- Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli
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2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.
- Horinouchi, Nobuyuki,Ogawa, Jun,Kawano, Takako,Sakai, Takafumi,Saito, Kyota,Matsumoto, Seiichiro,Sasaki, Mie,Mikami, Yoichi,Shimizu, Sakayu
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p. 1371 - 1378
(2008/02/11)
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- Purification and Characterization of Pyrophosphate: D-Fructose 6-Phosphate 1-Phosphotransferase from Rice Seedlings
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Pyrophosphate: D-fructose 6-phosphate 1-phosphotransferase was purified to homogeneity from rice seedlings.The enzyme had an apparent molecular weight of 100,000 and consisted of a single protein.The pH optimum for the forward and reverse reactions was 8.1 and 7.9, respectively.Fructose-2,6-bisphosphate (Fru-2,6-P2) shifted the pH optimum for both the forward and reverse reactions to slightly acidic.Half-maximum activation of the enzyme for Fru-2,6-P2 in the forward and reverse reactions was observed at 16 and 520 nM, respectively.The enzyme had normalMichaelis-Menten kinetics in both the presence and absence of Fru-2,6-P2.Fru-2,6-P2 stimulated the enzyme activity by increasing Vmax for Fru-6-P and PPi, and by raising the affinity for Fru-6-P, Fru-2,6-P2, and PPi.At a saturated concentration of Fru-2,6-P2, Pi strongly inhibited the enzyme in the forward reaction.The Dixon plots with Pi indicated that the inhibition behavior was of the mixed type with respect to both Fru-6-P and PPi with Ki of 1.46 and 1.53 mM, respectively.
- Enomoto, Toshiki,Ohyama, Hideo,Kodama, Miyuki
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p. 251 - 255
(2007/10/02)
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- Anomerization of Furanose Sugars and Sugar Phosphates
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Thermodynamic and kinetic parameters for the ring-opening and -closing reactions of several aldo- and ketofuanoses and their phosphate esters have been determined by NMR line-width and saturation-transfer methods.Cyclic forms interconvert via a single, acyclic carbonyl form under either acid or base catalysis.Ring-opening rates do not correlate with thermodynamic stability of the rings.For aldofuranose phosphates, α anomers open faster than β anomers; for ketofuranose phosphates the converse is observed.Intramolecular catalysis of anomerization by the phosphate group of sugar phosphates is documented.Biological and mechanistic implications of the observed kinetics are discussed.
- Pierce, John,Serianni, Anthony S.,Barker, Robert
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p. 2448 - 2456
(2007/10/02)
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- Synthesis of Sugars by Aldolase-Catalyzed Condensation Reactions
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Dihydroxyacetone phosphate was prepared in 200-mmol scale from dihydroxyacetone by two procedures: reaction with phosphorus oxytrichloride and glycerol kinase catalyzed phosphorylation using ATP with in situ regeneration of ATP by phosphoenolpyruvate or acetyl phosphate.Dihydroxyacetone phosphate was converted to fructose 6-phosphate in 80percent yield by exposure to a mixture of co-immobilized triosephosphate isomerase and aldolase followed by acid hydrolysis of the condensation product fructose 1,6-bisphosphate.Fructose 6-phosphate was subsequently converted by chemical and enzymatic schemes into fructose, glucose 6-phosphate, and glucose.Practical procedures are described for the preparation of D- and L-glyceraldehyde 3-phosphate and for several hexoses labeled with 13C in the C-2 and C-2,5 positions.
- Wong, Chi-Huey,Whitesides, George M.
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p. 3199 - 3205
(2007/10/02)
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- Chemical and Enzymatic Syntheses of 6-Deoxyhexoses. Conversion to 2,5-Dimethyl-4-hydroxy-2,3-dihydrofuran-3-one (Furaneol) and Analogues
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6-Deoxy-D-fructose 1-phosphate (6-deoxyF-1-P) forms when a solution containing D-fructose 1,6-diphosphate (FDP) and D-lactaldehyde is treated with the enzymes aldolase and triosephosphate isomerase (Scheme I).This transformation involves three reactions: aldolase-catalyzed cleavage of FDP to a mixture of dihydroxyacetone phosphate and D-glyceraldehyde phosphate, triosephosphate isomerase catalyzed equilibration of dihydroxyacetone phosphate and D-glyceraldehyde phosphate, and aldolase-catalyzed condensation of dihydroxyacetone phosphate and D-lactaldehyde to 6-deoxyF-1-P.An analogous process converts a mixture of FDP and L-lactaldehyde to 6-deoxysorbose 1-phosphate (6-deoxyS-1-P).Aldolase-catalyzed reaction of dihydroxyacetone phosphate, prepared separately, with D-lactaldehyde yields 6-deoxyF-1-P directly; similar reaction of dihydroxyacetone phosphate with α-hydroxybutyraldehyde yields a mixture of 6-methyl-6-deoxyhexose 1-phosphates.Acid-catalyzed hydrolysis of the sugar phosphates releases the corresponding free sugars.A mixture containing 6-deoxyhexoses is formed directly by base-catalyzed aldol condensation of dihydroxyacetone and D,L-lactaldehyde.Treatment of any of the 6-deoxyhexoses with acids generates 2,5-dimethyl-4-hydroxy-2,3-dihydrofuran-3-one (Furaneol, a flavor principle).Furaneol can also be prepared in moderate yields by hydrogenolysis of FDP and other hexose phosphates in alkaline media.
- Wong, Chi-Huey,Mazenod, Francois P.,Whitesides, George M.
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p. 3493 - 3497
(2007/10/02)
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