Pierre Monsan

Étude du mécanisme d'établissement des liaisons glutaraldéhyde-protéines

Summary Commercial aqueous 25 per cent glutaraldehyde solutions contain no stable derivative of this aldehyde, but compounds of variable molecular weight which easily revert to glutaraldehyde. The effect of pH on the reaction of glutaraldehyde with amino acids and on the stability of the products under acid conditions, shows the importance of the structure modification of the dialdehyde which occurs when pH increases, and even leads to precipitation in highly alkaline solutions. This precipitate results from aldol condensation of glutaraldehyde molecules. It contains aldehyd groups conjugated with ethylenic double bonds. Such a structure reacts with amino groups to give an imino bond, stabilized by resonance with the ethylenic bond, and does not undergo Michael-type addition reactions. Therefore, glutaraldehyde does not react with proteins under its free form, but as an unsaturated polymer, which gives imino bonds stabilized by conjugation.

Production and purification of alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355, for the synthesis of oligoalternans

Alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355, was produced by fed-batch cultures and purified by two-phase partition with polyethylene glycol. The contaminating dextransucrase activity was eliminated by heat treatment. Some properties of the enzyme were examined, in particular its transferase activity in the presence of maltose. A general procedure for oligoalternan production and purification is defined.

Purification and some characteristics of β-galactosidase from Aspergillus fonsecaeus

Abstract β-Galactosidase from Aspergillus fonsecaeus was purified 73-fold with a 40.5% yield and a specific activity of 314 U mg −1 protein. This enzyme had an approximate molecular weight of 126 kd and an isoelectric point of 4.2. The enzyme hydrolysed o -nitrophenol- β -d-galactopyranoside and lactose optimally in a pH zone from 2.6 to 4.5 and 2.6 to 3.0, respectively. K m values determined for these substrates were 1.78 m m and 61.3 m m , respectively. β-Galactosidase obtained from Aspergillus fonsecaeus had a higher thermal stability (half-life of 36 h at 65°C) than a commercially available preparation from Aspergillus oryzae . These characteristics render β-galactosidase from A. fonsecaeus particularly suitable for potential technological applications.

Small-Scale Production of Burkholderia cepacia ATCC21808 Lipase Adapted to High-Throughput Screening

The screening of variant libraries of recombinant Burkholderia cepacia ATCC21808 lipase generated in Escherichia coli is limited by expression difficulties that are mainly due to the formation of inclusion bodies. To circumvent these difficulties and provide an efficient small-scale screening protocol, the gene encoding the lipase from B. cepacia was expressed in various expression vectors. With the pFLAG-ATS-Lip-Hp construct, expression of up to 6807 U/L of culture was possible in Erlenmeyer flasks. The production protocol was miniaturized in 96 deep-well plates, yielding 1300 U/L of lipase in fusion with the FLAG tag. With this protocol, the activity was determined in less than 10 min for a full plate, with a coefficient of variance of about 25%. For validation, 18 mutants constructed by site-directed mutagenesis on position Valine 266 were screened. Nice variations of activity were detected and found to be in agreement with those obtained in Erlenmeyer flask cultures. The protocol enabled the identification of 5 mutants showing enhanced activity toward para-nitrophenyl butyrate. (Journal of Biomolecular Screening 2008;13:72-79)