Physicochemical parameters involved in the interaction of Saccharomyces cerevisiae cells with ion-exchange adsorbents in expanded bed chromatography.

Expanded bed adsorption (EBA) is an interesting primary technology allowing the adsorption of target proteins from unclarified feedstock in order to combine separation, concentration, and purification steps. However, interactions between cells and adsorbent beads during the EBA process can strongly reduce the performance of the separation. So, to minimize these interactions, the mechanisms of cell adsorption on the support were investigated. Adsorption kinetics of the baker's yeast Saccharomyces cerevisiae on the anion exchanger Q Hyper Z were directly performed under real EBA operating conditions, in a lab-scale UpFront 10 column. The yeast was marketed either as rod-shaped pellets (type I yeast) or as spherical pellets (type II yeast). For both types, a complete series of experiments for determining the adsorption profile versus time was performed, varying the superficial velocity or the pH. In parallel, the surface physicochemical properties of the cells (surface charge and electron-donor and electron-acceptor components) and of the support were determined. First of all, whatever the yeast types, the relation between cell adsorption and bed expansion has been highlighted, demonstrating the important role of hydrodynamic. However, for the type II yeast cells, adsorption increased dramatically, compared to the type I, even though it was shown that both types exhibited the same surface charge. In fact, there were strong differences in the Lewis acidic and basic components of the two yeasts. These differences explain the variable affinity toward the support, which was characterized by a strong electron-donor and a weak electron-acceptor component. These observed behaviors agreed with the colloidal theory. This work demonstrates that all kinds of interaction between the cells and the support (electrostatic, Lifshitz-van der Waals, acid/base) have to be taken into account together with hydrodynamic characteristics inside the bed.

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. The conversion rates, obtained with this enzyme at sucrose to acceptor molar ratio of 2:1 reached 81 and 61% for alpha-butylglucopyranoside and alpha-octylglucopyranoside, respectively. Analyses obtained from liquid chromatography coupled with mass spectrometry revealed that different series of alpha-alkylpolyglucopyranosides regioisomers of increasing polymerization degree can be formed depending on the specificity of the catalyst.

Role of the two catalytic domains of DSR-E dextransucrase and their involvement in the formation of highly alpha-1,2 branched dextran.

The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both alpha-1,6 and alpha-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by (13)C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only alpha-1,6 linkages. On the other hand, enzymes devoid of CD1 modified alpha-1,6 linear oligosaccharides and dextran acceptors through the formation of alpha-1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of alpha-1,6 glucosidic bonds and CD2 only catalyzing the formation of alpha-1,2 linkages. This finding permitted us to elucidate the mechanism of alpha-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of alpha-1,2 linkages. This enzyme will be very useful to control the rate of alpha-1,2 linkage synthesis in dextran or oligosaccharide production.

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. From sequence comparison with family 70 and alpha-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.