Efficient production of glycyrrhetic acid 3-O-mono-β-d-glucuronide by whole-cell biocatalysis in an ionic liquid/buffer biphasic system

Hydrolysis of glycyrrhizin (GL) to glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG) by whole-cell biocatalysts in a system containing non-conventional solvents was performed. Three whole-cell biocatalysts were used, including wild-type Penicillium purpurogenum Li-3 (w-PGUS) and recombinant strains Escherichia coli BL21 and Pichia pastoris GS115. The biotransformation of GL to GAMG by w-PGUS in a 1-butyl-3-methylimidazolium hexafluorophosphate ([Bmim]PF₆)/buffer biphasic system was the main focus of this study because w-PGUS showed a higher GAMG yield and a higher relative activity in this system than the other two whole-cell biocatalysts. Using the optimized reaction conditions determined as a pH 5.2 buffer, a 6.0 mM substrate concentration, a reaction temperature of 30 °C, and a 60 g/L (1.23 U/g) cell concentration, a GAMG yield of 87.63% was achieved after 60 h. After eight reaction cycles, [Bmim]PF₆ retained a high recovery percentage (85.48%)_[0], indicating the reusability of this IL. The biotransformation activity of w-PGUS was not significantly affected, even after two batch reaction cycles. Furthermore, the product GAMG and the byproduct glycyrrhetinic acid were spontaneously separated in the biphasic system. In conclusion, the combination of whole cells and ionic liquid is a promising approach for economical and industrial-scale production of GAMG.     

Asymmetric synthesis of (S)-3-chloro-1-phenyl-1-propanol using Saccharomyces cerevisiae reductase with high enantioselectivity

3-Chloro-1-phenyl-1-propanol is used as a chiral intermediate in the synthesis of antidepressant drugs. Various microbial reductases were expressed in Escherichia coli, and their activities toward 3-chloro-1-phenyl-1-propanone were evaluated. The yeast reductase YOL151W (GenBank locus tag) exhibited the highest level of activity and exclusively generated the (S)-alcohol. Recombinant YOL151W was purified by Ni-nitrilotriacetic acid (Ni-NTA) and desalting column chromatography. It displayed an optimal temperature and pH of 40°C and 7.5–8.0, respectively. The glucose dehydrogenase coupling reaction was introduced as an NADPH regeneration system. NaOH solution was occasionally added to maintain the reaction solution pH within the range of 7.0–7.5. By using this reaction system, the substrate (30 mM) could be completely converted to the (S)-alcohol product with an enantiomeric excess value of 100%. A homology model of YOL151W was constructed based on the structure of Sporobolomyces salmonicolor carbonyl reductase (Protein Data Bank ID: 1Y1P). A docking model of YOL151W with NADPH and 3-chloro-1-phenyl-1-propanone was then constructed, which showed that the cofactor and substrate bound tightly to the active site of the enzyme in the lowest free energy state and explained how the (S)-alcohol was produced exclusively in the reduction process.     

Two new isodrimene sesquiterpenes from the fungal culture broth of Polyporus arcularius

Two new isodrimene sesquiterpene derivatives, 2(S)-hydroxyalbicanol (1, =(2S,4aS,8S,8aS)-8-(hydroxymethyl)-4,4,8a-trimethyl-7-methylenedecahydronaphthalen-2-ol) and 2(S)-hydroxyalbicanol 11-acetate (2, =((1S,4aS,7S,8aS)-7-hydroxy-5,5,8a-trimethyl-2-methylenedecahydronaphthalen-1-yl)methyl acetate) were isolated from the culture broth of the fungus Polyporus arcularius, together with two phenylpropanediols, (1S,2S)- and (1R,2S)-1-phenyl-1,2-dihydroxypropane (3, 4). Compound 3 is reported as a naturally occurring compound for the first time. The structures of the compounds were elucidated on the basis of spectroscopic analysis. Compound 1 exhibited growth inhibition of lettuce seedlings with IC₅₀ values of 1.3 mM to hypocotyl and 1.7 mM to radicle.     

Extracting keratin from chicken feathers by using a hydrophobic ionic liquid

Keratin was extracted from chicken feathers by using a hydrophobic ionic liquid (IL), 1-hydroxyethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)amide ([HOEMIm][NTf₂]). Extracted keratin has good solubility in water while the ionic liquid is immiscible with water, and therefore the extracted keratin could be easily separated from the reaction system by water. The effects of ionic liquid, NaHSO₃, reaction temperature and time were investigated and extracting conditions were optimized. The maximum yield of keratin was up to about 21% with mass ratio of feathers to NaHSO₃1:1 and mass ratio of feathers to ionic liquid 1:40 at 80 °C for 4 h. Moreover, there was no obvious loss in the yield after ionic liquid was reused for five batches under optimized conditions. In addition, the recovery of ionic liquid was about 95% each time. The results indicated that [HOEMIm][NTf₂] was very efficient as catalyst and solvent for dissolving feathers and could be easily recovered due to its hydrophobicity.     

Chemical modifications by ionic liquid-inspired cations improve the activity and the stability of formate dehydrogenase in [MMIm][Me2PO4]

The formate dehydrogenase (FDH, EC: 1.2. 1.2) from Candida boidinii was found to be inactivated and unstable in the presence of high concentration (>50%) of the water soluble dimethylimidazolium dimethyl phosphate ([MMIm][Me₂PO₄]) ionic liquid. In order to circumvent this problem, the enzyme was chemically modified by cations usually present in ionic liquids: cholinium (1), hydroxyethyl-methylimidazolium (2) and hydroxypropyl-methylimidazolium (3) cations were activated with carbonyldiimidazole before being reacted with the FDH leading to a heterogeneous population of 6–7 biocatalysts. FDH modified by (1) or (3) led to 3–9 modifications while FDH modified by (2) led to 6 proteins presenting 7–12 grafted cations. Specific activity of the modified enzymes was decreased by a 2.5–3-fold factor (0.10–0.15 μmol min⁻¹ mg⁻¹) compared to the non-modified FDH (0.33 μmol min⁻¹ mg⁻¹) when assayed in carbonate buffer (pH 9.7, 25 mM). After modification, the FDH still present 0.06 μmol min⁻¹ mg⁻¹ in 70% [MMIm][Me₂PO₄] (v:v) (30–45% of their activity in aqueous buffer) while the native enzyme is inactive at this ionic liquid concentration, proving the efficiency of this strategy. The half-life of the modified enzyme is also increased by a 5-fold factor after modification by (1) (t_1/2 of 9 days) and by a 3-fold factor after modification by (2) or (3) (t_1/2 of 6 and 5 days respectively) in aqueous solution. When stored in 37.5% [MMIm][Me₂PO₄] (v:v), both modified and unmodified FDH have an increased half-life (t_1/2 of 6–9 days). This grafting strategy is found to be good methods to mimic and study the stabilizing effect of ionic liquids on enzymes.     

Cholinium carboxylate ionic liquids for pretreatment of lignocellulosic materials to enhance subsequent enzymatic saccharification

The present study is the first report demonstrating that ionic liquids consisting of cholinium cations and linear carboxylate anions ([Ch][CA] ILs) can be used for pretreatment of lignocellulosic materials to enhance subsequent enzymatic saccharification. Six variants of [Ch][CA] ILs were systematically prepared by combining cholinium cations with linear monocarboxylate anions ([C_nH_2n+1–COO]⁻, n = 0–2) or dicarboxylate anions ([HOOC–C_nH_2n+1–COO]⁻, n = 0–2). These [Ch][CA] ILs were analyzed for their toxicity to yeast cell growth and their ability to pretreat kenaf powder for subsequent enzymatic saccharification. When assayed against yeast growth, the EC₅₀ for choline acetate ([Ch][OAc]) was 510 mM, almost one order of magnitude higher than that for 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]). The cellulose saccharification ratio after pretreatment at 110 °C for 16 h with [Ch][OAc] (100.6%) was almost comparable with that after pretreatment with [Emim][OAc]. Therefore, [Ch][OAc] is a biocompatible alternative to [Emim][OAc] for lignocellulosic material pretreatment.     

Chemoenzymatic synthesis of (S)-duloxetine using carbonyl reductase from Rhodosporidium toruloides

A chemoenzymatic strategy was developed for (S)-duloxetine production employing carbonyl reductases from newly isolated Rhodosporidium toruloides into the enantiodetermining step. Amongst the ten most permissive enzymes identified, cloned, and overexpressed in Escherichia coli, RtSCR9 exhibited excellent activity and enantioselectivity. Using co-expressed E. coli harboring both RtSCR9 and glucose dehydrogenase, (S)-3-(dimethylamino)-1-(2-thienyl)-1-propanol 3a was fabricated with so far the highest substrate loading (1000 mM) in a space-time yield per gram of biomass (DCW) of 22.9 mmol L⁻¹ h⁻¹ g DCW⁻¹ at a 200-g scale. The subsequent synthetic steps from RtSCR9-catalyzed (S)-3a were further performed, affording (S)-duloxetine with 60.2% overall yield from 2-acethylthiophene in >98.5% ee.     

Biosynthesis of glycyrrhetic acid 3-O-mono-β-d-glucuronide catalyzed by β-d-glucuronidase with enhanced bond selectivity in an ionic liquid/buffer biphasic system

The bond selective hydrolysis of glycyrrhizin (GL) to glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG) catalyzed by recombinant β-d-glucuronidase from Escherichia coli BL21 (PGUS-E) was successfully performed in an ionic liquid (IL)/buffer biphasic system. Five ILs were analyzed, however, a hydrophobic IL 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) showed the best biocompatibility with PGUS-E. An obvious enhancement in the initial reaction rate, substrate conversion, GAMG yield and chemical bond selectivity (S_cb) was observed using 40% (v/v) [BMIM]PF₆/buffer as the reaction medium when compared to the acetate buffer medium. Under the optimized conditions (pH 6.0, temperature 50 °C, substrate concentration 6 mM and shaking speed 200 rpm), the initial reaction rate, the GAMG yield and the S_cb reached 3.15 mM h⁻¹, 74.36% and 98.12%, respectively. The recyclability of [BMIM]PF₆ was also studied and found to be reusable for five batches with high recovery percentage (≥92%). Furthermore, the desired product and byproduct were easily separated since they were distributed in different phases. Additionally, higher V_max (3.14 versus 2.24 mM h⁻¹), lower apparent K_m (1.21 versus 1.80 mM) and E_a (25.97 versus 32.60 kJ mol⁻¹) were achieved in [BMIM]PF₆/buffer biphasic system than that in monophasic buffer system.     

Asymmetric reduction of (4S)-(+)-carvone catalyzed by baker's yeast: A green method for monitoring the conversion based on liquid–liquid–liquid microextraction with polypropylene hollow fiber membranes

The α,β-unsaturated carbonyl compound (4S)-(+)-carvone was selectively reduced to (1R,2R,4S)-iso-dihydrocarveol using baker's yeasts. The conversion of the bioreduction reaction was monitored using a green hollow-fiber liquid–liquid–liquid microextraction (HF-LLLME) technique. Several parameters which may affect the bioreduction of (4S)-(+)-carvone, such as temperature, time, substrate/enzyme ratio, pH and buffer concentration, were evaluated. The effect of some additives, such as trehalose, DMSO and the ionic liquid [BMIm][PF₆], was also studied. The (1R,2R,4S)-iso-dihydrocarveol was recovered with 52.7% conversion and diastereoisomeric excess >99% after 48 h of reaction at 40 °C in an aqueous monophasic system, with 0.1 mol L^?1 buffer concentration (pH 7.5) and a substrate/yeast cell mass ratio of 8.0 mg g^?1. The HF-LLLME microextraction technique allowed the optimization of the reaction with a reduction of over 99.5% in relation to the use of organic solvents.     

Efficient production of (R)-(−)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp.

For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli. After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of (R)-(−)-mandelic acid after only 4 h of transformation reached 197 mM with an enantiomeric excess (ee_p) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure (R)-(−)-mandelic acid.     

Enantioselective synthesis of (S)-phenylephrine by whole cells of recombinant Escherichia coli expressing the amino alcohol dehydrogenase gene from Rhodococcus erythropolis BCRC 10909

(R)-phenylephrine [(R)-PE] is an α₁-adrenergic receptor agonist that is widely used in over-the-counter drugs to treat the common cold. We found that Rhodococcus erythropolis BCRC 10909 can convert detectable level of 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (S)-PE by high performance liquid chromatography tandem mass spectrometry analysis. An amino alcohol dehydrogenase gene (RE_AADH) which possesses the ability to convert HPMAE to (S)-PE was then isolated from R. erythropolis BCRC 10909 and expressed in Escherichia coli NovaBlue. The purified RE_AADH, tagged with 6×His, had a molecular mass of approximately 30 kDa and exhibited a specific activity of 0.19 μU/mg to HPMAE in the presence of NADPH, indicating this enzyme could be categorized as NADP⁺-dependent short-chain dehydrogenase reductase. E. coli NovaBlue cell expressing the RE_AADH gene was able to convert HPMAE to (S)-PE with more than 99% enantiomeric excess (ee), 78% yield and a productivity of 3.9 mmol (S)-PE/L h in 12 h at 30 °C and pH 7. The (S)-PE, recovered from reaction mixture by precipitation at pH 11.3, could be converted to (R)-PE (ee > 99%) by Walden inversion reaction. This is the first reported biocatalytic process for the production of (S)-PE from HPMAE.     

Characterization and PCR application of a new high-fidelity DNA polymerase from Thermococcus waiotapuensis

The family B DNA polymerase gene from the euryarchaeon Thermococcus waiotapuensis (Twa) contains an open reading frame of 4404 bases that encodes 1467 amino acid residues. The gene is split by two intein-coding sequences that forms a continuous open reading frame with the three polymerase exteins. Twa DNA polymerase genes with (whole gene) and without (genetically intein-spliced) inteins were expressed in Escherichia coli Rosetta(DE3)pLysS. The inteins of the expressed whole gene were easily spliced during purification. The molecular mass of the purified Twa DNA polymerase was about 90 kDa, as estimated by SDS-PAGE. The optimal pH for Twa DNA polymerase activity was 6.0 and the optimal temperature was 75 °C. The enzyme was activated by magnesium ions. The half-life of the enzyme at 99 °C was about 4 h. The optimal buffer for PCR with Twa DNA polymerase was 50 mM Tris–HCl (pH 8.2), 2.0 mM MgCl₂, 30 mM KCl, 2.0 mM (NH₄)₂SO₄, 0.01% Triton X-100, and 0.005% BSA. The PCR fidelity of Twa DNA polymerase was higher than Pfu, KOD and Vent DNA polymerases. A ratio of 15:1 Taq:Twa DNA polymerase efficiently facilitated long-range PCR.     

Enzymatic preparation of optically pure t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate by a novel alcohol dehydrogenase discovered from Klebsiella oxytoca

Alcohol dehydrogenases can catalyze the inter-conversion of aldehydes and alcohols. The t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate is a key chiral intermediate in the synthesis of statin-type drugs such as Crestor (rosuvastatin calcium) and Lipitor (atorvastatin). Herein, a novel alcohol dehydrogenase (named as KleADH) discovered from Klebsiella oxytoca by a genome mining method was cloned and characterized. The KleADH was functionally overexpressed in Escherichia coli Rosetta (DE3) and the whole cell biocatalyst was able to convert t-butyl 6-chloro-(5S)-hydroxy-3-oxohexanoate to t-butyl 6-chloro-(3R,5S)-dihydroxyhexanoate with more than 99% diastereomeric excess (de) and 99% conversion in 24 h without adding any expensive cofactors. Several factors influencing the whole cell catalyst activity such as temperature, pH, the effects of metal ions and organic solvent were determined. The optimum enzyme activity was achieved at 30 °C and pH 7.0 and it was shown that 1 mM Fe³⁺ can increase the enzyme activity by 1.2 times. N-hexane/water and n-heptane/water biphasic systems can also increase the activity of KleADH. Substrate specificity studies showed that KleADH also exhibited notable activity towards several aryl ketones with high stereoselectivity. Our investigation on this novel alcohol dehydrogenase KleADH reveals a promising biocatalyst for producing chiral alcohols for preparation of valuable pharmaceuticals.     

Enantioselective synthesis of (S)-phenylephrine by recombinant Escherichia coli cells expressing the short-chain dehydrogenase/reductase gene from Serratia quinivorans BCRC 14811

BackgroundAn amino alcohol dehydrogenase gene (RE_AADH) from Rhodococcus erythropolis BCRC 10909 has been used for the conversion of 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (S)-phenylephrine [(S)-PE]. However RE_AADH uses NADPH as cofactor, and only limited production of (S)-PE from HPMAE is achieved.MethodsA short-chain dehydrogenase/reductase gene (SQ_SDR) from Serratia quinivorans BCRC 14811 was expressed in Escherichia coli BL21 (DE3) for the conversion of HPMAE to (S)-PE.ResultsThe SQ_SDR enzyme was capable of converting HPMAE to (S)-PE in the presence of NADH and NADPH, with specific activities of 26.5 ± 2.3 U/mg protein and 0.24 ± 0.01 U/mg protein, respectively, at 30 °C and at a pH of 7.0. The E. coli BL21 (DE3), expressing NADH-preferring SQ_SDR, converted HPMAE to (S)-PE with more than 99% enantiomeric excess, a conversion yield of 86.6% and a productivity of 20.2 mmol/l h, which was much higher than our previous report using E. coli NovaBlue expressing NADPH-dependent RE_AADH as the biocatalyst.ConclusionThe SQ_SDR enzyme with its high catalytic activity and strong preference for NADH as a cofactor provided a significant advantage in bioreduction.     

Synthesis and antioxidant evaluation of (S,S)- and (R,R)-secoisolariciresinol diglucosides (SDGs)

Secoisolariciresinol diglucosides (SDGs) (S,S)-SDG-1 (major isomer in flaxseed) and (R,R)-SDG-2 (minor isomer in flaxseed) were synthesized from vanillin via secoisolariciresinol (6) and glucosyl donor 7 through a concise route that involved chromatographic separation of diastereomeric diglucoside derivatives (S,S)-8 and (R,R)-9. Synthetic (S,S)-SDG-1 and (R,R)-SDG-2 exhibited potent antioxidant properties (EC₅₀ = 292.17 ± 27.71 μM and 331.94 ± 21.21 μM, respectively), which compared well with that of natural (S,S)-SDG-1 (EC₅₀ = 275.24 ± 13.15 μM). These values are significantly lower than those of ascorbic acid (EC₅₀ = 1129.32 ± 88.79 μM) and α-tocopherol (EC₅₀ = 944.62 ± 148.00 μM). Compounds (S,S)-SDG-1 and (R,R)-SDG-2 also demonstrated powerful scavenging activities against hydroxyl [natural (S,S)-SDG-1: 3.68 ± 0.27; synthetic (S,S)-SDG-1: 2.09 ± 0.16; synthetic (R,R)-SDG-2: 1.96 ± 0.27], peroxyl [natural (S,S)-SDG-1: 2.55 ± 0.11; synthetic (S,S)-SDG-1: 2.20 ± 0.10; synthetic (R,R)-SDG-2: 3.03 ± 0.04] and DPPH [natural (S,S)-SDG-1: EC₅₀ = 83.94 ± 2.80 μM; synthetic (S,S)-SDG-1: EC₅₀ = 157.54 ± 21.30 μM; synthetic (R,R)-SDG-2: EC₅₀ = 123.63 ± 8.67 μM] radicals. These results confirm previous studies with naturally occurring (S,S)-SDG-1 and establish both (S,S)-SDG-1 and (R,R)-SDG-2 as potent antioxidants and free radical scavengers for potential in vivo use.     

Biophysical and enzymatic properties of aminoglycoside adenylyltransferase AadA6 from Pseudomonas aeruginosa

The gene coding for the aminoglycoside adenylyltransferase (aadA6) from a clinical isolate of Pseudomonas aeruginosa was cloned and expressed in Escherichia coli strain BL21(DE3)pLysS. The overexpressed enzyme (AadA6, 281 amino-acid residues) and a carboxy-terminal truncated variant molecule ([1-264]AadA6) were purified to near homogeneity and characterized. Light scattering experiments conducted under low ionic strength supported equilibrium between monomeric and homodimeric arrangements of the enzyme subunits. Circular Dichroism spectropolarimetry indicated a close structural relation to adenylate kinases. Both forms modified covalently the aminoglycosides streptomycin and spectinomycin. The enzyme required at least 5 mM MgCl₂ for normal Michaelis–Menten kinetics. Streptomycin exhibited a strong substrate inhibition effect at 1 mM MgCl₂. The truncated 17 residues at the C-terminus have little influence on protein folding, whereas they have a positive effect on the enzymic activity and stabilize dimers at high protein concentrations (>100 μM). Homology modelling and docking based on known crystal structures yielded models of the central ternary complex of monomeric AadA6 with ATP and streptomycin or spectinomycin.     

Cloning and characterization of a NADH-dependent aldo-keto reductase from a newly isolated Kluyveromyces lactis XP1461

An aldo-keto reductase gene (klakr) from Kluyveromyces lactis XP1461 was cloned and heterologously expressed in Escherichia coli. The aldo-keto reductase KlAKR was purified and found to be NADH-dependent with a molecular weight of approximately 36 kDa. It is active and stable at 30 °C and pH 7.0. The maximal reaction rate (v_max), apparent Michaelis–Menten constant (K_m) for NADH and t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a) and catalytic number (k_cat) were calculated as 7.63 U mg⁻¹, 0.204 mM, 4.42 mM and 697.4 min⁻¹, respectively. Moreover, the KlAKR has broad substrate specificity to a range of aldehydes, ketones and keto-esters, producing chiral alcohol with e.e. or d.e. >99% for the majority of test substrates.     

3-Aryl-4-acyloxyethoxyfuran-2(5H)-ones as inhibitors of tyrosyl-tRNA synthetase: Synthesis,molecular docking and antibacterial evaluation

Thirty-eight 3-aryl-4-acyloxyethoxyfuran-2(5H)-ones were designed, prepared and tested for antibacterial activities. Some of them showed significant antibacterial activity against Gram-positive organism, Gram-negative organism and fungus. Out of these compounds, 4-(2-(3-chlorophenylformyloxy)ethoxy)-3-(4-chlorophenyl)furan-2(5H)-one (d40) showed the widest spectrum of activity with MIC₅₀ of 2.0 μg/mL against Staphylococcus aureus, 4.3 μg/mL against Escherichia coli, 1.5 μg/mL against Pseudomonas aeruginosa and 1.2 μg/mL against Candida albicans. Our data disclosed that MIC₅₀ values against whole cell bacteria are positive correlation with MIC₅₀ values against tyrosyl-tRNA synthetase. Meanwhile, molecular docking of d40 into S. aureus tyrosyl-tRNA synthetase active site was also performed, and the inhibitor tightly fitting the active site might be an important reason why it has high antimicrobial activity.     

Synthesis and biological evaluation of enantiomerically pure glyceric acid derivatives as LpxC inhibitors

Inhibitors of the UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase (LpxC) represent a promising class of novel antibiotics, selectively combating Gram-negative bacteria. In order to elucidate the impact of the hydroxymethyl groups of diol (S,S)-4 on the inhibitory activity against LpxC, glyceric acid ethers (R)-7a, (S)-7a, (R)-7b, and (S)-7b, lacking the hydroxymethyl group in benzylic position, were synthesized. The compounds were obtained in enantiomerically pure form by a chiral pool synthesis and a lipase-catalyzed enantioselective desymmetrization, respectively. The enantiomeric hydroxamic acids (R)-7b (K_i = 230 nM) and (S)-7b (K_i = 390 nM) show promising enzyme inhibition. However, their inhibitory activities do not substantially differ from each other leading to a low eudismic ratio. Generally, the synthesized glyceric acid derivatives 7 show antibacterial activities against two Escherichia coli strains exceeding the ones of their respective regioisomes 6.     

AhpC (alkyl hydroperoxide reductase) from Anabaena sp. PCC 7120 protects Escherichia coli from multiple abiotic stresses

Alkyl hydroperoxide reductase (AhpC) is known to detoxify peroxides and reactive sulfur species (RSS). However, the relationship between its expression and combating of abiotic stresses is still not clear. To investigate this relationship, the genes encoding the alkyl hydroperoxide reductase (ahpC) from Anabaena sp. PCC 7120 were introduced into E. coli using pGEX-5X-2 vector and their possible functions against heat, salt, carbofuron, cadmium, copper and UV-B were analyzed. The transformed E. coli cells registered significantly increase in growth than the control cells under temperature (47 °C), NaCl (6% w/v), carbofuron (0.025 mg ml^?1), CdCl₂ (4 mM), CuCl₂ (1 mM), and UV-B (10 min) exposure. Enhanced expression of ahpC gene as measured by semi-quantitative RT-PCR under aforementioned stresses at different time points demonstrated its role in offering tolerance against multiple abiotic stresses.