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P Monsan

Publications and source records attributed to P Monsan.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cloning and sequencing of a gene coding for an extracellular dextransucrase (DSRB) from Leuconostoc mesenteroides NRRL B-1299 synthesizing only a alpha (1-6) glucan.

The coding region for a novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene (dsrB) was isolated and sequenced. Using degenerate primers homologous to a conserved region present in dextransucrases from Streptococcus (GTFs) and L. mesenteroides NRRL B-512F (DSRS) and conserved amino acid sequences located in the N-terminal catalytic region of these enzymes, about 60% of the DSRB encoding gene was isolated. Two sites, BamHI and HindIII, were identified which allowed one 0.5-kbp probe to be obtained to isolate the 5' and the 3' ends of dsrB. The nucleotide sequence of the dsrB gene was determined and found to consist of an open reading frame (ORF) of 4521 base pairs (bp) coding for a 1507-amino acid protein with an M1 of 168,511. The amino acid sequence is very close to that of DSRS. Like DSRS, the dextran produced appeared to be composed of only alpha (1-6) glucosidic bonds, and the oligosaccharides synthesized in the presence of acceptor maltose were also composed of alpha (1-6) linked glucosyl residues in addition to the maltosyl residue. DSRB thus appears to be a novel dextransucrase from L. mesenteroides NRRL B-1299. DSRB produces a dextran different from the typical dextran containing alpha (1-6) and alpha (1-2) linkages produced when this strain is grown in the presence of sucrose.

Amino Acid Sequence↗
PubMed1998

Characterization of the Different Dextransucrase Activities Excreted in Glucose, Fructose, or Sucrose Medium by Leuconostoc mesenteroides NRRL B-1299.

When grown in glucose or fructose medium in the absence of sucrose, Leuconostoc mesenteroides NRRL B-1299 produces two distinct extracellular dextransucrases named glucose glucosyltransferase (GGT) and fructose glucosyltransferase (FGT). The production level of GGT and FGT is 10 to 20 times lower than that of the extracellular dextransucrase sucrose glucosyltransferase (SGT) produced on sucrose medium (traditional culture conditions). GGT and FGT were concentrated by ultrafiltration before sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Their molecular masses were 183 and 186 kDa, respectively, differing from the 195 kDa of SGT. The structural analysis of the dextran produced from sucrose and of the oligosaccharides synthesized by acceptor reaction in the presence of maltose showed that GGT and FGT are two different enzymes not previously described for this strain. The polymer synthesized by GGT contains 30% alpha(1-->2) linkages, while FGT catalyzes the synthesis of a linear dextran only composed of alpha(1-->6) linkages.

Journal Article↗
PubMed1998

Effect of Leuconostoc mesenteroides NRRL B-512F dextransucrase carboxy-terminal deletions on dextran and oligosaccharide synthesis.

Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes synthesis of soluble dextran from sucrose. In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward glucooligosaccharide synthesis. Like glucosyltransferases from oral streptococci, DSR-S possesses a C-terminal glucan-binding domain composed of a series of tandem repeats. In order to determine the role of the C-terminal region of DSR-S in dextran or oligosaccharide synthesis, four DSR-S genes with deletions at the 3' end were constructed. The results showed that the C-terminal region modulated the initial velocity of dextran synthesis but that the K(m) for sucrose, the optimum pH, and the activation energy were all unaffected by the deletions. The C-terminal domain modulated the rate of oligosaccharide synthesis whatever acceptor molecule was used (a good acceptor molecule such as maltose or a poor acceptor molecule such as fructose). The C-terminal domain seemed to play no role in the catalytic process in dextran and oligosaccharide synthesis. In fact, it seems that the role of the C-terminal domain of DSR-S may be to facilitate the translation of dextran and oligosaccharides from the catalytic site.

Amino Acid Sequence↗
PubMed1997

Characterization of Leuconostoc mesenteroides NRRL B-512F dextransucrase (DSRS) and identification of amino-acid residues playing a key role in enzyme activity.

Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran from sucrose or oligosaccharides when acceptor molecules, like maltose, are present. The L. mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid. The characteristics of DSRS were found to be similar to the characteristics of the extracellular dextransucrase produced by L. mesenteroides NRRL B-512F. The enzyme also exhibited a high homology with other glucosyltransferases. In order to identify critical amino acid residues, the DSRS sequence was aligned with glucosyltransferase sequences and four amino acid residues were selected for site-directed mutagenesis experiments: aspartic acid 511, aspartic acid 513, aspartic acid 551 and histidine 661. Asp-511, Asp-513 and Asp-551 were independently replaced with asparagine and His-661 with arginine. Mutation at Asp-511 and Asp-551 completely suppressed dextran and oligosaccharide synthesis activities, showing that at least two carboxyl groups (Asp-511 and Asp-551) are essential for the catalysis process. However, glucan-binding properties were retained, showing that DSRS has a two-domain structure like other glucosyltransferases. Mutations at Asp-513 and His-661 resulted in greatly reduced dextransucrase activity. According to amino acid sequence alignments of glucosyltransferases, alpha-amylases or cyclodextrin glucanotransferases, His-661 may have a hydrogen-bonding function.

Amino Acid Sequence↗
PubMed1997

The self catalytic enzyme inactivation induced by solvent stirring: a new example of protein conformational change induction.

This article gives a new example of the protein conformational change induction. A well known example widely described in the literature is the prion, a protein whose the pathologic form, PrPsc, induces a conformational change of the normal form, PrPc, by interaction with it. This work highlights the existence of a self-catalytic conformation change for lysozyme. The functional modification of this protein is analysed in terms of irreversible loss of activity. Our experiments and the kinetic model derived from our results show that lysozyme inactivation is catalyzed by the inactivated lysozyme molecules; the lysozyme molecules with modified conformation induce a conformational change of native lysozyme molecules that in turn become inactive. This phenomenon is enhanced by stirring, which increases the probability and the efficiency of collisions between enzyme molecules. Furthermore, the self-catalytic inactivation kinetics of lysozyme is increased when salts are dissolved in the enzyme solution. Under these conditions, the protective effect due to the addition of salts, reported in previous literature, disappear. Salt-induced lysozyme destabilization effect can be observed. The salts enhance the aggregation of inactive enzyme molecules. A kinetic model of self catalytic inactivation of the enzyme has been developed, taking into account the results obtained with and without the addition of salts in aqueous solution.

Enzyme Activation↗
PubMed1996

Cloning and sequencing of a gene coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only alpha (1-6) and alpha (1-3) linkages.

The coding region for a Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene (dsrA) was isolated and sequenced. Using a pair of primers designed on the basis of two highly conserved amino-acid (aa) sequences in L. mesenteroides NRRL B-512F dextransucrase and streptococcal glucosyltransferases (GTFs), a fragment of dsrA was amplified by the polymerase chain reaction (PCR). This PCR product was used as an hybridization probe to isolate a 1.8-kb fragment identified as the central region of dsrA. Cleavage by Sac I of this fragment allowed two probes to be obtained to isolate the 5' and the 3' ends of dsrA. The nucleotide sequence of the dsrA gene was determined and found to consist of an open reading frame (ORF) of 4870 base pairs (bp) coding for a 1290-aa protein with an M(r) of 145590. The aa sequence exhibited a high similarity with other GTFs. The two domains previously described in GTFs are conserved in DSRA: an N-terminal conserved domain and a C-terminal domain composed of a series of repeats. Surprisingly, the expected signal peptide was not detected. The entire gene was reconstructed and the activity of DSRA was investigated. The dextran produced appeared to be composed of 85% alpha (1-6) and 15% alpha (1-3) linkages and the oligosaccharides synthesized in the presence of maltose were mainly composed of alpha (1-6) linkages. This enzyme is a novel dextransucrase from L. mesenteroides NRRL B-1299 producing no alpha (1-2) linkages and is the first glucosyltransferase having no signal peptide described.

Amino Acid Sequence↗
PubMed1995

Degradation and fermentation of alpha-gluco-oligosaccharides by bacterial strains from human colon: in vitro and in vivo studies in gnotobiotic rats.

The ability of several human gut bacteria to break down alpha-1,2 and alpha-1,6 glycosidic linkages in alpha-gluco-oligosaccharides (GOS) was investigated in vitro in substrate utilization tests. Bacteroides thetaiotaomicron, Bifidobacterium breve and Clostridium butyricum, which are usually found in the infant gut and have been associated with both beneficial and deleterious effects on health, were studied. Alpha-Gluco-oligosaccharide degradation was compared in vitro and in vivo in gnotobiotic rats associated with these organisms, inoculated alone or in combination. Oligomer breakdown and short chain fatty acid and gas production indicated hydrolysis and fermentation of the substrate. In vitro and in vivo, Cl. butyricum was the least efficient in utilizing GOS, whereas Bact. thetaiotaomicron was the most efficient. Kinetic studies on GOS hydrolysis in pH-regulated fermenters showed that alpha-1,2 glucosidic bonds, which characterize the substrate, were more resistant than alpha-1,6 linkages. Adaptation of gnotobiotic rats to a diet containing 2% (w/w) GOS significantly increased the hydrolysis of alpha-1,2 glucosidic bonds. Combination of bacteria in trixenic rats improved GOS degradation and inhibited Cl. butyricum metabolism. This inhibition was confirmed in pH-regulated fermenters containing GOS as the principal carbon source. The association of beneficial bacteria and GOS may therefore have a potential health-promoting effect in human neonates.

Animals↗
PubMed1994

Production and use of glucosyltransferases from Leuconostoc mesenteroides NRRL B-1299 for the synthesis of oligosaccharides containing alpha-(1-->2) linkages.

Glucosyltransferase activities, produced by batch culture of Leuconostoc mesenteroides NRRL B-1299, were recovered both in the culture supernatant (SGT) and associated with the insoluble part of the culture (IGT). A total glucosyltransferase activity of 3.5 U/mL was measured in batch culture. The enzymes from the supernatant were purified 313 times using aqueous two-phase partition between dextran and PEG phases, yielding a preparation with 18.8 U/mg protein. It was shown that both SGT and IGT preparations catalyze acceptor reactions and transfer the glucose unit from sucrose onto maltose to produce glucooligosaccharides. Some of the glucooligosaccharides synthesized (Ln series) contain alpha-(1-->6) osidic linkages and a maltose residue at the reducing end. They were completely hydrolyzed by glucoamylase and dextranase. The other glucooligosaccharides synthesized (Bn series) resisted the action of these enzymes. The tetrasaccharide of this series has been characterized by 13C NMR. Its structure was determined as 2-O-alpha-D-glucosylpanose. The oligosaccharides synthesized by the maltose acceptor reaction with the SGT and IGT preparations only differed in the relative amounts in which they were produced. The difference may arise from diffusional limitations appearing when the insoluble catalyst is used. Under the assay conditions, the glucanase resistant oligosaccharide yield was 35% with both glucosyltransferase preparations.

Carbohydrate Conformation↗
PubMed1993

Purification and characterization of an aminopeptidase A from Staphylococcus chromogenes and its use for the synthesis of amino-acid derivatives and dipeptides.

An aminopeptidase with original specificity was purified 3800-fold to homogeneity from a cellular extract of Staphylococcus chromogenes. The enzyme was specific for acidic amino acids (Asp and Glu) at the N-terminus of peptides and thus can be classified as an aminopeptidase A. However, its specificity was not restricted to acidic amino acids: alpha-hydroxy acids such as L-malic and L-lactic acids were also accepted in position P1. The enzyme had a broad specificity for the residue at position P' 1, accepting all types of amino acids, including Pro, in this position. The optimal conditions for the hydrolysis of Asp-Phe-NH2 were pH 9.5 and 60 degrees C. The enzyme was inhibited by chelating agents and serine-protease inhibitors. The activity lost by treatment with chelating agents could be restored by Mn2+ or Zn2+ which also stimulated the native enzyme. This suggests that it is a metalloprotease with a serine residue essential for the activity. The native enzyme had an apparent molecular mass of 430 kDa on gradient-gel electrophoresis and subunits of 43 kDa as determined by SDS/PAGE. The enzyme catalyzed the synthesis of peptide and amino acid derivatives such as Asp-Phe-OMe (Aspartame) and malyl-Tyr-OEt from L-Asp and L-malic acid as acyl donors and L-Phe-OMe and L-Tyr-OEt as nucleophiles, respectively. The use of the enzyme as a reagent in protease-catalyzed peptide synthesis, N-terminal protection and subsequent deprotection, is described.

Amino Acid Sequence↗
PubMed1993

Reversible enzymic protection of the alpha-amino group of amino acid derivatives using an aminopeptidase A.

In previous papers we have reported that an aminopeptidase A (EC 3.4.11.7) purified from Staphylococcus chromogenes was able to catalyse the introduction of L-malic acid at the N-terminus of Tyr and Phe derivatives. We now show that this enzyme can be used for selective alpha-amino protection of derivatives of probably all amino acids, except Gly and Pro, by the malyl group. The following L-malyl derivatives were synthesized in thermodynamically controlled reactions with yields ranging from 4 to 47%: L-malyl-Tyr-OEt, -ALA-OMe, -Ser-OEt, -Lys-OEt, -Phe-OMe, -Met-NH2, -Glu-MH2, Arg-NH2, -Tye-NH2, -Val-NH2, -Ala-Phe and -Ala-Phe-NH2 (OEt and OMe are ethyl and methyl esters respectively). The reactions were monitored by reverse-phase h.p.l.c.; the products were quantified by amino acid analysis, and their structure was confirmed by m.s. No synthesis was obtained with Gly and Pro derivatives as nucleophiles. The effects of pH, temperature, enzyme concentration, nucleophile concentration, reaction time and addition of an organic co-solvent were studied. An important shift towards synthesis was obtained by carrying out the reactions at 55 degrees C in the presence of 55% organic co-solvent Triglyme (2,4,8,11-tetraoxadodecane) [8-60-fold increase in Ksyn. ([product] [acyl-donor]-1 [nucleophile]-1)].

Amino Acid Sequence↗
PubMed1991

Production of homogeneous basic fibroblast growth factor by specific enzymatic hydrolysis of larger microheterogeneous molecular forms.

The 146-amino acid form of basic fibroblast growth factor (bFGF) was expressed in Escherichia coli and purified by a two step process including ion exchange and heparin-Sepharose chromatographies. However, the resulting protein consisted of a mixture of 146- and 145-amino acid forms, indicating that, besides the initial methionine, also the following residue (proline) was removed from the N-terminus. The same phenomenon was observed when the 155-amino acid form, which is biologically equivalent to the shorter one, was expressed in E. coli. Taking into account the previously known data concerning the possible mechanism of cleavage of the extended forms of bFGF in vivo, we developed an efficient enzymatic process that allows the production of an homogeneous 146-amino acid form from recombinant NH2-end extended forms. This process takes advantage of the protecting effect that heparin exerts on bFGF. Accordingly, when bFGF, complexed to heparin, is treated with pepsin A, an aspartic protease with a broad specificity, only the Leu9-Pro10 peptide bond is cleaved generating the 146-amino acid form. Quantitative yields of this reaction are also achieved when bFGF is bound to a heparin-Sepharose column, allowing the integration of this enzymatic step directly during purification of the recombinant extended forms of bFGF.

Amino Acid Sequence↗
PubMed1991

Synthesis of amino-acid derivatives and dipeptides with an original peptidase enzyme.

A peptidase from the non pathogenic Staphylococcus sp. strain BEC 299 was purified to a final specific activity of 84,400 U/mg protein. Its molecular weight is 450 kDa and optimum pH 10.0. This enzyme catalyzes the synthesis of dipeptides (aspartame) and alpha-amino acid derivatives (N-L-malyl-L-tyrosine ethyl ester). The influence of cosolvents and pH on dipeptides and alpha-amino acid derivative synthesis is described. Finally, we detail the use of the peptidase as a reagent in protease-catalyzed peptide synthesis.

Amino Acids↗