- Microfluidic multi-input reactor for biocatalytic synthesis using transketolase
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Biocatalytic synthesis in continuous-flow microreactors is of increasing interest for the production of specialty chemicals. However, the yield of production achievable in these reactors can be limited by the adverse effects of high substrate concentration on the biocatalyst, including inhibition and denaturation. Fed-batch reactors have been developed in order to overcome this problem, but no continuous-flow solution exists. We present the design of a novel multi-input microfluidic reactor, capable of substrate feeding at multiple points, as a first step towards overcoming these problems in a continuous-flow setting. Using the transketolase-(TK) catalysed reaction of lithium hydroxypyruvate (HPA) and glycolaldehyde (GA) to l-erythrulose (ERY), we demonstrate the transposition of a fed-batch substrate feeding strategy to our microfluidic reactor. We obtained a 4.5-fold increase in output concentration and a 5-fold increase in throughput compared with a single input reactor.
- Lawrence, James,O'Sullivan, Brian,Lye, Gary J.,Wohlgemuth, Roland,Szita, Nicolas
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Read Online
- L-erythrulose production by oxidative fermentation is catalyzed by PQQ-containing membrane-bound dehydrogenase.
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Thermotolerant Gluconobacter frateurii CHM 43 was selected for L-erythrulose production from mesoerythritol at higher temperatures. Growing cells and the membrane fraction of the strain rapidly oxidized mesoerythritol to L-erythrulose irreversibly with almost 100% of recovery at 37 degrees C. L-Erythrulose was also produced efficiently by the resting cells at 37 degrees C with 85% recovery. The enzyme responsible for mesoerythritol oxidation was found to be located in the cytoplasmic membrane of the organism. The EDTA-resolved enzyme required PQQ and Ca2+ for L-erythrulose formation, suggesting that the enzyme catalyzing meso-erythritol oxidation was a quinoprotein. Quinoprotein membrane-bound mesoerythritol dehydrogenase (QMEDH) was solubilized and purified to homogeneity. The purified enzyme showed a single band in SDS-PAGE of which the molecular mass corresponded to 80 kDa. The optimum pH of QMEDH was found at pH 5.0. The Michaelis constant of the enzyme was found to be 25 mM for meso-erythritol as the substrate. QMEDH showed a broad substrate specificity toward C3-C6 sugar alcohols in which the erythro form of two hydroxy groups existed adjacent to a primary alcohol group. On the other hand, the cytosolic NAD-denpendent meso-erythritol dehydrogenase (CMEDH) of the same organism was purified to a crystalline state. CMEDH showed a molecular mass of 60 kDa composed of two identical subunits, and an apparent sedimentation constant was 3.6 s. CMEDH catalyzed oxidoreduction between mesoerythritol and L-erythrulose. The oxidation reaction was observed to be reversible in the presence of NAD at alkaline pHs such as 9.0-10.5. L-Erythrulose reduction was found at pH 6.0 with NADH as coenzyme. Judging from the catalytic properties, the NAD-dependent enzyme in the cytosolic fraction was regarded as a typical pentitol dehydrogenase of NAD-dependent and the enzyme was independent of the oxidative fermentation of L-erythrulose production.
- Moonmangmee, Duangtip,Adachi, Osao,Shinagawa, Emiko,Toyama, Hirohide,Theeragool, Gunjana,Lotong, Napha,Matsushita, Kazunobu
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Read Online
- Enantioselective Reductive Oligomerization of Carbon Dioxide into l-Erythrulose via a Chemoenzymatic Catalysis
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A cell-free enantioselective transformation of the carbon atom of CO2has never been reported. In the urgent context of transforming CO2into products of high value, the enantiocontrolled synthesis of chiral compounds from CO2would be highly desirable. Using an original hybrid chemoenzymatic catalytic process, we report herein the reductive oligomerization of CO2into C3(dihydroxyacetone, DHA) and C4(l-erythrulose) carbohydrates, with perfect enantioselectivity of the latter chiral product. This was achieved with the key intermediacy of formaldehyde. CO2is first reduced selectively by 4e-by an iron-catalyzed hydroboration reaction, leading to the isolation and complete characterization of a new bis(boryl)acetal compound derived from dimesitylborane. In an aqueous buffer solution at 30 °C, this compound readily releases formaldehyde, which is then involved in selective enzymatic transformations, giving rise either (i) to DHA using a formolase (FLS) catalysis or (ii) to l-erythrulose with a cascade reaction combining FLS and d-fructose-6-phosphate aldolase (FSA) A129S variant. Finally, the nature of the synthesized products is noteworthy, since carbohydrates are of high interest for the chemical and pharmaceutical industries. The present results prove that the cell-freede novosynthesis of carbohydrates from CO2as a sustainable carbon source is a possible alternative pathway in addition to the intensely studied biomass extraction andde novosyntheses from fossil resources.
- Bontemps, Sébastien,Clapés, Pere,Desmons, Sarah,Dumon, Claire,Fauré, Régis,Grayson-Steel, Katie,Hurtado, John,Nu?ez-Dallos, Nelson,Vendier, Laure
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supporting information
p. 16274 - 16283
(2021/10/12)
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- PROCESSES FOR PREPARING C-4 SUGARS AND KETOSE SUGARS
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Various processes for preparing C4 aldoses and/or ketones thereof are described. Various processes are described for preparing C4 aldoses and/or ketones thereof from feed compositions comprising glycolaldehyde. Also, various processes for preparing useful downstream products and intermediates, such as erythritol and erythronic acid, from the C4 aldoses and/or ketones thereof are described.
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Page/Page column 37-39
(2021/11/20)
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- D -Serine as a Key Building Block: Enzymatic Process Development and Smart Applications within the Cascade Enzymatic Concept
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An efficient enzymatic method catalyzed by an enzyme from the d-threonine aldolase (DTA) family was developed for d-serine production at industrial scale. This process was used for the synthesis of two valuable ketoses, l-erythrulose and d-fructose, within the cascade enzymatic concept involving two other enzymes. Indeed, d-serine was used as a substrate of d-amino acid oxidase (DAAO) for the in situ generation of the corresponding α-keto acid, hydroxypyruvic acid (HPA), a key donor substrate of transketolase (TK). This enzyme catalyzed the irreversible transfer of the ketol group from HPA to an aldehyde acceptor to form a (3S)-ketose by stereoselective carbon-carbon bond formation. The compatibility of all enzymes and substrates allowed a sequential three-step enzymatic process to be performed without purification of the intermediates. This strategy was validated with two TK aldehyde substrates to finally obtain the corresponding (3S)-ketoses with high control of the stereoselectivity and excellent aldehyde conversion rates.
- Auffray, Pascal,Charmantray, Franck,Collin, Jér?me,Hecquet, Laurence,L'Enfant, Mélanie,Martin, Juliette,Ocal, Nazim,Pollegioni, Loredano
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p. 769 - 775
(2020/07/14)
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- One-Pot Cascade Synthesis of (3S)-Hydroxyketones Catalyzed by Transketolase via Hydroxypyruvate Generated in Situ from d-Serine by d-Amino Acid Oxidase
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We described an efficient in situ generation of hydroxypyruvate from d-serine catalyzed by a d-amino acid oxidase from Rhodotorula gracilis. This strategy revealed an interesting alternative to the conventional chemical synthesis of hydroxypyruvate starting from toxic bromopyruvate or to the enzymatic transamination from l-serine requiring an additional substrate as amino acceptor. Hydroxypyruvate thus produced was used as donor substrate of transketolases from Escherichia coli or from Geobacillus stearothermophilus catalyzing the stereoselective formation of a carbon?carbon bond. The enzymatic cascade reaction was performed in one-pot in the presence of d-serine and appropriate aldehydes for the synthesis of valuable (3S)-hydroxyketones, which were obtained with high enantio- and diastereoselectivity and in good yield. The efficiency of the process was based on the irreversibility of both reactions allowing complete conversion of d-serine and aldehydes. (Figure presented.).
- L'enfant, Mélanie,Bruna, Felipe,Lorillière, Marion,Ocal, Nazim,Fessner, Wolf-Dieter,Pollegioni, Loredano,Charmantray, Franck,Hecquet, Laurence
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p. 2550 - 2558
(2019/04/17)
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- Separating Thermodynamics from Kinetics—A New Understanding of the Transketolase Reaction
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Transketolase catalyzes asymmetric C?C bond formation of two highly polar compounds. Over the last 30 years, the reaction has unanimously been described in literature as irreversible because of the concomitant release of CO2 if using lithium hydroxypyruvate (LiHPA) as a substrate. Following the reaction over a longer period of time however, we have now found it to be initially kinetically controlled. Contrary to previous suggestions, for the non-natural conversion of synthetically more interesting apolar substrates, the complete change of active-site polarity is therefore not necessary. From docking studies it was revealed that water and hydrogen-bond networks are essential for substrate binding, thus allowing aliphatic aldehydes to be converted in the charged active site of transketolase.
- Marsden, Stefan R.,Gjonaj, Lorina,Eustace, Stephen J.,Hanefeld, Ulf
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p. 1808 - 1814
(2017/05/26)
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- Efficient Production of Biomass-Derived C4 Chiral Synthons in Aqueous Solution
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Carbohydrates are expected to replace petroleum and to become the base of industrial chemistry. Chirality is one particular area in which carbohydrates have a special potential advantage over petroleum resources. Herein, we report a catalytic approach for the direct production of d-tetroses [i.e., d-(?)-erythrose and d-(+)-erythrulose] from d-hexoses through a fast retro-aldol process at 190 °C that achieves a yield of 46 % and completely retains the chiral centers in the final chiral synthon. The d-tetrose products were further converted into their derivatives, thereby accomplishing transfer of chirality from natural chiral hexoses to high-value-added chiral chemicals. Our results also suggest that the product distribution for the conversion of d-hexoses was determined by their isomerization and epimerization trends that competed with their corresponding retro-aldol condensation processes.
- Lin, Shaoying,Guo, Xiao,Qin, Kai,Feng, Lei,Zhang, Yahong,Tang, Yi
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p. 4179 - 4184
(2017/12/02)
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- ISOMERISATION OF C4-C6 ALDOSES WITH ZEOLITES
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The present invention relates to isomerization of C4-C6 aldoses to their corresponding C4-C6 ketoses. In particular, the invention concerns isomerization of C4-C6 aldoses over solid zeolite catalysts free of any metals other than aluminum, in the presence of suitable solvent(s) at suitable elevated temperatures. C6 and C5 aldose sugars such as glucose and xylose, which are available in large amounts from biomass precursors, are isomerized to fructose and xylulose respectively, in a one or two-step process over inexpensive commercially available zeolite catalysts, containing aluminum as the only metal in the catalyst. The ketoses obtained are used as sweeteners in the food and/or brewery industry, or treated to obtain downstream platform chemicals such as lactic acid, HMF, levulinic acid, furfural, MMHB, and the like. FIG. 7
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Page/Page column 25-28
(2014/03/25)
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- Zeolite-catalyzed isomerization of tetroses in aqueous medium
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The isomerization of erythrose (ERO) was studied in water over commercially available large-pore zeolites, e.g. H-Y, H-USY and H-beta. Among the employed zeolites, H-USY(6) was found to efficiently isomerize the sugar, yielding 45% erythrulose (ERU), 42% ERO and 3% of the epimer threose (THO) (corresponding to the equilibrium mixture), i.e. total tetrose yield 90%, after reaction for 5-7 h at 120 °C. Changing the solvent from water to methanol decreased the yield of ERU markedly to 18% and gave only a total yield of tetroses of 27% which is significantly lower than that obtained in water. Hence, the results demonstrate that water is the preferred solvent compared to lower alcohols for zeolite-catalyzed tetrose isomerization, which is opposite to what has been found previously for analogous pentose and hexose isomerization. A reuse study revealed further that H-USY(6) could be applied for at least five reaction runs with essentially unchanged activity and without significant aluminum leaching from the catalyst. The use of benign reaction conditions and an industrially pertinent solid catalyst in combination with water establishes a new, green tetrose isomerization protocol. the Partner Organisations 2014.
- Saravanamurugan, Shunmugavel,Riisager, Anders
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p. 3186 - 3190
(2014/08/18)
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- Chemoselective Pd-catalyzed oxidation of polyols: Synthetic scope and mechanistic studies
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The regio- and chemoselective oxidation of unprotected vicinal polyols with [(neocuproine)Pd(OAc)]2(OTf)2 (1) (neocuproine = 2,9-dimethyl-1,10-phenanthroline) occurs readily under mild reaction conditions to generate α-hydroxy ketones. The oxidation of vicinal diols is both faster and more selective than the oxidation of primary and secondary alcohols; vicinal 1,2-diols are oxidized selectively to hydroxy ketones, whereas primary alcohols are oxidized in preference to secondary alcohols. Oxidative lactonization of 1,5-diols yields cyclic lactones. Catalyst loadings as low as 0.12 mol % in oxidation reactions on a 10 g scale can be used. The exquisite selectivity of this catalyst system is evident in the chemoselective and stereospecific oxidation of the polyol (S,S)-1,2,3,4-tetrahydroxybutane [(S,S)-threitol] to (S)-erythrulose. Mechanistic, kinetic, and theoretical studies revealed that the rate laws for the oxidation of primary and secondary alcohols differ from those of diols. Density functional theory calculations support the conclusion that β-hydride elimination to give hydroxy ketones is product-determining for the oxidation of vicinal diols, whereas for primary and secondary alcohols, pre-equilibria favoring primary alkoxides are product-determining. In situ desorption electrospray ionization mass spectrometry (DESI-MS) revealed several key intermediates in the proposed catalytic cycle.
- Chung, Kevin,Banik, Steven M.,De Crisci, Antonio G.,Pearson, David M.,Blake, Timothy R.,Olsson, Johan V.,Ingram, Andrew J.,Zare, Richard N.,Waymouth, Robert M.
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p. 7593 - 7602
(2013/07/05)
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- Chemoselective oxidation of polyols with chiral palladium catalysts
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Chiral palladium-based catalysts derived from pyridinyl oxazoline (pyOx) ligands catalyze the oxidation of alcohols, including 1,2-diols, triols, and tetraols, with high regio- and chemoselectivity. Screening of various chiral oxazoline-derived ligands for the oxidation of a model diol, 1,2-propanediol (1,2-PD), revealed that the nature of the ligand had a significant influence on the activity and chemoselectivity for oxidation of vicinal diols. The PyOx ligands containing an α-methyl substituent were the most active for the oxidation of 1,2-PD using benzoquinone as the terminal oxidant. Oxidation of vicinal diols and polyols occurs selectively at the secondary alcohol to afford α-hydroxy ketones in isolated yields of 62-87%. Chemoselective oxidation of meso-erythritol with the chiral [(S)-(α-Me(tert-Bu)PyOx)Pd(OAc)] 2[OTf]2 afforded (S)-erthyrulose in 62% yield and 24% ee.
- De Crisci, Antonio G.,Chung, Kevin,Oliver, Allen G.,Solis-Ibarra, Diego,Waymouth, Robert M.
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p. 2257 - 2266
(2013/05/21)
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- Thermostable transketolase from Geobacillus stearothermophilus: Characterization and catalytic properties
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Here we have characterized the first transketolase (TK) from a thermophilic microorganism, Geobacillus stearothermophilus, which was expressed from a synthetic gene in Escherichia coli. The G. stearothermophilus TK (mTK gst) retained 100% activity for one week at 50 °C and for 3 days at 65 °C, and has an optimum temperature range around 60-70 °C, which will be useful for preparative applications and for future biocatalyst development. The thermostability of the mTKgst allowed us to carry out an easy, one-step purification by heat shock treatment of crude cell extracts at 65 °C for 45min, directly yielding 132mg of pure mTK gst from 1L of culture. The reaction rate of mTKgst with glycolaldehyde was 14 times higher at 70 °C than at 20 °C, and 4 times higher at 50 °C when compared to E. coli TK under identical conditions. When tested at 50 °C with other aldehydes as acceptors, mTKgst activity was approximately 3 times higher than those obtained at 20 °C. Applications of this new TK in biocatalysis were performed with hydroxypyruvate as donor and three different aldehydes as acceptors-glycolaldehyde, D-glyceraldehyde and butyraldehyde-from which the corresponding products L-erythrulose 1, D-xylulose 2 and 1,3-dihydroxyhexan-2-one 3 were obtained, respectively. The optical rotations for products 1 and 2 indicate that the stereospecificity of mTKgst is identical to that of other TK sources, leading to a (3S) configuration. With the non-hydroxylated substrate, butanal, the ee value was 85% (3S), showing higher enantioselectivity than the E. coli TK (75% ee, 3S). Processes at elevated temperatures could offer opportunities to extend the applications of TK biocatalysis, by favoring hydrophobic aldehyde acceptor substrate solubility and tolerance towards non-conventional media. Copyright
- Abdoul-Zabar, Juliane,Sorel, Isabelle,Helaine, Virgil,Charmantray, Franck,Devamani, Titu,Yi, Dong,De Berardinis, Veronique,Louis, Dominique,Marliere, Philippe,Fessner, Wolf-Dieter,Hecquet, Laurence
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p. 116 - 128
(2013/03/13)
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- Modular microfluidic reactor and inline filtration system for the biocatalytic synthesis of chiral metabolites
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Biocatalytic synthesis is now well established amongst catalytic methodologies as an extremely useful approach for the industrial synthesis of high-value compounds, due to its many advantages such as high reaction specificity and selectivity. However, engineering a biocatalytic process can be complex and time-consuming. This paper presents a modular microfluidic reactor and in-line filtration system for the rapid and small-scale evaluation of biocatalytic reactions. The system combines a substrate with a biocatalyst in free solution, incubates the two components until full conversion to product has been achieved, before extracting the product. The system has been applied to the transketolase-catalysed reaction of hydroxypyruvate (HPA) and glycolaldehyde (GA) to l-erythrulose, demonstrating complete conversion of substrate to product, complete retention of the enzyme and an overall yield of approximately 65%. The complete conversion of HPA and propanal to (3S)-1,3-dihydroxypentan-2- one with a mutant transketolase further demonstrated the applicability of the microfluidic system for organic synthesis.
- O'Sullivan, Brian,Al-Bahrani, Homam,Lawrence, James,Campos, Maria,Cazares, Armando,Baganz, Frank,Wohlgemuth, Roland,Hailes, Helen C.,Szita, Nicolas
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experimental part
p. 1 - 8
(2012/06/29)
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- Efficient immobilization of yeast transketolase on layered double hydroxides and application for ketose synthesis
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Transketolase (TK) from S. cerevisiae was successfully immobilized on layered double hydroxides (LDH) using simple, affordable and efficient adsorption and coprecipitation based immobilization procedures. Optimization of the preparation was performed using zinc aluminium nitrate (Zn 2Al-NO3) and magnesium aluminium nitrate (Mg 2Al-NO3) LDH as immobilization supports, and the protein-to-LDH weight ratio (Q) was varied. The highest immobilization yields (98-99%) and highest relative specific activities (4.2-4.4 U·mg -1 for the immobilized enzyme compared to 4.5 U·mg -1 for the free enzyme) were both achieved when using a protein-to-LDH weight ratio (Q) of 0.38. Efficient lyophilization of the LDH-TK bionanocomposites thus synthesized was proven to allow easy use and storage of the supported TK with no significant loss of activity over a three-month period. The kinetic parameters of the LDH-TK enzyme were comparable to those of the free TK. The LDH-TK enzyme was finally tested for the synthesis of L-erythrulose starting from hydroxypyruvate lithium salt (Li-HPA) and glycolaldehyde (GA) as substrates. L-erythrulose was characterized and obtained with an isolated yield of 56% similar to that obtained with free TK. The reusability of the LDH-TK biohybrid material was then investigated, and we found no loss of enzymatic activity over six cycles. Copyright
- Benaissi, Karima,Helaine, Virgil,Prevot, Vanessa,Forano, Claude,Hecquet, Laurence
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experimental part
p. 1497 - 1509
(2011/08/03)
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- A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a 'mix and match' E. coli expression system with microscale experimentation
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This work describes an experimental 'toolbox' for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a 'mix and match' E. coli-based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables efficient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental 'toolbox', one-pot syntheses of the diastereoisomers (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3, 4-butanetriol (ABT) were designed and performed, which gave final product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specific activity and ee( enantiomeric excess) of >95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this 'toolbox' provides an efficient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors.
- Rios-Solis,Halim,Cazares,Morris,Ward,Hailes,Dalby,Baganz,Lye
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experimental part
p. 192 - 203
(2012/04/18)
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- A mutant D-fructose-6-phosphate aldolase (Ala129Ser) with improved affinity towards dihydroxyacetone for the synthesis of polyhydroxylated compounds
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A mutant of D-fructose-6-phosphate aldo-lase (FSA) of Escherichia coli, FSA A129S, with im-proved catalytic efficiency towards dihydroxyacetone (DHA), the donor substrate in aldol addition reac-tions, was explored for synthetic applications. The Kcat/KM value for DHA was 17-fold higher with FSA A129S than that with FSA wild type (FSA wt). On the other hand, for hydroxyacetone as donor sub-strate FSA A129S was found to be 3.5-fold less effi-cient than FSA wt. Furthermore, FSA A129S also ac-cepted glycolaldehyde (GA) as donor substrate with 3.3-fold lower affinity than FSA wt. This differential selectivity of both FSA wt and FSA A129S for GA makes them complementary biocatalysts allowing a control over donor and acceptor roles, which is par-ticularly useful in carboligation multi-step cascade synthesis of polyhydroxylated complex compounds. Production of the mutant protein was also improved for its convenient use in synthesis. Several carbohy-drates and nitrocyclitols were efficiently prepared, demonstrating the versatile potential of FSA A129S as biocatalyst in organic synthesis.
- Castillo, Jose A.,Guerard-Helaine, Christine,Gutierrez, Mariana,Garrabou, Xavier,Sancelme, Martine,Schuermann, Melanie,Inoue, Tomoyuki,Helaine, Virgil,Charmantray, Franck,Gefflaut, Thierry,Hecquet, Laurence,Joglar, Jesus,Clapes, Pere,Sprenger, Georg A.,Lemaire, Marielle
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experimental part
p. 1039 - 1046
(2010/07/14)
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- Application of a modified Mosher's method for the determination of enantiomeric ratio and absolute configuration at C-3 of chiral 1,3-dihydroxy ketones
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The enantiomeric ratio and absolute configuration of products of the transketolase reaction are typically determined by comparison of the specific rotation or derivatisation and HPLC or GC. A Mosher's ester method has been developed via ester formation at
- Galman, James L.,Hailes, Helen C.
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supporting information; body text
p. 1828 - 1831
(2010/01/06)
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- Reactivity of 1-deoxy-D-erythro-hexo-2,3-diulose; A key intermediate in the maillard chemistry of hexoses
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Degradation of 1-deoxyhexo-2,3-diulose, a key intermediate in Maillard chemistry, in the presence of L-alanine under moderate conditions (37 and 50 °C) was investigated. Different analytical strategies were accomplished to cover the broad range of product
- Voigt, Michael,Glomb, Marcus A.
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experimental part
p. 4765 - 4770
(2010/06/14)
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- Enhancing and reversing the stereoselectivity of Escherichia coli transketolase via single-point mutations
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Chiral auxiliary methodology and chiral assays have been developed to establish the enantiomeric purities of erythrulose and 1,3-dihydroxypentan-2-one generated using wild-type (WT) Escherichia coli transketolase (TK). L-Erythrulose was formed in 95% ee and (3S)-1,3-dihydroxypentan-2-one in 58% ee. Since the latter compound was formed in moderate ee, TK libraries were screened to identify higher performing mutants. A colorimetric screen and chiral assay were successfully applied to a 96-well format, and new active TK mutants were identified, which gave 1,3-dihydroxypentan-2-one in high stereoselectivities. Remarkably, activesite single-point mutants were identified that were able to both enhance and reverse the stereoselectivity of TK.
- Smith, Mark E. B.,Hibbert, Edward G.,Jones, Alexander B.,Dalby, Paul A.,Hailesa, Helen C.
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experimental part
p. 2631 - 2638
(2009/09/28)
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- Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases
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In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369-7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473-21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.
- Baykal, Ahmet,Chakraborty, Sumit,Dodoo, Afua,Jordan, Frank
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p. 380 - 393
(2008/02/09)
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- Methods for treating plants and enhancing plant growth using polyacylglycosides and/or polyalkylglycosides and formulations for same
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Methods and formulations for treating plants and enhancing plant growth and for safening high concentrations of one or more phytocatalysts, wherein one or more formulations, comprising, a high concentration of one or more phytocatalysts, and an effective amount of one or more polyacylglycosides and polyalkylglycosides; and isomers, and metabolites, salts, hydrates, esters, amines, and derivatives of the polyacylglycosides and polyalkylglycosides, and combinations thereof, is applied to the plants.
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- Electrooxidation of mesoerythritol on platinum, modified or not by adatoms, in acid medium
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The electrocatalytic oxidation of meso-erythritol has been studied in 0.1 M HClO4 on platinum and on adatoms modified platinum. Preliminary investigations by cyclic voltammetry showed that erythritol was not reactive on a Pt electrode. Underpotential deposition of lead or thallium adatoms at platinum allowed to increase significantly the current densities. Long-time electrolyses were carded out using a three potential plateau program with different values of the oxidation potentials. Chromatographic analyses showed that the oxidation of erythritol led mainly to erythrose, erythrulose and to erythronic acid. Otherwise, electrolysis of erythritol on a Pt-Tl modified electrode orientated selectively the distribution of the reaction products towards the formation of erythrulose.
- Cherqaoui,Chbihi,Takky,Kokoh,Leger,Lamy
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p. 510 - 521
(2007/10/03)
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- Glucose isomerase catalysed isomerisation reactions of (2 R,3 R)- configured aldofuranoses into the corresponding open-chain 2-ketoses
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Immobilised glucose isomerase (EC 5.3.1.5) accepted various (2 R,3R)- configured aldofuranoses such as D-erythrose, as well as homologous C-5- modified o-ribose derivatives, as substrates. In the case of D-erythrose, quantitative conversion into D-glycero-tetrulose took place. D-Ribofuranoses were converted into the corresponding open-chain 2-ketoses in isolated yields of 65%. Surprisingly, L-erythrose also turned out to be a substrate of this enzyme.
- Ebner, Michael,Stuetz, Arnold E.
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p. 331 - 336
(2007/10/03)
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- Transketolase from Escherichia coli: A practical procedure for using the biocatalyst for asymmetric carbon-carbon bond synthesis
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A practical procedure is reported for the use of the enzyme transketolase, from Escherichia coli, for asymmetric carbon-carbon bond synthesis. The reactions with the biocatalyst are conveniently carried out, on a gram scale, in unbuffered aqueous media by employing a pH autotitrator. An improved large scale synthesis of hydroxypyruvate is also reported.
- Morris, K. Gail,Smith, Mark E.B.,Turner, Nicholas J.,Lilly, Malcolm D.,Mitra, Robin K.,Woodley, John M.
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p. 2185 - 2188
(2007/10/03)
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- Study of the Specificity of a Spinach Transketolase on Achiral Substrates
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The behaviour and the specificity of a spinach transketolase towards achiral aldehydes was studied.We have shown that these aldehydes are accepted by the enzyme and yield the corresponding saccharides with good enantiomeric excess.
- Dalmas, Valerie,Demuynck, Colette
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p. 2383 - 2388
(2007/10/02)
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- Enzyme-catalysed Carbon-Carbon Bond Formation: Use of Transketolase from Escherichia coli
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Transketolase has been obtained in greater quantities from an over-expressed E. coli transformant carrying the transketolase gene.Crude extracts of this organism are suitable for use in small scale biotransformations to provide mmol quantities of product.Initial results indicate that the transketolase from E. coli is relatively non-specific for the aldehyde component of the reaction.
- Hobbs, Gordon R.,Lilly, Malcolm D.,Turner, Nicholas J.,Ward, John M.,Willets, Andrew J.,Woodley, John M.
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p. 165 - 166
(2007/10/02)
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- Substrate Specificity and Carbohydrate Synthesis Using Transketolase
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This paper describes the use of the enzyme transketolase as a catalyst in organic synthesis.The properties of transketolase from both yeast and spinach were investigated.The yeast enzyme was found to be more convenient for routine use.Examination of the substrate specificity of yeast tansketolase demonstrated that the enzyme accepts a wide variety of 2-hydroxy aldehydes as substrates.A practical protocol for tansketolase-catalyzed condensation of hydroxypyruvic acid with these aldehydes has been developed and used for the synthesis of four carbohydrates: L-idose, L-gulose, 2-deoxy-L-xylohexose, and L-xylose.
- Kobori, Yoshihiro,Myles, David C.,Whitesides, George M.
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p. 5899 - 5907
(2007/10/02)
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- ENZYME-CATALYZED SYNTHESIS OF CARBOHYDRATES: SYNTHETIC POTENTIAL OF TRANSKETOLASE
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The synthetic potential of yeast or spinach transketolases was studied, using hydroxypyruvate as a donor substrate and 31 aldehydes as acceptors.All of them react, including aromatic, heteroaromatic and α,β unsaturated aldehydes.
- Demuynck, Colette,Bolte, Jean,Hecquet, Laurence,Dalmas, Valerie
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p. 5085 - 5088
(2007/10/02)
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- UTILIZATION OF ENZYMES IN ORGANIC CHEMISTRY : TRANSKETOLASE CATALYZED SYNTHESIS OF KETOSES
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Substrate specificity of bakers' yeast transketolase has been studied: the enzyme appears to be enantioselective towards the acceptor aldehyde.L-erythrulose, D-xylulose and 5-deoxy-D-xylulose have been synthesized by spinach transketolase catalyzed condensation of hydroxypyruvate and the corresponding aldehyde.
- Bolte, Jean,Demdynck, Colette,Samaki, Hamid
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p. 5525 - 5528
(2007/10/02)
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- FORMATION OF 3-OCTULOSES BY A SELF-ALDOL REACTION OF D-ERYTHROSE
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When kept at 105 deg C for 2.5 h, weakly alkaline, syrupy D-erythrose was readily converted into a mixture containing mainly D-glycero-tetrulose, the previously unknown β-D-altro-L-glycero-3-octulofuranose (2), and α-D-gluco-L-glycero-3-octulopyranose, which were isolated as the corresponding acetates.Treatment of 2 with Dowex 50 (H+) resin yielded 3,8-anhydro-β-D-altro-L-glycero-octulopyranose, identified as its acetate.Previous discrepancies in the D values for D-erythrose appear partly to originate in the self-aldol reaction.The dimerisation of D-erythrose 4-phosphate is also described.
- Westerlund, Eric
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- Radical-Induced Dephosphorylation of Fructose Phosphates in Aqueous Solution
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Oxygen free N2O-saturated aqueous solutions of D-fructose-1-phosphate and D-fructose-6-phosphate were γ-irradiated.Inorganic phosphate and phosphate free sugars (containing four to six carbon atoms) were identified and their G-values measured.D-Fructose-1-phosphate yields (G-values in parentheses) inorganic phosphate (1.6), hexos-2-ulose (0.12), 6-deoxy-2,5-hexodiulose (0.16), tetrulose (0.05) and 3-deoxytetrulose (0.15).D-Fructose-6-phosphate yields inorganic phosphate (1.7), hexos-5-ulose (0.1), 6-deoxy-2,5-hexodiulose (0.36), 3-deoxy-2,5-hexodiulose and 2-deoxyhexos-5-ulose (together 0.18).On treatment with alkaline phosphate further deoxy sugars were recognized and in fructose-1-phosphate G(6-deoxy-2,5-hexodiulose) was increased to a G-value of 0.4.Dephosphorylation is considered to occur mainly after OH attack at C-5 and C-1 in fructose-1-phosphate and at C-5 and C-6 in fructose-6-phosphate.Reaction mechanisms are discussed. - Keywords: D-Fructose-1-phosphate, D-Fructose-6-phosphate, γ-Irradiation; Radical Reactions, Mass Spectrometry
- Zegota, Henryk,Sonntag, Clemens von
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p. 1331 - 1337
(2007/10/02)
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