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1.
Electron J Biotechnol ; 49: 56-63, Jan. 2021. tab, ilus, graf
Article in English | LILACS | ID: biblio-1291900

ABSTRACT

BACKGROUND: 2R,3R-butanediol dehydrogenase (R-BDH) and other BDHs contribute to metabolism of 3R/3S-Acetoin (3R/3S-AC) and 2,3-butanediol (2,3-BD), which are important bulk chemicals used in different industries. R-BDH is responsible for oxidizing the hydroxyl group at their (R) configuration. Bacillus species is a promising producer of 3R/3S-AC and 2,3-BD. In this study, R-bdh gene encoding R-BDH from Bacillus sp. DL01 was isolated, expressed and identified. RESULTS: R-BDH exerted reducing activities towards Diacetyl (DA) and 3R/3S-AC using NADH, and oxidizing activities towards 2R,3R-BD and Meso-BD using NAD+ , while no activity was detected with 2S,3S-BD. The R-BDH showed its activity at a wide range of temperature (25 C to 65 C) and pH (5.0­8.0). The R-BDH activity was increased significantly by Cd2+ when DA, 3R/3S-AC, and Meso-BD were used as substrates, while Fe2+ enhanced the activity remarkably at 2R,3R-BD oxidation. Kinetic parameters of the R-BDH from Bacillus sp. DL01 showed the lowest Km, the highest Vmax, and the highest Kcat towards the racemic 3R/3S-AC substrate, also displayed low Km towards 2R,3R-BD and Meso-BD when compared with other reported R-BDHs. CONCLUSIONS: The R-BDH from Bacillus sp. DL01 was characterized as a novel R-BDH with high enantioselectivity for R-configuration. It considered NAD+ and Zn2+ dependant enzyme, with a significant affinity towards 3R/3S-AC, 2R,3R-BD, and Meso-BD substrates. Thus, R-BDH is providing an approach to regulate the production of 3R/3S-AC or 2,3-BD from Bacillus sp. DL01.


Subject(s)
Bacillus subtilis/enzymology , Alcohol Oxidoreductases/isolation & purification , Alcohol Oxidoreductases/metabolism , Temperature , Kinetics , Hydrogen-Ion Concentration , Acetoin
2.
Chinese Journal of Biotechnology ; (12): 4277-4292, 2021.
Article in Chinese | WPRIM | ID: wpr-921505

ABSTRACT

The influence of different affinity tags on enzyme characteristics varies. The (S)-carbonyl reductase 2 (SCR2) from Candida parapsilosis can reduce 2-hydroxyacetophenone, which is a valuable prochiral ketones. Different affinity tags, i.e. his-tag, strep-tag and MBP-tag, were attached to the N terminus of SCR2. These tagged SCR2 enzymes, i.e. his6-SCR2, strep-SCR2 and MBP-SCR2, were heterologously expressed in Escherichia coli and purified to study their characteristics towards 2-hydroxyacetophenone reduction. Affinity tags did affect the characteristics of the recombinant SCR2 enzymes. Specifically, affinity tags affect the stability of recombinant SCR2 enzymes: 1) At pH 6.0, the remaining enzyme activities of his6-SCR2 and strep-SCR2 were only 95.2% and 90.0% of the untagged SCR2, while that of MBP-SCR2 was 1.2 times of the untagged SCR2 after incubating for 13 h at 30 °C. 2) The half-life of MBP-SCR2 at 50 °C was 26.6%-48.8% longer than those of strep-SCR2, his6-SCR2 and untagged SCR2. 3) The kcat of MBP-SCR2 was about 1.25-1.45 times of that of small affinity-tagged and untagged SCR2 after storing at -80 °C for 60 d. Structural informatics indicated that the α-helices at the C terminus of MBP-SCR2 contributed to the stability of the N terminus of fusion protein of SCR2. Data from circular dichroism showed that the MBP-tag has some influence on the secondary structure of SCR2, while melting temperature analysis demonstrated that the Tm of the recombinant MBP-SCR2 was about 5 °C higher than that of the untagged SCR2. This study obtained an efficient and stable recombinant SCR2, i.e. the MBP-SCR2. Moreover, this study could serve as a reference for other researchers to evaluate and select appropriate affinity tags for their research.


Subject(s)
Alcohol Oxidoreductases , Escherichia coli/genetics , Recombinant Fusion Proteins/genetics
3.
Chinese Journal of Biotechnology ; (12): 530-540, 2021.
Article in Chinese | WPRIM | ID: wpr-878580

ABSTRACT

One-carbon compounds such as methanol and methane are cheap and readily available feedstocks for biomanufacturing. Oxidation of methanol to formaldehyde catalyzed by methanol dehydrogenase (MDH) is a key step of microbial one-carbon metabolism. A variety of MDHs that depend on different co-factors and possess different enzymatic properties have been discovered from native methylotrophs. Nicotinamide adenine dinucleotide (NAD)-dependent MDHs are widely used in constructing synthetic methylotrophs, whereas this type of MDH usually suffers from low methanol oxidation activity and low affinity to methanol. Consequently, methanol oxidation is considered as a rate-limiting step of methanol metabolism in synthetic methylotrophs. To accelerate methanol oxidation, thereby improving the methanol utilization efficiency of synthetic methylotrophs, massive researches have focused on discovery and engineering of MDHs. In this review, we summarize the ongoing efforts to discover, characterize, and engineer various types of MDHs as well as the applications of MDHs in synthetic methylotrophs. Directed evolution of MDH and construction of multi-enzyme complexes are described in detail. In the future prospective part, we discuss the potential strategies of growth-coupled protein evolution and rational protein design for acquisition of superior MDHs.


Subject(s)
Alcohol Oxidoreductases/genetics , Carbon , Methane , Methanol
4.
Article in English | WPRIM | ID: wpr-773408

ABSTRACT

OBJECTIVE@#To investigate the molecular mechanisms of the adverse effects of exposure to sulfamonomethoxin (SMM) in pregnancy on the neurobehavioral development of male offspring.@*METHODS@#Pregnant mice were randomly divided into four groups: control- (normal saline), low- [10 mg/(kg•day)], middle- [50 mg/(kg•day)], and high-dose [200 mg/(kg•day)] groups, which received SMM by gavage daily during gestational days 1-18. We measured the levels of short-chain fatty acids (SCFAs) in feces from dams and male pups. Furthermore, we analyzed the mRNA and protein levels of genes involved in the mammalian target of rapamycin (mTOR) pathway in the hippocampus of male pups by RT-PCR or Western blotting.@*RESULTS@#Fecal SCFA concentrations were significantly decreased in dams. Moreover, the production of individual fecal SCFAs was unbalanced, with a tendency for an increased level of total fecal SCFAs in male pups on postnatal day (PND) 22 and 56. Furthermore, the phosphatidylinositol 3-kinase (PI3k)/protein kinase B (AKT)/mTOR or mTOR/ribosomal protein S6 kinase 1 (S6K1)/4EBP1 signaling pathway was continuously upregulated until PND 56 in male offspring. In addition, the expression of Sepiapterin Reductase (SPR), a potential target of mTOR, was inhibited.@*CONCLUSION@#In utero exposure to SMM, persistent upregulation of the hippocampal mTOR pathway related to dysfunction of the gut (SCFA)-brain axis may contribute to cognitive deficits in male offspring.


Subject(s)
Alcohol Oxidoreductases , Metabolism , Animals , Anti-Infective Agents , Toxicity , Fatty Acids, Volatile , Feces , Chemistry , Female , Hippocampus , Metabolism , Male , Memory , Mice, Inbred ICR , Pregnancy , Prenatal Exposure Delayed Effects , Sulfamonomethoxine , Toxicity , TOR Serine-Threonine Kinases , Metabolism
5.
Frontiers of Medicine ; (4): 104-111, 2019.
Article in English | WPRIM | ID: wpr-772708

ABSTRACT

Mitochondrion-localized retinol dehydrogenase 13 (Rdh13) is a short-chain dehydrogenase/reductase involved in vitamin A metabolism in both humans and mice. We previously generated Rdh13 knockout mice and showed that Rdh13 deficiency causes severe acute retinal light damage. In this study, considering that Rdh13 is highly expressed in mouse liver, we further evaluated the potential effect of Rdh13 on liver injury induced by carbon tetrachloride (CCl). Although Rdh13 deficiency showed no significant effect on liver histology and physiological functions under regular culture, the Rdh13 mice displayed an attenuated response to CCl-induced liver injury. Their livers also exhibited less histological changes and contained lower levels of liver-related metabolism enzymes compared with the livers of wild-type (WT) mice. Furthermore, the Rdh13 mice had Rdh13 deficiency and thus their liver cells were protected from apoptosis, and the quantity of their proliferative cells became lower than that in WTafter CCl exposure. The ablation of Rdh13 gene decreased the expression levels of thyroid hormone-inducible nuclear protein 14 (Spot14) and cytochrome P450 (Cyp2e1) in the liver, especially after CCl treatment for 48 h. These data suggested that the alleviated liver damage induced by CCl in Rdh13 mice was caused by Cyp2e1 enzymes, which promoted reductive CCl metabolism by altering the status of thyroxine metabolism. This result further implicated Rdh13 as a potential drug target in preventing chemically induced liver injury.


Subject(s)
Alcohol Oxidoreductases , Genetics , Animals , Carbon Tetrachloride Poisoning , Chemical and Drug Induced Liver Injury , Pathology , Cytochrome P-450 CYP2E1 , Metabolism , Female , Immunohistochemistry , Liver , Pathology , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins , Metabolism , Transcription Factors , Metabolism
6.
Article in Chinese | WPRIM | ID: wpr-247738

ABSTRACT

<p><b>OBJECTIVE</b>To explore pathogenic mutation in a family affected with 2-hydroxyglutaric aciduria.</p><p><b>METHODS</b>Exons of 3 candidate genes, including L2HGDH, D2HGDH and SLC25A1, were amplified with polymerase chain reaction and subjected to direct sequencing.</p><p><b>RESULTS</b>DNA sequencing has found that the proband and his affected younger brother have both carried a heterozygous mutation c.845G>A (p.R282Q) in the exon 7 of the L2HGDH gene. The same mutation was not detected in the his sister who was healthy. Pedigree analysis has confirmed that the above mutation was inherited from the mother. No mutation was detected in exons and flanking sequences of the D2HGDH and SLC25A1 genes.</p><p><b>CONCLUSION</b>Mutation of the L2HGDH gene probably underlies the 2-hydroxyglutaric aciduria in this family.</p>


Subject(s)
Alcohol Oxidoreductases , Genetics , Base Sequence , Brain , Diagnostic Imaging , Brain Diseases, Metabolic, Inborn , Diagnostic Imaging , Genetics , Child , Female , Humans , Male , Molecular Sequence Data , Mutation , Pedigree , Radiography , Young Adult
7.
Rev. bras. cir. cardiovasc ; 30(1): 24-32, Jan-Mar/2015. tab, graf
Article in English | LILACS | ID: lil-742904

ABSTRACT

Objective: A retrospective cohort study was preformed aiming to verify the presence of transient dysfunction of gas exchange in the postoperative period of cardiac surgery and determine if this disorder is linked to cardiorespiratory events. Methods: We included 942 consecutive patients undergoing cardiac surgery and cardiac procedures who were referred to the Intensive Care Unit between June 2007 and November 2011. Results: Fifteen patients had acute respiratory distress syndrome (2%), 199 (27.75%) had mild transient dysfunction of gas exchange, 402 (56.1%) had moderate transient dysfunction of gas exchange, and 39 (5.4%) had severe transient dysfunction of gas exchange. Hypertension and cardiogenic shock were associated with the emergence of moderate transient dysfunction of gas exchange postoperatively (P=0.02 and P=0.019, respectively) and were risk factors for this dysfunction (P=0.0023 and P=0.0017, respectively). Diabetes mellitus was also a risk factor for transient dysfunction of gas exchange (P=0.03). Pneumonia was present in 8.9% of cases and correlated with the presence of moderate transient dysfunction of gas exchange (P=0.001). Severe transient dysfunction of gas exchange was associated with patients who had renal replacement therapy (P=0.0005), hemotherapy (P=0.0001), enteral nutrition (P=0.0012), or cardiac arrhythmia (P=0.0451). Conclusion: Preoperative hypertension and cardiogenic shock were associated with the occurrence of postoperative transient dysfunction of gas exchange. The preoperative risk factors included hypertension, cardiogenic shock, and diabetes. Postoperatively, pneumonia, ventilator-associated pneumonia, renal replacement therapy, hemotherapy, and cardiac arrhythmia were associated with the appearance of some degree of transient dysfunction of gas exchange, which was a risk factor for reintubation, pneumonia, ventilator-associated pneumonia, and renal replacement therapy in the postoperative period ...


Objetivo: Estudo de coorte retrospectivo com objetivo de verificar a presença de disfunção transitória da troca gasosa no pós-operatório de cirurgia cardíaca e determinar se esse transtorno está relacionado a eventos cardiorrespiratórios. Métodos: Foram incluídos 942 pacientes consecutivos submetidos à cirurgia cardíaca e procedimentos cardíacos, encaminhados para a Unidade de Terapia Intensiva, entre junho de 2007 e novembro de 2011. Resultados: A síndrome do desconforto respiratório agudo foi observada em 15 (2%) pacientes, 199 (27,75%) pacientes apresentaram disfunção transitória da troca gasosa leve, disfunção transitória da troca gasosa moderada foi observada em 402 (56,1%) pacientes e disfunção transitória da troca gasosa grave em 39 (5,4%). A presença de hipertensão arterial sistêmica e choque cardiogênico foi associada ao surgimento de disfunção transitória da troca gasosa moderada no período pós-operatório (P=0,02 e P=0,019, respectivamente) e foram considerados fatores de risco para essa disfunção (P=0,0023 e P=0,0017, respectivamente). A presença de diabetes mellitus também foi considerada um fator de risco para disfunção transitória da troca gasosa (P=0,03). Houve correlação entre a presença de pneumonia e a presença de disfunção transitória da troca gasosa moderada em 8,9% dos casos (P=0,001). A presença de disfunção transitória da troca gasosa grave foi associada a pacientes que necessitaram de hemodiálise (P=0,0005), hemoterapia (P=0,0001), nutrição enteral (P=0,0012), ou arritmia cardíaca (P=0,0451). Conclusão: A presença de hipertensão arterial sistêmica pré-operatória e choque cardiogênico foi associada à ocorrência de disfunção transitória da troca gasosa pós-operatória. Os fatores de risco pré-operatórios foram hipertensão arterial sistêmica, choque cardiogênico e diabetes. No pós-operatório, pneumonia, pneumonia associada à ventilação, hemodiálise, hemoterapia e arritmia cardíaca foram associadas com certo grau de ...


Subject(s)
Animals , Humans , Rats , Alcohol Oxidoreductases/metabolism , Endothelial Cells/metabolism , NAD(P)H Dehydrogenase (Quinone)/metabolism , Phenanthrenes/metabolism , Aldehyde Reductase , CCAAT-Binding Factor/metabolism , Caspases/metabolism , Endoplasmic Reticulum/metabolism , Leupeptins/pharmacology , Oxidation-Reduction , Oxidative Stress , Poly(ADP-ribose) Polymerases/metabolism , Reactive Oxygen Species/metabolism
8.
Chinese Journal of Biotechnology ; (12): 220-230, 2015.
Article in Chinese | WPRIM | ID: wpr-345512

ABSTRACT

In this study, a fast carbonyl reductases colorimetric screening method for discovering stereoselective carbonyl reductases was established by combining the reverse alcohol oxidation with the azoreductase-catalyzed reduction of azo dye. When azo dye (Orange I , 4-(4-hydroxy-1-naphthylazo) benzenesulfonic acid) and azoreductase (AzoB) were added into the reaction system of alcohol oxidation catalyzed by carbonyl reductase, the produced NAD(P)H served as electron donor for the azoreductase to reduce the azo dye, resulting the color fade. Hence, the carbonyl reductases can be screened by the obvious color change. When chiral alcohol was used as the substrate, the activity and stereoselectivity of carbonyl reductases can be screened at the same time.


Subject(s)
Alcohol Oxidoreductases , Chemistry , Alcohols , Chemistry , Azo Compounds , Chemistry , Coloring Agents , Chemistry , High-Throughput Screening Assays , NADH, NADPH Oxidoreductases , Chemistry , NADP , Chemistry , Oxidation-Reduction , Stereoisomerism
9.
Salud colect ; 10(3): 325-337, sep.-dic. 2014. tab
Article in Spanish | LILACS | ID: lil-733293

ABSTRACT

En el marco de la creciente feminización de la profesión médica en México, el artículo indaga sobre las características de este proceso para el caso de la ginecobstetricia. Considerando la feminización como un proceso de cambio, que se analiza cuantitativa y cualitativamente, el artículo se detiene en especial en las experiencias de las mujeres ginecobstetras, experiencias que se dan en el seno de una especialidad que, desde sus orígenes, funcionó como un dispositivo de control del cuerpo de las mujeres. Basado en una investigación etnográfica, el artículo combina fuentes estadísticas, de archivo y de observación de campo. El material que surge de las entrevistas muestra las experiencias y tensiones que viven las ginecobstetras en este contexto.


In the framework of an increasing feminization of the medical profession in Mexico, this article explores the characteristics of this process in the obstetrics and gynecology specialty. Understanding feminization as a process of change to be analyzed both quantitatively and qualitatively, the article focuses special attention on the experiences of female obstetrician-gynecologists within a medical specialty that has since its origins functioned as a mechanism of control over women's bodies. Based on ethnographic research, the article combines statistical and archival sources and field observation. The interviews reveal the experiences and tensions women obstetrician-gynecologists encounter in this context.


Subject(s)
Alcohol Oxidoreductases/chemistry , Alcohol Oxidoreductases/metabolism , Arginine/chemistry , Pseudomonas putida/enzymology , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/isolation & purification , Binding, Competitive/genetics , Catalysis , Enzyme Activation/genetics , Flavin Mononucleotide/metabolism , Kinetics , Ligands , Mutagenesis, Site-Directed , Mandelic Acids/metabolism , Phenylacetates/metabolism , Protein Binding/genetics , Pseudomonas putida/genetics , Substrate Specificity/genetics , Sulfites/metabolism
10.
Rev. bras. parasitol. vet ; 23(4): 534-538, Oct-Dec/2014. tab
Article in English | LILACS | ID: lil-731264

ABSTRACT

With the aim of studying the endoparasite fauna of horses from the Formiga city, located in center-west region of the state of Minas Gerais, 25 animals that were naturally infected with helminths were evaluated. By means of parasitological necropsies, different endoparasites were found. The subfamily Cyathostominae presented the highest incidence, followed by Trichostrongylus axei, Oxyuris equi, Triodontophorus serratus, Strongyloides westeri, Strongylus edentatus, Habronema muscae, Parascaris equorum, Probstmayria vivipara, Strongylus vulgaris, Gasterophilus nasalis, Anoplocephala magna and Anoplocephala perfoliata. In the present study, if the species Probstmayria vivipara was not considered in the prevalence, the frequency of Cyathostominae was equivalent to 94.85%. The results obtained in this study allowed us to detect and identify different species of helminths in horses, and confirmed the high incidence of nematodes belonging to the subfamily Cyathostominae in the center-west region of Minas Gerais.


Com o objetivo de estudar a fauna de endoparasitas de equinos da Região Centro-Oeste do Estado de Minas Gerais, 25 animais naturalmente infectados por helmintos foram avaliados. Por meio de necropsias parasitológicas, diferentes endoparasitas foram identificados. A sub - família Cyathostominae apresentou maior incidência, seguido por Trichostrongylus axei, Oxyuris equi, Triodontophorus serratus, Strongyloides westeri, Strongylus edentatus, Habronema muscae, Parascaris equorum, Probstmayria vivipara, Strongylus vulgaris, Gasterophilus nasalis, Anoplocephala magna e Anoplocephala perfoliata. No presente estudo, se não for considerada a espécie Probstmayria vivipara na prevalência, a frequência de Cyathostominae é equivalente a 94,85%. Os resultados obtidos neste estudo, permitiu detectar e identificar diferentes espécies de helmintos em equinos, bem como confirmar a elevada incidência de nematódeos pertencentes à sub-família Cyathostominae na Região Centro-Oeste de Minas Gerais.


Subject(s)
Humans , Alcohol Oxidoreductases/antagonists & inhibitors , Pyrimidines/chemistry , Quantitative Structure-Activity Relationship , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Opportunistic Infections/drug therapy , Pneumocystis/enzymology , Pyrimidines/pharmacology , Regression Analysis
11.
Indian J Exp Biol ; 2014 Mar; 52(3): 252-260
Article in English | IMSEAR | ID: sea-150355

ABSTRACT

Activity differences of the first (phenylalanine ammonia lyase, PAL) and the last (cinnamyl alcohol dehydrogenase, CAD) enzymes of phenylpropanoid pathway in the roots of resistant (Yangambi Km5 and Anaikomban) and susceptible (Nendran and Robusta) banana cultivars caused by root lesion nematode, Pratylenchus coffeae, were investigated. Also, the accumulation of phenolics and deposition of lignin polymers in cell walls in relation to resistance of the banana cultivars to the nematode were analyzed. Compared to the susceptible cultivars, the resistant cultivars had constitutively significantly higher PAL activity and total soluble and cell wall-bound phenolics than in susceptible cultivars. The resistant cultivars responded strongly to the infection of the nematode by induction of several-time higher PAL and CAD enzymes activities, soluble and wall-bound phenolics and enrichment of lignin polymers in cell wall and these biochemical parameters reached maximum at 7th day postinoculation. In addition, profiles of phenolic acid metabolites in roots of Yangambi Km5 and Nendran were analyzed by HPLC to ascertain the underlying biochemical mechanism of bananas resistance to the nematode. Identification and quantification of soluble and cell wall-bound phenolic acids showed six metabolites and only quantitative, no qualitative, differences occurred between the resistant and susceptible cvs. and between constitutive and induced contents. A very prominent increase of p-coumaric, ferulic and sinapic acids, which are precursors of monolignols of lignin, in resistant cv. was found. These constitutive and induced biochemical alterations are definitely the chemical defenses of resistant cvs. to the nematode infection.


Subject(s)
Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Animals , Disease Resistance/genetics , Metabolic Networks and Pathways , Musa/enzymology , Musa/genetics , Musa/growth & development , Musa/parasitology , Nematoda/genetics , Nematoda/pathogenicity , Phenols/chemistry , Phenols/metabolism , Phenylalanine Ammonia-Lyase/genetics , Phenylalanine Ammonia-Lyase/metabolism , Plant Diseases/genetics , Plant Diseases/parasitology , Plant Roots/enzymology , Plant Roots/metabolism , Polymers/chemistry , Propanols/chemistry , Propanols/metabolism
12.
Chinese Medical Journal ; (24): 722-728, 2013.
Article in English | WPRIM | ID: wpr-342510

ABSTRACT

<p><b>BACKGROUND</b>Retinoic acid (RA) is a potent signaling molecule that plays pleiotropic roles in patterning, morphogenesis, and organogenesis during embryonic development. The synthesis from retinol (vitamin A) to retinoic acid requires two sequential oxidative steps. The first step involves the oxidation of retinol to retinal through the action of retinol dehydrogenases. Retinol dehydrogenases1l (RDH1l) is a novel zebrafish retinol dehydrogenase. Herein we investigated the role of zebrafish RDH1l in heart development and cardiac performance in detail.</p><p><b>METHODS</b>RDH1l specific morpholino was used to reduce the function of RDH1l in zebrafish. The gene expressions were observed by using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction (VSF).</p><p><b>RESULTS</b>The knock-down of RDH1l led to abnormal neural crest cells migration and reduced numbers of neural crest cells in RDH1l morphant embryos. The reduced numbers of cardiac neural crest cells also can be seen in RDH1l morphant embryos. Furthermore, the morpholino-mediated knock-down of RDH1l resulted in the abnormal heart loop. The left-right determining genes expression pattern was altered in RDH1l morphant embryos. The impaired cardiac performance was observed in RDH1l morphant embryos. Taken together, these data demonstrate that RDH1l is essential for the heart development and cardiac performance in zebrafish.</p><p><b>CONCLUSIONS</b>RDH1l plays a important role in the neural crest cells development, and then ultimately affects the heart loop and cardiac performance. These results show for the first time that an enzyme involved in the retinol to retinaldehyde conversion participate in the heart development and cardiac performance in zebrafish.</p>


Subject(s)
Alcohol Oxidoreductases , Genetics , Metabolism , Animals , Animals, Genetically Modified , Heart , Embryology , Zebrafish , Zebrafish Proteins , Genetics , Metabolism
13.
Protein & Cell ; (12): 598-606, 2013.
Article in English | WPRIM | ID: wpr-757785

ABSTRACT

The resolution of single molecule localization imaging techniques largely depends on the precision of localization algorithms. However, the commonly used Gaussian function is not appropriate for anisotropic dipoles because it is not the true point spread function. We derived the theoretical point spread function of tilted dipoles with restricted mobility and developed an algorithm based on an artificial neural network for estimating the localization, orientation and mobility of individual dipoles. Compared with fitting-based methods, our algorithm demonstrated ultrafast speed and higher accuracy, reduced sensitivity to defocusing, strong robustness and adaptability, making it an optimal choice for both two-dimensional and three-dimensional super-resolution imaging analysis.


Subject(s)
Alcohol Oxidoreductases , Genetics , Metabolism , Algorithms , Animals , COS Cells , Chlorocebus aethiops , Cytochrome P-450 Enzyme System , Genetics , Metabolism , HeLa Cells , Humans , Imaging, Three-Dimensional , Microscopy, Fluorescence , Normal Distribution , Plasmids , Metabolism
14.
Article in Chinese | WPRIM | ID: wpr-233269

ABSTRACT

Asymmetric reduction of bulky diaryl ketones is still one of the challenging tasks in biocatalysis. By genomic data mining, a putative carbonyl reductase gene pascr was found in Pichia pastoris GS115. pascr was cloned and over-expressed in Escherichia coli Rosseta2 (DE3). The recombinant enzyme was purified to homogeneity by Ni-NTA column and its catalytic properties were studied. PasCR strictly used NADPH as cofactor, gel filtration and SDS-PAGE analysis suggested that the native form of PasCR was a dimmer. PasCR exhibited the highest activity at 35 degrees C in phosphate buffer at pH 6.5. The enzyme catalyzed the reduction of some bulky diaryl ketones, such as 4-methylbenzophenone, 2-methylbenzophenone and 4-chlorobenzophenone, especially for 4-methylbenzophenone, the product S--alcohol was obtained with 85% ee.


Subject(s)
Alcohol Oxidoreductases , Genetics , Amino Acid Sequence , Catalysis , Cloning, Molecular , Ketones , Chemistry , Molecular Sequence Data , Pichia , Genetics , Stereoisomerism
15.
Chinese Journal of Biotechnology ; (12): 169-179, 2013.
Article in Chinese | WPRIM | ID: wpr-233257

ABSTRACT

Carbonyl reductases catalyze carbonyl compounds to chiral alcohols that are important building blocks in fine chemical industry. To study carbonyl reductase from Pichia pastoris GS115 (ppcr), we discovered a new gene (ppcr) encoding an NADPH-dependent carbonyl reductase by genomic data mining. It was amplified by PCR from the genomic DNA, and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity. The optimum temperature was 37 degrees C and the optimum pH of PPCR was 6.0. PPCR was stable below 45 degrees C. The Km and k(cat) value of the enzyme for ethyl 3-methyl-2-oxobutanoate were 9.48 mmol/L and 0.12 s, respectively. The enzyme had broad substrate specificity and high enantioselectivity. It catalyzed the reduction of aldehydes, a-ketoesters, beta-ketoesters and aryl ketones to give the corresponding alcohols with >97% ee with only a few exceptions, showing its application potential in the synthesis of chiral alcohols.


Subject(s)
Alcohol Oxidoreductases , Chemistry , Genetics , Amino Acid Sequence , Biotechnology , Methods , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Molecular Sequence Data , Pichia , Recombinant Proteins , Chemistry , Genetics , Stereoisomerism , Substrate Specificity , Temperature
16.
Chinese Journal of Biotechnology ; (12): 620-629, 2013.
Article in Chinese | WPRIM | ID: wpr-233215

ABSTRACT

Bacillus sp. TSH1 is a butanol-producing microorganism newly isolated in our laboratory; it can grow and ferment under facultative anaerobic conditions, while sharing similar fermentation pathways and products with Clostridium acetobutylicum. To illustrate the relationships between the products and the enzyme activities in Bacillus sp. TSH1, key butanol- and ethanol-forming enzymes were studied, including butyraldehyde dehydrogenase, butanol dehydrogenase and alcohol dehydrogenase. The activities of the three enzymes increased rapidly after the initiation of fermentation. Activities of three enzymes peaked before 21 h, and simultaneously, product concentrations also began to increase gradually. The maximum activity of alcohol dehydrogenase was 0.054 U/mg at 12 h, butyraldehyde dehydrogenase 0.035 U/mg at 21 h and butanol dehydrogenase 0.055 U/mg at 15 h. The enzyme activities then decreased, but remained constant at a low level after 24 h, while the concentrations of butanol, acetone, and ethanol continued increasing until the end of the fermentation. The results will attribute to the understanding of the butanol metabolic mechanism, and provide a reference for further study of a facultative Bacillus metabolic pathway.


Subject(s)
Alcohol Dehydrogenase , Metabolism , Alcohol Oxidoreductases , Metabolism , Aldehyde Oxidoreductases , Metabolism , Anaerobiosis , Bacillus , Classification , Genetics , Metabolism , Butanols , Metabolism , Fermentation , Metabolic Networks and Pathways
17.
Article in English | WPRIM | ID: wpr-172359

ABSTRACT

Adipose tissue, where various metabolic hormones are secreted, plays a role in metabolizing different substances including androgen. Within fat tissue, enzymes such as aromatase and aldo-keto reductase 1C are responsible for metabolizing testosterone into estrogen and 5-dihydrotestosterone into inactive metabolites. Adipose tissue can also affect the secretion of gonadotropin, which influences the formation of androgen in the testes. At the same time, androgen has an impact on the distribution and proliferation of adipose tissue. The adrenoreceptors for catecholamines, which have been proven to play an essential role in controlling lipolysis, function by being up-regulated by androgens. Furthermore, androgens regulate the activity of lipoprotein lipase, a key enzyme involved in intracellular esterification of adipose tissue.


Subject(s)
Adipose Tissue , Alcohol Oxidoreductases , Androgens , Aromatase , Catecholamines , Esterification , Estrogens , Gonadotropins , Humans , Lipolysis , Lipoprotein Lipase , Male , Oxidoreductases , Testis , Testosterone
18.
Mycobiology ; : 258-262, 2012.
Article in English | WPRIM | ID: wpr-729695

ABSTRACT

cDNA of the glx1 gene encoding glyoxal oxidase (GLX) from Phanerochaete chrysosporium was isolated and expressed in Pichia pastoris. The recombinant GLX (rGLX) produces H2O2 over 7.0 nmol/min/mL using methyl glyoxal as a substrate. Use of rGLX as a generator of H2O2 improved the coupled reaction with recombinant manganese peroxidase resulting in decolorization of malachite green up to 150 microM within 90 min.


Subject(s)
Alcohol Oxidoreductases , DNA, Complementary , Glyoxal , Manganese , Organometallic Compounds , Oxidoreductases , Peroxidase , Peroxidases , Phanerochaete , Pichia , Rosaniline Dyes
19.
Chinese Journal of Biotechnology ; (12): 1082-1091, 2011.
Article in Chinese | WPRIM | ID: wpr-324500

ABSTRACT

In order to successfully express the carbonyl reductase gene mldh in Bacillus subtilis and complete coenzyme regeneration by B. subtilis glucose dehydrogenase, the promoter PrpsD and the terminator TrpsD from B. subtilis rpsD gene were used as the expression cassette to be a recombinant plasmid pHY300plk-PrpsD-TrpsD. After that, the carbonyl reductase gene mldh was inserted into the previous plasmid and a plasmid pHY300plk-PrpsD-mldh-TrpsD was achieved, followed by transformed into B. subtilis Wb600 to obtain a recombinant B. subtilis Wb600 (pHY300plk-PrpsD-mldh-TrpsD). Subsequently, the results for whole-cell biotransformation from recombinant B. subtilis showed that it could be used to catalyze MAK (1-phenyl- 1-keto-2-methylaminopropane) to d-pseudoephedrine in the presence of glucose. The yield of d-pseudoephedrine could be up to 97.5 mg/L and the conversion rate of MAK was 24.1%. This study indicates the possibility of biotransformation production of d-pseudoephedrine from recombinant B. subtilis.


Subject(s)
Alcohol Oxidoreductases , Genetics , Bacillus subtilis , Genetics , Metabolism , Glucose 1-Dehydrogenase , Chemistry , Metabolism , Mutagenesis, Insertional , Pseudoephedrine , Metabolism , Recombinant Proteins , Genetics , Recombination, Genetic
20.
Acta Pharmaceutica Sinica ; (12): 778-784, 2009.
Article in Chinese | WPRIM | ID: wpr-344106

ABSTRACT

This study is aimed to clone and express human, rat alcohol dehydrogenase (ADH) and aldo-keto reductase. Then the enantioselective metabolism of mandelic acid (MA) was studied. Human alcohol dehydrogenase 2, rat alcohol dehydrogenase 1, human and rat aldo-keto reductase 1A1 were amplified using RT-PCR from human and rat liver samples. Then subcloned into pET-28a (+) and expressed in E. coli BL21 (DE3) stably. The protein was induced with IPTG and purified by affinity chromatography. Then the enzyme activities were measured. MA enantiomers were incubated with rat, human ADH and phenylglyoxylic acid (PGA) with AKR1A1, respectively. The metabolism was analyzed with HPLC. The proper genes were cloned and purified and proteins were obtained. All of the proteins obtained showed good activity. Stereoselective-metabolism of MA was observed in human ADH2, which favors for S-MA metabolism. The expression plasmids are constructed and the recombinant proteins are expressed successfully. The recombinant alcohol dehydrogenase and aldo-keto reductase have been employed to study MA metabolism.


Subject(s)
Alcohol Dehydrogenase , Genetics , Metabolism , Alcohol Oxidoreductases , Genetics , Metabolism , Aldehyde Reductase , Aldo-Keto Reductases , Animals , Cloning, Molecular , Humans , RNA, Messenger , Genetics , Rats , Rats, Sprague-Dawley , Rats, Wistar , Recombinant Proteins , Genetics , Metabolism
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