Michel Dion

A new oxygen-regulated operon in Escherichia coli comprises the genes for a putative third cytochrome oxidase and for pH 2.5 acid phosphatase (appA)

SummaryThe Escherichia coli acid phosphatase gene appA is expressed in response to oxygen deprivation and is positively controlled by the product of appR (katF) which encodes a putative new σ transcription-initiation factor. However, transcription of appA from its nearest promoter (P1) did not account for total pH 2.5 acid phosphatase expression and was not subject to regulation. The cloned region upstream of appA was extended and analyzed by insertions of transposon TnphoA and by fusions with lacZ. It contains two new genes, appC and appB, which both encode extracytoplasmic proteins. appC and appB are expressed from a promoter (P2) lying just upstream of appC. Both genes are regulated by oxygen, as is appA, and by appR gene product exactly as previously shown for appA. Analysis of the nucleotide sequence and of the origins of transcription have confirmed that the P2-appC-appB- (ORFX)-P1-appA region is organized on the chromosome as an operon transcribed clockwise from P2 and that P1 is a minor promoter for appA alone. Genes appC and appB encode proteins of Mr 58133 and 42377, respectively, which have the characteristics of integral membrane proteins. The deduced amino acid sequences of appC and appB show 60% and 57% homology, respectively, with subunits I and II of the E. coli cytochrome d oxidase (encoded by genes cydA and cydB). The notion that the AppC and AppB proteins constitute a new cytochrome oxidase or a new oxygen-detoxifying system is supported by the observation of enhanced sensitivity to oxygen of mutants lacking all three genes, cyo (cytochrome o oxidase), cyd (cytochrome d oxidase) and appB, compared to that of cyo cyd double mutants.

Converting a β-Glycosidase into a β-Transglycosidase by Directed Evolution

Directed evolution was applied to the β-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.

Cloning and expression of a beta-glycosidase gene from Thermus thermophilus. Sequence and biochemical characterization of the encoded enzyme.

A 3.2 kilobase pair DNA fragment from Thermus thermophilus HB27 coding for a β-galactosidase activity was cloned and sequenced. A gene and a truncated open reading frame orf1 encoding respectively a β-glycosidase (ttβ-gly) and probably a sugar permease were located directly adjacent to each other. The deduced aminoacid sequence of the enzyme Ttβ-gly showed strong identity with those of β-glycosidases belonging to the glycosyl hydrolase family 1. The enzyme was overexpressed in Escherichia coli and was purified by a two-step purification procedure. The recombinant enzyme is monomeric with a molecular mass of 49-kDa. It catalyzes the hydrolysis of β-D-galactoside, β-D-glucoside and β-D-fucoside derivatives. However, the kcat/Km ratio is much higher for p-nitrophenyl-β-D-glucoside and p-nitrophenyl-β-D-fucoside than for p-nitrophenyl-β-D-galactoside. The specificity towards linkage positions of the disaccharides tested decreased in the following order: β1-3 (100%) < β1-2 (71%) < β1-4 (40%) < β1-6 (10%). Ttβ-gly is a thermostable enzyme displaying an optimum temperature of 88°C and a half life of 10 min at 90°C. It performs transglycosylation reactions at high temperature with a yield exceeding 63% for transfucosylation reactions. On the basis of this work, the enzyme appears to be an attractive tool in the synthesis of fucosyl adducts and fucosyl sugars.

Comparative study of new α-galactosidases in transglycosylation reactions

Abstract We have studied the potential of several newly cloned α-galactosidases to catalyze the regioselective synthesis of disaccharides using 4-nitrophenylgalactoside as a donor. The kinetics of the reactions were followed by in situ NMR spectroscopy. The following thermophilic enzymes have been tested: Aga A and an isoenzyme Aga B obtained from the strain KVE39 and Aga 285 from the strain IT285 of Bacillus stearothermophilus; Aga T is an α-galactosidase from Thermus brockianus (strain IT360). Two other non-thermophilic α-galactosidases have also been evaluated: Aga 1 (Streptococcus mutans, strain Ingbritt) and Raf A (Escherichia coli, strain D1021). For all of the enzymes studied, high regioselectivity was observed leading to two (1→6)-disaccharides: 4-nitrophenyl α- d -galactopyranosyl-(1→6)-α- d -galactopyranoside and methyl α- d -galactopyranosyl-(1→6)-α- d -galactopyranoside, which were obtained in 54% (Aga B) and 20% (Aga T) yields, respectively.

The blue bronze Tl0.30MoO3 structure and physical properties

Abstract Crystals of the so-called blue bronze Tl 0.30 MoO 3 were prepared by tempering molten mixtures. The structure was refined in the space group C2/m from 2171 reflections corrected for absorption. The R and R w factors obtained from the last refinement cycle are 0.030 and 0.036 respectively for 114 refined parameters. The Zachariasen method enabled the 4d distribution over the three independent sites Mo1, Mo2 and Mo3 to be determined and gave 16.6 %, 39.7 % and 43.7 % respectively. For each octahedron a close correlation appears between the number of shared edges, the formal molybdenum charge and its eccentricity within the polyhedron. It is found that the molybdenum Mo1 has the highest formal charge ; this site does not seem to participate in the electrical conductivity which takes place mainly in the slabs thanks to the molybdenum sites Mo2 and Mo3 which represent 83.4 % of the electron density. Tl 0.30 MoO 3 shows a metal-to-semiconductor transition at 185 K, probably of Peierls type. We propose that the non-linear effects and metastability phenomena observed below 180 K, in the non-ohmic regime are very likely due to the depinning of a charge density wave, as it has been found in the blue bronze K 0.30 MoO 3

Semi-rational approach for converting a GH1 β-glycosidase into a β-transglycosidase

A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions, but little is known about what determines the balance between these two activities (transglycosylation/hydrolysis ratio). We previously obtained by directed evolution the mutants F401S and N282T of Thermus thermophilus β-glycosidase (Ttβ-gly, glycoside hydrolase family 1 (GH1)), which display a higher transglycosylation/hydrolysis ratio than the wild-type enzyme. In order to find the cause of these activity modifications, and thereby set up a generic method for easily obtaining transglycosidases from glycosidases, we determined their X-ray structure. No major structural changes could be observed which could help to rationalize the mutagenesis of glycosidases into transglycosidases. However, as these mutations are highly conserved in GH1 β-glycosidases and are located around the -1 site, we pursued the isolation of new transglycosidases by targeting highly conserved amino acids located around the active site. Thus, by single-point mutagenesis on Ttβ-gly, we created four new mutants that exhibit improved synthetic activity, producing disaccharides in yields of 68-90% against only 36% when native Ttβ-gly was used. As all of the chosen positions were well conserved among GH1 enzymes, this approach is most probably a general route to convert GH1 glycosidases into transglycosidases.

Correlation between thermostability and stability of glycosidases in ionic liquid

The activity and stability of a β-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][MeSO4] and 1,2,3-trimethylimidazolium methyl sulfate [TMIM][MeSO4]. 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 kcat was obtained. The results demonstrate that highly thermostable glycosidases are more suitable for biocatalytic reactions in water-miscible ILs.

Digital screening methodology for the directed evolution of transglycosidases

Engineering of glycosidases with efficient transglycosidases activity is an alternative to glycosyltransferases or glycosynthases for the synthesis of oligosaccharides and glycoconjugates. However, the engineering of transglycosidases by directed evolution methodologies is hampered by the lack of efficient screening systems for sugar-transfer activity. We report here the development of digital imaging-based high-throughput screening methodology for the directed evolution of glycosidases into transgalactosidases. Using this methodology, we detected transglycosidase mutants in intact Escherichia coli cells by digital imaging monitoring of the activation of non- or low-hydrolytic mutants by an acceptor substrate. We screened several libraries of mutants of beta-glycosidase from Thermus thermophilus using this methodology and found variants with up to a 70-fold overall increase in the transglycosidase/hydrolysis activity ratio. Using natural disaccharide acceptors, these transglycosidase mutants were able to synthesise trisaccharides, as a mixture of two regioisomers, with up to 76% yield.

A multi-channel bioluminescent bacterial biosensor for the on-line detection of metals and toxicity. Part I: design and optimization of bioluminescent bacterial strains

This study describes the construction of inducible bioluminescent strains via genetic engineering along with their characterization and optimization in the detection of heavy metals. Firstly, a preliminary comparative study enabled us to select a suitable carbon substrate from pyruvate, glucose, citrate, diluted Luria–Bertani, and acetate. The latter carbon source provided the best induction ratios for comparison. Results showed that the three constructed inducible strains, Escherichia coli DH1 pBzntlux, pBarslux, and pBcoplux, were usable when conducting a bioassay after a 14-h overnight culture at 30xa0°C. Utilizing these sensors gave a range of 12 detected heavy metals including several cross-detections. Detection limits for each metal were often close to and sometimes lower than the European standards for water pollution. Finally, in order to maintain sensitive bacteria within the future biosensor-measuring cell, the agarose immobilization matrix was compared to polyvinyl alcohol (PVA). Agarose was selected because the detection limits of the bioluminescent strains were not affected, in contrast to PVA. Specific detection and cross-detection ranges determined in this study will form the basis of a multiple metals detection system by the new multi-channel Lumisens3 biosensor.

Comparison of three microbial hosts for the expression of an active catalytic scFv

Antibodies represent an interesting protein framework on which catalytic functions can be grafted. In previous studies, we have reported the characterization of the catalytic antibody 4B2 obtained on the basis of the bait and switch strategy which catalyzes two different chemical reactions: the allylic isomerization of beta,gamma-unsaturated ketones and the Kemp elimination. We have cloned the antibody 4B2 and expressed it as a single-chain Fv (scFv) fragment in different expression systems, Escherichia coli and two yeasts species, in order to elicit the most suitable system to study its catalytic activity. The scFv4B2 was secreted as an active form in the culture medium of Pichia pastoris and Kluyveromyces lactis, which led respectively to 4 and 1.3mg/l after purification. In E. coli, different strategies were investigated to increase the cytoplasmic soluble fraction, which resulted, in all cases, in the expression of a low amount of functional antibodies. By contrast, substantial amount of scFv4B2 could be purified when it was expressed as inclusion bodies (12mg/l) and submitted to an in vitro refolding process. Its catalytic activity was measured and proved to be comparable to that of the whole IgG. However, the instability of the scFv4B2 in solution prevented from an exhaustive characterization of its activity and stabilization of this protein appears to be essential before designing strategies to improve its catalytic activity.