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

Ca(2+) signaling plays an important role in the function of dendritic cells (DC), the specialized antigen-presenting cells of the immune system. Here we describe functional ryanodine receptor (RyR) Ca(2+) release channels in murine, bone marrow-derived DC. RT-PCR analysis identified selective expression of the type 1 RyR, with higher levels detected in immature rather than mature DC. The RyR activators caffeine, FK506, ryanodine and 4-chloro-m-cresol mobilized Ca(2+) in DC, and responses to 4-chloro-m-cresol were inhibited by dantrolene. Furthermore, activation of RyRs both inhibited subsequent inositol trisphosphate-mediated Ca(2+) release and provoked store-operated Ca(2+) entry, suggesting a functional interaction between these intracellular Ca(2+) channels. Thus, the RyR1 channel may play an intrinsic role in Ca(2+) signaling in DC.     

Esterification by immobilized lipase in solvent-free media: kinetic and thermodynamic arguments

The aim of this study is to characterize, in solvent-free systems (SFS), the kinetic and thermodynamic performance of batch lipase-catalyzed esterification. SFS are compared to a conventional organic solvent, n-hexane. The esterification of oleic acid with ethanol was chosen as a model reaction. The TABEK (thermodynamic activity-based enzyme kinetics) approach was used to rationally analyze kinetics. Influence of the reaction medium on final conversions was also studied. Several factors, such as initial molar ratio of substrates, reactant availability, initial water content, and quantity of immobilized enzyme, were examined. Special attention was also turned to enzyme stability and reuse after reaction, this last item being a prerequisite in the development of industrial processes. SFS proved to be almost as efficient as n-hexane from a kinetic and thermodynamic point of view and offered a better volumetric production.     

Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity both in vivo and in vitro, and appear to act by cross-linking particular forms of FGF to appropriate FGF receptors. We have recently isolated and characterized two separate HS pools derived from immortalized embryonic day 10 mouse neuroepithelial 2.3D cells: one from cells in log growth phase, which greatly potentiates the activity of FGF-2, and the other from cells undergoing contact-inhibition and differentiation, which preferentially activates FGF-1. These two pools of HS have very similar functional activities to those species isolated from primary neuroepithelial cells at corresponding stages of active proliferation or differentiation. We present here a structural comparison between these cell line HS species to establish the nature of the changes that occur in the biosynthesis of HS. A combination of chemical and enzymatic cleavage, low pressure chromatography and strong anion-exchange HPLC were used to generate full chain models of each species. Overall, the HS pools synthesized in the dividing cell line pools possessed less complex sulfation than those derived from more differentiated, growth arrested cells.      

Heterologous expression of an engineered truncated form of human Lewis fucosyltransferase (Fuc-TIII) by the methylotrophic yeast Pichia pastoris

A stable GS115 Pichia pastoris recombinant strain was constructed to secrete a truncated form of the human alpha(1,3/4) fucosyltransferase (amino acids 45-361). Enzyme production resulted from a secretory pathway based on the pre-pro- alpha mating factor signal sequence of the yeast Saccharomyces cerevisiae . Following its transit through the Golgi apparatus, the enzyme accumulated in the periplasmic space before its release in the culture broth (about 30 mg/l). Cell-enclosed enzyme ( approximately 0.16%) proved to be fairly stable for many freezing and thawing cycles and could be used several times as an immobilized catalyst. Soluble enzyme (>99.8%) representing the main protein of the culture broth (10%) has been characterized by Western-blotting, substrate specificities and kinetic parameters. The two forms (cell- enclosed and soluble) of recombinant enzyme may be used for in vitro synthesis of Lewisadeterminants.      

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% α(1→2) linkages, while FGT catalyzes the synthesis of a linear dextran only composed of α(1→6) linkages.     

Kinetic modeling of oligosaccharide synthesis catalyzed by leuconostoc mesenteroides NRRL B-1299 dextransucrase

The kinetic behavior of soluble and insoluble forms of dextransucrase from Leuconostoc mesenteroides NRRL B-1299 was investigated with sucrose as substrate and maltose as acceptor. To study the parameters involved, a kinetic model was applied that was previously developed for L. mesenteroides NRRL B-512F dextransucrase. There are significant correlations between the parameters of the soluble form of B-1299 dextransucrase and those calculated for the B-512F enzyme; that is, their properties are comparable and differ from those of the insoluble form of B-1299 dextransucrase. Whereas the calculated parameters for high maltose concentrations describe the kinetic behavior very well, the time curves for low maltose concentrations were not described correctly. Therefore, the parameters were calculated separately for the two ranges. Copyright 1999 John Wiley & Sons, Inc.     

Influence of glucose on the kinetics of maltodextrin hydrolysis using free and immobilized glucoamylase

The study of the effect of glucose addition to the kinetics of maltodextrin hydrolysis catalyzed with free and immobilized Aspergillus niger glucoamylase does not show any significant glucose inhibitory effect. This result is in contradiction with data previously reported in the literature. On the contrary, a slight glucose-activating effect was observed. This effect was greater in the case of the immobilized enzyme. The glucose inhibitory effect may thus not be involved in the case of practical saccharification conditions catalyzed with glucoamylase when maltodextrins are used as substrate.     

Rational and Combinatorial Engineering of the Glucan Synthesizing Enzyme Amylosucrase

Rational engineering of amylosucrase required detailed investigations of the molecular basis of catalysis. Biochemical characterization of the enzyme coupled to structural analyses enabled the polymerization mechanism to be elucidated. This provided key information for successfully changing amylosucrase to a hydrolase or an improved polymerase using site-directed mutagenesis. In parallel, a combinatorial approach was developed to further improve the catalyst. The method, based on random mutagenesis and recombination of parent sequences, has generated libraries of variants. These were searched for improved polymer formation using a first step of selection followed by a screening test including a double detection method. Several mutants with desired properties have been isolated, their sequences revealed that they could not have been designed following a rational approach.     

Synthesis of dextrans with controlled amounts of α-1,2 linkages using the transglucosidase GBD–CD2

GBD–CD2 is an α-1,2 transglucosidase engineered from DSR-E, a glucansucrase naturally produced by Leuconostoc mesenteroides NRRL B-1299. This enzyme catalyses from sucrose, the α-1,2 transglucosylation of glucosyl moieties onto α-1,6 dextran chains. Steady-state kinetic studies showed that hydrolysis and transglucosylation reactions occurred at the early stage of the reaction in the presence of 70 kDa dextran as acceptor and sucrose. The transglucosylation reaction catalysed by GBD–CD2 follows a Ping Pong Bi Bi mechanism with a high k _cat value of 970 s⁻¹. The amount of the synthesised α-1,2 side chains was found to be directly dependent on the initial molar ratio [Sucrose]/[Dextran]. Dextrans with controlled α-1,2 linkage contents ranging from 13% to 40% were synthesised. The procedure resulted in the production of dextrans with the highest content of α-1,2 linkages ever reported.     

Enzymatic synthesis of alkyl-α-glucoside catalysed by a thermostable α-transglucosidase in solvent-free organic medium

α-Transglucosidase from Talaromycesduponti was used to synthesize different alkyl-α-d-glucosides from α(1-4) linked carbohydrate donors. The enzymatic preparation, purified by a single step, consisting of hydrophobic interaction chromatography, was sufficiently pure for very stereospecific synthesis to be achieved. Biphasic and homogeneous organic media could be compared for such purposes. Yields appeared to be two- to threefold higher in low-water biphasic media. High concentrations of the glucosyl donor were present in the aqueous phase, while water-immiscible alcohols were used as glucosyl acceptors. The high efficiency of the method was attributed to the shift of the thermodynamic equilibrium thanks to the extraction of the product from the aqueous phase, where the reaction occurs, into the organic phase. Operated in a continuous biphasic reactor, T. dupontiα-transglucosidase showed a good thermostability with a half-life of 23 days at 30 °C. Received: 26 January 1998 / Received revision: 15 April 1998 / Accepted: 19 April 1998