Glucosylation of alpha-butyl- and alpha-octyl-D-glucopyranosides by dextransucrase and alternansucrase from Leuconostoc mesenteroides.

For the first time, glucosylation of alpha-butyl- and alpha-octylglucopyranoside was achieved using dextransucrase (DS) of various specificities, and alternansucrase (AS) from Leuconostoc mesenteroides. All the glucansucrases (GS) tested used alpha-butylglucopyranoside as acceptor; in particular, DS produced alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside and alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside. In contrast, alpha-octylglucopyranoside was glucosylated only by AS which was shown to be the most efficient catalyst.

Effects of temperature and pressure on Rhizomucor miehei lipase stability.

Both high temperature and high hydrostatic pressure induce irreversible deactivation of enzymes. They enable the enzyme's thermodynamic parameters to be determined and are used to study the mechanisms involved in biochemical systems. The effect of these two factors on the stability of Rhizomucor miehei lipase have been investigated.

Genome-wide analysis of the response to cell wall mutations in the yeast Saccharomyces cerevisiae.

Perturbations of the yeast cell wall trigger a repair mechanism that reconfigures its molecular structure to preserve cell integrity. To investigate this mechanism, we compared the global gene expression in five mutant strains, each bearing a mutation (i.e.

Characterization of the CipA scaffolding protein and in vivo production of a minicellulosome in Clostridium acetobutylicum.

The cipA gene encoding the Clostridium acetobutylicum scaffolding protein CipA was cloned and expressed in Escherichia coli. CipA contains an N-terminal signal peptide, a family 3a cellulose-binding domain (CBD), five type I cohesin domains, and six hydrophilic domains. The uniqueness of CipA lies in the enchainment of cohesin domains that are all separated by a hydrophilic domain.

Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum.

A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824. Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp. Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes.

The influence of metabolic network structures and energy requirements on xanthan gum yields.

The metabolic network of Xanthomonas campestris is complex since a number of cyclic pathways are present making simple stoichiometric yield predictions difficult. The influence of certain pathway configurations and the resulting variations in flux have been examined as regards the maximum yield potential of this bacteria for xanthan gum production. These predictions have been compared with experimental results showing that the strain employed is functioning close to its theoretical maximum as regards yield criteria.

Anaerobic sugar catabolism in Lactococcus lactis: genetic regulation and enzyme control over pathway flux.

Lactic acid bacteria and particularly Lactococcus lactis are widely used for the production of lactic acid in fermented foods. Control of the catabolic rate in L. lactis, i.

Enzyme recovery during gas/liquid two-phase flow microfiltration of enzyme/yeast mixtures.

The effect of a gas/liquid two-phase flow on the recovery of an enzyme was evaluated and compared with standard crossflow operation when confronted with the microfiltration of a high-fouling yeast suspension. Ceramic tubular and flat sheet membranes were used. At constant feed concentration (permeate recycling) and transmembrane pressure, the results obtained with the tubular membrane were dependent on the two-phase flow pattern.

Molecular characterization of DSR-E, an alpha-1,2 linkage-synthesizing dextransucrase with two catalytic domains.

A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of alpha-1,6 and alpha-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases.

Carbon flux analysis in a pantothenate overproducing Corynebacterium glutamicum strain.

Carbon flux analysis during a pseudo-stationary phase of metabolite accumulation in a genetically engineered strain of Corynebacterium glutamicum, containing plasmids leading to over-expression of the ilvBNCD and panBC operons, has identified the basic metabolic constraints governing the potential of this bacterium to produce pantothenate. Carbon flux converging on pyruvate (75% of glucose uptake) is controlled by anabolic precursor requirements and NADPH demand provoking high carbon loss as CO2 via the pentose pathway. Virtually all the flux of pyruvate is directed into the branched pathway leading to both valine and pantothenate production, but flux towards valine is tenfold higher than that transformed to pantothenate, indicating that significant improvements will only be obtained if carbon flux at the ketoisovalerate branchpoint can be modulated.

Maltooligosaccharide disproportionation reaction: an intrinsic property of amylosucrase from Neisseria polysaccharea.

Amylosucrase from Neisseria polysaccharea (AS) is a remarkable transglycosidase of family 13 of the glycoside hydrolases that catalyses the synthesis of an amylose-like polymer from sucrose and is always described as a sucrose-specific enzyme. Here, we demonstrate for the first time the ability of pure AS to catalyse the disproportionation of maltooligosaccharides by cleaving the alpha-1,4 linkage at the non-reducing end of a maltooligosaccharide donor and transferring the glucosyl unit to the non-reducing end of another maltooligosaccharide acceptor. Surprisingly, maltose, maltotriose and maltotetraose are very poor glucosyl donors whereas longer maltooligosaccharides are even more efficient glucosyl donors than sucrose.

KNR4 is a member of the PKC1 signalling pathway and genetically interacts with BCK2, a gene involved in cell cycle progression in Saccharomyces cerevisiae.

In budding yeast, PKC1 plays an essential role in cell wall integrity and cell proliferation through a bifurcated PKC1/mitogen-activated protein (MAP) kinase pathway. The evidence that KNR4 is a member of the PKC1 pathway and genetically interacts with BCK2, a gene involved together with Cln3-Cdc28 in the G1 to S transition phase of the cell cycle, was as follows. Both KNR4 and BCK2 were isolated as a dosage suppressor of a calcofluor white hypersensitive ( cwh43) mutant.

Dynamic response of catabolic pathways to autoacidification in Lactococcus lactis: transcript profiling and stability in relation to metabolic and energetic constraints.

The dynamic response of the central metabolic pathways to autoacidification (accumulation of organic acid fermentation products) in Lactococcus lactis was investigated in a global manner by integrating molecular data (cellular transcript concentrations, mRNA turnover) within physiological investigations of metabolic and energetic parameters. The decrease in pH associated with the accumulation of organic acids modified the physiological state of the cell considerably. Cytoplasmic acidification led to inhibition of enzyme activities and, consequently, to a diminished catabolic flux through glycolysis and a decreased rate of biochemical energy synthesis.

amyP, a reporter gene to study strain degeneration in Clostridium acetobutylicum ATCC 824.

Clostridium acetobutylicum produces an extracellular alpha-amylase when grown on glucose as the sole carbon source. This enzyme was previously characterized from a biochemical point of view but its encoding gene was never identified. The 2283-bp amyP gene encodes a 83013-Da mature protein with an N-terminal domain that exhibits strong identity to the family 13 glycosyl hydrolases such as the Bacillus alpha-amylases.

Involvement of GFA1, which encodes glutamine-fructose-6-phosphate amidotransferase, in the activation of the chitin synthesis pathway in response to cell-wall defects in Saccharomyces cerevisiae.

Cell-wall damage caused by mutations of cell-wall-related genes triggers a compensatory mechanism which eventually results in hyperaccumulation of chitin reaching 20% of the cell-wall dry mass. We show that activation of chitin synthesis is accompanied by a rise, from 1.3-fold to 3.

Molecular characterization and transcriptional analysis of adhE2, the gene encoding the NADH-dependent aldehyde/alcohol dehydrogenase responsible for butanol production in alcohologenic cultures of Clostridium acetobutylicum ATCC 824.

The adhE2 gene of Clostridium acetobutylicum ATCC 824, coding for an aldehyde/alcohol dehydrogenase (AADH), was characterized from molecular and biochemical points of view. The 2,577-bp adhE2 codes for a 94.4-kDa protein.

Feasibility study of a compact process for biological treatment of highly soluble VOCs polluted gaseous effluent.

Volatile organic compounds (VOCs), representing a wide range of products mainly generated by industrial activity, are involved in air pollution. This study deals with a new biological treatment process of gaseous effluent combining a gas/liquid contactor called an "aero-ejector" and a membrane bioreactor. Combining these two innovative technologies enables a high elimination efficiency to be reached.

Transcript quantification based on chemical labeling of RNA associated with fluorescent detection.

A general method for RNA measurement, based on chemical labeling of RNA with digoxigenin (without retrotranscription), has been established. Labeled RNA is hybridized with nylon membranes containing spot blots of PCR-amplified gene fragments and the fluorescence detection is mediated via specific anti-digoxigenin antibody coupled to alkaline phosphatase. The method was optimized in order to be quantitative, and high precision (less than 24% error) was obtained, allowing analysis of relatively small changes in gene expression.

Growth rate influences reductive biodegradation of the organophosphorus pesticide demeton by Corynebacterium glutamicum.

The organophosphorous pesticide, demeton-S-methyl was transformed by Corynebacterium glutamicum in co-metabolism with more readily degradable substrates. Glucose, acetate and fructose were tested as growth substrates, and the highest demeton-S-methyl biotransformation average rate (0.78 mg l(-1) h(-1)) and maximum instantaneous rate (1.

Excess sludge reduction in activated sludge processes by integrating biomass alkaline heat treatment.

With new EC regulations, alternative treatment and disposal techniques of the excess sludge produced by activated sludge wastewater treatment plants have to be developed. To decrease activated sludge production yield, microbial cell lysis can be amplified to enhance cryptic growth (biomass growth on lysates). Cell breakage techniques (thermal, alkaline and a combination) were studied to generate Ralstonia eutropha (strain model) and waste activated sludge lysates and to evaluate their biodegradability.

Influence of a transverse flowrate on the oxygen transfer performance in heterogeneous aeration: case of hydro-ejectors.

This paper deals with the scaling of aeration devices, and more specifically hydro-ejectors, in the case of heterogeneous aeration. Because the transfer of oxygen only occurs in a very small part of the volume of the treatment basin, the transfer performance of the aerator depends on the device itself and on the surrounding flow characteristics. First experiments were achieved with a 10 L mechanically agitated reactor in order to operate at a known kLa and liquid flowrate Q.

Glucose metabolism and regulation of glycolysis in Lactococcus lactis strains with decreased lactate dehydrogenase activity.

The distribution of carbon flux at the pyruvate node was investigated in Lactococcus lactis under anaerobic conditions with mutant strains having decreased lactate dehydrogenase activity. Strains previously selected by random mutagenesis by H. Boumerdassi, C.

Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1-dependent cell wall integrity pathway.

The Saccharomyces cerevisiae cwh43-2 mutant, originally isolated for its Calcofluor white hypersensitivity, displays several cell wall defects similar to mutants in the PKC1-MPK1 pathway, including a growth defect and increased release of beta-1,6-glucan and beta-glucosylated proteins into the growth medium at increased temperatures. The cloning of CWH43 showed that it corresponds to YCR017c and encodes a protein with 14-16 transmembrane segments containing several putative phosphorylation and glycosylation sites. The N-terminal part of the amino acid sequence of Cwh43p shows 40% similarity with the mammalian FRAG1, a membrane protein that activates the fibroblast growth factor receptor of rat osteosarcoma (FGFR2-ROS) and with protein sequences of four uncharacterized ORFs from Caenorhabditis elegans and one from Drosophila melanogaster.

Interaction of Knr4 protein, a protein involved in cell wall synthesis, with tyrosine tRNA synthetase encoded by TYS1 in Saccharomyces cerevisiae.

The Knr4 protein, known to be involved in the regulation of cell wall assembly in Saccharomyces cerevisiae, strongly interacts with the tyrosine tRNA synthetase protein encoded by TYS1 as demonstrated by the genetic two-hybrid system and a biochemical pull-down experiment using GST--Tys1p fusion. Data reported here raise the possibility that this physical interaction between these proteins is required for dityrosine formation during the sporulation process. In addition, it is shown that the efficiency of spores formation was drastically reduced in diploid cells homozygous for the disruption of KNR4 or for a temperature-sensitive mutation of TYS1, although this effect could be independent of their protein interaction.

Kinetic analysis of growth and xanthan gum production with Xanthomonas campestris on sucrose, using sequentially consumed nitrogen sources.

A batch fermentation strategy using Xanthomonas campestris ATCC 13951 for xanthan gum production has been established in which all essential medium components are supplied at the onset. This has been achieved using sucrose as sole sugar feedstock. Sequential consumption of nitrogen sources (soybean hydrolysates, ammonium and nitrate salts) was observed to facilitate the further optimisation of the medium.