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J Boudrant

Publications and source records attributed to J Boudrant.

14 recordsLinked to original sources
PubMed2004

Pyrococcus abyssi alkaline phosphatase: the dimer is the active form.

Alkaline phosphatases (APs), E.C. 3.1.3.1, are non-specific phosphomonoesterases optimally active under alkaline conditions. They are classically known to be homodimeric metalloenzymes. This quaternary structure has been considered necessary for activity, although the relationship between quaternary structure and activity is not well understood. Recombinant Pyrococcus abyssi AP was previously isolated and characterized, appearing to have two active quaternary structures on native polyacrylamide gel electrophoresis: a monomer and a homodimer. The purpose of the present work was to determine the actual quaternary structure of P. abyssi AP in solution, by isolating each of the two quaternary forms and establishing the parameters governing the assembly and dissociation of the dimer. pH appeared to be an important parameter: in acidic media, the monomer/dimer ratio shifted towards monomer. Buffer composition also affected the quaternary structure: at the same pH, in potassium phosphate buffer, the two quaternary structures were observed, whereas in tris(hydroxymethyl)aminomethane hydrochloride buffer, only the dimer was observed. Metals bound to the enzyme were found to be involved in the stability of the quaternary structure. Indeed, the P. abyssi AP obtained upon removal of the metals was monomeric. Reactivation of the latter was achieved with variable efficiency. From these experiments, no active monomer could be isolated, leading the conclusion that the active form of P. abyssi AP is the homodimer.

Alkaline Phosphatase↗
PubMed2001

The effect of supplementation by different nitrogen sources on the production of lactic acid from date juice by Lactobacillus casei subsp. rhamnosus.

Production of lactic acid from date juice by fermentation has been studied using Lactobacillus casei subsp. rhamnosus as the producer organism. The optimum substrate concentration, expressed in its glucose content, was 60 g l(-1). Various nitrogen sources were compared with yeast extract in terms of their efficiency for lactic acid production. None of these nitrogen sources gave lactic acid concentrations as high as that obtained with yeast extract. As yeast extract supplementation was not economically attractive, different proportions of (NH4)2SO4 and yeast extract were used. When the elemental nitrogen ratio of(NH4)2SO4 to yeast extract was 4:1, the substrate use and efficiency of lactic acid production were the same as in date juice supplemented with 20 g l(-1) yeast extract (0:5).

Ammonium Sulfate↗
PubMed2001

Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi.

This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70 degrees C, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105 degrees C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiol-reducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intra- or interchain disulfide bonds. In addition, P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.

Alkaline Phosphatase↗
PubMed1999

Effects of pulse addition of carbon sources on continuous cultivation of Escherichia coli containing a recombinant E. coli gapA gene.

At high glucose concentrations, Escherichia coli produces acetate (Crabtree effect). To look for the influence of glucose and/or acetate in the medium on the expression of a recombinant gene in E. coli, the effect of a pulse addition of glucose, on transcription of a cloned E. coli gapA gene and the resulting glyceraldehyde-3P-dehydrogenase activity (GAPDH), was tested during continuous cultivation of E. coli HB101 transformed with the plasmid pBR::EcogapA. Stable continuous cultures were established in a semi-synthetic medium supplemented with 5 g/L of glucose. After the addition of 7 g of glucose within a few seconds, gapA gene expression was strongly and very rapidly induced. As shown by primer-extension analysis, promoter P1, one of the four transcriptional promoters of the gapA gene, was strongly activated, and GAPDH activity increased. However, after rapid glucose consumption, acetate was produced and acetate concentrations above 2 g/L induced stress conditions. This is shown by a strong activation of promoter P2, that is recognized by the stress specific Esigma32 RNA polymerase. During this period, the total cellular RNA content was strongly diminished. Later, when acetate was partially consumed a high level of total RNA was restored, translation was efficient and a regular increase of the GAPDH-specific activity was observed. The transitions between glucose metabolism, acetate production and the end of acetate consumption, were marked by large increases in RNase and protease activities. For comparison, pulse-addition experiments were also performed with serine and alanine. A transient increase of GAPDH production associated with an increase in biomass was also found for serine that can be utilized as an energy source, whereas the addition of alanine, which is only incorporated into newly synthesized proteins, did not increase GAPDH production. The implication of these data for overproduction of recombinant proteins in E. coli is discussed.

Acetic Acid↗
PubMed1994

Glucose and acetate influences on the behavior of the recombinant strain Escherichia coli HB 101 (GAPDH).

This study highlights data about the production of a recombinant protein (glyceraldehyde-3-phosphate dehydrogenase) by E. coli HB 101 (GAPDH) during batch and fed-batch fermentations in a complex medium. From a small number of experiments, this strain has been characterized in terms of protein production performance and glucose and acetate influences on growth and recombinant protein production. The present results show that this strain is suitable for recombinant protein production, in fed-batch culture 55 g L-1 of biomass and 6 g L-1 of GAPDH are obtained. However this strain, and especially GAPDH overproduction is sensitive to glucose availability. During fermentations, maximum yields of GAPDH production have been obtained in batch experiments for glucose concentration of 10 g L-1, and in fed-batch experiments for glucose availability of 10 g h-1 (initial volume 1.5 L). The growth of the strain and GAPDH overproduction are also inhibited by acetate. Moreover acetate has been noted as an activator of its own formation.

Acetates↗
PubMed1991

Metabolic roles of peptone and yeast extract for the culture of a recombinant strain of Escherichia coli.

The influence of complex compounds on the growth of a recombinant strain of Escherichia coli containing the gene encoding glyceraldehyde 3-phosphate dehydrogenase, as well as the production of this enzyme have been studied. Batchwise cultures led to an accumulation of acetate, which was not utilized in a yeast extract-free medium. After glucose exhaustion, growth stopped and enzyme activity decreased. Whereas yeast extract allowed acetate assimilation and growth, peptone stabilized the enzymatic activity. The addition of both compounds resulted in optimal performances for enzyme production.

Culture Media↗
PubMed1990

Microbial processes for ascorbic acid biosynthesis: a review.

L-Ascorbic acid is an important product currently made using the Reichstein process, which is mainly chemical. Recently, bacteria have been identified that are able to transform in a very efficient way glucose to 2,5-keto-D-gluconic acid and this product to 2-keto-L-idonic acid, precursor of L-ascorbic acid. When the corresponding strains are used together, it is possible to get 2-keto-L-idonic acid directly from glucose. Moreover, new strains have been constructed by introducing a gene from a strain responsible for the second step into a strain responsible for the first step. By using one of the new strains, the transformation can be performed in a single step with only one strain. However, the classical process still remains the most competitive.

Acetobacter↗
PubMed1979

Process characteristics of cell lysis mutants of Saccharomyces cerevisiae.

Several temperature-sensitive lysis mutants of Saccharomyces cerevisiae were selected according to their ability to release alkaline phosphatase when incubated at a nonpermissive temperature. For two mutants, cell lysis and release of alkaline phosphatase reached a maximum when cells in the logarithmic growth phase were shifted to the nonpermissive temperature. Morphological changes, as well as changes in macromolecular composition of the cells, were observed. Growth is necessary and oxygen is important for the expression of cell lysis at the nonpermissive temperature.

Alkaline Phosphatase↗
PubMed1976

Continuous Proteolysis with a stabilized stabilized protease. I. Chemical stabilization of an alkaline protease.

Due to the loss of enzymatic activity as a function of time, an alkaline protease, selected for the continuous preparation of protein hydrolysates (J. Boudrant and C. Cheftel, Biotechnol. Bioeng., 18,1735, 1976), was chemically stabilized by a simple treatment with glutaraldehyde. Two fractions, soluble and insoluble, were obtained. The activities of these two fractions were measured with casein and N-benzoyl-L-arginine ethyl ester (BAEE) as a function of glutaraldehyde concentration used. It was noted that the insoluble fraction was practically inactive with the first substrate and that the heat stability of the soluble form was likewise enhanced. Molecular weights of these two forms were unchanged, but the uv-spectrum of the soluble form was modified. From amino acid analysis, it appears that this treatment mainly provokes a decrease in lysine content.

Aldehydes↗
PubMed1976

Continuous proteolysis with a stabilized protease. II. Continuous experiments.

Batch proteolysis experiments were performed in order to choose a protein-protease system to prepare a correct hydrolysate suitable for the enrichment of soft-drinks. The system eventually studied was casein-Alcalase. Comparative batch and continuous proteolysis of casein by Alcalase showed that the reaction, which does not exactly follow first order kinetics with respect to the substrate concentration, is inhibited by the reaction products. Furthermore, experiments were done in order to determine the reaction conditions (pH8.8 in the reactor, casein concentration 5%, 40 degrees C). Determining the molecular weight of Alcalase (43,000) suggested the choice of ultrafiltration membrane PM 30. Sutdies of continuous proteolysis with the chemically stabilized enzyme retained by the ultrafiltration reactor showed that protease reuse for seven days at 40 degrees C is possible and that the growth of microorganisms is practically inhibited under these conditions. Gel chromatography showed the molecular weight.range of the peptides to be less than 2,000. Triangular taste tests showed that the threshold identification concentration of the dry hydrolysate in orange juice is about 0.65%.

Beverages↗
PubMed1975

[Application of the theory of oxygen transfer: determination of the Michaelis constant of glucose oxidase with respect to oxygen].

The oxidation of beta-D-glucose with glucose oxidase generally requires oxygen, which, under normal conditions is present at low concentrations in the reaction medium. Experiments show that glucose oxidase is no longer saturated by oxygen at enzyme concentrations greater than 0.4 mg.ml1. This is due to the decrease in the oxygen concentration of the solution. The value of the oxygen mass transfer coefficients and dissolved oxygen concentrations are determined. These dissolved oxygen concentrations are found to correlate with direct measurements with an oxygen electrode. From this, the Michaelis constant of glucose oxidase for oxygen is calculated. These experiments also show that oxygen is a limiting factor for this reaction.

Aspergillus niger↗
PubMedDate not supplied

Improved performances and control of beer fermentation using encapsulated alpha-acetolactate decarboxylase and modeling.

The use of the enzyme alpha-acetolactate decarboxylase allows the acceleration of beer fermentation/maturation because it shunts diacetyl formation, whose elimination is the rate-limiting step of the process. To obtain a cost reduction by using this exogenous enzyme, we propose a new process involving recoverable encapsulated alpha-acetolactate decarboxylase. The performance of traditional and new processes was investigated by a modeling approach. A simple model, focused on alpha-acetolactate and diacetyl profiles during beer fermentation, was set up. The simulated profiles are consistent with literature data. This study shows also that encapsulated alpha-acetolactate decarboxylase allows the acceleration of beer fermentation as efficiently as free alpha-acetolactate decarboxylase. The advantage of immobilized alpha-acetolactate decarboxylase versus free enzyme is that it is recoverable and reusable, which means a process cost reduction.

Acetoin Dehydrogenase↗