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1.
Bioorg Chem ; 80: 733-740, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30077176

RESUMO

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key chiral intermediate for the side chain synthesis of rosuvastatin. In this study, random mutagenesis, site-saturation mutagenesis and combinatorial mutagenesis methods were applied to improve the activity of a synthesized stereoselective short chain carbonyl reductase (SCR) to prepare (3R,5S)-CDHH. After screened by high-throughput screening method and high-performance liquid chromatography, mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser, were obtained, and the enzyme activities of mutants were improved by 1.60- and 1.91-fold compared with parent enzyme, respectively. The catalytically efficiencies (kcat/Km) of mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser exhibited 5.11- and 8.07-fold improvements in initial activity toward (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH), respectively. In the asymmetric reduction, mut-Phe145Tyr/Thr152Ser catalyzed 500 g L-1 of (S)-CHOH to produce (3R,5S)-CDHH with >99% yield and >99% e.e., and the highest space-time yield achieved at 752.76 mmol L-1 h-1 g-1 wet cell weight within 8 h bioconversion. This study provides a foundation for the preparation of (3R,5S)-CDHH by carbonyl reductase.

2.
Chem Commun (Camb) ; 54(67): 9356-9359, 2018 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-30079425

RESUMO

Photochromic [2]rotaxanes with bidirectional photoswitchability were fabricated, whose colored states exhibit remarkable visible-light and thermal stabilities as revealed by systematically spectroscopic investigations.

3.
Chem Commun (Camb) ; 53(39): 5396-5399, 2017 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-28451683

RESUMO

Linear tetrathiafulvalene (TTF) oligomers were synthesized, which could not only form a pleated TTF˙+ radical cation foldamer under oxidation conditions, but also interlocked with CBPQT4+ to form folded donor-acceptor [3]pseudorotaxane in the neutral state of TTF. Moreover, switchable transformation between these two folded supramolecular structures was achieved under the alternative regulation of the redox states of TTF units.

4.
Appl Microbiol Biotechnol ; 101(3): 987-1001, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28074225

RESUMO

Alcohol dehydrogenases (ADHs), which belong to the oxidoreductase superfamily, catalyze the interconversion between alcohols and aldehydes or ketones with high stereoselectivity under mild conditions. ADHs are widely employed as biocatalysts for the dynamic kinetic resolution of racemic substrates and for the preparation of enantiomerically pure chemicals. This review provides an overview of biotechnological applications for ADHs in the production of chiral pharmaceuticals and fine chemicals.


Assuntos
Álcool Desidrogenase , Biotecnologia , Tecnologia Farmacêutica , Álcoois/metabolismo , Aldeídos/metabolismo , Biocatálise , Biotecnologia/métodos , Biotecnologia/tendências , Cetonas/metabolismo , Estereoisomerismo
5.
Bioresour Technol ; 229: 26-32, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28092733

RESUMO

(4S)-3-[(5S)-5-(4-Fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one ((S)-ET-5) is an important chiral intermediate in the synthesis of chiral side chain of ezetimibe. Recombinant Escherichia coli expressing carbonyl reductase (CBR) was successfully constructed in this study. The total E. coli biomass and the specific activity of recombinant CBR in 5L fermenter culture were 10.9gDCWL-1 and 14900.3Ug-1DCW, respectively. The dual-enzyme coupled biocatalytic process in an aqueous-organic biphasic solvent system was first constructed using p-xylene as the optimal organic phase under optimized reaction conditions, and 150gL-1 (4S)-3-[5-(4-fluorophenyl)-1,5-dioxophentyl]-4-phenyl-1,3-oxazolidin-2-one (ET-4) was successfully converted to (S)-ET-5 with a conversion of 99.1% and diastereomeric excess of 99% after 24-h, which are the highest values reported to date for the production of (S)-ET-5.


Assuntos
Oxirredutases do Álcool/metabolismo , Ezetimiba/metabolismo , Glucose 1-Desidrogenase/metabolismo , Solventes/química , Água/química , Biocatálise/efeitos dos fármacos , Reatores Biológicos , Biotransformação , Tampões (Química) , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Fermentação/efeitos dos fármacos , Glucose/farmacologia , Concentração de Íons de Hidrogênio , NADP/farmacologia , Proteínas Recombinantes/metabolismo , Temperatura Ambiente
6.
Biotechnol Bioeng ; 114(4): 843-851, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27723097

RESUMO

L-methionine has attracted a great deal of attention for its nutritional, pharmaceutical, and clinical applications. In this study, Escherichia coli W3110 was engineered via deletion of a negative transcriptional regulator MetJ and over-expression of homoserine O-succinyltransferase MetA together with efflux transporter YjeH, resulting in L-methionine overproduction which is up to 413.16 mg/L. The partial inactivation of the L-methionine import system MetD via disruption of metI made the engineered E. coli ΔmetJ ΔmetI/pTrcA*H more tolerant to high L-ethionine concentration and accumulated L-methionine to a level 43.65% higher than that of E. coli W3110 ΔmetJ/pTrcA*H. Furthermore, deletion of lysA, which blocks the lysine biosynthesis pathway, led to a further 8.5-fold increase in L-methionine titer of E. coli ΔmetJ ΔmetI ΔlysA/pTrcA*H. Finally, addition of Na2 S2 O3 to the media led to an increase of fermentation titer of 11.45%. After optimization, constructed E. coli ΔmetJ ΔmetI ΔlysA/pTrcA*H was able to produce 9.75 g/L L-methionine with productivity of 0.20 g/L/h in a 5 L bioreactor. This novel metabolically tailored strain of E. coli provides an efficient platform for microbial production of L-methionine. Biotechnol. Bioeng. 2017;114: 843-851. © 2016 Wiley Periodicals, Inc.


Assuntos
Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Metionina/metabolismo , Técnicas de Cultura Celular por Lotes , Reatores Biológicos/microbiologia , Clonagem Molecular , Escherichia coli/genética , Fermentação , Técnicas de Inativação de Genes , Lisina/metabolismo , Redes e Vias Metabólicas , Metionina/análise , Plasmídeos/genética , Treonina/metabolismo
7.
Huan Jing Ke Xue ; 33(6): 1865-70, 2012 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-22946167

RESUMO

To study the endocrine disrupting effects and action mechanism of environmental levels of perfluorooctane sulfonate (PFOS) on the aquatic species, the research for the effects of PFOS exposure on vitellogenin (VTG) mRNA level in livers of zebrafish (Brachydanio rerio) was conducted. Zebrafish were exposed to PFOS at four environmental low concentrations (0.1, 1, 10, 100 microg x L(-1)) for 21 days. Livers from male and female zebrafish were collected for RNA extraction, VTG1 and VTG3 mRNA levels were measured respectively using real-time polymerase chain reaction (RT-PCR). The results show that: 1) The VTG1 and VTG3 mRNA level in the livers of male zebrafish increased after PFOS exposure. The VTG1 mRNA level increased with a positive dose response pattern, with the maximum response at 100 microg x L(-1) PFOS exposure where a significant difference compared with the control was observed. The VTG3 mRNA level increased as an inverted U-shaped dose response pattern, indicated as hormesis effects, where significant differences compared with the control were observed at 10 and 100 microg x L(-1) PFOS exposure. 2) The VTG1 mRNA level in the livers of female zebrafish increased where a significant difference compared with the control was observed at 10 microg x L(-1) PFOS exposure, but the standard errors for mRNA level at 10 and 100 microg x L(-1) PFOS exposure were distinct. The VTG3 mRNA level in the livers of female zebrafish increased at 10 microg L(-1) PFOS exposure but had no significant difference compared with the control. Thus, it deduced that PFOS exposure could be active on the endocrine system of zebrafish with the oestrogenic simulation action mechanism, and the VTG1 and VTG3 mRNA level in the livers of zebrafish might be sensitive biomarkers for the endocrine disrupting effects evaluation after PFOS exposure, with different responding patterns related to the gene subtypes and sex.


Assuntos
Ácidos Alcanossulfônicos/toxicidade , Exposição Ambiental/efeitos adversos , Fluorcarbonetos/toxicidade , Vitelogeninas/metabolismo , Peixe-Zebra/metabolismo , Animais , Disruptores Endócrinos/toxicidade , Feminino , Fígado/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vitelogeninas/genética
8.
Chemosphere ; 88(4): 514-23, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22469199

RESUMO

The Daphnia 21 d reproduction test is considered as a comprehensive and decisive test in the OECD Conceptual Framework for testing and assessment of endocrine disrupting chemicals (EDCs). However, how to interpret results of the Daphnia 21 d reproduction test for identification, risk assessment and testing strategy of EDCs remains an unsolved issue. This study analysed a total number of 135 published studies encompassing 86 known EDCs and non-EDCs with different modes of action. Our results show that the majority of effects on apical endpoints (survival, molting, growth, time to reproductive maturity, brood size, the number of broods, and the total number of offspring) do not seem to be EDC-specific. In contrast, the endpoint sex ratio is likely specific to juvenile hormones and their mimics. Variability is quantified for three most reported endpoints survival, the total number of offspring and sex ratio. Quantification of the endpoint sensitivity shows that the sensitivity of the sex ratio is lower than that of the total number of offspring. The Daphnia 21 d reproduction test gives insufficient information to conclude if a substance is an EDC or not. EDCs that are potent in assays in vitro may not be potent in the Daphnia 21 d reproduction test. We conclude that the Daphnia 21 d reproduction test is important for deriving No Observed Effect Concentrations for risk assessment but may produce false negatives in identification of EDCs when used on its own. A targeted testing strategy for selection of species, tests, and endpoints is suggested for identifying EDCs.


Assuntos
Daphnia/efeitos dos fármacos , Daphnia/fisiologia , Disruptores Endócrinos/análise , Disruptores Endócrinos/toxicidade , Animais , Relação Dose-Resposta a Droga , Determinação de Ponto Final , Feminino , Masculino , Reprodução/efeitos dos fármacos , Medição de Risco
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