Biosynthesis of ethyl caproate and other short ethyl esters catalyzed by cutinase in organic solvent

The main objective of this work was to study the enzymatic synthesis of short chain ethyl esters, a group of relevant aroma molecules, by Fusarium solani pisi cutinase in an organic solvent media (iso-octane), and to assess the influence of different parameters on the reaction yield.Cutinase displayed high initial esterification rates in iso-octane, which amounted to 1.15 μmol min⁻¹ mg⁻¹ for ethyl butyrate (C₄ acid chain) and 1.06 μmol min⁻¹ mg⁻¹ for ethyl valerate (C₅ acid chain). High product yields, 84% for ethyl butyrate and 96% for ethyl valerate, were observed after 6 h of reaction, for an initial equimolar concentration of substrates (0.1 M).The highest product yield (97%) was observed for ethyl caproate (C₆) synthesis, a compound which is a part of natural apple and pineapple flavour, for an alcohol:acid molar ratio of 2 (0.2 M ethanol concentration).Cutinase affinity for short chain length carboxylic acids (C₄–C₆) in ester synthesis in iso-octane confirmed previous observations in reversed micellar system.     

2,5-Diaryloxadiazoles and their precursors as novel inhibitors of cathepsins B,H and L

High levels of cathepsins indicated in various pathological conditions like arthritis, cancer progressions, and atherosclerosis explains the need to explore potential inhibitors of these proteases which can be of great therapeutic significance. We, in the present work, report the synthesis of some 2,5-diaryloxadiazoles from N-subsitutedbenzylidenebenzohydrazides. The synthesized compounds were screened for their inhibitory potential on cathepsins B, H and L. Structure Activity Relationship studies show that 2,5-diaryloxadiazoles were less inhibitory than their precursors. 1i and 2k have been found to be most inhibitory to cathepsins B and L. Their K_i values have been calculated as 11.38 × 10⁻⁸ M and 66.4 × 10⁻⁸ M for cathepsin B and 4.2 × 10⁻⁹ M and 47.31 × 10⁻⁹ M for cathepsin L, respectively. However, cathepsin H activity was maximally inhibited by compounds, 1e and 2c with K_i values of 4.4 × 10⁻⁷ M and 5.6 × 10⁻⁷ M, respectively. Enzyme kinetic studies suggest that these compounds are competitive inhibitors to the enzymes. The results have been compared with docking results obtained using iGemDock.     

Optimization studies to develop a low-cost medium for production of the lipases of Rhizopus microsporus by solid-state fermentation and scale-up of the process to a pilot packed-bed bioreactor

A low-cost lipase preparation is required for enzymatic biodiesel synthesis. One possibility is to produce the lipase in solid-state fermentation (SSF) and then add the fermented solids (FS) directly to the reaction medium for biodiesel synthesis. In the current work, we scaled up the production of FS containing the lipases of Rhizopus microsporus. Initial experiments in flasks led to a low-cost medium containing wheat bran and sugarcane bagasse (50:50 w/w, dry basis), supplemented only with urea. We used this medium to scale-up production of FS, from 10 g in a laboratory column bioreactor to 15 kg in a pilot packed-bed bioreactor. This is the largest scale yet reported for lipase production in SSF. During scale-up, the hydrolytic activity of the FS decreased 57%: from 265 U g⁻¹ at 18 h in the laboratory bioreactor to 113 U g⁻¹ at 20 h in the pilot bioreactor. However, the esterification activity decreased by only 14%: from 12.1 U g⁻¹ to 10.4 U g⁻¹. When the FS produced in the laboratory and pilot bioreactors were dried and added directly to a solvent-free reaction medium to catalyze the esterification of oleic acid with ethanol, both gave the same ester content, 69% in 48 h.     

Novel S-enantioselective lipase TALipB from Trichosporon asahii MSR54: Heterologous expression,characterization, conformational stability and homology modeling

A novel lipase encoding gene, TALipB from Trichosporon asahii MSR54 was heterologously expressed in Escherichia coli using three vectors, pET22b, pET28a & pEZZ18. The three recombinant proteins, viz. C-hexahistidine fused HLipB, N and C-hexahistidine fused HLipBH and ZZ-fused ZZLipB were purified using affinity chromatography. All the three enzymes were mid to long fatty acyl chain selective on p-NP esters and S-enantioselective irrespective of tags. HLipB had lowest activation energy (3.5 Kcal mol⁻¹) and highest catalytic efficiency (254 mM⁻¹ min⁻¹) on p-NP caprate followed by HLipBH and ZZLipB. However, ZZLipB demonstrated best pH stability (pH 6–10), thermostability (t_1/2 of 50 min at 70 °C) and stability toward the denaturant Guanidium chloride (300 mM). Far-UV CD and fluorescence studies confirmed the role of N-terminal ZZ-tag in stabilizing the protein by altering its secondary and tertiary structures. All the three proteins were thiol activated. ZZLipB required higher concentration of β-mercaptoethanol as compared to the other two proteins to attain similar velocity. This indicated the involvement of additional disulfide bonds in its conformational stability. In silico analysis suggested low sequence identity of the enzyme with the available database but a close structural homology with Candida antarctica lipase B (CALB) was revealed by PHYRE². MULTALIN with CALB predicted the active site residues (Ser137–Asp228–His261) which were confirmed by superimposition and site directed mutagenesis.     

Straightforward entry to pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones and their ADME properties

A straightforward synthesis of pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones was developed starting from 2-chloropyridine-3-carboxylic acid by esterification, nucleophilic aromatic substitution and amide formation in one step, and ring closure allowing their synthesis with two identical or two different group attached to nitrogen. The structural diversity of these [2,3-d]pyrimidine-2,4(1H,3H)-diones resulted in significant variation in the biopharmaceutical properties. This was reflected by the broad range in fasted state simulated intestinal fluid solubility values (12.6 μM to 13.8 mM), Caco-2 permeability coefficients (1.2 × 10⁻⁶ cm/s to 90.7 × 10⁻⁶ cm/s) and in vitro-predicted human in vivo intrinsic clearance values (0 to 159 ml/min/kg).     

Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.)

β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p-nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a V_max of 131.6 μmol min⁻¹ mg⁻¹ protein, K_m of 0.158 mM, K_cat of 144.8 s⁻¹, K_cat/K_m of 917.4 mM⁻¹ s⁻¹, and K_m/V_max of 0.0012 mM min mg μmole⁻¹, using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10⁻⁵ s⁻¹, and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material.     

Immobilization of lipase in organic solvent in the presence of fatty acid additives

In this study porcine pancreatic lipase (PPL) was covalently immobilized on cross-linked polyvinyl alcohol (PVA) in organic media in the presence of fatty acid additives in order to improve its immobilized activity. The effects of fatty acid additions to the immobilization media were investigated choosing tributyrin hydrolysis in water and ester synthesis by immobilized PPL in n-hexane. Various fatty acids which are also the substrates of lipases in esterification reactions were used as active site protecting agents during the immobilization process in an organic solvent. The obtained results showed that covalent immobilization carried out in the presence of fatty acids as protective ligands improved the hydrolytic and esterification activity of immobilized enzyme. A remarkable increase in activity of the immobilized PPL was obtained when octanoic acid was used as an additive and the hydrolytic activity was increased from 5.2 to 19.2 μmol min⁻¹ mg⁻¹ as compared to the non-additive immobilization method. With the increase of hydrolytic activity of immobilized lipase in the presence of octanoic acid, in an analogous manner, the rate of esterification for the synthesis of butyl octanoate was also increased from 7.3 to 26.3 μmol min⁻¹ g⁻¹ immobilized protein using controlled thermodynamic water activities with saturated salt solutions. In addition, the immobilized PPL activity was maintained at levels representing 63% of its original activity value after 5 repeated uses. The proposed method could be adopted for a wide variety of other enzymes which have highly soluble substrates in organic solvent such as other lipases and esterases.     

A new bioprocess for the production of prebiotic lactosucrose by an immobilized β-galactosidase

A new bioprocess for the synthesis of lactosucrose was studied using a covalently immobilized β-galactosidase on macrospheres of chitosan. The effects of temperature and pH on the production of lactosucrose and other oligosaccharides were evaluated. At 30 °C and pH 7.0, the maximum concentration of lactosucrose reached to 79 g L⁻¹. The change of the reaction conditions allowed to modify the qualitative profile of the final products without quantitative change in the total of oligosaccharides produced. At pH 7 and 30 °C, products profile was 79 g L⁻¹ of lactosucrose, 37 g L⁻¹ of galactooligosaccharides and 250 g L⁻¹ of total oligosaccharides, while at pH 5 and 64 °C the concentrations for the same compounds were 40, 62 and 250 g L⁻¹, respectively. The immobilization increased the thermal stability up to 260-fold. Using 300 g L⁻¹ of sucrose and 300 g L⁻¹ of lactose, and 8.5 mg of chitosan mL⁻¹, 30 cycles of reuse were performed and the biocatalyst kept the maximal lactosucrose synthesis. These results fulfill some important aspects for the enzyme immobilization and oligosaccharides synthesis: the simplicity of the protocols, the high operational stability of the enzyme and the possibility of driving the final products.     

Effect of high salt concentrations on the stability of immobilized lipases: Dramatic deleterious effects of phosphate anions

We have analyzed the effects of the buffer nature on the stability of immobilized lipases. Commercial phospholipase Lecitase Ultra (LU), lipase B from Candida antarctica (CALB) and lipase from Thermomyces lanuginosus (TLL) have been immobilized on octyl-glyoxyl agarose beads. The enzymes were readily inactivated using 4 M sodium phosphate but 6 M NaCl did not inactivate them. Using 2 M of sodium phosphate, the inactivation of the 3 immobilized enzymes still was very significant even at 25 °C but at lower rate than with higher phosphate concentration. Thermal stress inactivations of the immobilized enzymes revealed that even 100 mM sodium phosphate produced a significant decrease in enzyme stability; this effect was less pronounced for Lecitase but dramatic for CALB. While 6 M NaCl presented slightly positive (LU) or negative (TLL) effects on their thermal stabilities of, CALB was thermally stabilized under the same conditions. Results were very different using free enymes. Fluorescence spectroscopy revealed dramatic structural rearrangements of the immobilized enzymes in the presence of high phosphate concentration. From these results, the use of sodium phosphate does not seem to be recommended for studies on thermal stability of lipases, although this should be verified for each enzyme and immobilized preparation.     

A new type of neuron-specific aminopeptidase NAP-2 in rat brain synaptosomes

A novel neutral aminopeptidase (NAP-2) was found exclusively in the rat central nervous system (CNS). It was separated from the ubiquitous puromycin-sensitive aminopeptidase (PSA) and the neuron-specific aminopeptidase (NAP) by an automated FPLC-aminopeptidase analyzer. The activity of the neuronal aminopeptidase enriched in the synaptosomes is different from NAP and PSA in distribution and during brain development. The enzyme was purified 2230-fold to apparent homogeneity from rat brain cytosol with 4% recovery by ammonium sulfate fractionation, followed by column chromatography successively on Phenyl-Sepharose, Q-Sepharose, Sephadex G-200, and Mono Q. The single-chain enzyme with a molecular mass of 110 kDa has an optimal pH of 7.0 and a pI of 5.6. It splits β-naphthylamides of amino acid with aliphatic, polar uncharged, positively charged, and aromatic side chain. Leucyl β-naphthylamide (Leu βNA) is the best substrate with the highest hydrolytic coefficiency followed by Met βNA = Arg βNA = Lys βNA > Ala βNA > Tyr βNA > Phe βNA. The cysteine-, metallo-, glyco-aminopeptidase releases the N-terminal Tyr from Leu-enkephalin with a K_m 82 μM and a k_cat of 1.08 s⁻¹, and Met-enkephalin with a K_m of 106 μM and a k_cat of 2.6 s⁻¹. The puromycin-sensitive enzyme is most susceptible to amastatin with an IC₅₀ of 0.05 μM. The data indicate that the enzyme is a new type of NAP found in rodent. Its possible function in neuron growth, neurodegeneration, and carcinomas is discussed.     

Synthesis,anti-inflammatory activity and modeling studies of cycloartane-type terpenes derivatives isolated from Parthenium argentatum

The 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema model in mice determined the anti-inflammatory activities in vivo of argentatins A, B and D, the main cycloartenol-type triterpenes present in Parthenium argentatum. Our results showed that argentatin B (ED₅₀ = 1.5 × 10⁻⁴ mmol/ear) and argentatin A (ED₅₀ = 2.8 × 10⁻⁴ mmol/ear) were more potent anti-inflammatory agents than indomethacin (ED₅₀ = 4.5 × 10⁻⁴ mmol/ear), the reference drug. Based on these findings, we decided to evaluate 13 derivatives of argentatins A and B. All the derivatives showed anti-inflammatory activity in the TPA-induced edema model in mice. The most active compound was 25-nor-cycloart-3, 16-dione-17-en-24-oic acid, obtained from argentatin A (ED₅₀ = 1.4 × 10⁻⁴ mmol/ear). Argentatin B was assayed as inhibitor of COX-2 activity one of the key enzymes involved in the TPA assay. The results showed that argentatin B at 15 μM doses inhibited 77% COX-2 activity. Docking studies suggest that argentatin B interacts with Arg 120, a key residue for COX-2 activity.     

Osmoregulation in Dunaliella,Part II: Photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta

In response to an osmotic stress, Dunaliella tertiolecta osmoregulates by metabolizing intracellular glycerol as compatible solute. Upon the application of a salt stress to 0.17 M or 0.7 M NaCl grown D. tertiolecta cells, rates of total glycerol synthesis were substantially higher than that arising from photosynthetic ¹⁴CO₂ fixation into glycerol. The source of this extra carbon is the reserve starch pool. The contribution of carbon from the starch breakdown to glycerol synthesis was estimated from the difference between the total glycerol synthesized and that arising from ¹⁴CO₂ fixation. The maximum observed flux of carbon from ¹⁴CO₂ to glycerol from photosynthesis was of the order of 15–20 μmol ¹⁴C-glycerol mg⁻¹ Chl h⁻¹, whereas the total glycerol synthesis reached about 70 μmol glycerol mg⁻¹ Chl h⁻¹. The contribution of products of starch breakdown to glycerol synthesis increased progressively with increasing salt stress. In light, contrary to prevailing assumptions, both the photosynthesis and the starch breakdown contribute carbon to glycerol biosynthesis. The relative contributions of these two processes in the light, while cells were actively photosynthesizing, depended on the magnitude of the salt stress. On application of dilution stress, the flux of carbon from newly photosynthetically fixed ¹⁴CO₂ into glycerol was reduced progressively with increasing dilution stress that was also accompanied by a decline in total glycerol contents of the cell. The maximum observed rate of glycerol dissimilation was about 135 μmol glycerol mg⁻¹ Chl h⁻¹.     

Immobilized Candida antarctica lipase B on ZnO nanowires/macroporous silica composites for catalyzing chiral resolution of (R,S)-2-octanol

ZnO nanowires were successfully introduced into a macroporous SiO₂ by in situ hydrothermal growth in 3D pores. The obtained composites were characterized by SEM and XRD, and used as supports to immobilize Candida antarctica lipase B (CALB) through adsorption. The high specific surface area (233 m²/g) and strong electrostatic interaction resulted that the average loading amount of the composite supports (196.8 mg/g) was 3–4 times of that of macroporous SiO₂ and approximate to that of a silica-based mesoporous material. Both adsorption capacity and the activity of the CALB immobilized on the composite supports almost kept unchanged as the samples were soaked in buffer solution for 48 h. The chiral resolution of 2-octanol was catalyzed by immobilized CALB. A maximum molar conversion of 49.1% was achieved with 99% enantiomeric excess of (R)-2-octanol acetate under the optimal condition: a reaction using 1.0 mol/L (R,S)-2-octanol, 2.0 mol/L vinyl acetate and 4.0 wt.% water content at 60 °C for 8 h. After fifteen recycles the immobilized lipase could retain 96.9% of relative activity and 93.8% of relative enantioselectivity.     

Isolation of hexavalent chromium resistant bacteria from industrial saline effluents and their ability of bioaccumulation

The mixed cultures has been isolated from industrial saline wastewater contaminated with chromium(VI), using enrichment in the presence of 50 mg l⁻¹ chromium(VI) and 4% (w/v) NaCl at pH 8. In this study, the molasses (M) medium was selected a suitable medium for the effective chromium bioaccumulation by the mixed cultures. Eleven pure isolates obtained from mixed cultures and some of them showed high bioaccumulation in the M media containing about 100 mg l⁻¹ chromium(VI) and 4% NaCl. The strain 8 (99.3%) and 10 (99.1%) were able to bioaccumulate more efficient than the mixed culture (98.9%) in this media. But the highest specific Cr uptake was obtained by the mixed cultures followed by strain 8 and 10 with 56.71, 33.14 and 21.7 mg g⁻¹, respectively. Bioaccumulation of chromium(VI) ions by the strain 8 growing in the media with chromium(VI) and NaCl was studied in a batch system as a function of initial chromium(VI) (86.6–547.6 mg l⁻¹) and NaCl (0, 2, 4, 6% w/v) concentrations. During all the experiments, the uptake yield of the strain 8 was highly affected from NaCl concentrations in the medium at high initial chromium(VI) concentrations. But at low chromium(VI) concentration, strain 8 was not affected from NaCl concentrations in the medium. The maximum uptake yield were obtained in the M media with 2% NaCl as 98.8% for 110.0 mg l⁻¹, 98.6% for 217.1 mg l⁻¹, 98.6% for 381.7 mg l⁻¹ and 98.2% for 547.6 mg l⁻¹ initial chromium(VI) concentrations. The strain 8 tolerated a 6% (w/v) NaCl concentration was able to bioaccumulate more than 95% of the applied chromium(VI) at the 97.6–224.4 mg l⁻¹ initial chromium(VI) concentrations. The results presented in this paper was shown that these pure and mixed cultures might be of use for the bioaccumulation of chromium(VI) from saline wastewater.     

Enhancement of polysaccharide production in suspension cultures of protocorm-like bodies from Dendrobium huoshanense by optimization of medium compositions and feeding of sucrose

A strategy that optimization of medium compositions for maximum biomass followed by feeding of sucrose for maximum polysaccharide synthesis was developed for enhancing polysaccharide production in suspension culture of protocorm-like bodies (PLBs) of Dendrobium huoshanense C.Z. Tang et S.J. Cheng. In growth stage, the original half-strength MS medium was optimized with carbon sources, nitrogen sources and metal ion combinations. The effects of different carbon sources on PLBs growth were remarkable and sucrose at 35 g l⁻¹ was the most suitable. Sole nitrate nitrogen of 30 mmol l⁻¹ was the best for PLBs growth. Metal ions (Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺) showed different influences on PLBs growth. The optimal concentration of Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ was 4.5 mmol l⁻¹, 0.1 mmol l⁻¹, 0.5 mmol l⁻¹ and 0.06 mmol l⁻¹, respectively. In the optimized medium (sucrose, nitrate, Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ concentration as described above, the other component concentration seen in half-strength MS), 33.9 g DW l⁻¹ PLBs were harvested after 30 days of culture and biomass increase was improved 245% as compared with that in the original medium. In production stage, polysaccharide synthesis was significantly improved by the feeding sucrose. The maximum polysaccharide production (22 g l⁻¹) was obtained in the case of 50 g l⁻¹ sucrose feeding at day 30 of culture, which was about 109-fold higher than that in the original medium without feeding of sucrose.     

Multiple effects of anthracene-9-carboxylic acid on the TMEM16B/anoctamin2 calcium-activated chloride channel

Ca^2 +-activated Cl⁻ currents (CaCCs) play important roles in many physiological processes. Recent studies have shown that TMEM16A/anoctamin1 and TMEM16B/anoctamin2 constitute CaCCs in several cell types. Here we have investigated for the first time the extracellular effects of the Cl⁻ channel blocker anthracene-9-carboxylic acid (A9C) and of its non-charged analogue anthracene-9-methanol (A9M) on TMEM16B expressed in HEK 293T cells, using the whole-cell patch-clamp technique. A9C caused a voltage-dependent block of outward currents and inhibited a larger fraction of the current as depolarization increased, whereas the non-charged A9M produced a small, not voltage dependent block of outward currents. A similar voltage-dependent block by A9C was measured both when TMEM16B was activated by 1.5 and 13 μM Ca^2 +. However, in the presence of 1.5 μM Ca^2 + (but not in 13 μM Ca^2 +), A9C also induced a strong potentiation of tail currents measured at − 100 mV after depolarizing voltages, as well as a prolongation of the deactivation kinetics. On the contrary, A9M did not produce potentiation of tail currents, showing that the negative charge is required for potentiation. Our results provide the first evidence that A9C has multiple effects on TMEM16B and that the negative charge of A9C is necessary both for voltage-dependent block and for potentiation. Future studies are required to identify the molecular mechanisms underlying these complex effects of A9C on TMEM16B. Understanding these mechanisms will contribute to the elucidation of the structure and functional properties of TMEM16B channels.     

Salt effects on β-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl⁻ salts) the effectiveness follows the series: Cu⁺², Fe⁺² > Zn⁺² > Li⁺ > Ca⁺² > Mg⁺² > Cs⁺ > NH₄⁺ > Rb⁺ > K⁺ > Na⁺ and for anions (Na⁺ salts) the series is: I⁻ > ClO₄⁻ > ⁻SCN > Br⁻ ≈ NO₃⁻ > Cl⁻ ≈ ⁻OAc > F⁻ ≈ SO₄^− 2. The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k_cat/K_m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pK_as and not to an effect on the pH-independent second-order rate constant, (k_cat/K_m)^lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pK_a1 increases (from 3.7 to 4.9) and the apparent pK_a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (k_cat/K_m)^lim (= 9 × 10⁴ M^− 1 s^− 1) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pK_as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pK_a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pK_a of 3.7 and dissociated from the group with a pK_a of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two pK_as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (k_cat/K_m)^lim, of the monoprotonated species. For other enzymes which may show such salt-induced pK_a shifts, this provides a convenient test for the role of reverse protonation.     

N − 3PUFA differentially modulate palmitate-induced lipotoxicity through alterations of its metabolism in C2C12 muscle cells

Excessive energy intake leads to fat overload and the formation of lipotoxic compounds mainly derived from the saturated fatty acid palmitate (PAL), thus promoting insulin resistance (IR) in skeletal muscle. N − 3 polyunsaturated fatty acids (n − 3PUFA) may prevent lipotoxicity and IR. The purpose of this study was to examine the differential effects of n − 3PUFA on fatty acid metabolism and insulin sensitivity in muscle cells. C2C12 myotubes were treated with 500 μM of PAL without or with 50 μM of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for 16 h. PAL decreased insulin-dependent AKT activation and glucose uptake and increased the synthesis of ceramides and diglycerides (DG) derivatives, leading to protein kinase Cθ activation. EPA and DHA, but not ALA, prevented PAL-decreased AKT activation but glucose uptake was restored to control values by all n − 3PUFA vs. PAL. Total DG and ceramide contents were decreased by all n − 3PUFA, but only EPA and DHA increased PAL β-oxidation, decreased PAL incorporation into DG and reduced protein kinase Cθ activation. EPA and DHA emerge as better candidates than ALA to improve fatty acid metabolism in skeletal muscle cells, notably via their ability to increase mitochondrial β-oxidation.     

Synthesis of benzyl β-d-galactopyranoside by transgalactosylation using a β-galactosidase produced by the over expression of the Kluyveromyces lactis LAC4 gene in Arxula adeninivorans

The LAC4 gene of Kluyveromyces lactis encoding for β-galactosidase was overexpressed in the yeast Arxula adeninivorans to produce the enzyme, which can be used for the synthesis of β-d-galactosides. These compounds play a major role as precursors for the synthesis of glycolipids and glycoproteins in medicine or for the production of tensides.The Xplor^®2 transformation/expression platform was used because it enabled stable integration of the gene in the Arxula genome and the production of high levels of the enzyme. The recombinant β-galactosidase, fused with C-terminal His-tag region (Lac4-6hp), was purified by precipitation with ammonium sulphate and FPLC using hydroxylapatite. The enzyme exhibited optimal activity at 37 to 40 °C, pH 6.5 in 50 mM sodium acetate buffer. Activity was measured by the formation of p-nitrophenol at 405 nm from the hydrolyzed chromogenic substrate, p-nitrophenyl-β-d-gal. Biochemical characterization included the calculation of K_M and apparent k_cat values of the enzyme. The formation of benzyl β-d-gal by 0.1 U enzyme from A. adeninivorans with transgalactosylation was six times higher than that for the prokaryotic enzyme from E. coli. Moreover, the partially purified enzyme was used for the selective hydrolysis of allyl β-d-gal in a mixture of allyl β- and allyl α-d-gal, with 4 g l⁻¹ being hydrolysed within one day by 1 U ml⁻¹. Thus, the recombinant β-galactosidase produced in A. adeninivorans is of potential interest for the enzymatic synthesis of benzyl β-d-gal and other galactosides as well as the selective hydrolysis of anomeric mixtures and could be used to replace difficult chemical procedures.