; all of these proteins are members of cellulase family E . Alignment of the amino acid sequences of Clostridium thermocellum celD, E1, E4, and four other members of family E demonstrates a clear relationship between their catalytic domains, although there is as little as 25% identity between some of them . Residues in celD that have been identified by site-directed mutagenesis and chemical modification to be important for catalytic activity are conserved in all seven proteins . The catalytic domains of E1 and E4 are not similar to those of T . fusca E2 or E5, but all four enzymes share similar cellulose-binding domains and have the same 14-bp inverted repeat upstream of their initiation codons . This sequence has been identified previously as the binding site for a protein that regulates induction. Anal Biochem, 1993 Jul, 212(1), 259 - 62 Simultaneous high-performance liquid chromatographic determination of both the cleavage pattern and the stereochemical outcome of the hydrolysis reactions catalyzed by various glycosidases; Braun C et al.; A high-performance liquid chromatographic method for the simultaneous determination of both the stereochemical outcome and the cleavage pattern of enzymatic action on unmodified sugar substrates is described . Three different enzymes were investigated by this method . Human pancreatic alpha-amylase hydrolyzed maltopentaose with retention of anomeric configuration, with the cleavage position being two glucose units from the reducing end . Cellulomonas fimi endoglucanase D hydrolyzed cellopentaose with retention of anomeric configuration and predominantly two glucose units from the reducing end . beta-D-Xylosidase from Butyrivibrio fibrisolvens hydrolyzed o-nitrophenyl beta-D-xylopyranoside with inversion of anomeric configuration. J Bacteriol, 1993 Apr, 175(7), 1910 - 8 Cellulose-binding polypeptides from Cellulomonas fimi: endoglucanase D (CenD), a family A beta-1,4-glucanase; Meinke A et al.; Five cellulose-binding polypeptides were detected in Cellulomonas fimi culture supernatants . Two of them are CenA and CenB, endo-beta-1,4-glucanases which have been characterized previously; the other three were previously uncharacterized polypeptides with apparent molecular masses of 120, 95, and 75 kDa . The 75-kDa cellulose-binding protein was designated endoglucanase D (CenD) . The cenD gene was cloned and sequenced . It encodes a polypeptide of 747 amino acids . Mature CenD is 708 amino acids long and has a predicted molecular mass of 74,982 Da . Analysis of the predicted amino acid sequence of CenD shows that the enzyme comprises four domains which are separated by short linker polypeptides: an N-terminal catalytic domain of 405 amino acids, two repeated sequences of 95 amino acids each, and a C-terminal domain of 105 amino acids which is > 50% identical to the sequences of cellulose-binding domains in Cex, CenA, and CenB from C . fimi . Amino acid sequence comparison placed the catalytic domain of CenD in family A, subtype 1, of beta-1,4-glycanases . The repeated sequences are more than 40% identical to the sequences of three repeats in CenB and are related to the repeats of fibronectin type III . CenD hydrolyzed the beta-1,4-glucosidic bond with retention of anomeric configuration . The activities of CenD towards various cellulosic substrates were quite different from those of CenA and CenB. Biotechnology (N Y), 1993 Apr, 11(4), 491 - 5 Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose binding domains; Francisco JA et al.; The entire Cex exoglucanase from Cellulomonas fimi and the Cex Cellulose Binding Domain (CBDCex) were expressed in Escherichia coli as fusions to an Lpp-OmpA hybrid which had been shown earlier to direct a heterologous protein to the cell surface . Both Cex and CBDCex were readily localized on the cell surface and could be detected by immunofluorescence microscopy, whole cell ELISAs and functional assays . In cells expressing the entire Cex, about 90% of the total cellobiose hydrolase activity was anchored on the external side of the outer membrane and was susceptible to protease (papain) added in the extracellular fluid . Cells expressing either Cex or CBDCex bound tightly and rapidly to cellulosic materials such as cotton fibers . This property can be exploited for the preparation of immobilized microbial biocatalysts via adsorption to cellulose and for cell separation through specific agglutination on inexpensive cellulosic materials . In addition, our results demonstrate the general utility of fusions to lpp-ompA for the efficient display of proteins and the engineering of the surface topology of Gram-negative bacteria. FEMS Microbiol Lett, 1993 Jan 1, 106(1), 77 - 83 The role of conserved tryptophan residues in the interaction of a bacterial cellulose binding domain with its ligand; Poole DM et al.; The five conserved tryptophan residues in the cellulose binding domain of xylanase A from Pseudomonas fluorescens subsp . cellulosa were replaced with alanine and phenylalanine . The mutated domains were fused to mature alkaline phosphatase, and the capacity of the hybrid proteins to bind cellulose was assessed . Alanine substitution of the tryptophan residues, in general, resulted in a significant decrease in the capacity of the cellulose binding domains to bind cellulose . Mutant domains containing phenylalanine substitution retained some affinity for cellulose . The C-terminal proximal tryptophan did not play an important role in ligand binding, while Trp13, Trp34 and Trp38 were essential for the cellulose binding domain to retain cellulose binding capacity . Data presented in this study suggest major differences in the mechanism of cellulose attachment between Pseudomonas and Cellulomonas cellulose binding domains. Can J Microbiol, 1993 Jan, 39(1), 70 - 80 An "all-purpose" cellulase reporter for gene fusion studies and application to the paracrystalline surface (S)-layer protein of Caulobacter crescentus; Bingle WH et al.; The secreted endoglucanase (CenA) from the Gram-positive bacterium Cellulomonas fimi and a deletion derivative (delta CenA) lacking the N-terminal leader peptide of native CenA were used to explore the potential of delta CenA as a reporter molecule in Caulobacter crescentus . Expression of cenA in C . crescentus yielded extracellular endoglucanase activity, suggesting that the N-terminal leader peptide of CenA could direct the enzyme to the periplasm where it subsequently leaked into the medium . In contrast, expression of delta cenA yielded only cell-associated endoglucanase activity; this suggested that the enzyme retained activity in the C . crescentus cytoplasm . Using the putative cytoplasmic and periplasmic forms of delta CenA as markers, a simple assay for periplasmic delta CenA hybrids was developed . This assay indicated that delta CenA activity was largely independent of cellular location . To facilitate the use of delta CenA as a reporter, a broad host range translational fusion vector (pEC215) incorporating delta cenA was constructed . This vector was used to investigate factors important to the expression of the gene (rsaA) encoding the paracrystalline surface protein (S-layer) of the bacterium . It was found that altering the 5' untranslated region of the rsaA mRNA reduced gene expression by 70% . One rsaA:delta cenA gene fusion resulting from these experiments that incorporated only rsaA translation initiation information was further modified to serve as a general reporter for creating transcriptional gene fusions with other promoters . Gene fusions between alkaline phosphatase (phoA) and either cenA or lacZ were used to supplement information about RsaA secretion derived from rsaA:phoA gene fusions . It was found that linkage of the N-terminal leader peptide of CenA to PhoA yielded 50-100 times more cell-associated PhoA activity in C . crescentus than linkage of the RsaA N terminus . Taken together, these experiments indicated that delta CenA was useful for tagging proteins localized to the cytoplasm, exported to the periplasm, or secreted from the cell, as well as for monitoring events in the cytoplasm such as examining factors important to the level of gene expression . Further, because delta CenA was active in all cell compartments, it could be used to estimate the efficiency of hybrid protein export-secretion from enzyme activity measurements alone . In short, delta CenA possessed many of the attributes of an "all-purpose" reporter. J Bacteriol, 1992 Dec, 174(24), 7996 - 8002 Cellobiose chemotaxis by the cellulolytic bacterium Cellulomonas gelida; Hsing W et al.; In the course of a study on the bacterial degradation of plant cell wall polysaccharides, we observed that growing cells of motile cellulolytic bacteria accumulated, without attachment, near cellulose fibers present in the cultures . Because it seemed likely that the accumulation was due to chemotactic behavior, we investigated the chemotactic responses of one of the above-mentioned bacteria (Cellulomonas gelida ATCC 488) . We studied primarily the responses toward cellobiose, which is the major product of cellulose hydrolysis by microorganisms, and toward hemicellulose hydrolysis products . We found that cellobiose, cellotriose, D-glucose, xylobiose, and D-xylose, as well as other sugars that are hemicellulose components, served as chemoattractants for C . gelida, as determined by a modification of Adler's capillary assay . Competition and inducibility experiments indicated that C . gelida possesses at least two types of separately regulated cellobiose chemoreceptors (Cb1 and cellobiose, cellotriose, xylobiose, and D-glucose, and it is constitutively synthesized . The presence in C . gelida of a constitutive response toward cellobiose and of at least two distinct cellobiose chemoreceptors has implications for the survival of this cellulolytic bacterium in nature . A possible mechanism for cellobiose-mediated bacterial chemotaxis toward cellulose is proposed . We suggest that, in natural environments, motile cellulolytic bacteria migrate toward plant materials that contain cellulose and hemicellulose by swimming up cellobiose concentration gradients and/or concentration gradients of other sugars (e.g., xylobiose, D-xylose, and D-glucose) formed by enzymatic hydrolysis of plant cell wall polysaccharides. Mol Microbiol, 1992 Dec, 6(23), 3611 - 21 The gene encoding the cellulase (Avicelase) Cel1 from Streptomyces reticuli and analysis of protein domains; Schlochtermeier A et al.; Streptomyces reticuli produces an unusual cellulase (Avicelase), with an apparent molecular weight of 82 kDa, which is solely sufficient to degrade crystalline cellulose . During cultivation the processing of the Avicelase to a truncated enzyme (42 kDa) and an inactive protein (40 kDa) correlated with the occurrence of an extracellular protease . After its purification this 36 kDa protease cleaved the S . reticuli Avicelase in vitro in the same manner . Using antibodies raised against the Avicelase and its truncated form (42 kDa) and gene libraries of S . reticuli DNA in the Escherichia coli phage vectors lambda gt11 and Charon 35, the Avicelase gene (cel1) was identified . Further subcloning and DNA-sequencing revealed a G+C rich (72%) reading frame of 2238 bp encoding a protein of 746 amino acids . The transcriptional start site was mapped about 180 bp upstream from the GTG start codon . A signal sequence of 29 amino acids was identified by aligning the deduced amino acids with the characterized N-terminus of the 82 kDa Avicelase . Comparison of the N-terminal amino acids from the purified proteins with the amino acid sequence derived from the Avicelase gene revealed that the truncated enzyme (42 kDa) corresponds to the C-terminal region whereas the inactive proteolytically derived protein (40 kDa) represents the N-terminal part of the 82 kDa Avicelase . Comparisons with amino acid sequences deduced from known cellulase genes indicated the presence of three putative protein domains: (i) an N-terminal part showing significant similarity with a repeat region of endoglucanase C from Cellulomonas fimi, recently shown to be a cellulose-binding domain; (ii) an adjoining region sharing homology with the N-terminal domains with unknown function of endoglucanase A from Pseudomonas fluorescens, endoglucanase D from Clostridium thermocellum and a cellodextrinase from Butyrivibrio fibrisolvens, and (iii) a C-terminal catalytic domain belonging to cellulase family E. J Mol Biol, 1992 Nov 20, 228(2), 693 - 5 Crystallization and preliminary X-ray diffraction analysis of the catalytic domain of Cex, an exo-beta-1,4-glucanase and beta-1,4-xylanase from the bacterium Cellulomonas fimi; Bedarkar S et al.; Single crystals of the catalytic domain of Cex, an exo-beta-1,4-glucanase and beta-1,4-xylanase from the cellulolytic bacterium Cellulomonas fimi, have been grown in the presence of polyethylene glycol 4000 using the vapour diffusion technique . The crystals, which diffract to better than 2.0 A resolution, belong to space group P4(1)2(1)2 or P4(3)2(1)2 and have cell constants: a = b = 88.21 A, c = 81.10 A; alpha = beta = gamma = 90 degrees. Gene, 1992 Nov 2, 121(1), 143 - 7 Streptomyces lividans glycosylates an exoglucanase (Cex) from Cellulomonas fimi; MacLeod AM et al.; Exoglucanase Cex from Cellulomonas fimi is a glycoprotein {Langsford et al., J . Gen . Microbiol . 130 (1984) 1367-1376} . Cex produced by Streptomyces lividans from the cloned cex gene is also glycosylated . The extent and nature of glycosylation are similar for Cex from both organisms . The glycosylation affords protection against proteolysis for the enzymes from both organisms when they are bound to cellulose, but not in solution . The ability to glycosylate cloned gene products enhances the utility of Streptomyces as a host for the production of heterologous polypeptides. Gene, 1992 Sep 21, 119(1), 17 - 28 Sequence analysis of a gene cluster encoding cellulases from Clostridium cellulolyticum; Bagnara-Tardif C et al.; The sequence of a 5633-bp EcoRI-PvuII DNA fragment from Clostridium cellulolyticum was determined . This fragment contains two complete endo-beta-1,4-glucanase-encoding genes, designated celCCC and celCCG . These two genes are flanked by two other partial open reading frames (ORF1 and celCCE) that probably encode two cellulases or related enzymes . The celCCC and celCCG genes appear to be present in a polycistronic transcriptional unit . Northern blot hybridisations with intragenic probes derived from celCCC and celCCG gave similar patterns . Two transcripts of about 5 and 6 kb were identified . The celCCC and celCCG ORFs extend over 1380 bp and 2175 bp, respectively . They are separated by only 87 nt . A typical signal sequence is present at the N terminus of the deduced polypeptides . The mature CelCCC and CelCCG proteins have M(r)s 47,201 and 76,101, respectively . Comparisons between their amino acid (aa) sequences and other known cellulase sequences revealed that: first, they both contain the repeated 24-aa sequence characteristic of clostridial beta-glycanases, secondly, the N-terminal catalytic domains of CelCCC and CelCCG can be classified into the D and E2 families, respectively, and thirdly, the largest CelCCG contains an additional internal domain which is very similar to that of the Bacillus-type cellulose-binding domain (CBD) . The ORF1-C-terminal-encoded sequence also contains the clostridial 24-aa repeat . The CelCCE N-terminus consists of a typical signal sequence followed by a 168-aa domain homologous to the N-terminal repeated domain of Cellulomonas fimi CenC . This domain is connected to an incomplete catalytic domain of family E1 by a Pro-rich junction linker. Glycobiology, 1992 Aug, 2(4), 321 - 6 The tertiary structure of endo-beta-1,4-glucanase B (CenB), a multidomain cellulase from the bacterium Cellulomonas fimi; Meinke A et al.; Endo-beta-1,4-glucanase B (CenB) is a large (110 kDa) extracellular enzyme from the cellulolytic bacterium Cellulomonas fimi . CenB contains five domains, including a typical C.fimi cellulose-binding domain, separated by distinctive linker polypeptides (Meinke et al., 1991b) . X-ray scattering analyses show that CenB has a highly elongated shape resembling beads on a string . The sizes of the polypeptides produced by treatment of CenB with proteases, together with their N-terminal amino acid sequences, show that at least two of the four linkers connecting the five domains of CenB are more sensitive to proteolysis than the domains themselves . It is concluded that the beads represent the domains of CenB, the string represents the linkers. Appl Microbiol Biotechnol, 1992 Aug, 37(5), 621 - 5 In-vitro cleavage of a fusion protein bound to cellulose using the soluble yscFs (Kex2) variant; Seeboth PG et al.; In order to show site-specific cleavage of fusion proteins with an engineered soluble yscF variant, we have constructed a fusion gene encoding eglinC from Hirudo medicinalis and the cellulose-binding domain from the Cellulomonas fimi exoglucanase (Cex) . The two fusion partners were separated by a Lys-Arg-containing recognition sequence for the yeast endoprotease yscF (Kex2) . The fusion protein (eglinC-Cex) was expressed intracellularly in Saccharomyces cerevisiae . After disruption of the cells eglin-C-Cex was shown to bind to cellulose when present in total crude cell lysates . This step efficiently removed the majority of contaminating host proteins . While immobilized on cellulose, eglin-C-Cex could be cleaved into the expected fragments . Upon cleavage the eglinC part was released from the cellulose, while the purification tag, i.e . the cellulose binding domain, stayed bound to the cellulose matrix. Mol Microbiol, 1992 May, 6(9), 1243 - 52 The binding of Cellulomonas fimi endoglucanase C (CenC) to cellulose and Sephadex is mediated by the N-terminal repeats; Coutinho JB et al.; Endoglucanase C (CenC) from Cellulomonas fimi binds to cellulose and to Sephadex . The enzyme has two contiguous 150-amino-acid repeats (N1 and N2) at its N-terminus and two unrelated contiguous 100-amino-acid repeats (C1 and C2) at its C-terminus . Polypeptides corresponding to N1, N1N2, C1, and C1C2 were produced by expression of appropriate cenC gene fragments in Escherichia coli . N1N2, but not N1 alone, binds to Sephadex; both polypeptides bind to Avicel, (a heterogeneous cellulose preparation containing both crystalline and non-crystalline components) . Neither C1 nor C1C2 binds to Avicel or Sephadex . N1N2 and N1 bind to regenerated ('amorphous') cellulose but not to bacterial crystalline cellulose; the cellulose-binding domain of C . fimi exoglucanase Cex binds to both of these forms of cellulose . Amino acid sequence comparison reveals that N1 and N2 are distantly related to the cellulose-binding domains of Cex and C . fimi endoglucanases A and B. FEMS Microbiol Lett, 1992 Apr 15, 71(2), 199 - 203 Endoglucanase A from Cellulomonas fimi in which the hinge sequence of human IgA1 is substituted for the linker connecting its two domains is hydrolyzed by IgA proteases from Neisseria gonorrhoeae; Miller PB et al.; The hinge in IgA1 and the linker in endoglucanase A (CenA) are quite similar . The IgA1 hinge is 18 amino acids long and contains only proline, threonine and serine . The linker in CenA is 27 amino acids long and contains only proline, threonine and a single serine . IgA proteases from Neisseria gonorrhoeae cleave Pro-Ser and Pro-Thr bonds within the IgA1 hinge sequence, but they do not attack CenA . When the linker sequence of CenA is replaced with the hinge sequence of IgA1, the hybrid polypeptide is susceptible to the N . gonorrhoeae proteases . It is cleaved within the hinge sequence at the same sites as IgA1. J Biol Chem, 1992 Apr 5, 267(10), 6743 - 9 The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose; Gilkes NR et al.; CenA is a bacterial cellulase (beta-1,4-glucanase) comprised of a globular catalytic domain joined to an extended cellulose-binding domain (CBD) by a short linker peptide . The adsorption of CenA and its two isolated domains to crystalline cellulose was analyzed . CenA and CBD.PTCenA' (the CBD plus linker) adsorbed rapidly to cellulose at 30 degrees C, and no net desorption of protein was observed during the following 16.7 h . There was no detectable adsorption of the catalytic domain . Scatchard plots of adsorption data for CenA and for CBD.PTCenA were nonlinear (concave upward) . The adsorption of CenA and CBD.PTCenA exceeded 7 and 8 mumol/g cellulose, respectively, but saturation was not attained at the highest total protein concentrations employed . A new model for adsorption was developed to describe the interaction of a large ligand (protein) with a lattice of overlapping potential binding sites (cellobiose residues) . A relative equilibrium association constant (Kr) of 40.5 and 45.3 liter.g cellulose-1 was estimated for CenA and CBD.PTCenA, respectively, according to this model . A similar Kr value (33.3 liter.g-1) was also obtained for Cex, a Cellulomonas fimi enzyme which contains a related CBD but which hydrolyzes both beta 1,4-xylosidic and beta-1,4-glucosidic bonds . It was estimated that the CBD occupies approximately 39 cellobiose residues on the cellulose surface. J Bacteriol, 1992 Feb, 174(4), 1403 - 9 Characterization and comparison of Clostridium cellulovorans endoglucanases-xylanases EngB and EngD hyperexpressed in Escherichia coli; Foong FC et al.; By the use of a T7 expression system, endoglucanases-xylanases EngB and EngD from Clostridium cellulovorans were hyperexpressed and purified from Escherichia coli . The two enzymes demonstrated both endoglucanase and xylanase activities . The substrate specificities of both endoglucanases were similar except that EngD had four-times-greater p-nitrophenyl beta-1,4-cellobiosidase activity . The two proteins were very homologous (80%) up to the Pro-Thr-Thr region which divided the protein into -NH2- and -COOH-terminals . The -COOH- region of EngB has high homology to the endoglucanases and a xylanase from Clostridium thermocellum and to an endoglucanase from Clostridium cellulolyticum and did not show strong binding to cellulose (Avicel) . However, the -COOH- region of EngD, which had homology to the cellulose-binding domains of Cellulomonas fimi exo- and endoglucanases and to Pseudomonas fluorescens endoglucanase, demonstrated binding ability to cellulose even when the domain was fused to the N-terminal domain of EngB . By probing the Avicel-purified cellulase complex (F8) with anti-EngB and anti-EngD antibodies, both EngB and EngD were shown to be present on the cellulase complex of C . cellulovorans . Many proteins homologous to EngB and EngD were also present on the complex. Mol Gen Genet, 1992 Feb, 231(3), 472 - 9 Analysis of functional domains of endoglucanases from Clostridium cellulovorans by gene cloning, nucleotide sequencing and chimeric protein construction; Hamamoto T et al.; The nucleotide sequence of engD, an endo-beta-1,4-glucanase gene from Clostridium cellulovorans was determined (Genbank Accession No . M37434) . The COOH-terminal part of the gene product, EngD, contained a Thr-Thr-Pro repeated sequence followed by a region that has homology to the exoglucanase of Cellulomonas fimi . EngD and EngB, another C . cellulovorans endoglucanase, show 75% amino acid sequence homology at their NH2-termini, in contrast to their carboxyterminal domains which show no homology . EngD had endoglucanase activity on carboxymethylcellulose (CMC), cellobiosidase activity on p-nitrophenyl-cellobioside (p-NPC), and partial hydrolytic activity on crystalline cellulose (Avicel), while EngB showed hydrolytic activity against only CMC . Chimeric proteins between EngB and EngD were constructed by exchanging the non-homologous COOH-terminal regions . Chimeric proteins that contained the NH2-terminus of EngD retained cellobiosidase activity but chimeras with the EngB NH2-terminus showed no cellobiosidase activity . Hydrolysis of crystalline cellulose (Avicelase activity) was observed only with the enzyme containing the EngD NH2-terminus and EngD COOH-terminus. Eur J Biochem, 1991 Dec 5, 202(2), 367 - 77 Structural and functional relationships in two families of beta-1,4-glycanases; Gilkes NR et al.; CenA and Cex are beta-1,4-glycanases produced by the cellulolytic bacterium Cellulomonas fimi . Both enzymes are composed of two domains and contain six Cys residues . Two disulfide bonds were assigned in both enzymes by peptide analysis of the isolated catalytic domains . A further disulfide bond was deduced in both cellulose-binding domains from the absence of free thiols under denaturing conditions . Corresponding Cys residues are conserved in eight of nine other known C . fimi-type cellulose-binding domains . CenA and Cex belong to families B and F, respectively, in the classification of beta-1,4-glucanases and beta-1,4-xylanases based on similarities in catalytic domain primary structure . Disulfide bonds in the CenA catalytic domain correspond to the two disulfide bonds in the catalytic domain of Trichoderma reesei cellobiohydrolase II (family B) which stabilize loops forming the active-site tunnel . Sequence alignment indicates the probable occurrence of disulfides at equivalent positions in the two other family B enzymes . Partial resequencing of the gene encoding Streptomyces KSM-9 beta-1,4-glucanase CasA (family B) revealed five errors in the original nucleotide sequence analysis . The corrected amino acid sequence contains an Asp residue corresponding to the proposed proton donor in hydrolysis catalysed by cellobiohydrolase II . Cys residues which form disulfide bonds in the Cex catalytic domain are conserved in XynZ of Clostridium thermocellum and Xyn of Cryptococcus albidus but not in the other eight known family F enzymes . Like other members of its family, Cex catalyses xylan hydrolysis . The catalytic efficiency (kcat/Km) for hydrolysis of the heterosidic bond of p-nitrophenyl-beta-D-xylobioside is 14,385 min-1.mM-1 at 25 degrees C; the corresponding kcat/Km for p-nitrophenyl-beta-D-cellobioside hydrolysis is 296 min-1.mM-1. Biotechnol Appl Biochem, 1991 Dec, 14(3), 365 - 77 Immunological versatility and carbon regulation of Cellulomonas fimi endo-1,4-beta-glucanases; Kolios G et al.; More than 10 protein molecules with endo-1,4-beta-glucanase activity were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram in Cellulomonas fimi culture supernatants, grown in CMC as carbon source . These molecules are shown to belong to at least four immunologically different groups, against three of which polyclonal antibodies were raised . The protein species used as antigens showed significant differences in cross reactivity, carbon regulation, and affinity to crystalline cellulose . Three intracellular precursors of the first group were detected, two of which were under carbon catabolite control with the third apparently being synthesized constitutively . In the extracellular environment this group showed the largest versatility in protein molecules . The second group appeared to originate from two intracellular precursors both synthesized constitutively and subject to minor extracellular modifications as compared to the first group . The main extracellular protein of this group showed high affinity toward crystalline cellulose . One intracellular precursor was identified for the third group, which was subject to carbon catabolite control . Only one extracellular molecule without binding ability to crystalline cellulose corresponded to this precursor, indicating that the latter was resistant to proteolytic modifications after excretion . It appears that the C . fimi cellulases are more complex than expected and reconstitution of the whole system will be difficult. J Bacteriol, 1991 Nov, 173(22), 7126 - 35 Multiple domains in endoglucanase B (CenB) from Cellulomonas fimi: functions and relatedness to domains in other polypeptides; Meinke A et al.; Endoglucanase B (CenB) from the bacterium Cellulomonas fimi is divided into five discrete domains by linker sequences rich in proline and hydroxyamino acids (A . Meinke, C . Braun, N . R . Gilkes, D . G . Kilburn, R . C . Miller, Jr., and R . A . J . Warren, J . Bacteriol . 173:308-314, 1991) . The catalytic domain of 608 amino acids is at the N terminus . The sequence of the first 477 amino acids in the catalytic domain is related to the sequences of cellulases in family E, which includes procaryotic and eucaryotic enzymes . The sequence of the last 131 amino acids of the catalytic domain is related to sequences present in a number of cellulases from different families . The catalytic domain alone can bind to cellulose, and this binding is mediated at least in part by the C-terminal 131 amino acids . Deletion of these 131 amino acids reduces but does not eliminate activity . The catalytic domain is followed by three domains which are repeats of a 98-amino-acid sequence . The repeats are approximately 50% identical to two repeats of 95 amino acids in a chitinase from Bacillus circulans which are related to fibronectin type III repeats (T . Watanabe, K . Suzuki, K . Oyanagi, K . Ohnishi, and H . Tanaka, J . Biol . Chem . 265:15659-15665, 1990) . The C-terminal domain of 101 amino acids is related to sequences, present in a number of bacterial cellulases and xylanases from different families, which form cellulose-binding domains (CBDs) . It functions as a CBD when fused to a heterologous polypeptide . Cells of Escherichia coli expressing the wild-type cenB gene accumulate both native CenB and a stable proteolytic fragment of 41 kDa comprising the three repeats and the C-terminal CBD . The 41-kDa polypeptide binds to cellulose but lacks enzymatic activity. Res Microbiol, 1991 Nov-Dec, 142(9), 927 - 36 Nucleotide sequence of the cellulase gene celF of Clostridium thermocellum; Navarro A et al.; The nucleotide sequence of the celF gene of Clostridium thermocellum was determined . The open reading frame extended over 2217 bp . The encoded 739-aa polypeptide, CelF, with a Mw = 82,015, was an endoglucanase with activity against carboxymethylcellulose . The N terminus showed a typical signal peptide, and a cleavage site after Ala-27 was predicted . From residues 28 to 470, the sequence of CelF was related to the catalytic domains of type E2 endoglucanases, with a strong homology to the endoglucanases CelZ of Clostridium stercorarium and CenB of Cellulomonas fimi . The catalytic region was followed by a 134-aa segment also present in C . stercorarium CelZ and in C . fimi CenB, and belonging to the family of non-catalytic, presumably cellulose-binding domains first identified in Bacillus subtilis endoglucanase . A 21-aa segment rich in Pro/Thr/Ser residues separated the putative cellulose-binding region from the COOH-terminal region, which contained two conserved stretches of 24 amino acids closely similar to those previously described in endoglucanases CelA, CelB, CelD, CelE, CelH and CelX, and xylanase XynZ of C . thermocellum. FEMS Microbiol Lett, 1991 Oct 15, 67(3), 305 - 9 Multiple xylanases of Cellulomonas fimi are encoded by distinct genes; Clarke JH et al.; Cellulomonas fimi genomic DNA encoding xylanase activity has been cloned and expressed in Escherichia coli . As judged by DNA hybridization and restriction analysis, twelve xylanase-positive clones carried a minimum of four different xylanase (xyn) genes . The encoded enzymes were devoid of cellulase activity but three of the four bound to Avicel. J Gen Microbiol, 1991 Oct, 137 ( Pt 10), 2299 - 305 Molecular cloning and nucleotide sequence of the gene encoding an endo-1,4-beta-glucanase from Bacillus sp . KSM-330; Ozaki K et al.; The gene encoding an acid endo-1,4-beta-glucanase from Bacillus sp . KSM-330 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101 . The recombinant plasmid contained a 3.1 kb HindIII insert, 1.8 kb of which was sufficient for the expression of endoglucanase activity in E . coli HB101 . Nucleotide sequencing of this region (1816 bp) revealed an open reading frame of 1389 bp . The protein deduced from this sequence was composed of 463 amino acids with an Mr of 51882 . The deduced amino acid sequence from amino acids 56 through 75 coincided with the amino-terminal sequence of the endoglucanase, Endo-K, purified from culture of Bacillus sp . KSM-330 . The deduced amino acid sequence of Endo-K had 30% homology with that of the celA enzyme from Clostridium thermocellum NCIB 10682 and 25% homology with that of the enzyme from Cellulomonas uda CB4 . However, the Endo-K protein exhibited no homology with respect to either the nucleotide or the amino acid sequences of other endoglucanases from Bacillus that had been previously characterized . These results indicate that the gene for Endo-K in Bacillus sp . KSM-330 has evolved from an ancestral gene distinct from that of other Bacillus endoglucanases. Gene, 1991 Sep 30, 106(1), 109 - 14 Sequence analysis of the cellulase-encoding celY gene of Erwinia chrysanthemi: a possible case of interspecies gene transfer; Guiseppi A et al.; The Erwinia chrysanthemi (strain 3937) celY gene encoding the minor endoglucanase (EGY) was sequenced . The analysis of the upstream region allowed us to identify an in vivo active promoter recognized by the NtrA (sigma 54) holoenzyme . No similarity was found between the predicted amino acid (aa) sequences of EGY and either the Er . chrysanthemi major endoglucanase, EGZ, or the Er . carotovora CelS endoglucanase . In contrast, a very high level of identity, both at the nucleotide and the predicted aa levels, was found between celY and an EG-encoding gene from Cellulomonas uda, a Gram + bacterium taxonomically distant from Er . chrysanthemi . By comparing the molar G + C% of the cellulase-encoding genes and that of Er . chrysanthemi and C . uda chromosomal DNAs, we speculate that celY was transferred from Er . chrysanthemi to C . uda. J Biol Chem, 1991 Sep 5, 266(25), 16886 - 91 Biosynthetic regulation of monobutyrin, an adipocyte-secreted lipid with angiogenic activity; Wilkison WO et al.; 1-Butyrylglycerol (monobutyrin) is a novel angiogenic compound that is synthesized and secreted during the differentiation of 3T3-F442A preadipocytes into adipocytes . To study the regulation of monobutyrin biosynthesis we have developed an assay utilizing the lgycerol kinase enzyme from Cellulomonas to quantitate the levels of this compound in cell-conditioned medium . Analysis of several cultured cell types, including tumor cell lines, indicated that monobutyrin production is detectable only from adipocytes, reaching a steady-state concentration of approximately 1.0 microM in conditioned medium . Monobutyrin synthesis was demonstrated in vitro using {14C}butyryl-CoA with total homogenate or particulate fractions from adipocytes . Similar fractions from non-adipocyte cell lines failed to synthesize monobutyrin . This biosynthetic activity was shown to be distinct by substrate competition studies from the microsomal sn-glycerol-3-phosphate acyltransferase, whose activity is known to increase during adipocyte differentiation . The production of monobutyrin was hormonally regulated, as the addition of epinephrine to adipocytes caused a 10-fold increase in the amount of monobutyrin secreted . These results indicate that monobutyrin synthesis is adipocyte specific, occurs through an apparently novel particulate enzyme system, and is regulated in a hormone-dependent manner . The implications of these results for adipose physiology and angiogenesis are discussed. J Biol Chem, 1991 Aug 25, 266(24), 15621 - 5 Glutamic acid 274 is the nucleophile in the active site of a "retaining" exoglucanase from Cellulomonas fimi; Tull D et al.; In addition to its known substrate activity with p-nitrophenyl beta-cellobioside, the exoglucanase from Cellulomonas fimi has been shown to utilize substituted phenyl beta-glucosides as substrates, of which the best is 2',4'-dinitrophenyl beta-D-glucopyranoside . The enzyme can be inactivated by treatment with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside, by trapping of the covalent intermediate in catalysis, as has been shown for a beta-glucosidase (Withers, S.G., and Street, I.P . (1988) J . Am . Chem . Soc . 110, 8551-8553) . The intermediate formed is stable but can undergo turnover in the presence of cellobiose, reactivating the enzyme by transglycosylation . Using a tritium-labeled inactivator it has been possible to isolate and sequence a radiolabeled peptide from this enzyme, and the active site nucleophile has been identified as glutamic acid residue 274 . This glutamic acid residue and its sequentially proximal amino acids are absolutely conserved in the homologous family F of cellulases. Agric Biol Chem, 1991 Aug, 55(8), 1969 - 75 Purification and characterization of a novel lyase from Cellulomonas sp . that degrades Fusarium and Gibberella acidic polysaccharides; Takegawa K et al.; A Cellulomonas sp . isolated from soil produced a novel lyase that degraded the acidic polysaccharide of Fusarium sp . M7-1 with the formation of mannose and O-beta-D-mannopyranosyl-(1----2)-D-mannose . DEAE-Toyopearl 650M column chromatography showed three lyase activity peaks (fractions I, II, and III) . The major fraction was purified to homogeneity by polyacrylamide gel electrophoresis analysis, and its molecular weight was 74,000 . The optimum pH was 6.5 to 8.0 and the stable pH range was 6.0 to 8.0 . The purified enzyme did not degrade glucuronic or galacturonic acid-containing polysaccharides such as chondroitin, hyaluronic acid, pectin, or pectic acid . However, the purified enzyme specifically degraded various Fusarium and Gibberella acidic polysaccharides, and unsaturated sugars were produced with the release of mannose and O-beta-D-mannopyranosyl-(1----2)-D-mannose . These results suggest that the acidic polysaccharides derived from Fusarium and Gibberella have similar structures. J Biol Chem, 1991 Jun 15, 266(17), 11335 - 40 Deletion of the linker connecting the catalytic and cellulose-binding domains of endoglucanase A (CenA) of Cellulomonas fimi alters its conformation and catalytic activity; Shen H et al.; The Pro-Thr box is a linker of 23 amino acids ((PT)4T(PT)7) connecting the catalytic domain and the cellulose-binding domain (CBD) of endoglucanase A (CenA) from the bacterium Cellulomonas fimi . Deletion of the Pro-Thr box alters the conformation of CenA by changing the relative orientation of the catalytic domain and the CBD . The tertiary structures of the catalytic domain and the CBD appear to be unchanged . The change in conformation reduces the catalytic efficiency of the enzyme and masks one of two protease-sensitive sites between the domains . The deletion does not affect the adsorption of the enzyme to microcrystalline cellulose, but it does affect its desorption from cellulose . The results suggest that the Pro-Thr box in CenA has an extended, kinked, and rigid conformation. J Bacteriol, 1991 Jun, 173(11), 3397 - 407 DNA sequences of three beta-1,4-endoglucanase genes from Thermomonospora fusca; Lao G et al.; The DNA sequences of the Thermomonospora fusca genes encoding cellulases E2 and E5 and the N-terminal end of E4 were determined . Each sequence contains an identical 14-bp inverted repeat upstream of the initiation codon . There were no significant homologies between the coding regions of the three genes . The E2 gene is 73% identical to the celA gene from Microbispora bispora, but this was the only homology found with other cellulase genes . E2 belongs to a family of cellulases that includes celA from M . bispora, cenA from Cellulomonas fimi, casA from an alkalophilic Streptomyces strain, and cellobiohydrolase II from Trichoderma reesei . E4 shows 44% identity to an avocado cellulase, while E5 belongs to the Bacillus cellulase family . There were strong similarities between the amino acid sequences of the E2 and E5 cellulose binding domains, and these regions also showed homology with C . fimi and Pseudomonas fluorescens cellulose binding domains. Mol Microbiol, 1991 May, 5(5), 1221 - 33 Nucleotide sequence of the endoglucanase C gene (cenC) of Cellulomonas fimi, its high-level expression in Escherichia coli, and characterization of its products; Coutinho JB et al.; The cenC gene of Cellulomonas fimi, encoding endoglucanase CenC, has an open reading frame of 1101 codons closely followed by a 9 bp inverted repeat . The predicted amino acid sequence of mature CenC, which is 1069 amino acids long, is very unusual in that it has a 150-amino-acid tandem repeat at the N-terminus and an unrelated 100-amino-acid tandem repeat at the C-terminus . CenC belongs to subfamily E1 of the beta-1,4-glycanases . High-level expression in Escherichia coli of cenC from a 3.6 kbp fragment of C . fimi DNA leads to levels of CenC which exceed 10% of total cell protein . Most of the CenC is in the cytoplasm in an inactive form . About 60% of the active fraction of CenC is in the periplasm . The catalytic properties of the active CenC are indistinguishable from those of native CenC from C . fimi . The Mr of CenC from E . coli and C . fimi is approximately 130 kDa . E . coli and C . fimi also produce an endoglucanase, CenC', of approximate Mr 120kDa and with the same N-terminal amino acid sequence and catalytic properties as CenC . CenC' appears to be a proteolytic product of CenC . CenC and CenC' can bind to cellulose and to Sephadex . CenC is the most active component of the C . fimi cellulase system isolated to date. J Bacteriol, 1991 Jan, 173(1), 308 - 14 Unusual sequence organization in CenB, an inverting endoglucanase from Cellulomonas fimi; Meinke A et al.; The nucleotide sequence of the cenB gene was determined and used to deduce the amino acid sequence of endoglucanase B (CenB) of Cellulomonas fimi . CenB comprises 1,012 amino acids and has a molecular weight of 105,905 . The polypeptide is divided by so-called linker sequences rich in proline and hydroxyamino acids into five domains: a catalytic domain of 607 amino acids at the N terminus, followed by three repeats of 98 amino acids each which are greater than 60% identical, and a C-terminal domain of 101 amino acids which is 50% identical to the cellulose-binding domains of C . fimi cellulases Cex and CenA . A deletion mutant of the cenB gene encodes a polypeptide lacking the C-terminal 333 amino acids of CenB . The truncated polypeptide is catalytically active and, like intact CenB, binds to cellulose, suggesting that CenB has a second cellulose-binding site . The sequence of amino acids 1 to 461 of CenB is 35% identical, with a further 15% similarity, to that of a cellulase from avocado, which places CenB in cellulase family E . CenB releases mostly cellobiose and cellotetraose from cellohexaose . Like CenA, CenB hydrolyzes the beta-1,4-glucosidic bond with inversion of the anomeric configuration . The pH optimum for CenB is 8.5, and that for CenA is 7.5. J Gen Microbiol, 1990 Oct, 136 ( Pt 10), 2089 - 97 Cloning and sequencing of the celA gene encoding endoglucanase A of Butyrivibrio fibrisolvens strain A46; Hazlewood GP et al.; Genomic DNA from Butyrivibrio fibrisolvens strain A46 was digested with EcoRI and ligated into lambda gt11 . Two recombinant phages isolated from the gene bank hydrolysed carboxymethylcellulose and were shown to contain the same 2.3 kb EcoRI restriction fragment, which was cloned into pUC12 to generate pBA46 . Escherichia coli JM83 harbouring pBA46 expressed an endoglucanase (EGA) which hydrolysed a range of other substrates including barley beta-glucan, Avicel, filter paper and p-nitrophenyl beta-D-cellobioside . Nucleotide sequencing of the B . fibrisolvens strain A46 DNA cloned in pBA46 revealed a single open reading frame (ORF) of 1296 bp, encoding a protein of 48,863 Da . Confirmation that the ORF coded for EGA was obtained by comparing the N-terminal sequence of the purified endoglucanase with that deduced from the nucleotide sequence . EGA contains a typical prokaryotic signal peptide at its N-terminus and shows some homology with the Bacillus family of cellulases . The enzyme does not contain distinct functional domains, which are prevalent in cellulases from Pseudomonas fluorescens subsp . cellulosa and Cellulomonas fimi. Biochem J, 1990 Oct 1, 271(1), 277 - 80 The tertiary structure of a bacterial cellulase determined by small-angle X-ray-scattering analysis; Pilz I et al.; CenA from Cellulomonas fimi is a beta-1,4-endoglucanase that binds tightly to cellulose . X-ray-scattering analyses show that the enzyme is tadpole-shaped: the previously identified catalytic and cellulose-binding domains comprise the head and tail respectively . It appears that this structural and functional organization is common to several cellulases from bacteria and fungi. J Bacteriol, 1990 Jul, 172(7), 3837 - 41 DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene; Teather RM et al.; The DNA sequence of a mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase {EC 3.2.1.73}) gene from Fibrobacter succinogenes cloned in Escherichia coli was determined . The general features of this gene are very similar to the consensus features for other gram-negative bacterial genes . The gene product was processed for export in E . coli . There is a high level of sequence homology between the structure of this glucanase and the structure of a mixed-linkage beta-glucanase from Bacillus subtilis . The nonhomologous region of the amino acid sequence includes a serine-rich region containing five repeats of the sequence Pro-Xxx-Ser-Ser-Ser-Ser-(Ala or Val) which may be functionally related to the serine-rich region observed in Pseudomonas fluorescens cellulase and the serine- and/or threonine-rich regions observed in Cellulomonas fimi endoglucanase and exoglucanase, in Clostridium thermocellum endoglucanases A and B, and in Trichoderma reesei cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I. Agric Biol Chem, 1990 Feb, 54(2), 419 - 25 Beta-eliminative cleavage of the acidic polysaccharide of Fusarium sp . M7-1 by an enzyme preparation of Cellulomonas sp; Iwahara S et al.; By digestion with an enzyme preparation derived from a culture filtrate of Cellulomonas sp., an unsaturated disaccharide was produced accompanied by the release of mannose and beta 1----2 mannobiose from the acidic oligomer mixture that was obtained from the acidic polysaccharide of Fusarium sp . M7-1 by acetolysis . The unsaturated disaccharide produced was isolated and identified as 4-deoxy-L-threo-hex-4-enopyranouronosyl alpha(1----2)-D-galactose . The same unsaturated sugar linked to the polysaccharide was also produced accompanied by the release of mannose and beta 1----2 mannobiose from the acidic polysaccharide by the enzyme digestion. Arch Microbiol, 1990, 153(2), 129 - 33 Expression of the Cellulomonas fimi cellulase genes cex and cenA from the divergent tet promoters of transposon Tn10; Din N et al.; A cartridge was constructed which contained the divergent tet promoters of transposon Tn10 between an exoglucanase gene (cex) and an endoglucanase gene (cenA) of Cellulomonas fimi . When carried in a broad-host-range vector, the cartridge gave expression of cex and cenA in Escherichia coli, Rhodobacter capsulatus and Klebsiella pneumoniae. J Biol Chem, 1989 Oct 25, 264(30), 17802 - 8 Structural and functional analysis of a bacterial cellulase by proteolysis; Gilkes NR et al.; CenA is an endo-beta 1,4-glucanase from the cellulolytic bacterium Cellulomonas fimi . It is a bifunctional enzyme comprising an amino-terminal cellulose-binding domain and a carboxyl-terminal catalytic domain joined by a short sequence of prolyl and threonyl residues (the Pro-Thr box) . Additional structural and functional information was revealed by a detailed analysis of the products generated by proteolytic cleavage of a nonglycosylated form of CenA . An extracellular C . fimi protease attacked nonglycosylated CenA at the junctions between the Pro-Thr box and the two functional domains . A stable "core" peptide (p30), corresponding to the catalytic domain, remained after extensive proteolysis . p30 was resistant to further attack even in the presence of 2-mercaptoethanol plus urea or dithiothreitol, but treatment in the presence of sodium dodecyl sulfate allowed complete fragmentation to small peptides . Stable peptides, identical, or closely related to p30, were generated by alpha-chymotrypsin or papain . These results indicated that the catalytic domain adopts a tightly folded conformation affording protection from proteolytic attack . In contrast, the cellulose-binding domain showed a relatively loose conformation . Progressive proteolytic truncation from the amino terminus was apparent during incubation with alpha-chymotrypsin or papain, or with C . fimi protease under reducing conditions . Affinity for cellulose was retained by products missing up to 64 amino-terminal amino acids . The remaining carboxyl-proximal region of the cellulose-binding domain with affinity (47 amino acids) contained sequences highly conserved in analogous domains from other bacterial endo-beta 1,4-glucanases . By analogy with other systems, the properties of the Pro-Thr box are consistent with an elongated conformation . The results of this investigation suggest that CenA has a tertiary structure which resembles that of certain fungal cellulases. Appl Environ Microbiol, 1989 Oct, 55(10), 2480 - 7 Purification and characterization of endoglucanase C of Cellulomonas fimi, cloning of the gene, and analysis of in vivo transcripts of the gene; Moser B et al.; Two nonglycosylated endoglucanases which bind to Sephadex were purified from culture supernatants of Cellulomonas fimi grown on microcrystalline cellulose . Their Mrs were 120,000 and 130,000 . The N-terminal amino acid sequences of the enzymes were identical, suggesting that the enzymes were related . A DNA fragment encoding this N-terminal sequence was cloned in Escherichia coli . The nucleotide sequence corresponding to the N-terminal amino acid sequence was preceded by a sequence encoding a typical leader peptide . Transcripts hybridizing to the cloned fragment were detected in total RNA isolated from C . fimi cells grown on carboxymethyl cellulose but not from cells grown on glycerol or glucose . Transcription started at a cluster of sites 53 to 59 nucleotides upstream of a GUG translation initiation codon and terminated at either of two closely spaced C residues immediately downstream of a region of potential secondary structure . The size of the transcript was approximately 3.5 kilobases, sufficient to encode a polypeptide of 130 kilodaltons . The 130-kilodalton polypeptide is designated endoglucanase C (CenC), and the gene encoding it is designated cenC. Gene, 1989 Sep 1, 81(1), 83 - 95 Cellulase families revealed by hydrophobic cluster analysis; Henrissat B et al.; The amino acid sequences of 21 beta-glycanases have been compared by hydrophobic cluster analysis . Six families of cellulases have been identified on the basis of primary structure homology: (A) endoglucanases B, C and E of Clostridium thermocellum; endoglucanases of Erwinia chrysanthemi and Bacillus sp.; endoglucanase III of Trichoderma reesei; endoglucanase I of Schizophyllum commune; (B) cellobiohydrolase II of T . reesei; endoglucanases of Cellulomonas fimi and Streptomyces sp; (C) cellobiohydrolases I of T . reesei and of Phanerochaete chrysosporium; endoglucanase I of T . reesei; (D) endoglucanase A of C . thermocellum and an endoglucanase from Ce . uda; (E) endoglucanase D of C . thermocellum and an endoglucanase from Pseudomonas fluorescens; (F) xylanases of C . thermocellum and of Cryptococcus albidus and the cellobio-hydrolase of Ce . fimi . For each family, conserved potentially catalytic residues have have been listed and previous allocations of the active-site residues are evaluated in the light of the alignment of the amino acid sequences . A strong homology is also reported for the putative cellulose-binding domains of cellulases of Ce . fimi and of P . fluorescens. FEMS Microbiol Lett, 1989 May, 50(1-2), 167 - 72 Homologues of catalytic domains of Cellulomonas glucanases found in fungal and Bacillus glycosidases; West CA et al.; We demonstrate homology between the catalytic domains of exoglucanase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) from Cellulomonas fimi and those of endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8) from Bacillus sp . strain C-125 and the fungus Cryptococcus albidus; and between the catalytic domains of endoglucanase (1,4-(1,3:1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) from Cellulomonas fimi and exoglucanase II from Trichoderma reesei . These five enzymes apparently evolved by reshuffling of two catalytic domains and several substrate-binding domains. FEBS Lett, 1989 Feb 13, 244(1), 127 - 31 Fusion to an endoglucanase allows alkaline phosphatase to bind to cellulose; Greenwood JM et al.; Endoglucanase CenA of Cellulomonas fimi comprises an N-terminal cellulose-binding domain and a C-terminal catalytic domain joined together by a sequence of 23 proline and threonine residues (the Pro-Thr box) . The domains function independently when separated by proteolysis . TnphoA has been used to generate cenA'-'phoA fusions . CenA'-'PhoA fusion polypeptides which contain the entire cellulose-binding domain of CenA bind to cellulose, allowing their purification from periplasmic extracts in a single, facile step . This result has implications for purification or immobilisation of chimeric proteins on a cheap cellulose matrix. Gene, 1988 Dec 30, 74(2), 549 - 53 Multigene families of Cellulomonas flavigena encoding endo-beta-1,4-glucanases (CM-cellulases); Akhtar MW et al.; Multiple genes coding for endo-beta-1,4-glucanases (CM-cellulases) have been isolated from a newly discovered highly cellulolytic strain of Cellulomonas flavigena . Clones of C . flavigena DNA were isolated in Escherichia coli and screened for gene expression on CM-cellulose plates staining with congo red . Six clones produced CM-cellulase activity as detected in liquid assays, and on activity gels . They fell into three groups within which the sequences cross-hybridised . There were small differences in the pH and temperature optima of the enzymes encoded by representatives of the three groups of clones. J Biol Chem, 1988 Jul 25, 263(21), 10401 - 7 Precise excision of the cellulose binding domains from two Cellulomonas fimi cellulases by a homologous protease and the effect on catalysis; Gilkes NR et al.; An endo-beta-1,4-glucanase (CenA) and an exo-beta-1,4-glucanase (Cex) were prepared from Escherichia coli expressing recombinant DNA of the cellulolytic bacterium Cellulomonas fimi . Purification was facilitated by the high affinities of these enzymes for cellulose . An extracellular C . fimi protease cleaved both enzymes in vivo in a highly specific manner . The affinity of the parent enzyme for cellulose was contained independently in an amino-terminal fragment (p20) of CenA and a carboxyl-terminal fragment (p8) of Cex . These fragments contained homologous amino acid sequences which were proposed to comprise cellulose binding domains . Corresponding fragments, p30 from CenA and p35 from Cex, which were unable to bind to cellulose, contained catalytic domains . In both enzymes, the two functional domains were joined by a hinge region consisting solely of prolyl and threonyl residues . The binding domain was excised from CenA by proteolytic cleavage immediately adjacent to the carboxyl terminus of this hinge . Cex was cleaved at an exactly analogous site . p30 and p35 retained several of the catalytic functions of their parent enzymes . However, p30 was less active than intact CenA against microcrystalline cellulose implying a critical role for the binding domain of CenA in the hydrolysis of crystalline substrate. J Bacteriol, 1988 Jun, 170(6), 2827 - 31 Effects of end-product inhibition of Cellulomonas uda anaerobic growth on cellobiose chemostat culture; Dermoun Z et al.; Cellulomonas uda was grown anaerobically in a chemostat with 3.33 and 11.41 mM cellobiose in the feed medium at dilution rates varying from 0.017 to 0.29/h . Unusual results obtained were analyzed by using curves simulating the steady-state biomass . This unusual behavior could be accounted for by a classical growth model taking end-product inhibition into account . Acetate has been identified to be the major inhibitor in the experimental conditions used . Parameters calculated from experimental observations gave theoretical curves of biomass production versus dilution rate which fitted the experimental points very well. Gene, 1988 May 30, 65(2), 229 - 38 Cloning and nucleotide sequence of a cellulase gene, casA, from an alkalophilic Streptomyces strain; Nakai R et al.; A gene encoding an endo-type semi-alkaline cellulase was cloned from an alkalophilic Streptomyces strain in Streptomyces lividans, and its nucleotide sequence was determined . Downstream from the transcriptional start point, which was determined by high-resolution S1 mapping, an open reading frame of 388 amino acids (aa) was present . The N-terminal amino acid sequence of the mature enzyme determined by an Edman degradation procedure suggested that the cellulase had an extraordinarily long leader sequence of about 70 aa . Comparison with the leader sequences of endoglucosidase H from Streptomyces plicatus and the cellulase from Cellulomonas fimi suggested that the semi-alkaline cellulase was processed in two steps during maturation. FEBS Lett, 1987 Dec 10, 225(1-2), 163 - 7 Glycosylation of bacterial cellulases prevents proteolytic cleavage between functional domains; Langsford ML et al.; Glycosylated cellulases from Cellulomonas fimi were compared with their non-glycosylated counterparts synthesized in Escherichia coli from recombinant DNA . Glycosylation of the enzymes does not significantly affect their kinetic properties, or their stabilities towards heat and pH . However, the glycosylated enzymes are protected from attack by a C . fimi protease when bound to cellulose, while the non-glycosylated enzymes yield active, truncated products with greatly reduced affinity for cellulose. J Bacteriol, 1987 Oct, 169(10), 4674 - 7 Regulation and initiation of cenB transcripts of Cellulomonas fimi; Greenberg NM et al.; We characterized the in vivo transcription of the Cellulomonas fimi cenB gene, which encodes an extracellular endo-beta-1,4-glucanase (EC 3.2.1.4) . By Northern blot (RNA blot) analysis, cenB mRNA was detected in C . fimi RNA preparations from glycerol-, glucose-, and carboxymethyl cellulose (CMC)-grown cells . The relative abundance of the specific mRNAs in these preparations appeared to depend on the carbon source provided, with the preparations from CMC-grown cells having the greatest amount of cenB transcripts, followed by glycerol- and glucose-grown cells . Therefore, the transcription of this gene could be regulated by the carbon source provided to C . fimi . High-resolution nuclease S1 protection studies were used to map cenB mRNA 5' termini with a unique 5'-labeled DNA probe and C . fimi RNA isolated in vivo . With this procedure, three 5' termini were found in abundance upstream of the translational initiation ATG codon in RNA preparations from C . fimi grown on CMC, while less-abundant 5' termini were found 52 bases closer to the ATG codon in RNA prepared from C . fimi grown on any one of the three substrates . These results are indicative of a tandem promoter arrangement, with the ATG-proximal promoter directing constitutive low-level cenB transcription and the more distal promoter directing higher levels of transcription under the inducing effects of the cellulosic substrate . The corresponding transcripts were not detected in S1 mapping experiments with RNA isolated in vivo from Escherichia coli clones harboring recombinant plasmids carrying C . fimi genomic inserts . Comparative analysis of the 5' -flanking DNA sequences of the cenB gene and the cenA and cex genes