共查询到20条相似文献,搜索用时 31 毫秒
1.
2.
Ren J 《Obesity (Silver Spring, Md.)》2007,15(12):2932-2941
Objective: Consumption of high‐fat diet and alcohol is associated with obesity, leading to enhanced morbidity and mortality. This study was designed to examine the interaction between high‐fat diet and the alcohol metabolizing enzyme alcohol dehydrogenase (ADH) on ethanol‐induced cardiac depression. Research Methods and Procedures: Mechanical and intracellular Ca2+ properties were measured in cardiomyocytes from ADH transgenic and Friend Virus‐B type (FVB) mice fed a low‐ or high‐fat diet for 16 weeks. Expression of protein kinase B (Akt) and Foxo3a, two proteins essential for cardiac survival, was evaluated by Western blot. Cardiac damage was determined by carbonyl formation. Results: High fat but not ADH induced obesity without hyperglycemia or hypertension, prolonged time‐to‐90% relengthening (TR90), and depressed peak shortening (PS) and maximal velocity of shortening/relengthening (± dL/dt) without affecting intracellular Ca2+ properties. Ethanol suppressed PS and intracellular Ca2+ rise in low‐fat‐fed FVB mouse cardiomyocytes. ADH but not high‐fat diet shifted the threshold of ethanol‐induced inhibition of PS and ± dL/dt to lower levels. The amplitude of ethanol‐induced cardiac depression was greater in the high‐fat but not the ADH group without additive effects. Ethanol down‐ and up‐regulated Akt and Foxo3a expression, respectively, and depressed intracellular Ca2+ rise, the effects of which were exaggerated by ADH, high‐fat, or both. High‐fat diet, but not ADH, enhanced Foxo3a expression and carbonyl content in non‐ethanol‐treated mice. Ethanol challenge significantly enhanced protein carbonyl formation, with the response being augmented by ADH, high‐fat, or both. Discussion: Our data suggest that high‐fat diet and ADH transgene may exaggerate ethanol‐induced cardiac depression and protein damage in response to ethanol. 相似文献
3.
We examined expression of two plant genes encoding coclaurine N-methyltransferase (CMT) and norcoclaurine synthase (NCS) in Escherichia coli from the Salmonella enterica prpBCDE promoter (PprpB) and compared it to that from the strongest IPTG-inducible promoter, PT7. In contrast to our previous study showing slightly higher production of green fluorescent protein (GFP) from the pPro system compared to that from the T7 system, production of two plant proteins CMT and NCS from PprpB was 2- to 4-fold higher than that from PT7. Unlike PT7, expression from PprpB did not reduce cell growth even when highly induced, indicating that this propionate-inducible system is more efficient for overproduction of proteins that result in growth inhibition. In an auto-induction experiment, which does not require monitoring the culture or adding inducer during cell growth, the pPro system exhibited much higher protein production than the T7 system. These results strongly indicate that the pPro system is well-suited for overproduction of recombinant proteins. 相似文献
4.
The PsADH2-promoter of Pichia stipitis alcohol dehydrogenase II (ADH II) gene was employed to control the expression of Vitreoscilla hemoglobin (VHb) gene in Pichia pastoris. As in P. stipitis, the promoter was also induced microaerobically in P. pastoris. The expression level of VHb in P. pastoris at low O2 tension (<5% air saturation) was 16 nmol/g dry cell wt, i.e. about 24-fold higher than that at 60% air saturation. The expressed
VHb enhanced growth of P. pastoris under microaerobic conditions. The application of O2-regulated promoter in P. pastoris revealed that induction of high-level expression of heterologous protein is feasible without addition of supplementary compounds. 相似文献
5.
Objectives
Binge drinking and alcohol toxicity are often associated with myocardial dysfunction possibly due to accumulation of the ethanol metabolite acetaldehyde although the underlying mechanism is unknown. This study was designed to examine the impact of accelerated ethanol metabolism on myocardial contractility, mitochondrial function and apoptosis using a murine model of cardiac-specific overexpression of alcohol dehydrogenase (ADH).Methods
ADH and wild-type FVB mice were acutely challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Myocardial contractility, mitochondrial damage and apoptosis (death receptor and mitochondrial pathways) were examined.Results
Ethanol led to reduced cardiac contractility, enlarged cardiomyocyte, mitochondrial damage and apoptosis, the effects of which were exaggerated by ADH transgene. In particular, ADH exacerbated mitochondrial dysfunction manifested as decreased mitochondrial membrane potential and accumulation of mitochondrial O2 •−. Myocardium from ethanol-treated mice displayed enhanced Bax, Caspase-3 and decreased Bcl-2 expression, the effect of which with the exception of Caspase-3 was augmented by ADH. ADH accentuated ethanol-induced increase in the mitochondrial death domain components pro-caspase-9 and cytochrome C in the cytoplasm. Neither ethanol nor ADH affected the expression of ANP, total pro-caspase-9, cytosolic and total pro-caspase-8, TNF-α, Fas receptor, Fas L and cytosolic AIF.Conclusions
Taken together, these data suggest that enhanced acetaldehyde production through ADH overexpression following acute ethanol exposure exacerbated ethanol-induced myocardial contractile dysfunction, cardiomyocyte enlargement, mitochondrial damage and apoptosis, indicating a pivotal role of ADH in ethanol-induced cardiac dysfunction possibly through mitochondrial death pathway of apoptosis. 相似文献6.
D. C. La Grange I. S. Pretorius W. H. van Zyl 《Applied microbiology and biotechnology》1997,47(3):262-266
A genomic DNA library of the bacterium Bacillus pumilus PLS was constructed and the β-xylosidase gene (xynB) was amplified from a 3-kb genomic DNA fragment with the aid of the polymerase chain reaction technique. The amplified xynB gene was inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2
P
) and terminator (ADH2
T
) sequences on a multicopy episomal plasmid (pDLG11). The xynB gene was also fused in-frame to the secretion signal sequence of the yeast mating pheromone α-factor (MFα1
S
) before insertion between the ADH2
P
and ADH2
T
sequences on a similar multicopy episomal plasmid (pDLG12). The resulting construct ADH2
P
-MFα1
S
-xynB-ADH2
T
was designated XLO1. Both plasmids pDLG11 and pDLG12 were introduced into Saccharomyces cerevisiae but only the expression of the XLO1 gene yielded biologically functional β-xylosidase. The total β-xylosidase activity remained cell-associated with a maximum
activity of 0.09 nkat/ml obtained when the recombinant S. cerevisiae strain was grown for 143 h in synthetic medium. The temperature and pH optima of the recombinant Xlo1 enzyme were 45–50 °C
and pH 6.6 respectively. The enzyme was thermostable at 45 °C; however, at 60 °C most of the Xlo1 was inactive after 5 min.
Received: 11 July 1996 / Received revision: 23 October 1996 / Accepted: 25 October 1996 相似文献
7.
HELGA N. PALMA‐GUTIÉRREZ JOSÉ SALUD RODRÍGUEZ‐ZAVALA RICARDO JASSO‐CHÁVEZ RAFAEL MORENO‐SÁNCHEZ EMMA SAAVEDRA 《The Journal of eukaryotic microbiology》2008,55(6):554-561
ABSTRACT. Euglena gracilis is a freshwater free‐living organism able to grow with ethanol as carbon source; to facilitate this metabolism several alcohol dehydrogenase (ADH) activities have been detected. We report the gene cloning, over‐expression, and biochemical characterization of a medium‐chain NAD+‐dependent ADH from E. gracilis (EgADH). The enzyme's amino acid sequence displayed the highest percentages of similarity and identity with ADHs of bacteria and fungi. In the predicted three‐dimensional model, all the residues involved in Zn2+, cofactor, and substrate binding were conserved. A conventional signal peptide for import into mitochondria could not be clearly identified. The protein of 37 kDa was over‐expressed, purified to homogeneity, and kinetically characterized. The enzyme's optimal pH was 7.0 for ethanol oxidation displaying a Vm of 11.7±3.6 U/mg protein and a Km of 3.2±0.7 mM for this substrate. Isopropanol and isopentanol were also utilized, although with less efficiency. It showed specificity for NAD+ with a Km value of 0.39±0.1 mM and Mg2+ or Zn2+ were essential for activity. The recombinant EgADH reported here may help to elucidate the roles that different ADHs have on the metabolism of short‐ and long‐chain alcohols in this microorganism. 相似文献
8.
Cao Y Liao L Xu XW Oren A Wang C Zhu XF Wu M 《Extremophiles : life under extreme conditions》2008,12(3):471-476
Alcohol dehydrogenase (ADH; EC: 1.1.1.1) is a key enzyme in production and utilization of ethanol. In this study, the gene
encoding for ADH of the haloalkaliphilic archaeon Natronomonas pharaonis (NpADH), which has a 1,068-bp open reading frame that encodes a protein of 355 amino acids, was cloned into the pET28b vector
and was expressed in Escherichia coli. Then, NpADH was purified by Ni-NTA affinity chromatography. The recombinant enzyme showed a molecular mass of 41.3 kDa by SDS-PAGE.
The enzyme was haloalkaliphilic and thermophilic, being most active at 5 M NaCl or 4 M KCl and 70°C, respectively. The optimal
pH was 9.0. Zn2+ significantly inhibited activity. The K
m value for acetaldehyde was higher than that for ethanol. It was concluded that the physiological role of this enzyme is likely
the catalysis of the oxidation of ethanol to acetaldehyde. 相似文献
9.
Genetic modification of industrial yeast strains to obtain controllable NewFlo flocculation property and lower diacetyl production 总被引:3,自引:0,他引:3
The expression cassette I10 containing the new-found flocculation gene, FLONS, was transformed into an industrial strain Saccharomyces cerevisiae YSF5. Upstream activating sequences of the S. cerevisiae alcohol dehydrogenase II (ADH2) gene promoter (PU-ADH2) were used to regulate the expression of FLONS; α-acetolactate synthase gene ILV2 was chosen for homologous recombination of I10 to the YSF5 chromosome; copper binding metallothionein (encoded by CUP1) was used for selection of transformants. Ten randomly selected transformants exhibited increased flocculation ability of
1.5 to 2.3 fold more than the original strain. Based on their sensitivity to glucose, maltose and sucrose, flocculation property
of the transformants was supported to be NewFlo-type. After successive subculture, the introduced CUP1 remained in the transformants. At the end of simulated fermentation test, diacetyl content of the culture media of 5I-1 was
0.45 g l−1, lower than YSF5 (0.48 g l−1). 相似文献
10.
Ethanol tolerance, alcohol dehydrogenase (ADH;EC1.1.1.1) activity, and tissue-specific expression wereexamined in species of the cardini group ofDrosophila using D. melanogaster as astandard of comparison. In contrast to most fruit-breeding species, allcardini species examined, two from the cardini subgroupand five from the dunni subgroup, were ethanol sensitive(LC50 2.05%) and the mean ADH activityof males ranges from only 8 to 16% that of D.melanogaster AdhFF. Among all sevencardini species, there were small but significantdifferences in ethanol tolerance and ADH activity.Differences in enzyme mobility were in accordance with the proposedphylogeny for the dunni-subgroup species. ADH isexpressed in the fat body and midgut. Males of D.acutilabella and of D. belladunni havesignificantly less ethanol tolerance and express less ADH activitythan females in zymograms and histologicalpreparations. 相似文献
11.
12.
13.
Erika Nahomy Marino-Marmolejo Antonio De León-Rodríguez Ana Paulina Barba de la Rosa Leticia Santos 《Molecular biotechnology》2009,42(1):61-67
Analysis of the Thermoplasma acidophilum DSM 1728 genome identified two putative alcohol dehydrogenase (ADH) open reading frames showing 50.4% identity against each
other. The corresponding genes Ta0841 and Ta1316 encode proteins of 336 and 328 amino acids with molecular masses of 36.48 and 36.01 kDa, respectively. The genes were expressed
in Escherichia coli and the recombinant enzymes were functionally assessed for activity. Throughout the study only Ta1316 ADH resulted active
in the oxidative reaction in the pH range 2–8 (optimal pH 5.0) and temperatures from 25 to 90°C (optimal 75°C). This ADH catalyzes
the oxidation of several alcohols such as ethanol, methanol, 2-propanol, butanol, and pentanol during the reduction of the
cofactor NAD+. The highest activity was found in the presence of ethanol producing optically pure acetaldehyde. The specific enzyme activity
of the purified Ta1316 ADH with ethanol as a substrate in the optimal conditions was 628.7 U/mg. 相似文献
14.
Hongling Liu Xihui Wang Shaojie Yang Ruiming Wang Tengfei Wang 《Biotechnology progress》2019,35(4):e2826
Trehalose is a nonreducing disaccharide synthesized by trehalose synthase (TreS), which catalyzes the reversible interconversion of maltose and trehalose. We aimed to enhance the catalytic conversion of maltose to trehalose by saturation mutagenesis, and constructed a self-inducible TreS expression system by generating a robust Bacillus subtilis recombinant. We found that the conversion yield and enzymatic activity of TreS was enhanced by saturation mutations, especially by the combination of V407M and K490L mutations. At the same time, these saturation mutations were contributing to reducing by-products in the reaction. Compared to WT TreS, the conversion yield of maltose to trehalose was increased by 11.9%, and the kcat/Km toward trehalose was 1.33 times higher in the reaction catalyzed by treSV407M-K490L. treSV407M-K490L expression was further observed in the recombinant B. subtilis W800N(ΔσF) under the influence of PsrfA, Pcry3Aa, and PsrfA-cry3Aa promoters without an inducer. It was shown that PsrfA-cry3Aa was evidently a stronger promoter for treSV407M-K490L expression, with the intracellular enzymatic activity of recombinant treSV407M-K490L being over 5,800 U/g at 35 hr in TB medium. These results suggested the combination of two mutations, V407M and K490L, was conducive for the production of trehalose. In addition, the self-inducible TreSV407M/K490L mutant in the B. subtilis host provides a low-cost choice for the industrial production of endotoxin-free trehalose with high yields. 相似文献
15.
Xiangxian Ying Amy M. Grunden Lin Nie Michael W. W. Adams Kesen Ma 《Extremophiles : life under extreme conditions》2009,13(2):299-311
The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both
enzymes appeared to be homotetramers with a subunit size of 45 ± 1 kDa as revealed by SDS-PAGE, which was close to the calculated
value (44.8 kDa). The recombinant ADH contained 1.0 ± 0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with
a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105 ± 2 U mg−1, which was very similar to that of the native enzyme (110 ± 3 U mg−1). The optimal pH-values for both enzymes for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively.
Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there
was no activity towards methanol and secondary alcohols. Kinetic parameters of the enzymes showed lower K
m-values for acetaldehyde and NADPH and higher K
m-values for ethanol and NADP+. It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile. 相似文献
16.
The final activity of the alcohol dehydrogenase (E.C.1.1.1.1, abbreviated ADH) from germinating pea, isolated by fractionating with ammonium sulphate, chromatography on DEAE cellulose and gel filtration, was 80,000, from bean 25,000 and from lentil 13,500 units per mg protein. Molecular weights of the ADHs are close to each other: pea and bean ADH 60,000, lentil ADH 70,000. The Km values are mutually similar with three enzymes, i.e. of the order of 10−4M for NAD and 10−2M for ethanol. The pH optima lie in the alkaline region. These enzymes catalyse oxidation of a number of monovalent alcohols. At temperatures above 60°C the enzymes are thermally unstable. Stability is enhanced slowly by ethanol but not by NAD. Pyrazol, imidazol and pyridine inhibit plant ADH similarly to the enzyme from horse liver. There is a similarity between plant alcohol dehydrogenases and animal and yeast enzymes. 相似文献
17.
《Bioscience, biotechnology, and biochemistry》2013,77(12):2720-2726
The function of mitochondrial Adh3 in the thermotolerant yeast Kluyveromyces marxianus was investigated. An ADH3-disrupted mutant exhibited growth retardation on non-fermentable carbon sources, except for ethanol, and this was suppressed by supplementation with antioxidants. Detailed analysis of the phenotype revealed that the mutant showed an increase in the activity of NADH dehydrogenase, sensitivity to H2O2, and accumulation of reactive oxygen species (ROS), and that these carbon sources increased the activity of succinate dehydrogenase. The increase in both activities may reflect enhanced expression of both dehydrogenases by elevation of their substrate levels. The ROS level became low when antioxidants were added. These findings suggest that the ADH3 mutation and such carbon sources cause an elevation of the substrate level of the respiratory chain and eventually of the ROS level via increased expression of primary dehydrogenases, which in turn causes cell growth retardation. Adh3 might thus play a crucial role in the control of the NADH/NAD+ balance in mitochondria. 相似文献
18.
The synthetic antibody model “M41” was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine CK and CH 1γ1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P212121 with unit cell dimensions a = 104.7 Å, b = 113.9 Å, c = 98.8 Å and diffracted X-rays to a nominal resolution of 2.5 Å. © 1995 Wiley-Liss, Inc. 相似文献
19.
20.
Surisa Suwannarangsee Doo-Byoung Oh Jeong-Woo Seo Chul Ho Kim Sang Ki Rhee Hyun Ah Kang Warawut Chulalaksananukul Ohsuk Kwon 《Applied microbiology and biotechnology》2010,88(2):497-507
The thermotolerant methylotrophic yeast Hansenula polymorpha has recently been gaining interest as a promising host for bioethanol production due to its ability to ferment xylose, glucose,
and cellobiose at elevated temperatures up to 48 °C. In this study, we identified and characterized alcohol dehydrogenase
1 of H. polymorpha (HpADH1). HpADH1 seems to be a cytoplasmic protein since no N-terminal mitochondrial targeting extension was detected. Compared
to the ADHs of other yeasts, recombinant HpADH1 overexpressed in Escherichia coli exhibited much higher catalytic efficiency for ethanol oxidation along with similar levels of acetaldehyde reduction. HpADH1
showed broad substrate specificity for alcohol oxidation but had an apparent preference for medium chain length alcohols.
Both ADH isozyme pattern analysis and ADH activity assay indicated that ADH1 is the major ADH in H. polymorpha DL-1. Moreover, an HpADH1-deleted mutant strain produced less ethanol in glucose or glycerol media compared to wild-type. Interestingly, when the ADH1 mutant was complemented with an HpADH1 expression cassette, the resulting strain produced significantly increased amounts of ethanol compared to wild-type, up to
36.7 g l−1. Taken together, our results suggest that optimization of ADH1 expression would be an ideal method for developing H. polymorpha into an efficient bioethanol production strain. 相似文献