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.
Alcohol Dehydrogenase ; metabolism ; Alcohol Oxidoreductases ; metabolism ; Aldehyde Oxidoreductases ; metabolism ; Anaerobiosis ; Bacillus ; classification ; genetics ; metabolism ; Butanols ; metabolism ; Fermentation ; Metabolic Networks and Pathways

BACKGROUNDRetinoic 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.

METHODSRDH1l 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).

RESULTSThe 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.

CONCLUSIONSRDH1l 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.

Alcohol Oxidoreductases ; genetics ; metabolism ; Animals ; Animals, Genetically Modified ; Heart ; embryology ; Zebrafish ; Zebrafish Proteins ; genetics ; metabolism

Through studying the process of glycerol fermentation to 1, 3-propanediol(1, 3-PD) by Klebsiella pneumoniae, it was found that the cell growth and product (or by-product) production were under salt stress. Cell growth and product formation kept high rate at low salt concentration. High salt concentration led to low growth of cells, final concentration of 1, 3-PD and conversion from glycerol to 1, 3-PD, and, 1, 3-propanediol oxidoreductase activity decreased. When the salt concentration in 5 m3 bioreactor was controlled under appropriate manner, the concentration of 1, 3-PD production was markedly enhanced. The final 1, 3-PD concentration ,the conversion of glycerol to 1, 3-PD and productivity were 64 g/L, 61% and 2.1 g/(L x h).
Alcohol Dehydrogenase ; Alcohol Oxidoreductases ; metabolism ; Culture Media ; Culture Techniques ; Fermentation ; Glycerol ; metabolism ; Klebsiella pneumoniae ; growth & development ; metabolism ; physiology ; Propylene Glycols ; metabolism ; Sodium Chloride ; pharmacology ; Stress, Physiological

Using response surface method, we optimized the medium for the asymmetric whole cell biotransformation by Candida tropicalis 104. This strain was used for microbial reduction of 1-[3,5-bis(trifluoromethyl)phenyl] ethanone to (S)-l-[3,5-bis(trifluoromethyl)phenyl] alcohol, with enantiomeric excess(e.e.) reached more than 99.9%. Fractional factorial design was used to evaluate the effects of medium components on carbonyl reductase activity of Candida tropicalis 104. Yeast extract, glucose and NH4Cl were the most important factors among six tested variables that influence the enzyme activity for the biotransformation process. Based on the experimental results, the path of steepest ascent was undertaken to approach the optimal region of these factors. Central composite design and response surface analysis were subsequently employed for further optimization. The optimal medium for Candida tropicalis 104 was composed of (in g/L): glucose 47.14, yeast extract 13.25, NH4Cl 2.71, MgSO4.7H2O 0.4, KH2PO4 1, K2HPO4 1. Under the optimum conditions, the maximum enzyme activity of 852.75 U/L in theory and 851.13 U/L in the experiment were obtained, with an increase of 65.2% compared to the original medium components.
Alcohol Oxidoreductases ; biosynthesis ; metabolism ; Ammonium Chloride ; pharmacology ; Candida tropicalis ; growth & development ; metabolism ; Culture Media ; Fermentation ; Models, Theoretical

Under the catalysis of a variety of metabolic enzymes in vivo, such as UDP-glucuronyl transferases, cytochrome P450, carboxylesterase, sulfotransferase, butyrylcholinesterase, catechol-O-methyl transferase and 6-morphine dehydrogenase, the drugs perform glucuronidation, hydrolysis, oxidation, sulfonation and other reactions, then translate into active or inactive metabolites, which are excreted through urination, bile or the other pathways at last. Different drugs own their different metabolic pathways. This paper introduces the studies about the metabolism of drugs in human and animal in recent years, such as morphine-like drugs, amphetamine, ketamine, cannabis and cocaine, and reviews the research progress about the sites of metabolism, metabolic enzymes, metabolites and physiological activity of those drugs metabolic in vivo.
Alcohol Oxidoreductases/metabolism* ; Animals ; Carboxylesterase/metabolism* ; Catechol O-Methyltransferase/metabolism* ; Cholinesterases/metabolism* ; Cytochrome P-450 Enzyme System/metabolism* ; Glucuronosyltransferase/metabolism* ; Humans ; Illicit Drugs/metabolism* ; Oxidation-Reduction ; Sulfotransferases/metabolism*

OBJECTIVETo study the activities of the key enzymes in reteplase production by Pichia pastoris.

METHODSIn shaking flasks, a series of samples were maintained after methanol induction. The cells were sonicated to prepare cell-free suspensions, in which the activities of AOX, FAD, PDC, G-6-PD, ID, alpha-KGD and SD were measured.

RESULTSThe specific activity of AOX increased during the initial 6 h, reaching the maximum of 44.5 U/mg protein. The activity decreased quickly between 6 and 24 h, followed by increment in the following 24 h and decreased afterwards. The specific activity of FAD increased gradually in the initial 48 h and then decreased, with the peak level of 6.72 U/mg protein occurred at 48 h. The specific activity of G-6-PD increased at in 2-6 h and 24-48 h, but decreased in 6-24 h and after 48 h. The specific activity of PDC decreased during the initial 6 h and increased slowed afterwards. The specific activities of ID, alpha-KGD and SD all showed a rapid decrease in the initial 6 h and a slow decrease in 6-24 h. After 24 h, the activity of ID continued to decrease, but the other two increased in the following 24 h and then decreased, reaching the maximum at 48 h.

CONCLUSIONSAccording to the changes of these enzyme activities, the whole induction phase can be divided into 4 stages: the methanol-adaptive period in the initial 6 h, the fast growth period between 6 and 24 h, the product accumulation period in 24-48 h and the metabolism lag period in 48-72 h. In the methanol-adaptive period, complete oxidation of methanol is the dominant pathway. But in the following two stages, the metabolic pathway shifts towards glycolysis and TCA cycle.

Alcohol Oxidoreductases ; metabolism ; Fermentation ; Genetic Vectors ; Glucosephosphate Dehydrogenase ; metabolism ; Methanol ; pharmacology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Tissue Plasminogen Activator ; biosynthesis ; genetics ; metabolism

The 1,3-propanediol oxidoreductase isoenzyme encoding gene (yqhD) from E. coli was amplified by PCR. yqhD was inserted in pEtac to yield the recombinant expression vector pEtac-yqhD. Over-expression of yqhD in E. coli JM109 was achieved with pEtac-yqhD. SDS-PAGE analysis showed an over-expressed recombinant product at about 43 kD, consistent with the molecular weight predicted from gene sequence. Compared with E. coli JM109 (pEtac), the 1,3-propanediol oxidoreductase isoenzyme activity of the recombinant E. coli (pEtac-yqhD) reached 120 u/mg protein under the induction of 1.0 mmol/L IPTG at 37 degrees C for 4 hours; at similar conditions, enzyme activity of E. coli JM109 (pEtac) was only 0.5 u/mg protein. The recombinant E. coli JM109 (pUCtac-dhaB, pEtac-yqhD) was constructed. After induction with 1.0 mmol/L IPTG, the recombinant strain could transform 50 g/L glycerol to 38 g/L 1,3-propanediol under aerobic conditions. This work demonstrated firstly that the 1,3-propanediol oxidoreductase isoenzyme could show high activity under aerobic conditions.
Aerobiosis ; Alcohol Dehydrogenase ; Alcohol Oxidoreductases ; genetics ; metabolism ; Aldehyde Reductase ; genetics ; metabolism ; Escherichia coli ; enzymology ; genetics ; Escherichia coli Proteins ; genetics ; metabolism ; Genetic Engineering ; methods ; Isoenzymes ; Propylene Glycols ; metabolism ; Recombinant Proteins ; genetics ; metabolism

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.
Alcohol Oxidoreductases ; genetics ; Bacillus subtilis ; genetics ; metabolism ; Glucose 1-Dehydrogenase ; chemistry ; metabolism ; Mutagenesis, Insertional ; Pseudoephedrine ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic

The present study was designed to analyze the metabolites of all-trans-retinal (atRal) and compare the cytotoxicity of atRal versus its derivative all-trans-retinoic acid (atRA) in human retinal pigment epithelial (RPE) cells. We confirmed that atRA was produced in normal pig neural retina and RPE. The amount of all-trans-retinol (atROL) converted from atRal was about 2.7 times that of atRal-derived atRA after incubating RPE cells with 10 μmol/L atRal for 24 h, whereas atRA in medium supernatant is more plentiful (91 vs. 29 pmol/mL), suggesting that atRA conversion facilitates elimination of excess atRal in the retina. Moreover, we found that mRNA expression of retinoic acid-specific hydroxylase CYP26b1 was dose-dependently up-regulated by atRal exposure in RPE cells, indicating that atRA inactivation may be also initiated in atRal-accumulated RPE cells. Our data show that atRA-caused viability inhibition was evidently reduced compared with the equal concentration of its precursor atRal. Excess accumulation of atRal provoked intracellular reactive oxygen species (ROS) overproduction, heme oxygenase-1 (HO-1) expression, and increased cleaved poly(ADP-ribose) polymerase 1 (PARP1) expression in RPE cells. In contrast, comparable dosage of atRA-induced oxidative stress was much weaker, and it could not activate apoptosis in RPE cells. These results suggest that atRA generation is an antidotal metabolism pathway for atRal in the retina. Moreover, we found that in the eyes of ABCA4^-/-RDH8^-/- mice, a mouse model with atRal accumulation in the retina, the atRA content was almost the same as that in the wild type. It is possible that atRal accumulation simultaneously and equally promotes atRA synthesis and clearance in eyes of ABCA4^-/-RDH8^-/- mice, thus inhibiting the further increase of atRA in the retina. Our present study provides further insights into atRal clearance in the retina.
ATP-Binding Cassette Transporters/physiology* ; Alcohol Oxidoreductases/physiology* ; Animals ; Cell Survival/drug effects* ; Cells, Cultured ; Humans ; Inactivation, Metabolic ; Mice ; Retina/metabolism* ; Retinal Pigment Epithelium/metabolism* ; Swine ; Tretinoin/pharmacology*

Objective To explore the change rules of blood ethanol and blood acetaldehyde concentration, the impairment of psychomotor functions of different acetaldehyde dehydrogenase （ALDH） 2 genotype individuals after alcohol consumption and the relationship among them. Methods The ALDH2 genotypes in seventy-nine healthy volunteers were obtained by SNaPshot^TM method, then divided into ALDH2*1/*1 （wild type） and ALDH2*1/*2 （mutant type） group. After volunteers consumed 1.0 g/kg of alcohol, blood ethanol concentration and blood acetaldehyde concentration at a series of time points before and after alcohol consumption and psychomotor functions, such as, visual selective response time, auditory simple response time and tracking experiment were detected. Biphasic alcohol response questionnaires were collected. Results After alcohol consumption, ALDH2*1/*2 group's blood ethanol and blood acetaldehyde concentration reached the peak earlier than ALDH2*1/*1 group. Its blood acetaldehyde concentration was higher than that of ALDH2*1/*1 group, 1-6 h after alcohol consumption. The psychomotor functions, such as visual selective response time and auditory simple response time in ALDH2*1/*2 group were more significantly impaired than those in ALDH2*1/*1 group after alcohol consumption. There was no statistical significance between the two groups in excitement or sedation reactions （P>0.05）. Pearson correlation coefficient test showed that blood acetaldehyde concentration was related with psychomotor function. Conclusion There are significant differences between the psychomotor function of ALDH2 wild type and mutant type individuals after alcohol consumption estimated to be related to the difference in blood acetaldehyde concentration after alcohol consumption.
Acetaldehyde/metabolism* ; Alcohol Drinking/blood* ; Aldehyde Dehydrogenase/genetics* ; Aldehyde Dehydrogenase, Mitochondrial ; Aldehyde Oxidoreductases ; Ethanol/metabolism* ; Genotype ; Humans ; Polymorphism, Genetic/genetics* ; Psychomotor Performance/physiology*