Correlation between thermostability and stability of glycosidases in ionic liquid.

The activity and stability of a ß-glycosidase (Thermus thermophilus) and two α-galactosidases (Thermotoga maritima and Bacillus stearothermophilus) were studied in different hydrophilic ionic liquid (IL)/water ratios. For the ILs used, the glycosidases showed the best stability and activity in 1,3-dimethylimidazolium methyl sulfate [MMIM][MeSO(4)] and 1,2,3-trimethylimidazolium methyl sulfate [TMIM][MeSO(4)]. A close correlation was observed between the thermostability of the enzymes and their stability in IL media. At high IL concentration (80%), a time-dependent irreversible denaturing effect was observed on glycosidases while, at lower concentration (<30%), a reversible inactivation affecting mainly the k (cat) was obtained. The results demonstrate that highly thermostable glycosidases are more suitable for biocatalytic reactions in water-miscible ILs.

Rational design of a GH1 beta-glycosidase to prevent self-condensation during the transglycosylation reaction.

Mutant N282T of a thermostable beta-glycosidase from GH1 family (TtbetaGly) presenting a high transglycosidase activity was previously obtained by directed evolution. However, it displays a self-condensation activity with the donor 2-nitrophenyl-beta-d-galactopyranoside (oNPGal) which competes with the condensation reaction and entails undesirable effects. In order to prevent this reaction, we rationally modified this enzyme at the [+1]/[+2] subsites so that oNPGal would bind less tightly. Molecular modeling (MM) suggested the mutation A221W, which decreased the affinity of the donor at these sites and moved it away from the bound galactose at the -1 subsite. A single (A221W) and a double mutant (A221W/N282T) were constructed, and they gave rise to a drastic decrease in self-condensation. The A221W mutant had no transglycosylation activity whereas the A221W/N282T mutant still displayed a condensation activity, comparable to that of the N282T mutant for the transfer on pNPGlcNAc. MM revealed that the double mutant A221W/N282T could induce the synthesis of a glycosidic bond between a donor and an acceptor displaying an equatorial 4-position. Moreover, it is suggested that mutation N282T could change the orientation of residue N219, leading to a stabilization of the acceptor with a new hydrogen bond. This finding opens the way to further improvements of evolved transglycosidases.

Evolved beta-galactosidases from Geobacillus stearothermophilus with improved transgalactosylation yield for galacto-oligosaccharide production.

A mutagenesis approach was applied to the beta-galactosidase BgaB from Geobacillus stearothermophilus KVE39 in order to improve its enzymatic transglycosylation of lactose into oligosaccharides. A simple screening strategy, which was based on the reduction of the hydrolysis of a potential transglycosylation product (lactosucrose), provided mutant enzymes possessing improved synthetic properties for the autocondensation product from nitrophenyl-galactoside and galacto-oligosaccharides (GOS) from lactose. The effects of the mutations on enzyme activity and kinetics were determined. An change of one arginine to lysine (R109K) increased the oligosaccharide yield compared to that for the wild-type BgaB. Subsequently, saturation mutagenesis at this position demonstrated that valine and tryptophan further increased the transglycosylation performance of BgaB. During the transglycosylation reaction with lactose of the evolved beta-galactosidases, a major trisaccharide was formed. Its structure was characterized as beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->4)-D-glucopyranoside (3'-galactosyl-lactose). At the lactose concentration of 18% (wt/vol), this trisaccharide was obtained in yields of 11.5% (wt/wt) with GP21 (BgaB R109K), 21% with GP637.2 (BgaB R109V), and only 2% with the wild-type BgaB enzyme. GP643.3 (BgaB R109W) was shown to be the most efficient mutant, with a 3'-galactosyl-lactose production of 23%.

Engineering of glucoside acceptors for the regioselective synthesis of beta-(1-->3)-disaccharides with glycosynthases.

Glycosynthase mutants obtained from Thermotogamaritima were able to catalyze the regioselective synthesis of aryl beta-D-Galp-(1-->3)-beta-D-Glcp and aryl beta-D-Glcp-(1-->3)-beta-D-Glcp in high yields (up to 90 %) using aryl beta-D-glucosides as acceptors. The need for an aglyconic aryl group was rationalized by molecular modeling calculations, which have emphasized a high stabilizing interaction of this group by stacking with W312 of the enzyme. Unfortunately, the deprotection of the aromatic group of the disaccharides was not possible without partial hydrolysis of the glycosidic bond. The replacement of aryl groups by benzyl ones could offer the opportunity to deprotect the anomeric position under very mild conditions. Assuming that benzyl acceptors could preserve the stabilizing stacking, benzyl beta-d-glucoside firstly assayed as acceptor resulted in both poor yields and poor regioselectivity. Thus, we decided to undertake molecular modeling calculations in order to design which suitable substituted benzyl acceptors could be used. This study resulted in the choice of 2-biphenylmethyl beta-D-glucopyranoside. This choice was validated experimentally, since the corresponding beta-(1-->3) disaccharide was obtained in good yields and with a high regioselectivity. At the same time, we have shown that phenyl 1-thio-beta-D-glucopyranoside was also an excellent substrate leading to similar results as those obtained with the O-phenyl analogue. The NBS deprotection of the S-phenyl group afforded the corresponding disaccharide quantitatively.

Directed evolution of the alpha-L-fucosidase from Thermotoga maritima into an alpha-L-transfucosidase.

The alpha-L-fucosidase from Thermotoga maritima (Tm alpha fuc) was converted into alpha-L-transfucosidase variants by directed evolution. The wild-type enzyme catalyzes oligosaccharide synthesis by transfer of a fucosyl residue from a pNP-fucoside donor to pNP-fucoside (self-condensation) with alpha-(1-->3) regioselectivity or pNP-galactoside (transglycosylation) with alpha-(1-->2) regioselectivity at low yields (7%). The wild-type enzyme was submitted to one cycle of mutagenesis, followed by rational recombination of the selected mutations, which allowed identification of variants with improved transferase activity. The transferase and hydrolytic kinetics of all the mutants were assessed by NMR methods and capillary electrophoresis. It was shown that the best mutant exhibited a dramatic 32-fold increase in the transferase/hydrolytic kinetic ratio, while keeping 60% of the overall wild-type enzyme activity. Accordingly, the maximum yield of a specific transglycosylation product [pNP-Gal-alpha-(1-->2)-Fuc] reached more than 60% compared to 7% with WT enzyme at equimolar and low concentrations of donor and acceptor (10 mM). Such an improvement was obtained with only three mutations (T264A, Y267F, L322P), which were all located in the second amino acid shell of the fucosidase active site. Molecular modeling suggested that some of these mutations (T264A, Y267F) cause a reorientation of the amino acids that are in direct contact with the substrates, resulting in a better docking energy. Such mutants with high transglycosidase activity may constitute novel enzymatic tools for the synthesis of fucooligosaccharides.

In vivo selection for the enhancement of Thermotoga maritima exopolygalacturonase activity at neutral pH and low temperature.

The aim of this study was to develop an Escherichia coli-based metabolic selection system for the uncovering of new oligogalacturonate-active enzymes. Based on the expression of the specific permease TogMNAB, this system enabled the entry of oligogalacturonates into the cytoplasm of E. coli thus providing a modified strain usable for this purpose. This tool was used for the metabolic selection of Thermotoga maritima exopolygalacturonase (TmGalU) mutants enabling the uptake of sodium trigalacturonate as the sole carbon source by the bacterium. In only one round of error-prone PCR and selection, mutants of TmGalU with a 4-fold increased turnover at pH 7.0 and 2-fold more active at 37 degrees C than wild-type enzyme were isolated. These results show the versatility of this strain for the evolution of oligogalacturonate-active enzymes.

Converting a {beta}-glycosidase into a {beta}-transglycosidase by directed evolution.

Directed evolution was applied to the beta-glycosidase of Thermus thermophilus in order to increase its ability to synthesize oligosaccharide by transglycosylation. Wild-type enzyme was able to transfer the glycosyl residue with a yield of 50% by self-condensation and of about 8% by transglycosylation on disaccharides without nitrophenyl at their reducing end. By using a simple screening procedure, we could produce mutant enzymes possessing a high transferase activity. In one step of random mutagenesis and in vitro recombination, the hydrolysis of substrates and of transglycosylation products was considerably reduced. For certain mutants, synthesis by self-condensation of nitrophenyl glycosides became nearly quantitative, whereas synthesis by transglycosylation on maltose and on cellobiose could reach 60 and 75%, respectively. Because the most efficient mutations, F401S and N282T, were located just in front of the subsite (-1), molecular modeling techniques were used to explain their effects on the synthesis reaction; we can suggest that repositioning of the glycone in the (-1) subsite together with a better fit of the acceptor in the (+1) subsite might favor the attack of a glycosyl acceptor in the mutant at the expense of water. Thus these new transglycosidases constitute an interesting alternative for the synthesis of oligosaccharides by using stable and accessible donor substrates.

New methods for chemo-enzymatic galactosidation of 2S rapeseed protein.

Two chemo-enzymatic methodologies to synthesize neoglycoproteins from rapeseed 2S protein (napin) were developed. In the first approach, glycosidases were used to catalyse 1-O-glycosylation of serine residues, whereas in the second one, 6-N-galactosylation was examined using an amino-reduction reaction between the epsilon-NH2 of lysine residues and 6-oxogalactosides (readily available by means of the oxidation reaction of the corresponding galactosides mediated by galactose oxidase). Our results indicated that glycosidases were unable to glycosylate native proteins. Conversely, this reaction was possible, although in low yields (10%), after the introduction of a hydroxyethylene spacer. The latter modified proteins were obtained via the condensation of epsilon-NH2 of lysines with ethylene carbonate in basic medium (40% yield). The second approach was much more efficient, as 61% of the lysine residues were shown to be 6-N-galactosylated using sodium cyanoborohydride as a reduction reagent.

Cloning expression and characterization of a thermostable exopolygalacturonase from Thermotoga maritima.

A gene encoding for a thermostable exopolygalacturonase (exo-PG) from hyperthermophilic Thermotoga maritima has been cloned into a T7 expression vector and expressed in Escherichia coli. The gene encoded a polypeptide of 454 residues with a molecular mass of 51,304 Da. The recombinant enzyme was purified to homogeneity by heat treatment and nickel affinity chromatography. The thermostable enzyme had maximum of hydrolytic activity for polygalacturonate at 95 degrees C, pH 6.0 and retains 90% of activity after heating at 90 degrees C for 5 h. Study of the catalytic activity of the exopolygalacturonase, investigated by means of 1H NMR spectroscopy revealed an inversion of configuration during hydrolysis of alpha-(1-->4)-galacturonic linkage.

Behaviors of bovine serum albumin and rapeseed proteins at the air/water interface after grafting aliphatic or aromatic chains.

The influence of grafting aliphatic or aromatic groups on the behaviors of bovine serum albumin (BSA) and rapeseed proteins (napin and cruciferin) at the air/water interface is studied. From compression isotherms, it is shown that the chemical modification induces an increase in the interfacial molecular areas of the three proteins. The more hydrophobic the groups grafted, the more important this increase is. The dilatational modulus clearly emphasized that the grafting of hydrophobic groups also leads to an increase of the collapse pressure, demonstrating a higher cohesiveness and resistance to pressure of the interfacial films. These results are discussed on the basis of the physicochemical changes due to these chemical modifications, especially the conformation, the surface hydrophobicity, and the flexibility of the modified proteins. The improvement of surface properties obtained by grafting aliphatic or aromatic chains onto these proteins looks very promising in regard to emulsifying and foaming properties.

Exopolygalacturonate lyase from Thermotoga maritima: cloning, characterization and organic synthesis application.

A new exopolygalacturonate lyase (Pel) gene of the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli cells. A 42 kDa monomeric Pel was shown to undergo N-terminal processing by cleavage at a putative site between alanine and serine residues. The enzyme catalyzes selectively a beta-4,5 elimination at the third galacturonic unit from the reducing end of polygalacturonic acid by producing (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid)-(1-->4)-(alpha-D-galactopyranosyluronic acid)-(1-->4)-alpha-D-galactopyranuronic acid (3) with a 60% yield. The optimum activity of the enzyme was detected at pH 9.5 and T> or=95 degrees C. The highly thermostable enzyme constitutes a useful catalyst for a simplified synthesis of 4,5-unsaturated trigalacturonic acid 3, a trisaccharide which is extremely difficult to obtain via chemical synthesis.

Alpha-galactosyl fluoride in transfer reactions mediated by the green coffee beans alpha-galactosidase in ice.

We show that the yields in saccharide synthesis by tranglycosylation with alpha-galactosidase from green coffee beans can be greatly enhanced when working in ice. Thus, methyl alpha-D-galactopyranosyl-(1-->3)-alpha-D-galactopyranoside (3a) produced by reaction of alpha-D-galactopyranosyl fluoride 1 with methyl alpha-D-galactopyranoside (2) is obtained with 51% yield in ice while only 29% is synthesized at 37 degrees C. This result, already previously found by others with proteases and by us with a beta-galactosidase appears to be a general property of hydrolases.