Investigation on the Synthesis of Shigella flexneri Specific Oligosaccharides Using Disaccharides as Potential Transglucosylase Acceptor Substrates.

Chemo-enzymatic strategies hold great potential for the development of stereo- and regioselective syntheses of structurally defined bioactive oligosaccharides. Herein, we illustrate the potential of the appropriate combination of a planned chemo-enzymatic pathway and an engineered biocatalyst for the multistep synthesis of an important decasaccharide for vaccine development. We report the stepwise investigation, which led to an efficient chemical conversion of allyl α-d-glucopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→3)-2-deoxy-2-trichloroacetamido-ß-d-glucopyranoside, the product of site-specific enzymatic α-d-glucosylation of a lightly protected non-natural disaccharide acceptor, into a pentasaccharide building block suitable for chain elongation at both ends. Successful differentiation between hydroxyl groups features the selective acylation of primary alcohols and acetalation of a cis-vicinal diol, followed by a controlled per-O-benzylation step. Moreover, we describe the successful use of the pentasaccharide intermediate in the [5 + 5] synthesis of an aminoethyl aglycon-equipped decasaccharide, corresponding to a dimer of the basic repeating unit from the O-specific polysaccharide of Shigella flexneri 2a, a major cause of bacillary dysentery. Four analogues of the disaccharide acceptor were synthesized and evaluated to reach a larger repertoire of O-glucosylation patterns encountered among S. flexneri type-specific polysaccharides. New insights on the potential and limitations of planned chemo-enzymatic pathways in oligosaccharide synthesis are provided.

Essential role of amino acid position 226 in oligosaccharide elongation by amylosucrase from Neisseria polysaccharea.

Amylosucrase from Neisseria polysaccharea is a remarkable transglucosylase that synthesizes an insoluble amylose-like polymer from sole substrate sucrose. One particular amino acid, Arg226, was proposed from molecular modeling studies to play an important role in the formation of the active site topology and in the accessibility of ligands to the catalytic site. The systematic mutation of this Arg residue by all 19 other possible amino acids revealed that all single-mutants had a higher activity on sucrose compared to the wild-type enzyme. An extensive kinetic investigation showed that catalytic efficiencies are greatly impacted by the presence of natural acceptors in the reaction media, their chain length and the nature of the amino acid at position 226. Compared to the wild-type enzyme, the R226N mutant showed a 10-fold enhancement in the catalytic efficiency and a nearly twofold higher production of an insoluble amylose-like polymer that can be of interest for biotechnological applications.

Tolerance engineering in Deinococcus geothermalis by heterologous efflux pumps.

Producing industrially significant compounds with more environmentally friendly represents a challenging task. The large-scale production of an exogenous molecule in a host microfactory can quickly cause toxic effects, forcing the cell to inhibit production to survive. The key point to counter these toxic effects is to promote a gain of tolerance in the host, for instance, by inducing a constant flux of the neo-synthetized compound out of the producing cells. Efflux pumps are membrane proteins that constitute the most powerful mechanism to release molecules out of cells. We propose here a new biological model, Deinococcus geothermalis, organism known for its ability to survive hostile environment; with the aim of coupling the promising industrial potential of this species with that of heterologous efflux pumps to promote engineering tolerance. In this study, clones of D. geothermalis containing various genes encoding chromosomal heterologous efflux pumps were generated. Resistant recombinants were selected using antibiotic susceptibility tests to screen promising candidates. We then developed a method to determine the efflux efficiency of the best candidate, which contains the gene encoding the MdfA of Salmonella enterica serovar Choleraesuis. We observe 1.6 times more compound in the external medium of the hit recombinant than that of the WT at early incubation time. The data presented here will contribute to better understanding of the parameters required for efficient production in D. geothermalis.

Rationally engineered double substituted variants of Yarrowia lipolytica lipase with enhanced activity coupled with highly inverted enantioselectivity towards 2-bromo phenyl acetic acid esters.

Inverting enzyme enantioselectivity by protein engineering is still a great challenge. Lip2p lipase from Yarrowia lipolytica, which demonstrates a low S-enantioselectivity (E-value = 5) during the hydrolytic kinetic resolution of 2-bromo-phenyl acetic acid octyl esters (an important class of chemical intermediates in the pharmaceutical industry), was converted, by a rational engineering approach, into a totally R-selective enzyme (E-value > 200). This tremendous change in selectivity is the result of only two amino acid changes. The starting point of our strategy was the prior identification of two key positions, 97 and 232, for enantiomer discrimination. Four single substitution variants were recently identified as exhibiting a low inversion of selectivity coupled to a low-hydrolytic activity. On the basis of these results, six double substituted variants, combining relevant mutations at both 97 and 232 positions, were constructed by site-directed mutagenesis. This work led to the isolation of one double substituted variant (D97A-V232F), which displays a total preference for the R-enantiomer. The highly reversed enantioselectivity of this variant is accompanied by a 4.5-fold enhancement of its activity toward the preferred enantiomer. The molecular docking of the R- and S-enantiomers in the wild-type enzyme and the D97A-V232F variant suggests that V232F mutation provides a more favorable stacking interaction for the phenyl group of the R-enantiomer, that could explain both the enhanced activity and the reversal of enantioselectivity. These results demonstrate the potential of rationally engineered mutations to further enhance enzyme activity and to modulate selectivity.

Conjugated autoxidizable triene (CAT) assay: a novel spectrophotometric method for determination of antioxidant capacity using triacylglycerol as ultraviolet probe.

Described here is a novel spectrophotometric method for estimating antioxidant capacity in a 96-well microplate using as UV probes the conjugated triene triacylglycerols (TAGs) naturally present in tung oil. The TAGs of this commercially available oil contain around 86% eleostearic acid, an octadecatrienoic acid with conjugated trienes exhibiting strong UV absorption at 273 nm. In an oil-in-water emulsion at 37 degrees C, the azo initiator 2,2'-azobis(2-amidinopropane) dihydrochloride generated a constant flux of peroxy radicals, which destroyed the conjugated trienes. The absorbance decay at 273 nm, related to oxidative degradation of conjugated triene TAGs, was monitored in the absence and presence of various concentrations of antioxidants. To validate this new method, the antioxidant capacity of four phenolic compounds (gallic acid, (-)-epicatechin, chlorogenic acid, and rosmarinic acid) was measured using an area-under-the-curve calculation and expressed as Trolox equivalents, the value for Trolox being taken as reference. The order of efficacy was found to be rosmarinic acid > chlorogenic acid approximately epicatechin>Trolox>gallic acid, which can be partially explained by the number of catecholic moieties and the polarity of these antioxidants. Moreover, the conjugated autoxidizable triene (CAT) assay provided good insight into antioxidant behavior (i.e., retarder or chain breaker). The same molecules were then analyzed in 2,2-diphenyl-1-picrylhydrazyl and fluorescein-based oxygen radical absorbance capacity assays, and the results are discussed critically with respect to those obtained with the CAT assay. This new method may constitute an easy-to-use, sensitive, and high-throughput in vitro protocol for evaluation of the antioxidant capacity of pure molecules and natural extracts in lipid oxidation.

Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase.

A computer-aided engineering approach recently enabled to deeply reshape the active site of N. polysaccharea amylosucrase for recognition of non-natural acceptor substrates. Libraries of variants were constructed and screened on sucrose allowing the identification of 17 mutants able to synthesize molecules from sole sucrose, which are not synthesized by the parental wild-type enzyme. Three of the isolated mutants as well as the new products synthesized were characterized in details. Mutants contain between 7 and 11 mutations in the active site and the new molecules were identified as being a sucrose derivative, named erlose (α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→2)-ß-d-Fructose), and a new malto-oligosaccharide named panose (α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-(1→4)-α-d-Glucose). These product specificities were never reported for none of the amylosucrases characterized to date, nor their engineered variants. Optimization of the production of these trisaccharides of potential interest as sweeteners or prebiotic molecules was carried out. Molecular modelling studies were also performed to shed some light on the molecular factors involved in the novel product specificities of these amylosucrase variants.

Engineering of anp efficient mutant of Neisseria polysaccharea amylosucrase for the synthesis of controlled size maltooligosaccharides.

Amylosucrase from Neisseria polysaccharea naturally catalyzes the synthesis of α-1,4 glucans from sucrose. The product profile is quite polydisperse, ranging from soluble chains called maltooligosaccharides to high-molecular weight insoluble amylose. This enzyme was recently subjected to engineering of its active site to enable recognition of non-natural acceptor substrates. Libraries of variants were constructed and screened on sucrose, allowing the identification of a mutant that showed a 6-fold enhanced activity toward sucrose compared to the wild-type enzyme. Furthermore, its product profile was unprecedented, as only soluble maltooligosaccharides of controlled size chains (2

Comparison of the lipase activity in hydrolysis and acyl transfer reactions of two latex plant extracts from babaco (Vasconcellea x Heilbornii Cv.) and Plumeria rubra: Effect of the Aqueous microenvironment.

The enzymatic properties of Plumeria rubra latex have been evaluated for the first time, showing a high activity in both hydrolysis and synthesis reactions, and compared to the biocatalytic behavior of babaco (Vasconcellea x Heilbornii cv.) latex. Both biocatalysts have been optimized by studying the various parameters that influence reaction kinetics. The optimum temperatures for hydrolysis reactions were 50 and 55 degrees C for babaco and Plumeria, respectively. The optimum pH for babaco latex was 7, whereas for Plumeria latex, two optimal pH values (4 and 7) were observed. With regard to esterification and acyl transfer reactions such as alcoholysis and interesterification, the influence of thermodynamic water activity on reaction yields was determined and correlated with water sorption and desorption isotherms. When babaco latex is used as a biocatalyst, optimal synthesis reaction yields are obtained when the enzymatic extract is stabilized at a water activity value of 0.38, which corresponds to a water content of 5.7%. This optimal level of hydration is located on the linear portion of the biocatalyst's sorption isotherm, where the water molecules exhibit high-energy interactions with the protein network. In synthesis reactions (esterification, alcoholysis, and interesterification) biocatalyzed by Plumeria latex, correlation between best reaction yields and water activity cannot be done. Indeed, the sorption isotherm plot has an atypical shape, indicating that water might be trapped in the latex matrix and, consequently, that the water content of the biocatalyst is highly dependent on the hydration history of the latex.

Programmed chemo-enzymatic synthesis of the oligosaccharide component of a carbohydrate-based antibacterial vaccine candidate.

The powerful chemo-enzymatic synthesis of the pentadecasaccharide hapten involved in the first synthetic carbohydrate-based vaccine candidate against endemic shigellosis is reported. The high yielding site-selective α-D-glucosylation of a lightly protected disaccharide by an engineered transglucosylase-sucrose system gave a trisaccharide, which was chemically elongated by an efficient [5+5] process.

Engineering and production of laccase from Trametes versicolor in the yeast Yarrowia lipolytica.

The lcc1 gene coding for the laccase from Trametes versicolor DSM11269 was cloned into the genome of Yarrowia lipolytica using either single or multiple integration sites. The levels of the recombinant laccase activity secreted in the culture media were 0.25 and 1 U ml(-1) for single and multiple integrations, respectively. The strain with a single integration was successfully used to express variant libraries which were screened on ABTS substrate. The strain encoding the double mutant L185P/Q214K (rM4A) showed a sixfold enhancement in secreted enzyme activity. The catalytic efficiency of the purified rM-4A laccase was respectively increased 2.4- and 2.8-fold towards ABTS and 2,6-dimethoxyphenol, compared to the rWT. Culture supernatants containing either rWT or rM-4A catalyzed the almost complete decolorization of an Amaranth solution (70 nMs(-1)). Taken together, our results open new perspectives for the use of Y. lipolytica as a molecular evolution platform to engineer laccases with improved properties.