- Construction of an L-isoleucine overproducing strain of Escherichia coli K-12
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The genes for a threonine deaminase that is resistant to feedback inhibition by L-isoleucine and for an active acetohydroxyacid synthase II were introduced by a plasmid into a L-threonine-producing recombinant strain of Escherichia coli K-12. Analysis of culture broth of the strain using 13C nuclear magnetic resonance suggested that α, β-dihydroxy-β-methylvalerate (DHMV) and α-keto-βmethylvalerate (KMV), the third and the fourth intermediates in the L-isoleucine biosynthetic pathway from L-threonine, respectively, accumulated in the medium in amounts comparable to that of L-isoleucine. The ratio of accumulated L-isoleucine:DHMV:KMV were approximately 2:1:1. The concentration of accumulated L-isoleucine increased by twofold after the additional introduction of the genes for dihyroxyacid dehydratase (DH) and transaminase-B (TA-B), and the intermediates no longer accumulated. The resultant strain TVD5 accumulated 10 g/1 of L-isoleucine from 40 g/1 of glucose.
- Hashiguchi, Ken-Ichi,Takesada, Hiroko,Suzuki, Eiichiro,Matsui, Hiroshi
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- Production of α-Ketoisocaproate and α-Keto-β-Methylvalerate by Engineered L-Amino Acid Deaminase
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This study aimed to develop an efficient enzymatic strategy for industrial production of α-ketoisocaproate (α-KIC) and α-keto-β-methylvalerate (α-KMV) from L-leucine and L-isoleucine, respectively. L-amino acid deaminase from Proteus mirabilis (PmLAAD) was heterologously expressed in E. coli BL21(DE3) and modified to increase its catalytic efficiency by engineering the PmLAAD substrate-binding cavity and entrance tunnel. Four essential residues (Q92, M440, T436, and W438) were identified from structural analysis and molecular dynamics simulations. Residue Q92 was mutated to alanine, and the volume of the binding cavity, enzyme activity, and the kcat/Km value of mutant PmLAAD Q92A increased to 994.2 ?3, 191.36 U mg?1, and 1.23 mM?1 min?1, respectively; consequently, the titer and conversion rate of α-KIC from L-leucine were 107.1 g L?1 and 98.1 %, respectively. For mutant PmLAADT436/W438A, the entrance tunnel, enzyme activity, and the kcat/Km value increased to 1.71 ?, 170.12 U mg?1, and 0.70 mM?1 min?1, respectively; consequently, the titer and conversion rate of α-KMV from L-isoleucine were 98.9 g L?1 and 99.7 %, respectively. Therefore, augmentation of the substrate-binding cavity and entrance tunnel of PmLAAD can facilitate efficient industrial synthesis of α-KIC and α-KMV.
- Yuan, Yuxiang,Song, Wei,Liu, Jia,Chen, Xiulai,Luo, Qiuling,Liu, Liming
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- Heterocyclic Compounds from the Mushroom Albatrellus confluens and Their Inhibitions against Lipopolysaccharides-Induced B Lymphocyte Cell Proliferation
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Eight hetereocyclic compounds conflamides B-I with an unprecedented skeleton and their precursor conflamide A were isolated from the mushroom Albatrellus confluens. Their structures and absolute configurations were determined by use of NMR studies, total synthesis, and calculated ECD spectra. Conflamides D and E were found to exhibit potent inhibition against LPS-induced B lymphocyte cell proliferation with IC50 values 1.48 and 5.71 μM, respectively.
- Zhang, Shuaibing,Huang, Ying,He, Shijun,Chen, Heping,Wu, Bin,Li, Shanyong,Zhao, Zhenzhu,Li, Zhenghui,Wang, Xian,Zuo, Jianping,Feng, Tao,Liu, Jikai
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- Asymmetric C-Alkylation by the S-Adenosylmethionine-Dependent Methyltransferase SgvM
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S-Adenosylmethionine-dependent methyltransferases (MTs) play a decisive role in the biosynthesis of natural products and in epigenetic processes. MTs catalyze the methylation of heteroatoms and even of carbon atoms, which, in many cases, is a challenging reaction in conventional synthesis. However, C-MTs are often highly substrate-specific. Herein, we show that SgvM from Streptomyces griseoviridis features an extended substrate scope with respect to the nucleophile as well as the electrophile. Aside from its physiological substrate 4-methyl-2-oxovalerate, SgvM catalyzes the (di)methylation of pyruvate, 2-oxobutyrate, 2-oxovalerate, and phenylpyruvate at the β-carbon atom. Chiral-phase HPLC analysis revealed that the methylation of 2-oxovalerate occurs with R selectivity while the ethylation of 2-oxobutyrate with S-adenosylethionine results in the S enantiomer of 3-methyl-2-oxovalerate. Thus SgvM could be a valuable tool for asymmetric biocatalytic C-alkylation reactions.
- Sommer-Kamann, Christina,Fries, Alexander,Mordhorst, Silja,Andexer, Jennifer N.,Müller, Michael
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- Chemoenzymatic Production of Enantiocomplementary 2-Substituted 3-Hydroxycarboxylic Acids from l-α-Amino Acids
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A two-enzyme cascade reaction plus in situ oxidative decarboxylation for the transformation of readily available canonical and non-canonical l-α-amino acids into 2-substituted 3-hydroxycarboxylic acid derivatives is described. The biocatalytic cascade consisted of an oxidative deamination of l-α-amino acids by an l-α-amino acid deaminase from Cosenzaea myxofaciens, rendering 2-oxoacid intermediates, with an ensuing aldol addition reaction to formaldehyde, catalyzed by metal-dependent (R)- or (S)-selective carboligases namely 2-oxo-3-deoxy-l-rhamnonate aldolase (YfaU) and ketopantoate hydroxymethyltransferase (KPHMT), respectively, furnishing 3-substituted 4-hydroxy-2-oxoacids. The overall substrate conversion was optimized by balancing biocatalyst loading and amino acid and formaldehyde concentrations, yielding 36–98% aldol adduct formation and 91–98% ee for each enantiomer. Subsequent in situ follow-up chemistry via hydrogen peroxide-driven oxidative decarboxylation afforded the corresponding 2-substituted 3-hydroxycarboxylic acid derivatives. (Figure presented.).
- Pickl, Mathias,Marín-Valls, Roser,Joglar, Jesús,Bujons, Jordi,Clapés, Pere
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p. 2866 - 2876
(2021/04/14)
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- Biocatalytic Construction of Quaternary Centers by Aldol Addition of 3,3-Disubstituted 2-Oxoacid Derivatives to Aldehydes
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The congested nature of quaternary carbons hinders their preparation, most notably when stereocontrol is required. Here we report a biocatalytic method for the creation of quaternary carbon centers with broad substrate scope, leading to different compound classes bearing this structural feature. The key step comprises the aldol addition of 3,3-disubstituted 2-oxoacids to aldehydes catalyzed by metal dependent 3-methyl-2-oxobutanoate hydroxymethyltransferase from E. coli (KPHMT) and variants thereof. The 3,3,3-trisubstituted 2-oxoacids thus produced were converted into 2-oxolactones and 3-hydroxy acids and directly to ulosonic acid derivatives, all bearing gem-dialkyl, gem-cycloalkyl, and spirocyclic quaternary centers. In addition, some of these reactions use a single enantiomer from racemic nucleophiles to afford stereopure quaternary carbons. The notable substrate tolerance and stereocontrol of these enzymes are indicative of their potential for the synthesis of structurally intricate molecules.
- Marín-Valls, Roser,Hernández, Karel,Bolte, Michael,Parella, Teodor,Joglar, Jesús,Bujons, Jordi,Clapés, Pere
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supporting information
p. 19754 - 19762
(2020/12/01)
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- The pseudoalteromonas luteoviolacea L-amino acid oxidase with antimicrobial activity is a flavoenzyme
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The marine environment is a rich source of antimicrobial compounds with promising pharmaceutical and biotechnological applications. The Pseudoalteromonas genus harbors one of the highest proportions of bacterial species producing antimicrobial molecules. For decades, the presence of proteins with L-amino acid oxidase (LAAO) and antimicrobial activity in Pseudoalteromonas luteoviolacea has been known. Here, we present for the first time the identification, cloning, characterization and phylogenetic analysis of Pl-LAAO, the enzyme responsible for both LAAO and antimicrobial activity in P. luteoviolacea strain CPMOR-2. Pl-LAAO is a flavoprotein of a broad substrate range, in which the hydrogen peroxide generated in the LAAO reaction is responsible for the antimicrobial activity. So far, no protein with a sequence similarity to Pl-LAAO has been cloned or characterized, with this being the first report on a flavin adenine dinucleotide (FAD)-containing LAAO with antimicrobial activity from a marine microorganism. Our results revealed that 20.4% of the sequenced Pseudoalteromonas strains (specifically, 66.6% of P. luteoviolacea strains) contain Pl-laao similar genes, which constitutes a well-defined phylogenetic group. In summary, this work provides insights into the biological significance of antimicrobial LAAOs in the Pseudoalteromonas genus and shows an effective approach for the detection of novel LAAOs, whose study may be useful for biotechnological applications.
- Andreo-Vidal, Andrés,Sanchez-Amat, Antonio,Campillo-Brocal, Jonatan C.
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- A method for preparing calcium isoleucine racemization alkone
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The invention discloses a method for preparing calcium 3-methyl-2-oxovalerate. The method comprises the following steps of: dripping diethyl oxalate into an alcoholic solution of sodium alkoxide; dripping 2-methyl butyraldehyde, keeping the temperature, stirring, adding an alkali solution, regulating the acid after heat insulation is ended, extracting, and adding a certain amount of water into an extracting solution; adding the alkali solution to regulate the pH, dripping an aqueous solution of calcium chloride for salifying to obtain the coarse 3-methyl-2-oxovalerate product; and refining the coarse 3-methyl-2-oxovalerate product in a mixed solvent of purified water and organic solvent, and obtaining the refined 3-methyl-2-oxovalerate product. The process is mild in reaction conditions, easy to operate, fewer in steps, high in yield and high in quality, the adopted raw materials are cheap and basically do not cause pollution, waste gas and lots of waste residues are avoided, and the method is suitable for industrial production.
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Page/Page column 0032; 0033
(2016/12/22)
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- NOVEL COMPOUND, PHARMACEUTICALLY ACCEPTABLE SALT OR OPTICAL ISOMER THEREOF, METHOD FOR PREPARING SAME, AND PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF VIRAL DISEASES CONTAINING SAME AS ACTIVE INGREDIENT
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The present invention relates to a novel compound, to a pharmaceutically acceptable salt or optical isomer thereof, to a method for preparing same, and to a pharmaceutical composition for the prevention or treatment of viral diseases containing same as an active ingredient. The novel compound according to the present invention not only has low cytotoxicity but also has excellent antiviral activity against picornavirus such as coxsackievirus, enterovirus, echovirus, poliovirus and rhinovirus, and thus can be effectively used as a pharmaceutical composition for the prevention or treatment of viral diseases such as infantile paralysis, acute hemorrhagic conjunctivitis, viral meningitis, hand-foot-and-mouth disease, vesicular disease, hepatitis A, myitis, myocarditis, pancreatitis, diabetes, epidemic myalgia, encephalitis, cold, herpangina, foot-and-mouth disease, asthma, chronic obstructive pulmonary disease, pneumonia, sinus infection, or otitis media.
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Paragraph 0105; 107
(2015/11/16)
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- Stereoselective synthesis of l-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum
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A leucine dehydrogenase from Exiguobacterium sibiricum (EsLeuDH) was discovered by genome mining approach. The EsLeuDH was overexpressed in Escherichia coli BL21, purified to homogeneity and characterized. This enzyme showed good thermostability with a half-life of 3.1 h at 60 °C. Furthermore, EsLeuDH has a broad spectrum of substrate specificity, showing activities toward many aliphatic α-keto acids and L-amino acids, in addition to some aryl α-keto acids and aryl α-amino acids, such as α-oxobenzeneacetic and l-phenylglycine. The EsLeuDH was successfully coexpressed with Bacillus megaterium glucose dehydrogenase (BmGDH) in Escherichia coli BL21 for the production of l-tert-leucine. By using the coexpressed whole cells, a decagram preparation of l-tert-leucine was performed at a substrate concentration of 0.6 M (78.1 g L-1) in 1 L scale with 99% conversion after 5.5 h, resulting in 80.1% yield and > 99% ee (enantiomeric excess).2014 Published by Elsevier B.V.
- Li, Jing,Pan, Jiang,Zhang, Jie,Xu, Jian-He
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- Biocatalytic asymmetric synthesis of unnatural amino acids through the cascade transfer of amino groups from primary amines onto keto acids
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Flee to the hills: An unfavorable equilibrium in the amino group transfer between amino acids and keto acids catalyzed by α-transaminases was successfully overcome by coupling with a ω-transaminase reaction as an equilibrium shifter, leading to efficient asymmetric synthesis of diverse unnatural amino acids, including L-tert-leucine and D-phenylglycine. Copyright
- Park, Eul-Soo,Dong, Joo-Young,Shin, Jong-Shik
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p. 3538 - 3542
(2014/01/06)
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- Characterization of d-amino acid aminotransferase from Lactobacillus salivarius
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We searched a UniProt database of lactic acid bacteria in an effort to identify d-amino acid metabolizing enzymes other than alanine racemase. We found a d-amino acid aminotransferase (d-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between d-alanine and α-ketoglutarate. This is the first characterization of a d-AAT from a lactic acid bacterium. L. salivarius d-AAT is a homodimer that uses pyridoxal-5′-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60 °C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 °C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and d-alanine concentrations in the presence of several fixed concentrations of α-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The Km values for d-alanine and α-ketoglutarate were 1.05 and 3.78 mM, respectively. With this enzyme, d-allo-isoleucine exhibited greater relative activity than d-alanine as the amino donor, while α-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than α-ketoglutarate. The substrate specificity of L. salivarius d-AAT thus differs greatly from those of the other d-AATs so far reported.
- Kobayashi, Jyumpei,Shimizu, Yasuhiro,Mutaguchi, Yuta,Doi, Katsumi,Ohshima, Toshihisa
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- Isolation, purification, and characterization of phenylpyruvate transaminating enzymes of Erwinia carotovora
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Enzymes of Erwinia carotovora that transaminate phenylpyruvate were isolated, purified, and characterized. Two aromatic aminotransferases (PAT1 and PAT2) and an aspartic aminotransferase (PAT3) were found. According to gel filtration, these enzymes have molecular weights of 76, 75, and 78 kDa. The enzymes consist of two identical subunits of molecular weights of 31.4, 31, and 36.5 kDa, respectively. The isoelectric points of PAT1, PAT2, and PAT3 were determined as 3.6, 3.9, and 4.7, respectively. The enzyme preparations considerably differ in substrate specificity. All three of the enzymes productively interacted with the following amino acids: L-aspartic acid, L-leucine (except PAT3), L-isoleucine (except PAT3), L-serine, L-methionine, L-cysteine, L-phenylalanine, L-tyrosine, and L-tryptophane. The aromatic aminotransferases display higher specificity to the aromatic amino acids and the leucine-isoleucine pair, whereas the aspartic aminotransferase displays higher specificity to L-aspartic acid and relatively low specificity to the aromatic amino acids. The aspartic aminotransferase does not use L-leucine or L-isoleucine as a substrate. PAT1, PAT2, and PAT3 show the highest activity at pH 8.9 and at 48, 53, and 58°C, respectively.
- Paloyan,Hambardzumyan,Halebyan
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scheme or table
p. 98 - 104
(2012/06/29)
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- Synthesis-guided structure revision of the sarcodonin, sarcoviolin, and hydnellin natural product family
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A sweeping structural revision of the sarcodonin natural product family (published structures 1a-13a) is proposed after extensive studies aimed at their chemical synthesis. Key features of revised structure 1b include replacement of the N,N-dioxide moiety with an oxime, ring-opening of the central diketopiperazine, and transposition of the terphenyl wing from the 1β-2β position of 1a to the 2β-3β position of 1b. This structure revision arose from the serendipitous synthesis of a benzodioxane aminal (44) whose structure was unambiguously determined by X-ray crystallography and whose spectral properties bore considerable resemblance to the published data for the sarcodonins. A versatile new method for O-arylation of hydroxamic acids is also reported herein, as well as a manganese(III)- mediated α-oxidation of hydroxamic acids to aminals.
- Lin, David W.,Masuda, Takeshi,Biskup, Moritz B.,Nelson, Jonathan D.,Baran, Phil S.
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experimental part
p. 1013 - 1030
(2011/04/15)
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- Syntheses of isotopically labelled L-?±-amino acids with an asymmetric centre at C-3
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Approaches are described to the synthesis of a series of isotopically labelled L-a-amino acids each with an asymmetric centre at C-3, including isoleucine, allo-isoleucine, threonine and allo-threonine. The methods may be simply adapted for the selective incorporation of an isotopic label at each site of L-valine including the selective labelling of either diastereotopic methyl group with carbon-13 and/or deuterium and labelling of the amine with nitrogen-15. ? The Royal Society of Chemistry 2000.
- Harding, John R.,Hughes, Rachael A.,Kelly, Nicholas M.,Sutherland, Andrew,Willis, Christine L.
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p. 3406 - 3416
(2007/10/03)
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- Concerted base-promoted elimination in the decomposition of N-halo amino acids
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N-Chloroamino acids are unstable in aqueous solution and decompose through different pathways depending on the reaction conditions, yielding precursors of carcinogenic and/or mutagenic compounds. One of these pathways is a 1,2-elimination process, which has scarcely received any attention and for which no systematic analysis is available. The process is first order relative to the N-chloroamino acid and to that of hydroxide ion. The use of 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoropropan-2-ol buffer solutions established that the process is general-base catalysed. The reaction rate is affected by the presence of a methyl group on the nitrogen atom and the nature of the leaving group. The results show an important steric effect due to the alkyl substituents on the α-carbon. With bulky alkyl substitueras on the α-carbon, and in particular in the case of N-alkylamino acids, the catalytic effect increases as the base strength decreases. To characterize the transition state, Brtonsted's β and βlg were used. A More O'Ferrall-Jencks diagram shows the transition state structure changing from carbanion-like to nitrenium-like, a large perpendicular effect being evident. The reaction proceeds through a concerted mechanism AxhDHDN instead of the stepwise AxhDH? + DN proposed earlier.
- Armesto,Canle L,Garcia,Losada,Santaballa
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p. 552 - 560
(2007/10/03)
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- Halogen-induced aqueous oxidation of (L)-isoleucine
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The halogen-induced oxidation of the essential α-amino acid (L)-isoleucine has been studied as a model for similar compounds.Different reactions take place during this oxidation.First, α-amino acids suffer a very fast halogenation in aqueous medium, yielding (N-X)-α-amino acids.The so-formed (N-X)-α-amino acids undergo decomposition.Different pathways are possible depending mainly on the acidity of the medium.Grob fragmentation is the main process in mild acid, neutral and mild basic conditions, yielding aldehydes or ketones and ammonia or primary amines, while a concerted elimination is the most important reaction in basic medium, leading to α-keto acids and, again, ammonia or primary amines.Both pathways are discussed and a kinetic model is presented for the overall oxidation that explains the behavior of other amino acids.On the basis of these conclusions, the organic-nitrogen charge and the conditions of the medium should be strictly controlled in water-halogenation disinfection processes in order not to generate further unhealthy organic compounds. - Keywords: water disinfection / chlorination / N-chlorinated amino acid / N-brominated amino acid / concerted fragmentation / concerted elimination / solvent effect
- Armesto, Xose Luis,Canle, L. Moises,Garcia, Maria Victoria,Losada, Manuel,Santaballa, J. A.
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p. 1061 - 1068
(2007/10/02)
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- Kinetics of oxidation of amino acids by hexachloroiridate(IV) in aqueous acid medium
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The title reaction studied in the pH range of 2.5 to 3.5 is pseudo-first order in in the presence of excess .The rate increases with increase in and the order in is fractional.The rate also increases with increase in +> and the order in +> is unity.Added salts and change in dielectric constant of the medium do not affect the rate appreciably.However, added acrylamide induces polymerisation.A suitable mechanism has been proposed.
- Kumar, Ch. Sudheer,Chandraiah, U.,Siddiqui, M. A. A.,Kandlikar, Sushma
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p. 714 - 716
(2007/10/02)
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- Synthesis of dihydro-2,3-furandiones from diethyl oxalate and aldehydes through the action of sodium methoxide
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Dihydro-4,4-dimethyl-2,3-furandione (4e) was synthesized from diethyl oxalate, methylpropanal (1a) and formaldehyde in the presence of sodium methoxide. In a similar manner, analogs of dihydro-2,3-furandione 4 were prepared using other aldehydes.
- Hata,Morishita,Akutsu,Kawamura
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p. 289 - 291
(2007/10/02)
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- An Evaluation of the Substrate Specificity, and of Its Modification by Site-Directed Mutagenesis, of the Cloned L-Lactate Dehydrogenase from Bacillus stearothermophilus
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The L-lactate dehydrogenase of Bacillus stearothermophilus (BSLDH) is a stable, thermophilic oxidoreductase.It has been selected as a model of enzymes with considerable future promise in assymetric synthesis in that it has been cloned to ensure a plentiful and inexpensive supply and because of the potential for tailoring its specificity to accept unnatural substrate structures via the site-directed mutagenesis techniques of moleculer biology.In this study, the specificity of BSLDH toward representative α-keto acids possessing straight- and branched-chain alkyl,cycloalkyl, or aromatic side chains has been evaluated.The results show that substrates that are sterically bulky in the region of the α-keto group to be reduced are poorly accepted by the enzyme.Graphics analyses indicated that the low activities of these hindered substrates might be partly due to a bad interaction of the active site residue Gln102 with large or branched substituents adjacent to the α-keto group.Accordingly, Gln102 has been replaced by the smaller Asn residue by site-directed mutagenesis in an attempt to expand the active site volume available to receive substrates larger than the natural pyruvate.However, the kinetic data show that bulky α-keto acids are only marginally better accommodated by the Gln102 -> Asn mutant than by the wild-type enzyme.
- Luyten, Marcel A.,Bur, Daniel,Wynn, Hla,Parris, Wendy,Glod, Marvin,et al.
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p. 6800 - 6804
(2007/10/02)
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