Modeling Tetrahymena thermophila growth and protease production

Oxygen concentrations stimulated growth (maximum number of cells) and protease secretion by Tetrahymena thermophila. Agitation and aeration conditions for growth and protease secretion were optimised by a central composite design. The best optimised combination was a stirrer speed of 338 rpm and an aeration of 1 vvm. Journal of Industrial Microbiology & Biotechnology (2000) 25, 58–61. Received 24 September 1999/ Accepted in revised form 06 March 2000     

In vitro binding ofBifidobacterium bifidum DSM 20082 to mucosal glycoproteins and hemagglutinating activity

Adhesive properties ofBifidobacterium bifidum strain DSM 20082 were studied by the hemagglutination test and an enzyme-linked immunosorbent assay (ELISA).B. bifidum caused agglutination of human A, B, and O erythrocytes and rabbit erythrocytes, but the interactions were not specific of blood group antigens. The hemagglutination was inhibited by porcine gastric mucin and rat intestinal and colonic mucin.B. bidifum was shown to adhere to different immobilized mucosal glycoproteins and to glycophorin A, a specific erythrocyte membrane glycoprotein. The data obtained with many glycosylated components indicated thatB. bifidum receptors involved in the hemagglutination test were not the same as those that adhere to mucus glycoproteins. The results suggest that the mucosal preparations contain receptors for specific bacterial adhesins, but their structures remain to be determined.     

First evidence of human meconium glycoasparagines

During a systematic study of carbohydrate material present inhuman meconium, in addition to the previously described mucins,glycolipids and free oligosaccharides, we have now characterizeda significant quantity of free glycoasparagines. These glycoasparagineshave been isolated from human meconium by a combination of ion-exchange,concanavalin A (ConA)-affinity and high-performance liquid (HPLC)chromatographies. Their structures have been established by400 MHz ¹H-NMR spectroscopy. These compounds are related toN-acetyllactosaminic type structures and are based on the commoncore These glycoasparagines are probably derived from both proteaseand partial exoglycosidase hydrolysis of fetal gastrointestinalN-glycosyl proteins. Their structures are discussed in the contextof the known catabolic pathways of N-glycans glycoasparagine N-glycosyl protein catabolism meconium NMR     

Primary structure of ten galactosides formed by transglycosylation during lactose hydrolysis by Bifidobacterium bifidum

Oligosaccharides formed by a transgalactosylation reaction during lactose hydrolysis with Bifidobacterium bifidum were separated into eight fractions by gel-permeation chromatography and their structures studies determined by trimethylsilylation analysis, methylation analysis, f.a.b.-m.s., g.l.c.-m.s. and enzymic hydrolysis as beta-D-Galp-(1----3)-D-Glc, beta-D-Galp-(1----6)-D-Glc, beta-D-Galp-(1----6)-D-Gal, beta-D-Galp-(1----3)-beta-D-Galp-(1----4)-D-Glc, beta-D-Galp-(1----6)[beta-D-Galp-(1----4)]-D-Glc, beta-D-Galp-(1----2)[beta-D-Galp-(1----6)]-D-Glc, beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Galp-(1----4)-D-Glc, beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Ga lp- (1----4)-D-Glc, beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-DGalp-(1----3)-beta -D-Galp-(1----3)-beta-D-Galp-(1----4)-D-Glc, and beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Galp-(1----3)-beta-D-Ga lp-(1----3)-beta-D-G-alp-(1----3) beta-D-Galp-(1----4)-D-Glc.     

Industrial media and fermentation processes for improved growth and protease production by Tetrahymena thermophila BIII

Tetrahymena thermophila was cultivated on industrial by-product media. The composition of the best medium (with milk proteins) was optimised by a central composite design for growth and protease secretion. The optimal combination [1.07% (w/v) of yeast extract and 0.99% (w/v) of skimmed milk] improved biomass production by 46%. In a fermentation strategy, the pH must be regulated to produce no cell damage, lengthening the stationary phase and resulting in a more abundant protease production. To increase cell concentration and protease secretion, a continuous culture with cell recycling by microfiltration was successfully tested on ciliated protozoa. Journal of Industrial Microbiology & Biotechnology (2000) 24, 285–290. Received 28 July 1999/ Accepted in revised form 20 January 2000     

Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Isolation and characterization of the potential receptor for wheat germ agglutinin from human neutrophils

Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.     

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine metabolism in <Emphasis Type="Italic">Bifidobacterium</Emphasis>

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism. S. Foley and E. Stolarczyk contributed equally to this work     

Glucose and Galactose Transport in Bifidobacterium bifidum DSM 20082

Sugar uptake was measured with ³H-galactose and ¹⁴C-glucose. Galactose transport system was not modified by inhibitors of known translocases and did not present a saturation kinetic with high concentration of galactose. Glucose incorporation was inhibited by lasalocid (cation symport inhibitor) and increased by KCl. The kinetic parameters K _M and V _max were respectively 9.16 mM and 26.56 nmol/min/mg cell protein. On the basis of this study, galactose crossed through the membrane by diffusion, and glucose was incorporated by a cation symport which is regulated by K⁺ ions. Received: 19 February 1997 / Accepted: 20 March 1997