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1.
Oncogene ; 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831835

RESUMO

The original version of this Article omitted the following from the Acknowledgements: Professor Stebbing sits on SABs for Celltrion, Singapore Biotech, Vor Biopharma, TLC Biopharmaceuticals and Benevolent AI, has consulted with Lansdowne partners, Vitruvian and Social Impact Capital and Chairs the Board of Directors for BB Biotech Healthcare Trust and Xerion Healthcare. This has now been corrected in both the PDF and HTML versions of the Article.

2.
Curr Microbiol ; 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31832840

RESUMO

Bacillus sp. TYF-LIM-B05, which is isolated from spoilage vinegar, is resistant to high temperature, high concentrated alcohol, acid, and salt, and can produce ethanol from mono-, di-, polysaccharide, and complex biomass as the sole carbon source. Thus, this strain is a potential candidate for consolidated bioprocessing (CBP) of fermenting lignocellulose to ethanol in a single step. To provide insight into the key enzymes involved in lignocellulose degradation and ethanol production, a draft genome of TYF-LIM-B05 was developed in this study. The results indicated that 348 genes are related to carbohydrate transport and metabolism according to the clusters of orthologous groups of proteins and annotated 187 CAZy domains from a total of 61 different families. The presence of genes encoding laccases, quinone oxidoreductases/reductases, and aryl-alcohol dehydrogenases further implies that TYF-LIM-B05 has the potential to degrade lignin. Remarkably, this strain has the ability to catalyze the oxidative decarboxylation of pyruvate to acetyl-CoA by pyruvate dehydrogenase complexes. The genomic information provided in this study will help researchers to better understand the mechanisms of the lignocellulose degradation and ethanol production pathway in thermophiles.

3.
Opt Express ; 27(20): 28123-28132, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31684570

RESUMO

This paper reports a torsion sensor based on the multimode interference theory. The sensor is fabricated by sandwiching a section of perfluorinated polymer optical fiber (POF) between two silica single mode fibers to construct a single-mode-multimode-single-mode (SMS) structure. The perfluorinated POF is easily connected to the optical fiber via the precise alignment of ceramic ferrules and ceramic mating sleeve. With the considerable flexibility and deformability of the perfluorinated POF, the proposed sensor is especially suitable for torsion measurement. Experimental results show that a wavelength sensitivity of 106.762 pm/(rad/m) and an intensity sensitivity of 0.165 dBm/(rad/m) are obtained within a large torsion rate of -100∼100 rad/m.

4.
Oncogene ; 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31754213

RESUMO

EGFR-mutant non-small-cell lung cancer (NSCLC) patients inevitably develop drug resistance when treated with EGFR tyrosine kinase inhibitors (TKIs). Systematic genetic analysis is important to understand drug-resistant mechanisms; however, the clinical significance of co-occurring genetic alterations at baseline, co-acquired mutations at progressive disease (PD), and the clonal evolution remain underinvestigated. We performed targeted sequencing of pre-treatment and PD tumor samples from 54 EGFR-mutant NSCLC patients. Ten additional patients were sequenced using whole-exome sequencing to infer the clonal evolution patterns. We observed a domain-dependent effect of PIK3CA mutation at baseline on patient progression-free survival (PFS). In addition, at baseline, 9q34.3/19p13.3 (NOTCH1/STK11/GNA11) showed a co-deletion pattern, which was associated with a significantly worse PFS (p = 0.00079). T790M-postive patients with other concurrent acquired oncogenic mutations had a significantly shorter PFS (p = 0.005). Besides acquired T790M mutation, chromosomal instability (CIN) related genes, including AURKA and TP53 alterations, were the most frequently acquired events. CIN significantly increased during TKI treatment in T790M-negative patients and is a candidate resistance mechanism to the first-generation TKIs. Clonal evolution analyses suggest that the composition and relationship among resistant subclones, particularly relationship with T790M subclone, affect patients' outcomes. Overall, our findings of novel co-occurring alterations and clonal evolution patterns can be served as predictive biomarkers to stratify patients and help to better understand the drug-resistant mechanism to TKIs.

5.
Appl Opt ; 58(26): 7251-7257, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31504001

RESUMO

In this paper, a structural damage identification algorithm based on a single fiber Bragg grating (FBG) sensor is proposed. The signal detected by the FBG can be analyzed by the wavelet packet decomposition and back propagation neural network to obtain the damage location information. A high-speed FBG demodulation system based on a tunable Fabry-Perot filter and unbalanced Mach-Zehnder (M-Z) interferometer is designed to respond to a signal with a frequency range from 0 to 4 kHz, which will increase the sensing accuracy. This algorithm is verified by the aluminum plate model, which can simulate the generation of damage in reality. The experimental results show that a single FBG sensor is enough to realize accurate damage location identification according to this algorithm, and identification accuracy can reach 90.0%.

6.
Microbiol Resour Announc ; 8(31)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31371531

RESUMO

Rummeliibacillus sp. strain TYF005 is a thermophilic bacterium with high ethanol (8% vol/vol) and salt (13% wt/vol) tolerance that was isolated from spoilage vinegar. Here, we report the draft genome sequence of this strain, which has 117 scaffolds with a total genome size of 3.7 Mb and a 34.4% GC content.

7.
Indoor Air ; 29(2): 215-230, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30474277

RESUMO

Thermal comfort, self-reported acute health symptoms, cognitive performance, and physiological reactions were examined at four temperatures (26, 30, 33, and 37°C) at a relative humidity of 70%. Thirty-two sub-tropically acclimatized subjects experienced each condition for 175 minute, in balanced order, in a climatic chamber. The perception of heat gradually increased with increasing temperature, but the subjects felt hot only at 37°C. The temperature of 33°C was on average rated as acceptable and only just uncomfortable. The acceptability of air quality decreased linearly with increasing temperature. The intensity of acute health symptoms reported by the subjects increased with increasing temperature, but it was no more than moderate even at the highest temperature; dryness of skin and eye were alleviated. The eardrum temperature, skin temperature and moisture, heart rate, end-tidal carbon dioxide, and weight loss increased significantly with increasing temperature, whereas the percentage of adjacent heart inter-beat intervals differing by >50 ms decreased significantly. These results suggest that the perceived heat, self-reported symptoms, and physiological reactions occurred concurrently. They show additionally that acclimatization to heat may shift the boundary of thermal discomfort to a higher temperature. The role of psychological adaptation and of the contextual aspects of this process still requires clarification in future experiments.

8.
Opt Express ; 26(18): 22944-22953, 2018 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-30184951

RESUMO

The thermal cycling process experienced by spacecraft during orbital operation would lead to deterioration of the demodulation performance of fiber Bragg grating (FBG). A new demodulation method based on Fabry-Perot (F-P) filter and hydrogen cyanide (HCN) gas is proposed to improve the performance. The method skillfully utilizes the self-marked HCN absorption lines as absolute wavelength references. In the thermal cycling environment whose temperature ranging from 5°Cto 65°C,the fluctuation of demodulation wavelength reduces to ± 2.6 pm, which is improved by 3.1 times compared with traditional method. The proposed method also shows a good robustness in the cases of weak light source intensity and poor signal-to-noise ratio (SNR) of HCN spectrum.

9.
Sci Rep ; 8(1): 3313, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29463811

RESUMO

In clear-cell renal cell carcinoma (ccRCC), loss of von Hippel-Lindau (VHL) tumour suppressor gene and reduced oxygen tension promote stabilisation of hypoxia-inducible factor (HIF) family of transcription factors, which promote changes in the expression of genes that contribute to oncogenesis. Multiple studies have demonstrated significant perturbations in DNA methylation in ccRCC via largely unclear mechanisms that modify the transcriptional output of tumour cells. Here, we show that the methylation status of the CpG dinucleotide within the consensus hypoxia-responsive element (HRE) markedly influences the binding of HIF and that the loss of VHL results in significant alterations in the DNA methylome. Surprisingly, hypoxia, which likewise promotes HIF stabilisation and activation, has relatively few effects on global DNA methylation. Gene expression analysis of ccRCC patient samples highlighted expression of a group of genes whose transcription correlated with methylation changes, including hypoxic responsive genes such as VEGF and TGF. These results suggest that the loss of VHL alters DNA methylation profile across the genome, commonly associated with and contributing to ccRCC progression.


Assuntos
Carcinoma de Células Renais/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , DNA de Neoplasias/genética , Humanos , Hipóxia/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
10.
Clin Cancer Res ; 24(2): 445-459, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29084921

RESUMO

Purpose: Regulated in development and DNA damage response-1 (REDD1) is a stress-related protein and is involved in the progression of cancer. The role and regulatory mechanism of REDD1 in bladder urothelial carcinoma (BUC), however, is yet unidentified.Experimental Design: The expression of REDD1 in BUC was detected by Western blot analysis and immunohistochemistry (IHC). The correlation between REDD1 expression and clinical features in patients with BUC were assessed. The effects of REDD1 on cellular proliferation, apoptosis, autophagy, and paclitaxel sensitivity were determined both in vitro and in vivo Then the targeted-regulating mechanism of REDD1 by miRNAs was explored.Results: Here the significant increase of REDD1 expression is detected in BUC tissue, and REDD1 is first reported as an independent prognostic factor in patients with BUC. Silencing REDD1 expression in T24 and EJ cells decreased cell proliferation, increased apoptosis, and decreased autophagy, whereas the ectopic expression of REDD1 in RT4 and BIU87 cells had the opposite effect. In addition, the REDD1-mediated proliferation, apoptosis, and autophagy are found to be negatively regulated by miR-22 in vitro, which intensify the paclitaxel sensitivity via inhibition of the well-acknowledged REDD1-EEF2K-autophagy axis. AKT/mTOR signaling initially activated or inhibited in response to silencing or enhancing REDD1 expression and then recovered rapidly. Finally, the inhibited REDD1 expression by either RNAi or miR-22 sensitizes BUC tumor cells to paclitaxel in a subcutaneous transplant carcinoma model in vivoConclusions: REDD1 is confirmed as an oncogene in BUC, and antagonizing REDD1 could be a potential therapeutic strategy to sensitize BUC cells to paclitaxel. Clin Cancer Res; 24(2); 445-59. ©2017 AACR.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Paclitaxel/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Neoplasias da Bexiga Urinária/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Biotechnol Lett ; 40(2): 349-358, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29124518

RESUMO

OBJECTIVES: To investigate the efficiency of a new cascade biocatalysis system for the conversion of R, S-ß-amino alcohols to enantiopure vicinal diol and ß-amino alcohol. RESULTS: An efficient cascade biocatalysis was achieved by combination of a transaminase, a carbonyl reductase and a cofactor regeneration system. An ee value of > 99% for 2-amino-2-phenylethanol and 1-phenyl-1, 2-ethanediol were simultaneously obtained with 50% conversion from R, S-2-amino-2-phenylethanol. The generality of the cascade biocatalysis was further demonstrated with the whole-cell approaches to convert 10-60 mM R, S-ß-amino alcohol to (R)- and (S)-diol and (R)- and (S)-ß-amino alcohol in 90-99% ee with 50-52% conversion. Preparative biotransformation was demonstrated at a 50 ml scale with mixed recombinant cells to give both (R)- and (S)-2-amino-2-phenylethanol and (R)- and (S)-1-phenyl-1, 2-ethanediol in > 99% ee and 40-42% isolated yield from racemic 2-amino-2-phenylethanol. CONCLUSIONS: This cascade biocatalysis system provides a new practical method for the simultaneous synthesis of optically pure vicinal diol and an ß-amino alcohol.


Assuntos
Oxirredutases do Álcool/metabolismo , Amino Álcoois/química , Amino Álcoois/metabolismo , Biotecnologia/métodos , Amino Álcoois/análise , Proteínas de Bactérias/metabolismo , Biocatálise , Sistema Livre de Células , Escherichia coli/enzimologia , Estereoisomerismo , Transaminases/metabolismo
12.
Appl Biochem Biotechnol ; 184(1): 12-24, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28577192

RESUMO

Chitinases are glycosyl hydrolases that catalyze the hydrolysis of ß-(1,4)-glycosidic bonds in chitin, the major structural polysaccharide presented in the cuticle and gut peritrophic matrix of insects. Two aspartate residues (D143, D145) and one tryptophan (W146) in the Lymantria dispar chitinase are highly conserved residues observed within the second conserved motif of the family 18 chitinase catalytic region. In this study, a chitinase cDNA, LdCht5, was cloned from L. dispar, and the roles of the three residues were investigated using site-directed mutagenesis and substituting them with three other amino acids. Seven mutant proteins, D143E, D145E, W146G, D143E/D145E, D143E/W146G, D145E/W146G, and D143E/D145E/W146G, as well as the wild-type enzyme, were produced using the baculovirus-insect cell line expression system. The enzymatic and kinetic properties of these mutant enzymes were measured using the oligosaccharide substrate MU-(GlcNAc)3. Among the seven mutants, the D145E, D143E/D145E, and D145E/W146G mutations kept some extant catalytic activity toward MU-(GlcNAc)3, while the D143E, W146G, D143E/W146G, and D143E/D145E/W146G mutant enzymes were inactivated. Compared with the mutant enzymes, the wild-type enzyme had higher values of k cat and k cat / K m . A study of the multiple point mutations in the second conserved catalytic region would help to elucidate the role of the critical residues and their relationships.


Assuntos
Quitinases/metabolismo , Lepidópteros/enzimologia , Aminoácidos/química , Animais , Baculoviridae/genética , Sequência de Bases , Biocatálise , Domínio Catalítico , Quitinases/química , Quitinases/genética , Clonagem Molecular , Primers do DNA , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Mutagênese Sítio-Dirigida , Filogenia , Temperatura Ambiente
13.
J Exp Clin Cancer Res ; 36(1): 110, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28810896

RESUMO

BACKGROUND: Radiotherapy has been used increasingly to treat primary hepatocellular carcinoma. Clinically, the main cause of radiotherapy failure is cellular radioresistance, conferred via glycolytic metabolism. Our previous study demonstrated that Girdin is upregulated in primary hepatocellular carcinoma and promotes the invasion and metastasis of tumor cells. However, whether Girdin underlies the radio-sensitivity of hepatocellular carcinoma remains unclear. METHODS: A short hairpin RNA (shRNA) was used to silence CCDC88A (encoding Girdin), and real-time PCR was performed to determine CCDC88A mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of Girdin silencing on cellular radiosensitivity. Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the signaling pathway downstream of Girdin. Finally, animal experiments were performed to demonstrate the effect of CCDC88A silencing on the radiosensitivity of hepatoma in vivo. RESULTS: shRNA-induced Girdin silencing suppressed glycolysis and enhanced the radio-sensitivity of hepatic cell lines, HepG2 and Huh-7. Furthermore, silencing of Girdin inhibited the PI3K/AKT/HIF-1α signaling pathway, which is a central regulator of glycolysis. CONCLUSION: Girdin can regulate glycolysis in hepatocellular carcinoma cells through the PI3K/AKT/HIF-1α signaling pathway, which decreases the sensitivity of tumor cells to radiotherapy.


Assuntos
Carcinoma Hepatocelular/radioterapia , Neoplasias Hepáticas/radioterapia , Proteínas dos Microfilamentos/genética , Tolerância a Radiação/genética , Proteínas de Transporte Vesicular/genética , Adulto , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Glicólise/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Proteínas dos Microfilamentos/antagonistas & inibidores , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Proteínas de Transporte Vesicular/antagonistas & inibidores
14.
Appl Biochem Biotechnol ; 181(3): 972-985, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27714638

RESUMO

Four uncharacterized ω-transaminases (ωTAs) from Pseudomonas putida NBRC 14164 have been identified and cloned from the pool of fully sequenced genomes. The genes were functionally expressed in Escherichia coli BL21, and the enzymes were purified and characterized. Four TAs showed highly (S)-selective ωTA activity and converted (S)-α-methylbenzylamine and pyruvate to acetophenone and L-Ala. The maximum activity of cloned enzymes was in the pH range of 8.0-8.5 (Pp36420), 8.5-9.5 (Pp21050), 9.0-9.5 (PpspuC), and 9.5-10.5 (PpbauA), and the optimal temperatures were at 35 °C (Pp36420, Pp21050, and PpspuC) and 50 °C (PpbauA), respectively, with K M of 161.3 mM (Pp21050), 136.7 mM (PpbauA), 398.5 mM (Pp36420), and 130.9 mM (PpspuC) and yielding a catalytic efficiency k cat/K M of 0.015, 0.003, 0.012, and 0.023 mM-1 s-1. Several racemic amines and amino alcohols were resolved by the cloned ωTAs; perfect conversions (48-50 %) were obtained by at least one enzyme, and the residual substrates were left with 97-99 % ee. Kinetic resolution of racemic phenylglycinol was done with PpspuC in a 100-mL scale. Enaniomeric excess of (S)-phenylglycinol reached 99 % with 45 % isolated yield. The high enantioselectivity and large substrate spectra of the cloned PpTAs showed an attractive potency for biotechnology application in production of chiral amines and amino alcohols.


Assuntos
Amino Álcoois/química , Proteínas de Bactérias/química , Pseudomonas putida/enzimologia , Transaminases/química , Proteínas de Bactérias/genética , Concentração de Íons de Hidrogênio , Cinética , Pseudomonas putida/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Estereoisomerismo , Transaminases/genética
15.
J Biotechnol ; 243: 1-9, 2017 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-28011130

RESUMO

Optically pure 1-phenyl-1,2-ethanediol is a very important chiral building block and intermediate in fine chemical and pharmaceutical industries. Reduction of 2-hydroxyacetophenone provides a straightforward approach to access these important compounds. In this study, two enantiocomplementary carbonyl reductases, BDHA (2,3-butanediol dehydrogenase from Bacillus subtilis) and GoSCR (polyol dehydrogenase from Gluconobacter oxydans) were discovered for the first time to convert 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED) and (S)-1-phenyl-1,2-ethanediol ((S)-PED) with excellent stereochemical selectivity, respectively. The two enzymes were purified and characterized. In vitro bioreduction of 2-HAP catalyzed by BDHA and GoSCR coupled with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration were demonstrated, affording both (R)-PED and (S)-PED in>99% ee and 99% conversion. Recombinant Escherichia coli whole cells co-expressing both GDH and BDHA or GoSCR genes were used to asymmetric reduction of 2-HAP to (R)-PED or (S)-PED. Under the optimized conditions, the bioreduction of 400mM (54g/L) substrate was proceeded smoothly without the external addition of cofactor, and the product (R)-PED and (S)-PED were obtained with 99% yield, >99% ee and 18.0g/L/h volumetric productivity. These results offer a practical biocatalytic method for the preparation of both (R)-PED and (S)-PED with high volumetric productivity.


Assuntos
Acetofenonas/metabolismo , Oxirredutases do Álcool/metabolismo , Etilenoglicóis/metabolismo , Acetofenonas/química , Oxirredutases do Álcool/química , Bacillus subtilis/enzimologia , Biotransformação , Butileno Glicóis/metabolismo , Clonagem Molecular , Ativação Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Etilenoglicóis/química , Gluconobacter oxydans/enzimologia , Gluconobacter oxydans/genética , Glucose 1-Desidrogenase/metabolismo , L-Iditol 2-Desidrogenase/metabolismo , Chaperonas Moleculares , Estereoisomerismo , Especificidade por Substrato
16.
Anal Biochem ; 518: 94-101, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27899283

RESUMO

Chiral vicinal amino alcohols are important chiral building blocks and intermediates in the pharmaceutical industry. The transaminase (TAm) catalyzed kinetic resolution of racemic amino alcohols provides a straightforward approach to access these important compounds. This study describes the development of a novel microtiter plate assay to screen vicinal amino alcohol-specific TAms using a tetrazolium red-based colorimetric assay to monitor the rate of α-hydroxy ketone formation at 510 nm. This approach is the first to determine the Michaelis-Menten parameters for a recombinant TAm (PpbauA) from Pseudomonas putida NBRC14164. The corresponding Vmax and KM values for both enantiomers of 2-amino-1-propanol and 2-amino-1-butanol were obtained, and the calculated kinetic E-factors of PpbauA toward 2-amino-1-propanol and 2-amino-1-butanol are 3 (S) and 6 (R), respectively. The method is sensitive and exhibits low level background coloration. Moreover, this method can be used to detect transaminase activity and enantioselectivity toward amino alcohols in a high-throughput format. Additionally, this simple method is compatible with the most widely used (R)- and (S)-selective transaminases and may be a broadly applicable tool for screening transaminases from a transaminase mutant library.


Assuntos
Amino Álcoois/química , Proteínas de Bactérias/química , Propanolaminas/química , Pseudomonas putida/enzimologia , Transaminases/química , Amino Álcoois/metabolismo , Proteínas de Bactérias/metabolismo , Propanolaminas/metabolismo , Especificidade por Substrato/fisiologia , Transaminases/metabolismo
17.
Biotechnol Lett ; 38(9): 1559-64, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27233513

RESUMO

OBJECTIVES: To investigate the efficiency of a cofactor regeneration enzyme co-expressed with a glycerol dehydrogenase for the production of 1,3-dihydroxyacetone (DHA). RESULTS: In vitro biotransformation of glycerol was achieved with the cell-free extracts containing recombinant GlyDH (glycerol dehydrogenase from Escherichia coli), LDH (lactate dehydrogenase form Bacillus subtilis) or LpNox1 (NADH oxidase from Lactobacillus pentosus), giving DHA at 1.3 g l(-1) (GlyDH/LDH) and 2.2 g l(-1) (GlyDH/LpNox1) with total turnover number (TTN) of NAD(+) recycling of 6039 and 11100, respectively. Whole cells of E. coli (GlyDH-LpNox1) co-expressing both GlyDH and LpNox1 were constructed and converted 10 g glycerol l(-1) to DHA at 0.2-0.5 g l(-1) in the presence of zero to 2 mM exogenous NAD(+). The cell free extract of E. coli (GlyDH-LpNox) converted glycerol (2-50 g l(-1)) to DHA from 0.5 to 4.0 g l(-1) (8-25 % conversion) without exogenous NAD(+). CONCLUSIONS: The disadvantage of the expensive consumption of NAD(+) for the production of DHA has been overcome.


Assuntos
Di-Hidroxiacetona/metabolismo , Glicerol/metabolismo , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Desidrogenase do Álcool de Açúcar/metabolismo
18.
Huan Jing Ke Xue ; 37(1): 35-40, 2016 Jan 15.
Artigo em Chinês | MEDLINE | ID: mdl-27078938

RESUMO

PM2.5 samples were collected in six different functional zones in Nanchang City during autumn in 2013. PM2.5 mass concentration and enrichment characteristics of eighteen metal elements (Mg, Al, K, Ca, Ti, V, Ba, Co, Cr, Mn, Fe, Ni, Cu, Zn, Cd, Pb, As and Hg) were analyzed. The pollution sources of the above elements in PM2.5 were discussed based on the results of multivariate statistical analysis. The results showed that the average daily mass concentration of PM2.5 during autumn in Nanchang City met the secondary standard limit (≤ 75 µg · m⁻³) of National Ambient Air Quality Standards (GB 3095-2012). The enrichment factors of Mn, Ti, Al and V were lower than 1.0, indicating that these elements were barely enriched. The enrichment factors of Fe, Cr, Co, K, Mg, Ba, Ca, Cu and As ranged from 1.7 to 7.8, suggesting the influence of both natural sources and anthropogenic sources. Hg, Zn, Pb, Ni and Cd were obviously affected by anthropogenic emissions since their enrichment factors ranged from 21. 9 to 481.2. The combined results of correlation analysis, principal components analysis and cluster analysis revealed the pollution sources of these metals in PM2.5: Mg, K, Al, Ca and Ti mainly came from natural soil and building material dust; As and Hg were mainly from coal combustion; Ba, Ni and Mn were mainly from industrial emission of metal smelting; V, Cu, Fe, Cd, Pb, Cr and Co mainly came from traffic sources; Zn was influenced by metal smelting and coal burning.


Assuntos
Poluentes Atmosféricos/análise , Monitoramento Ambiental , Metais/análise , Material Particulado/análise , Estações do Ano , China , Cidades , Poeira , Poluição Ambiental , Análise Multivariada , Solo
19.
Bioprocess Biosyst Eng ; 39(4): 603-11, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26801669

RESUMO

Two uncharacterized nicotinamide adenine dinucleotide (NADH) oxidases (named as LpNox1, LpNox2) from Lactobacillus pentosus ATCC 8041 were cloned and overexpressed in Escherichia coli BL21 (DE3). The sequence analysis revealed that the two enzymes are water-forming Noxs with 64 % and 52 % identity to LbNox from Lactobacillus brevis DSM 20054. The optimal pH and temperature of the purified LpNox1 and LpNox2 were 7.0 and 8.0 and 35 and 40 °C, respectively, with K M of 99.0 µM (LpNox1) and 27.6 µM (LpNox2), and yielding catalytic efficiency k cat/K M of 1.0 and 0.2 µM(-1) s(-1), respectively. Heat inactivation studies revealed that the two enzymes are relatively instable. The application of LpNox1 for the regeneration of NAD(+) was demonstrated by coupling with a glycerol dehydrogenase-catalyzed oxidation of glycerol to 1,3-dihydroxyacetone. The characteristics of the LpNox1 could prove to be of interest in industrial application such as NAD(+) regeneration in dehydrogenase-catalyzed oxidations.


Assuntos
Proteínas de Bactérias , Lactobacillus pentosus , NADPH Oxidases , NAD/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Clonagem Molecular , Concentração de Íons de Hidrogênio , Lactobacillus pentosus/enzimologia , Lactobacillus pentosus/genética , NAD/genética , NADPH Oxidases/biossíntese , NADPH Oxidases/química , NADPH Oxidases/genética , Oxirredução
20.
Biochemistry (Mosc) ; 80(2): 242-50, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25756539

RESUMO

Insect chitinase plays essential roles in chitin catabolism involved in digestion and molting during insect development. In the current work, we cloned a chitinase cDNA, LrCht5, from the apple leaf miner moth Lithocolletis ringoniella and characterized its amino acid sequence and protein properties. The L. ringoniella chitinase cDNA was 2136 bp in length with an open reading frame of 1737 bp that encodes a polypeptide of 579 amino acid residues with a predicted molecular mass of 64.4 kDa and pI of 5.49. The catalytic domain has several phosphorylation and glycosylation sites. The recombinant LrCht5 was expressed in Escherichia coli and the Spodoptera frugiperda cell line Sf9, and the LrCht5 expressed in insect cells exhibited chitinolytic activity. LrCht5 was most stable at pH 6.0 and 45°C. This work has potential application in the development of novel and more specific synthetic chitinase inhibitors for use as bioinsecticides.


Assuntos
Quitinases/química , Mariposas/genética , Sequência de Aminoácidos , Animais , Domínio Catalítico , Quitinases/genética , Quitinases/metabolismo , Clonagem Molecular , Glicosilação , Dados de Sequência Molecular , Fosforilação , Filogenia , Conformação Proteica
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