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.

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.

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.

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-β-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.

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.

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.

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.

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.

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.

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.