- Identification of 2, 3-dihydrodipicolinate as the product of the dihydrodipicolinate synthase reaction from Escherichia coli
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Dihydrodipicolinate synthase (DHDPS) catalyzes the first step in the pathway for the biosynthesis of L-lysine in most bacteria and plants. The substrates for the enzyme are pyruvate and L-aspartate-β-semialdehyde (ASA). The product of the reaction was originally proposed to be 2,3-dihydrodipicolinate (DHDP), but has now generally been assumed to be (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinate (HTPA). ASA is unstable at high pH and it is proposed that ASA reacts with itself. At high pH ASA also reacts with Tris buffer and both reactions are largely reversible at low pH. It is proposed that the basic un-protonated form of the amine of Tris or the α-amine of ASA reacts with the aldehyde functional group of ASA to generate an imine product. Proton NMR spectra of ASA done at different pH values shows new NMR peaks at high pH, but not at low pH, confirming the presence of reaction products for ASA at high pH. The enzymatic product of the DHDPS reaction was examined at low pH by proton NMR starting with either 3 h-pyruvate or 3 d-pyruvate and identical NMR spectra were obtained with four new NMR peaks observed at 1.5, 2.3, 3.9 and 4.1 ppm in both cases. The NMR results were most consistent with DHDP as the reaction product. The UV-spectral studies of the DHDPS reaction shows the formation of an initial product with a broad spectral peak at 254 nM. The DHDPS reaction product was further examined by reduction of the enzymatic reaction components with borohydride followed by GC-MS analysis of the mixture. Three peaks were found at 88, 119 and 169 m/z, consistent with pyruvate, homoserine (reduction product of ASA), and the reduction product of DHDP (1,2,3,6-tetrahydropyridine-2,6-dicarboxylate). There was no indication for a peak associated with the reduced form of HTPA.
- Karsten, William E.,Nimmo, Susan A.,Liu, Jianguo,Chooback, Lilian
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- Energy- And cost-effective non-sterilized fermentation of 2,3-butanediol by an engineered: Klebsiella pneumoniae OU7 with an anti-microbial contamination system
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Microbial contamination is a serious challenge that needs to be overcome for the successful biosynthesis of 2,3-butanediol (2,3-BD). However, traditional strategies such as antibiotic administration or sterilization are costly, have high energy demands, and may increase the risk of antibiotic resistance. Here, we intend to develop a robust strategy to achieve non-sterilized fermentation of 2,3-BD. Briefly, the robust strain can metabolize unconventional chemicals as essential growth nutrients, and therefore, outcompete contaminant microbes that cannot use unconventional chemicals. To this end, Klebsiella pneumoniae OU7, a robust strain, was confirmed to rapidly exploit urea and phosphite (unconventional chemicals) as the primary sources of nitrogen (N) and phosphorus (P), and withstand deliberate contamination in the possibly contaminated systems. Secondly, metabolic engineering, pathogenicity elimination and adaptive laboratory evolution were successively performed, endowing the best strain with an excellent fermentation performance for safe 2,3-BD production. Finally, 84.53 g L-1 of 2,3-BD was synthesized with a productivity of 1.17 g L-1 h-1 and a yield of 0.38 g g-1 under the non-sterilized system. In summary, our technique reduces labor and energy costs and simplifies the fermentation process because sterilization does not need to be performed. Thus, our work will be beneficial for the sustainable synthesis of 2,3-BD. This journal is
- Guo, Ze-Wang,Ou, Xiao-Yang,Xu, Pei,Gao, Hui-Fang,Zhang, Liao-Yuan,Zong, Min-Hua,Lou, Wen-Yong
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- Modelling of the periodic anaerobic baffled reactor (PABR) based on the retaining factor concept
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The fact that the active biomass is continuously removed from the continuously stirred anaerobic digesters, leading to long retention times, has been overcome in a number of high rate systems based on immobilization of the active biomass, such as the Upflow Anaerobic Sludge Blanket Reactor (UASBR) and the Anaerobic Baffled Reactor (ABR). A kinetic model of glucose consumption, which was developed based on a batch kinetic experiment, was used for the development of a dynamic model for the prediction of the behaviour of the recently developed flexible reactor called the Periodic Anaerobic Baffled Reactor (PABR). The PABR may be operated as a UASBR, an ABR or at an intermediate mode. The key assumption of the model is that the hydraulic behaviour of a PABR is equivalent with the behaviour of CSTRs in series as concerning the dissolved matter, whereas the biomass is allowed to be retained in the PABR through a retention factor accounting for precipitation. The model adequately predicted the experimental behaviour of a glucose fed PABR. The model was subsequently used to examine the behaviour of the PABR as a function of operating conditions, both for constant and varying loading rates. It was shown that for different cases, the reactor should best be operated as a UASBR or as an ABR. The fact that the active biomass is continuously removed from the continuously stirred anaerobic digesters, leading to long retention times, has been overcome in a number of high rate systems based on immobilisation of the active biomass, such as the Upflow Anaerobic Sludge Blanket Reactor (UASBR) and the Anaerobic Baffled Reactor (ABR). A kinetic model of glucose consumption, which was developed based on a batch kinetic experiment, was used for the development of a dynamic model for the prediction of the behaviour of the recently developed flexible reactor called the Periodic Anaerobic Baffled Reactor (PABR) [(1998) Wat. Sci. Technol. 38(8-9), 401- 408]. The PABR may be operated as a UASBR, an ABR or at an intermediate mode. The key assumption of the model is that the hydraulic behaviour of a PABR is equivalent with the behaviour of CSTRs in series as concerning the dissolved matter, whereas the biomass is allowed to be retained in the PABR through a retention factor accounting for precipitation. The model adequately predicted the experimental behaviour of a glucose fed PABR. The model was subsequently used to examine the behaviour of the PABR as a function of operating conditions, both for constant and varying loading rates. It was shown that for different cases, the reactor should best be operated as a UASBR or as an ABR. (C) 2000 Elsevier Science Ltd.
- Skiadas,Gavala,Lyberatos
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- Halogenation of Acetone. A Method for Determining pKas of Ketones in Aqueous Solution, with an Examination of the Thermodynamics and Kinetics of Alkaline Halogenation and a Discussion of the Best Value for the Rate Constant for a "Diffusion-Controlled Reaction". Energetic ...
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The pKa of a simple ketone can be determined by analysis of the kinetics of halogenation in alkaline solution (J.Am.Chem.Soc. 1982, 104, 895).Details of the determination of the pKa of acetone are reported.The stoichiometry of chlorination of acetone is 2.15 hypohalites per acetone; bromination consumes 2.83 hypobromites.The major product from chlorination is lactate and not acetate; lactate is a minor product from bromination of acetone.The kinetic significance of the possible side reactions is discussed.The determination of the pKa requires knowledge of the rate constant for a diffusion-controlled reactionof nonspherically symmetrical molecules in water; the state of our knowledge of this rate constant is reviewed critically.It is shown that for reactions involving diffusion together of reactants which then undergo direct heavy-atom bond making it is very unusual for the diffusion limit to be reached, although the observed rate constant may become insensitive to the thermodynamic rectivity of the reactants.
- Guthrie, J. Peter,Cossar, John,Klym, Alex
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- Efficient transfer hydrogenation of carbonate salts from glycerol using water-soluble iridium N-heterocyclic carbene catalysts
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The transfer hydrogenation of CO2and carbonates from biomass-derived alcohols, such as glycerol, to afford formic and lactic acid is a highly attractive path to valorizing two waste streams, and is significantly more thermodynamically favorable than direct carbonate hydrogenation. Expanding on our seminal report of the first homogeneous catalyst for this process, here we show that thermally-robust and water-soluble Ir(i) and Ir(iii) N-heterocyclic carbene (NHC) complexes with sulfonate-functionalized wingtips are highly prolific and robust catalysts for carbonate transfer hydrogenation from glycerol, requiring no additives in aqueous media. The most prolific catalyst of the nine examined, [Ir(NHC-Ph-SO3?)2CO2]Na (cat7), effectively facilitates the reaction at low catalyst loading (10 ppm) at 150 °C using microwave or conventional heating. The cation of the carbonate salt significantly impacts catalytic activity, with highest activity observed with Cs2CO3(27?850 and 13?350 TONs for lactate and formate respectively in 6 hours, compared to 15?400 and 8120 with K2CO3). Catalytic amounts of Cs+were found to significantly enhance activity with K2CO3. Catalyst7is even more prolific with conventional heating under a positive N2pressure, reaching TOFs of >3000 h?1and >2100 h?1respectively for lactate and formate with K2CO3. The high activity of this catalyst compared to non-sulfonated and cyclooctadiene analogs is attributed to a combination of catalyst solubility in aqueous media and presence of π-acceptor carbonyl ligands. A catalytic mechanism is proposed for7involving O-H oxidative addition of glycerol, β-hydride elimination, bicarbonate dehydroxylation, insertion and reductive elimination.
- Ainembabazi, Diana,Finn, Matthew,Ridenour, James,Voutchkova-Kostal, Adelina,Wang, Kai
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- Ni2 +-activated glyoxalase i from Escherichia coli: Substrate specificity, kinetic isotope effects and evolution within the βαβββ superfamily
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The Escherichia coli glyoxalase system consists of the metalloenzymes glyoxalase I and glyoxalase II. Little is known regarding Ni 2 +-activated E. coli glyoxalase I substrate specificity, its thiol cofactor preference, the presence or absence of any substrate kinetic isotope effects on the enzyme mechanism, or whether glyoxalase I might catalyze additional reactions similar to those exhibited by related βαβ ββ structural superfamily members. The current investigation has shown that this two-enzyme system is capable of utilizing the thiol cofactors glutathionylspermidine and trypanothione, in addition to the known tripeptide glutathione, to convert substrate methylglyoxal to non-toxic d-lactate in the presence of Ni2 + ion. E. coli glyoxalase I, reconstituted with either Ni2 + or Cd2 +, was observed to efficiently process deuterated and non-deuterated phenylglyoxal utilizing glutathione as cofactor. Interestingly, a substrate kinetic isotope effect for the Ni 2 +-substituted enzyme was not detected; however, the proton transfer step was observed to be partially rate limiting for the Cd 2 +-substituted enzyme. This is the first non-Zn 2 +-activated GlxI where a metal ion-dependent kinetic isotope effect using deuterium-labelled substrate has been observed. Attempts to detect a glutathione conjugation reaction with the antibiotic fosfomycin, similar to the reaction catalyzed by the related superfamily member FosA, were unsuccessful when utilizing the E. coli glyoxalase I E56A mutein.
- Mullings, Kadia Y.,Sukdeo, Nicole,Suttisansanee, Uthaiwan,Ran, Yanhong,Honek, John F.
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- Room temperature, near-quantitative conversion of glucose into formic acid
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Herein, a facile and efficient method was developed to selectively transform glucose into formic acid at room temperature. After parameter optimization, formic acid was obtained in an unprecedented 91.3% yield with a reaction time of 8 h in lithium hydroxide aqueous solution with hydrogen peroxide as the oxidant. The synergistic effects of the base and the oxidant promoted the glucose conversion at room temperature and enhanced the selectivity towards FA. Besides, the employed mild conditions have suppressed FA decomposition that often occurred under harsh conditions, which further improved the FA selectivity. A series of model compound tests were conducted to probe the possible intermediates based on which a plausible reaction pathway was proposed. In addition, the process is applicable to various carbohydrates such as cellobiose, starch, xylan, etc. This work opens up a simple, mild but effective method to produce FA from renewable biomass resources, which would remarkably alleviate the energy consumption, capital costs, handling risks, etc.
- Wang, Can,Chen, Xi,Qi, Man,Wu, Jianeng,G?zaydin, G?kalp,Yan, Ning,Zhong, Heng,Jin, Fangming
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- Hydrogen and chemicals from alcohols through electrochemical reforming by Pd-CeO2/C electrocatalyst
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The development of low-cost and sustainable hydrogen production is of primary importance for a future transition to sustainable energy. In this work, the selective and simultaneous production of pure hydrogen and chemicals from renewable alcohols is achieved using an anion exchange membrane electrolysis cell (electrochemical reforming) employing a nanostructured Pd-CeO2/C anode. The catalyst exhibits high activity for alcohol electrooxidation (e.g. 474 mA cm?2 with EtOH at 60 °C) and the electrolysis cell produces high volumes of hydrogen (1.73 l min?1 m?2) at low electrical energy input (Ecost = 6 kWh kgH2?1 with formate as substrate). A complete analysis of the alcohol oxidation products from several alcohols (methanol, ethanol, 1,2-propandiol, ethylene glycol, glycerol and 1,4-butanediol) shows high selectivity in the formation of valuable chemicals such as acetate from ethanol (100%) and lactate from 1,2-propandiol (84%). Importantly for industrial application, in batch experiments the Pd-CeO2/C catalyst achieves conversion efficiencies above 80% for both formate and methanol, and 95% for ethanol.
- Bellini, Marco,Pagliaro, Maria V.,Marchionni, Andrea,Filippi, Jonathan,Miller, Hamish A.,Bevilacqua, Manuela,Lavacchi, Alessandro,Oberhauser, Werner,Mahmoudian, Jafar,Innocenti, Massimo,Fornasiero, Paolo,Vizza, Francesco
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- Xylitol Hydrogenolysis over Ruthenium-Based Catalysts: Effect of Alkaline Promoters and Basic Oxide-Modified Catalysts
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The aqueous-phase hydrogenolysis of xylitol into glycols over Ru/C was performed in the presence and absence of a wide range of concentrations of Ca(OH)2 to investigate the reaction pathway. Without base, epimerization and cascade decarbonylation were the predominant reactions with high selectivities to C5 and C4 alditols and light alkanes at full conversion. Glycol production was obtained by the addition of Ca(OH)2 to promote the retro-aldol reaction. It competed with reactions without base and became the main reaction for a OH?/ xylitol molar ratio Rmol(OH/xylitol) of 0.13, and high selectivities to glycols (56 %) and glycerol (16 %) were observed. However, lactate was a byproduct at up to 27 % with a high base amount (Rmol(OH/xylitol)=0.68). Bifunctional Ru/metal oxide/C catalysts (metal: Zn, Sn, Mn, Sr, W) were synthesized and were able to cleave the C?C bond into glycols without a base promoter. The 3.1 wt %Ru/MnO(4.5 %)/C catalyst was the most active (220 h?1) with reasonable selectivity to glycols (22 %) and glycerol (10 %) and a low production of lactate (1 %). Nevertheless, metal oxide leaching of the catalyst was observed likely because of the production of traces of lactate.
- Rivière, Maxime,Perret, Noémie,Cabiac, Amandine,Delcroix, Damien,Pinel, Catherine,Besson, Michèle
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p. 2145 - 2159
(2017/06/28)
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- The crystal structure of a homodimeric Pseudomonas glyoxalase I enzyme reveals asymmetric metallation commensurate with half-of-sites activity
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The Zn inactive class of glyoxalase I (Glo1) metalloenzymes are typically homodimeric with two metal-dependent active sites. While the two active sites share identical amino acid composition, this class of enzyme is optimally active with only one metal per homodimer. We have determined the X-ray crystal structure of GloA2, a Zn inactive Glo1 enzyme from Pseudomonas aeruginosa. The presented structures exhibit an unprecedented metal-binding arrangement consistent with half-of-sites activity: one active site contains a single activating Ni2+ ion, whereas the other contains two inactivating Zn2+ ions. Enzymological experiments prompted by the binuclear Zn2+ site identified a novel catalytic property of GloA2. The enzyme can function as a Zn2+/Co2+ -dependent hydrolase, in addition to its previously determined glyoxalase I activity. The presented findings demonstrate that GloA2 can accommodate two distinct metal-binding arrangements simultaneously, each of which catalyzes a different reaction.
- Bythell-Douglas, Rohan,Suttisansanee, Uthaiwan,Flematti, Gavin R.,Challenor, Michael,Lee, Mihwa,Panjikar, Santosh,Honek, John F.,Bond, Charles S.
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p. 541 - 544
(2015/10/12)
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- Fermentation process using yeast cells having disrupted pathway from dihydroxyacetone phosphate to glycerol
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In a fermentation process, a cell of a pre-whole genome duplication yeast, which is genetically modified to delete or disrupt a native metabolic pathway from dihydroxyacetone phosphate to glycerol, is cultivated under fermentation conditions and in the presence of a carbon source to produce a desired fermentation product, wherein the glycerol yield is less than 2% based on the weight of the carbon source that is consumed by the cell, and whereby glycerol is added to the fermentation medium.
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Paragraph 0127; 0128; 0129; 0130
(2013/05/09)
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- Synthesis of α-amino and α-hydroxy acids under volcanic conditions: implications for the origin of life
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Facile synthesis of α-hydroxy and α-amino acids is observed at temperatures from 145 to 280 °C with catalytic Ni2+, with cyano ligands as source for C and N, and with CO as a reductant and as a source for C. Implications for the problem of the origin of life are discussed.
- Huber, Claudia,Eisenreich, Wolfgang,W?chtersh?user, Günter
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scheme or table
p. 1069 - 1071
(2010/04/05)
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- Substrate and reaction intermediate mimics as inhibitors of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase
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3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAH7P) synthase catalyses the aldol-like addition of phosphoenolpyruvate (PEP) to d-erythrose 4-phosphate in the first step of the shikimate pathway to aromatic amino acids. A series of compounds, designed to m
- Walker, Scott R.,Cumming, Hemi,Parker, Emily J.
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supporting information; experimental part
p. 3031 - 3035
(2011/02/25)
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