Thierry Maugard

Microwave-assisted synthesis of galacto-oligosaccharides from lactose with immobilized β-galactosidase from Kluyveromyces lactis

Galacto-oligosaccharides (GOS) were synthesized from lactose by immobilized and free β-galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP-G) using either focused microwave irradiation or conventional heating. Immobilization of the β-galactosidase on to Duolite A-568 increased the synthesis of GOS. GOS selectivity (GOS synthesis/lactose hydrolysis ratio) increased when the water activity of the media was reduced, notably with a high initial lactose concentration but also by using co-solvents in the media. The advantage of microwave heating on GOS formation was also examined. Addition of solvent and carrying out the reaction under microwave irradiation resulted an increase in the production of GOS. The selectivity for GOS synthesis can be increased by 217-fold under microwave irradiation, using immobilized β-glucosidase and with added co-solvents such as hexanol.

Study of vitamin ester synthesis by lipase-catalyzed transesterification in organic media.

Immobilized lipase from Candida antarctica (Novozym 435) was used in organic media to catalyze esterifications of vitamins (ascorbic acid and retinol) from hydroxy acid. We described the synthesis of retinyl L−lactate by transesterification between retinol and L−methyl lactate with yield reaching 90% and the synthesis of ascorbyl L−lactate by transesterification between ascorbic acid and L−methyl lactate with yield reaching 80%. The kinetic study of the esterification of vitamins with L−methyl lactate in organic media has been carried out and agrees with ping‐pong‐ordered Bi‐Bi when the initial vitamin concentration is low. When initial vitamin concentration is high, the kinetic is similar to a hybrid ping‐pong‐ordered Bi Bi or hybrid ping‐pong‐random Bi Bi mechanism. However, with high initial substrate concentration, change of the kinetic by other phenomena, such as interaction of substrates with molecular sieves, adsorption of the methanol formed, and decreases of substrate diffusion, could be considered. It is obvious that in these conditions, classical enzymology (i.e., Michaelian enzymology) cannot be used for the interpretation of results.

β-Galactosidase Catalyzed Selective Galactosylation of Aromatic Compounds

A new approach to galacto‐oligosaccharides and galacto‐conjugates synthesis performed by the β‐galactosidase from Kluyveromyces lactisis reported. The enzymatic galactosylation of eight kinds of adsorbed aromatic primary alcohols, in particular the two drugs guaifenesin and chlorphenesin, gave the corresponding β‐d‐galacto‐pyranosides in yields ranging between approximately 10% and 96%. For the first time, we have showed that the adsorption of acceptor substrates onto solid supports such as silica gel influences the yield and the selectivity of galacto‐conjugates synthesis. In particular, we observed that adsorption of acceptor favored the synthesis of digalactosylated compounds.

Synthesis of Water‐Soluble Retinol Derivatives by Enzymatic Method

Retinoids (vitamin A and derivatives) are of great commercial potential in cosmetics and pharmaceuticals such as skin care products. However, the clinical effectiveness of these retinoids is limited by skin irritation, water insolubility, and except for retinyl‐esters, extreme instability. In this paper, an enzymatic method for preparing water‐soluble retinol derivatives catalyzed by immobilized lipase is described. The synthesis is based on a unique strategy of two‐step enzymatic acylation. Among the different synthesized compounds, the most water‐soluble are the disaccharide derivatives such as saccharose retinyl adipate (nonionic water‐soluble retinol derivative) and the sodium salt of retinyl diacids such as retinyl succinate sodium salt (ionic water‐soluble retinol derivative).

Enzymatic synthesis of derivatives of vitamin A in organic media

The present article provides an enzymatic method of retinol esterification to reduce photodestruction and irritation problems characteristic of retinol. More specifically, it relates to a method of synthesising retinyl adipate, retinyl succinate, retinyl oleate and retinyl lactate greatly appreciated by cosmetic manufacturer. Desired compounds can be synthesised directly using Candida antarctica lipase and Rhizomucor miehei lipase in organic media.

β-Glucosidase-Catalyzed Hydrolysis of Indican from Leaves of Polygonum tinctorium

In this article, a HPLC method to identify and quantify the dyes and the indigo precursors produced in Polygonum tinctorium is described. Using this technique, indican has been positively identified in extracts of P. tinctorium. Our work with two cultivars of P. tinctorium has confirmed that the quantity of indican is dependent on the cultivars, harvest period, and age of the leaves. Two enzymes, Novozym 188 (cellobiase) and Novarom G ( β‐glucosidase), are compared on the basis of their activities to hydrolyze the indican at several pH values. We observed that Novarom G is more active than Novozym 188 whatever the pH and that optimum pH of both enzymes for indican hydrolysis is 3. Liberated indoxyl can be oxidized in alkaline media and transformed into indigo and indirubin.

Biochemical Composition and Changes of Extracellular Polysaccharides (ECPS) Produced during Microphytobenthic Biofilm Development (Marennes-Oléron, France)

The main goal of this work was to study the dynamics and biochemical composition of extracellular polysaccharides (ECPS), a fraction of the extracellular polymeric substances (EPS) produced during the development of a microphytobenthic biofilm in a European intertidal mudflat (Marennes-Oléron Bay, France) during winter. Microphytobenthic biomass was surveyed during four consecutive emersion periods to confirm the biofilm growth. Bacteria abundance was also checked considering the importance of heterotrophic bacteria observed by various authors in the dynamics of EPS. Various colorimetric assays, coupled to biochemical chromatographic analysis, were used to characterize the three main fractions of extracted EPS: colloidal, bound, and residual. The monosaccharide distribution of colloidal ECPS highlighted their role of carbon source for bacteria (>50% of glucose) even if no increase of colloidal carbohydrate amounts was observed during the tidal exposure. Bound ECPS were composed of deoxy or specific sugars (30% rhamnose) and uronic acids (18% galacturonic acid). Their levels and dynamics could be correlated to the development of the microphytobenthic biofilm, enhancing the stabilization of the sediment or increasing binding forces accordingly. Residual fractions, containing refractory bound ECPS and other internal polymeric substances, were composed of various carbohydrates. The high ratio of glucose in these fractions (18% to 43%) was interesting, as it was once attributed to colloidal sugars due to poor extraction procedures. Finally, the presence of inositol (15%) was significant since no author has highlighted it before, knowing that inositol is a major growth factor for heterotrophic bacteria.

A comparative study of the regioselectivity of the β-galactosidases from Kluyveromyces lactis and Bacillus circulans in the enzymatic synthesis of N-Acetyl-lactosamine in aqueous media

The enzymatic synthesis of N‐acetyl‐lactosamine (LacNAc) was studied in aqueous media with high substrate concentrations using the transgalactosylation of N‐acetyl‐D‐glucosamine (GlcNAc), starting from lactose as a galactosyl donor. The efficiency and regioselectivity of the β‐galactosidases from Kluyveromyces lactis (KlβGal) and Bacillus circulans (BcβGal) were compared. The reaction was optimized by varying the experimental conditions (pH, catalytic activity concentration, and mass concentration ratio of the substrates), which enhanced the synthesis yields with both enzymes and especially with BcβGal. BcβGal catalyzed the formation of the maximal LacNAc concentration obtained (101 mM or 39 g L−1, corresponding to a yield of 11% on the basis of GlcNAc conversion), after 5 h at pH 6.5 and for a substrate mass concentration ratio of 1. This enzyme also gave an optimal synthesis yield of about 17.5%. No change in regioselectivity was observed when using KlβGal, whereas the regioselectivity of BcβGal proved to be subject to variations, the 1–4 and 1–6 linkages being favored under kinetic and thermodynamic control conditions, respectively. Finally, it was demonstrated that the N‐acetyl‐allolactosamine synthesized during the GlcNAc transgalactosylation catalyzed by BcβGal was a thermodynamic product and did not result from a chemical and/or enzymatic isomerization of LacNAc.

Measuring Angiotensin-I Converting Enzyme Inhibitory Activity by Micro Plate Assays: Comparison Using Marine Cryptides and Tentative Threshold Determinations with Captopril and Losartan

To determine the angiotensin-I converting enzyme (ACE) inhibitory activity of marine cryptides, different methods were tested. ACE inhibition was measured using two synthetic substrates, (N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) and N-hippuryl-His-Leu hydrate salt (HHL)), and a natural one, angiotensin-I. The IC50 value (defined as the concentration of inhibitory molecule needed to inhibit 50% of the ACE activity) of the reference synthetic inhibitor captopril was in the nanomolar range (1.79-15.1 nM) when synthetic substrates were used, whereas it exhibited IC50 of micromolar range (16.71 μM) with angiotensin-I. We chose losartan, an antagonist of angiotensin-II receptor as negative control for the ACE inhibition. Losartan was also able to inhibit ACE whatever the substrate tested, with IC50 of micromolar range (17.13-146 μM). We defined this value as a limit above which molecules are not showing in vitro ACE inhibitory activity. Val-Trp (VW), Val-Tyr (VY), Lys-Tyr (KY), Lys-Trp (KW), Ile-Tyr (IY), Ala-Pro (AP), Val-Ile-Tyr (VIY), Leu-Lys-Pro (LKP), Gly-Pro-Leu (GPL), Ala-Lys-Lys (AKK), and Val-Ala-Pro (VAP) were tested as inhibitors of ACE with synthetic and natural substrates. IC50 displayed were substrate-dependent. With FAPGG as substrate, IW, VAP, KY, IY, AP, AKK, and VIY show IC50 values over the IC50 value of losartan and should not be considered as inhibitors of ACE. VY, VW, KW, and LKP exhibited IC50 value lower than the IC50 value of losartan for all substrates tested and were thus considered as good candidates for effectively decreasing hypertension. It appears that the comparison of IC50 is not consistent when IC50 values are obtained with different substrates and different methods. In vitro ACE inhibitory activity assays should always include various ACE substrates and references such as captopril and a negative control to obtain data reliable to discriminate ACE inhibitory peptides.

Role of water activity and temperature on activity and stability of dried whole cells of Saccharomyces cerevisiae in a continuous solid-gas bioreactor

Abstract The conversion of gaseous substrates by whole cells represents a new biotechnological concept with applications in gas–solid bioreactor. In this paper, the gas phase continuous production of hexanol from hexanal using dried baker’s yeast (Saccharomyces cerevisiae) was studied. Influence of temperature and water activity on reaction rate and stability of the biocatalyst was investigated. An increase of one of these parameters induces an increase in the reaction rate associated to a dramatic decrease in half-life time of the biocatalyst. Moreover, efficiency of ethanol, butanol, and pentanol have been compared for regeneration of the cofactor. It has been observed that initial rate of hexanal reduction decreases as the chain length of the alcohol used for regeneration increases. Efficiency in bioconversion ability is linked to the nature of the yeast used as catalyst.