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71.
A putative long-chain mannitol-1-phosphate 5-dehydrogenase from Aspergillus fumigatus (AfM1PDH) was overexpressed in Escherichia coli to a level of about 50% of total intracellular protein. The purified recombinant protein was a approximately 40-kDa monomer in solution and displayed the predicted enzymatic function, catalyzing NAD(H)-dependent interconversion of d-mannitol 1-phosphate and d-fructose 6-phosphate with a specific reductase activity of 170 U/mg at pH 7.1 and 25 degrees C. NADP(H) showed a marginal activity. Hydrogen transfer from formate to d-fructose 6-phosphate, mediated by NAD(H) and catalyzed by a coupled enzyme system of purified Candida boidinii formate dehydrogenase and AfM1PDH, was used for the preparative synthesis of d-mannitol 1-phosphate or, by applying an analogous procedure using deuterio formate, the 5-[2H] derivative thereof. Following the precipitation of d-mannitol 1-phosphate as barium salt, pure product (>95% by HPLC and NMR) was obtained in isolated yields of about 90%, based on 200 mM of d-fructose 6-phosphate employed in the reaction. In situ proton NMR studies of enzymatic oxidation of d-5-[2H]-mannitol 1-phosphate demonstrated that AfM1PDH was stereospecific for transferring the deuterium to NAD+, producing (4S)-[2H]-NADH. Comparison of maximum initial rates for NAD+-dependent oxidation of protio and deuterio forms of D-mannitol 1-phosphate at pH 7.1 and 25 degrees C revealed a primary kinetic isotope effect of 2.9+/-0.2, suggesting that the hydride transfer was strongly rate-determining for the overall enzymatic reaction under these conditions. 相似文献
72.
J. G. Shirani Ranasinghe Ming-Cheh Liu Yoichi Sakakibara Yumiko Takeshita Nobuhiro Fukuda Tetsuo Nasu Masahito Suiko 《Journal of Molecular Catalysis .B, Enzymatic》2001,12(1-6)
In our previous studies, we have demonstrated the stereoselective and manganese-dependent sulfation of tyrosine and Dopa isomers by human monoamine (M)-form phenol sulfotransferase (PST). In the present study, we investigated the occurrence of these phenomena in vivo using Sprague–Dawley rats as an experimental model. Three groups of six male rats were orally introduced with 1 ml of, respectively, 3 mM, 10 mM and 30 mM MnCl2 with a constant level of 0.2 mM
-m-tyrosine per day for 7 days. Their urine was collected and analyzed for the presence of sulfated
-m-tyrosine (
-m-TyrS) by ion-pair HPLC using a C18 reversed-phase column. The level of urinary
-m-TyrS, which was detected in the urine of the MnCl2-treated rats but not control rats, appeared to increase proportionally to the amount of MnCl2 administered. Chiral HPLC was employed to differentiate the
-form and
-form m-TyrS present in the urine sample of MnCl2-treated rats. Both
-m-TyrS and
-m-TyrS were detected, with the
-form being present at significantly higher level than the
-form. 相似文献
73.
Undheim K 《Amino acids》2008,34(3):357-402
The focus has been on the development of methodology for stereoselective preparation of spiroannulated intermediates of the Sch?llkopf chiron and further transformations to cyclic alpha-amino acids. The spiroannulations are effected by Ru(II)-catalysed ring-closing metathesis reactions, by Ru(II)- and Pd(0)-catalysed cycloisomerisations, by Rh(II)-carbenoid cyclisation reactions and by intramolecular aldol condensations. Hydrolytic reactions of the spirane intermediates have provided several groups of highly novel and functionalised five-, six- and seven-membered cyclic alpha-quaternary-alpha-amino acid derivatives as well as alicyclic derivatives. The novel cyclic amino acid derivatives can be regarded as cyclic constrained analogues of corresponding common amino acids, or in some cases as intermediates for further preparation of such amino acids. Some emphasis has been on the preparation of cyclic serine analogues. Major efforts have been on the preparation of cyclic alpha-quaternary bis(alpha-amino acid) derivatives as conformationally constrained dicarba-analogues of cystine. 相似文献
74.
An efficient and stereoselective synthesis of d,l-gulose was described. The key step of the synthetic route is represented by a multicomponent enyne cross metathesis—hetero Diels-Alder reaction which allows the formation of the pyran ring from cheap and commercially available substrates in a single synthetic step. The synthesis of d,l-gulose was accomplished without the use of protecting groups making this approach highly desirable also in terms of atom economy. 相似文献
75.
Marko D. Mihovilovic Radka Snajdrova Birgit Grtzl 《Journal of Molecular Catalysis .B, Enzymatic》2006,39(1-4):135-140
Screening of 4,4-disubstituted and 3,4,5-polysubstituted cyclohexan- and cyclohexenones with eight different overexpression systems of microbial monooxygenases in recombinant Escherichia coli provided valuable information about substrate acceptance and enantioselectivity of this enzyme family, which are responsible for the stereoselective Baeyer–Villiger biooxidation of ketones. For this purpose whole-cell mediated biotransformations were realized to overcome some limitations in the application of cofactor dependent biocatalysts. The different behavior of various enzymes reflects a recent hypothesis about two distinct clusters of biooxidation catalysts. In contrast to isolated enzyme biooxidations, recombinant cells did not yield unsaturated lactone products derived from cycloalkenones. They rather displayed reductase activity to reduce such precursors to saturated ketones, which were subsequently oxidized to the corresponding Baeyer–Villiger products in a sequential two-step biotransformation. 相似文献
76.
J.D. Carballeira M.A. Quezada P. Hoyos Y. Sime M.J. Hernaiz A.R. Alcantara J.V. Sinisterra 《Biotechnology advances》2009,27(6):686-714
Enzyme catalyzed reactions are commonly used at laboratory or industrial scale. Contrarily, the whole cell catalyzed reactions are restricted to special cases. The tremendous advances in the last years in Molecular Biology and more specifically in Metabolic Engineering and Directed Enzyme Evolution have opened the door to create tailor-made microorganisms or “designer bugs” for industrial purposes. Whole cell catalysts can be much more readily and inexpensively prepared than purified enzymes and the enzymes – inside the cells – are protected from the external environment and stabilized by the intracellular medium. Three situations have traditionally been considered convenient to select the use of whole cell catalyzed processes against the free enzyme catalyzed process: i) when the enzyme is intracellular; ii) when the enzyme needs a cofactor to carry out the catalytic act and iii) in the development of multienzymatic processes. Red–ox reactions represent the molecular basis for energy generation in the cell. These reactions are catalyzed by intracellular enzymes and are cofactor dependent as red–ox reactions need electron carriers as helpers in reduction reactions (gain of electrons) or oxidation (loss of electrons).In this review we present an overview of the state of the art of red–ox biotransformations catalyzed by whole cells — wild-type or genetically engineered microorganisms. Stereoselective reductions, hydroxylations of arenes and unfunctionalized alkanes, alkene monooxygenation, and Baeyer–Villiger reactions are among the processes described along the text, focusing in their chemo-, regio- and stereoselectivity. 相似文献
77.
Ruud van Deursen Wolfgang Stampfer Klaus Edegger Kurt Faber Wolfgang Kroutil 《Journal of Molecular Catalysis .B, Enzymatic》2004,31(4-6):159-163
Biocatalytic reduction of the keto-moiety of α,β-unsaturated ketones (enones) was achieved with absolute chemo- and stereo-selectivity employing whole lyophilized cells of Rhodococcus ruber DSM 44541 to furnish the corresponding allylic alcohols in e.e. up to >99%. It was shown that a stereocenter in γ-position of the ketone moiety to be reduced is too distant from the reaction center to induce any significant diastereoselectivity, thus no kinetic resolution of an racemic ketone occurred. 相似文献