Production Of Alcohol Dehydrogenase Research Articles

Production of N-glycosylated alcohol dehydrogenase in Escherichia coli

N-glycosylation of recombinant proteins using bacterial glycosylation system has proven to be a valuable although developing tool ultimately applicable to various industries. When used for enzyme engineering, it offers the possibility of increased stability or immobilization route and thus increasing effectiveness of e.g. biotransformation or other biocatalysis procedures. One such promising enzyme is alcohol dehydrogenase (ADH) for use in redox biotransformation reactions. Given the current possibilities of recombinant enzyme production, including major advances in glycoengineering and glycoprotein production in bacterial organisms, the aim of this work was the production of thermotolerant ADH from Rhodococcus ruber (RrADH) in glycosylated form in Escherichia coli. We have successfully developed a dual plasmid expression system enabling glycosylation of target proteins utilizing a glyco-tag approach. We were able to produce RrADH in soluble form and at the same time we detected a bacterial glycan conjugated to RrADH as well as the activity of the enzyme. The glycan bound to recombinant enzyme can be used for oriented covalent immobilization of the enzyme, which would increase the potential for its practical application in biotransformation of various compounds.

Investigating human-derived lactic acid bacteria for alcohol resistance

BackgroundExcessive alcohol consumption has been consistently linked to serious adverse health effects, particularly affecting the liver. One natural defense against the detrimental impacts of alcohol is provided by alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), which detoxify harmful alcohol metabolites. Recent studies have shown that certain probiotic strains, notably Lactobacillus spp., possess alcohol resistance and can produce these critical enzymes. Incorporating these probiotics into alcoholic beverages represents a pioneering approach that can potentially mitigate the negative health effects of alcohol while meeting evolving consumer preferences for functional and health-centric products.ResultsFive lactic acid bacteria (LAB) isolates were identified: Lactobacillus paracasei Alc1, Lacticaseibacillus rhamnosus AA, Pediococcus acidilactici Alc3, Lactobacillus paracasei Alc4, and Pediococcus acidilactici Alc5. Assessment of their alcohol tolerance, safety, adhesion ability, and immunomodulatory effects identified L. rhamnosus AA as the most promising alcohol-tolerant probiotic strain. This strain also showed high production of ADH and ALDH. Whole genome sequencing analysis revealed that the L. rhamnosus AA genome contained both the adh (encoding for ADH) and the adhE (encoding for ALDH) genes.ConclusionsL. rhamnosus AA, a novel probiotic candidate, showed notable alcohol resistance and the capability to produce enzymes essential for alcohol metabolism. This strain is a highly promising candidate for integration into commercial alcoholic beverages upon completion of comprehensive safety and functionality evaluations.

No increase in alcohol dehydrogenase levels following repeated ethanol exposure in young honeybee workers

AbstractSome workers of the honeybee show high alcohol dehydrogenase (ADH) levels and high resistance to the sedative effects of alcohol, yet it is unknown whether these two issues are directly related. Here we looked for a link between ADH levels and sedation latency in response to alcohol exposure. We used molecular and immunoblotting methods to investigate a possible induction of ADH production in young, ADH‐lacking workers in response to repeated ethanol vapour presentation. Although we found increased sedation latency in individuals after several ethanol encounters, this was not accompanied by detectibly increased ADH levels in such workers. The lack of ethanol‐induced ADH production indicated that it was not needed for increased coping with alcohol inebriation. In this work, we expanded current knowledge about the effects of alcohol on honeybee workers and related it to the existing literature on the subject.

Enhanced Production of Recombinant Alcohol Dehydrogenase Using the Genetically Engineered Escherichia coli Strain that Heterologously Expresses Carrot Heat Shock Protein 70.

Escherichia coli (E. coli) has been widely used as a host organism for producing recombinant proteins such as biocatalysts, antibody fragments, and therapeutic hormones. To enhance recombinant protein production, many E. coli strains have been genetically engineered on practical purposes. In this study, we developed the engineered E. coli strain expressing Heat shock protein 70, DcHsp70, from carrot (Daucus carota L.). The DNA construct for DcHsp70 expression, Lipoprotein promoter-DcHsp70 gene-Flippase recognition target cassette, which is flanked by the insertion site yddE pseudogene sequences, was generated by overlap PCR and inserted into the E. coli genome by lambda Red-mediated homologous recombination. To examine if the engineered E. coli cells can effectively produce recombinant proteins, the alcohol dehydrogenase (ADH) gene from a thermophile, Geobacillus stearothermophilus, was cloned into a pET11a expression vector and expressed by isopropyl β-D-1-thiogalactopyranoside treatment. Compared to wild type, the genetically engineered E. coli expressing DcHsp70 exhibited up to approximately 11-fold higher production of his-tagged ADH, mostly in soluble forms. The his-ADH protein that was purified from the engineered cells exhibited the enzyme activity. The genetically engineered E. coli developed in this study can be useful for the efficient production of recombinant proteins, such as recombinant ADH.

Effects of Exercise Intensity on Alcohol Dehydrogenase Gene Expression in the Rat Large IntestineExercise and ADH Gene Expression

Background and Objective
 The aim of this study was to examine the relationship between the intensity of treadmill exercise and alcohol dehydrogenase (ADH) expression in the large intestine.
 Material and Methods
 Thirty Sprague-Dawley white male rats were randomly assigned to a control group (CON; no exercise), low-intensity exercise group (LIG; 30-min exercise at 8 m/min 5 times a week for 4 weeks), or high-intensity exercise group (HIG; 30-min exercise at 28 m/min 5 times a week for 4 weeks).
 Results
 Microarray analysis was conducted to evaluate ADH gene expression levels in large intestinal tissue, significant changes in the expression of four ADH genes (Adh1, Adh4, Adh6a, and Adh7) related to exercise intensity. In addition, pooled samples of the exercise groups showed decreased expression levels of these four genes compared with those of the control group. These findings were confirmed by reverse transcription-polymerase chain reaction. In addition, differences were detected with respect to exercise intensity: Adh1, Adh4, and Adh6a levels were significantly decreased in the LIG compared with those in the HIG, whereas Adh7 expression showed an opposite trend.
 Conclusion
 In conclusion, this study suggests that regular exercise can decrease the incidence of alcohol-related disease by suppressing ADH production in the digestive system.

The Optimization of Alcohol Dehydrogenase Production from Pseudomonas aeruginosa NW

The locally isolated Pseudomonas aeruginosa NW was tested for their ability to produce alcohol dehydrogenase (ADH) enzyme by growing the bacteria on three different aromatic hydrocarbons: naphthalene, xylene and toluene. Following five days incubation the bacterium showed a good ability to utilize xylene, limited ability for toluene utilization and failed to degrade naphthalene. The ADH activity was assayed following each day of incubation on xylene as sole source of carbon and energy and it was found that a maximum ADH volume activity was 2.86 and 4.11 U/ml following the third and fourth day of incubation, respectively. The production of ADH was optimized under different conditions involving: temperature, pH, xylene concentration and incubation period. Results indicated that the optimal conditions for ADH production were by incubating the bacterium in MSM medium containing 1% xylene, pH: 8 for four days and incubation at 37ºC.

Modelling of the alcohol dehydrogenase production in baker's yeast

A mathematical model was formulated to simulate cell growth and enzyme production during the aerobic and micro-aerobic culture of the yeast Saccharomyces cerevisiae. Model development was based on three simplified metabolic events in the yeast: glucose fermentation, glucose oxidation and ethanol oxidation. Cell growth was expressed as a composite of these metabolic events. Their contributions to the total specific growth rate depended on the activities of the pacemaker enzyme pools of the individual pathways. The effect of substrate concentrations on the specific growth rate was described by a Michaelis–Menten equation. It was assumed that enzyme formation is cell growth associated. The model successfully predicted the dynamics of cell growth, glucose consumption, ethanol metabolism and alcohol dehydrogenase (ADH) production. A good agreement between model simulations and experimental data was achieved. It was observed that ADH production depends on the available oxygen concentration in the medium. In general, the proposed model appears to be useful for the design, scale-up, control and optimization of alcohol dehydrogenase production.

Generation mechanism and purification of an inactive form convertible in vivo to the active form of quinoprotein alcohol dehydrogenase in Gluconobacter suboxydans.

Alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound quinohemoprotein-cytochrome c complex involved in vinegar production. In Gluconobacter suboxydans grown under acidic growth conditions, it was found that ADH content in the membranes was largely increased but the activity was not much changed, suggesting that such a condition produces an inactive form of ADH (inactive ADH). A similar phenomenon could be also observed in Acetobacter aceti, another genus of acetic acid bacteria. Furthermore, aeration conditions were also shown to affect ADH production; the ADH level was increased and was present as an active form under low-aeration conditions, while the ADH level was decreased and was present mainly as an inactive form under high-aeration conditions. Inactive ADH was solubilized from the membranes of G. suboxydans grown in acidic and high-aeration conditions and was purified separately from the normal, active form of ADH (active ADH). In spite of having 10 times less enzyme activity than active ADH, inactive ADH could not be distinguished from active ADH with respect to their subunit compositions, molecular sizes, and prosthetic groups. Inactive ADH, however, had a relatively loose conformation with a partially oxidized state, while active ADH had a tight conformation with a completely reduced state, suggesting that inactive ADH may lack a right subunit's interaction and that one of the heme c components may be inactivated. Reactivation from such an inactive ADH occurred either by shifting of the pH of the culture medium up during the cultivation or by incubation of the resting cells at the neutral pH region in the presence of an energy source such as D-sorbitol. Such an activation of ADH was repressed by the addition of a proton uncoupler and could not occur in the spheroplasts. Thus, the results suggest that inactive ADH could be generated abundantly under acidic growth conditions and converted to the active form at a neutral culture pH. The data also suggest that some periplasmic component may be involved in the conversion of inactive ADH into the active form by consuming some forms of energy.

ACTIVITY OF ADH AND ETHANOL PRODUCTION IN CARNATION FLOWERS TRANSFERRED TO N2 FROM AIR AND LOW O2

Freshly harvested carnation flowers (Dianthus caryophyllus L. cv. White's Sim) were kept for 4 days either in air or low O2 before they were transferred to N2. Low O2 in the range of 1.2–2.7% resulted, depending on the concentration, in a decrease in respiration and 3–5 fold increase in alcohol dehydrogenase (ADH) activity, without a concomitant increase in ethanol production. Anoxia initially, within 4 hours, depressed by about 40% the rate of CO2 evolution in air, but had no effect on low O2-treated flowers. Anoxia induced in all treatments an increase in the activity of ADH, but the levels of ADH were 1.5 fold higher in the low O2-treated flowers than those kept in air. This difference increased to 10-fold after 6 days because by then, the air treated flowers were almost dead. Prior exposure to hypoxia enhanced the anoxic life of flowers by 3–4 days. Anoxia also induced an increase in ethanol production in both air and low O2-treated flowers. The peak value of ethanol evolution was about 1.5-fold higher in the low O2 than in air-treated flowers. The data are discussed in terms of the effect of hypoxia on carnation flower metabolism and longevity.

Retinoic acid activation and thyroid hormone repression of the human alcohol dehydrogenase gene ADH3.

Mammalian alcohol dehydrogenase (ADH) catalyzes the oxidation of retinol to retinaldehyde, the rate-limiting step in the synthesis of retinoic acid. There exists a family of ADH isozymes encoded by unique genes, and it is unclear which isozymes are most important for regulation of retinoic acid synthesis during differentiation or development. A region in the human ADH3 promoter from -328 to -272 base pairs was shown previously to function as a retinoic acid response element (RARE), prompting an hypothesis for a positive feedback mechanism controlling retinoic acid synthesis (Duester, G., Shean, M. L., McBride, M. S., and Stewart, M. J. (1991) Mol. Cell. Biol. 11, 1638-1646). The ADH3 RARE contains three direct AGGTCA repeats which constitute the critical nucleotides of RAREs present in other genes. We dissected the ADH3 RARE and determined that receptor binding as well as transactivation are dependent upon only the two downstream AGGTCA motifs separated by 5 base pairs, a structure noticed previously for a RARE in the promoter for the retinoic acid receptor beta (RAR beta) gene. ADH3 and RAR beta RAREs functioned similarly in transfection assays, suggesting that the feedback mechanisms controlling ADH3 and RAR beta utilize a common RARE. We also found that the normal functioning of the ADH3 RARE was abrogated by thyroid hormone receptor in the presence of thyroid hormone. A negative thyroid hormone response element in the human ADH3 promoter was found to colocalize with the RARE. Since ADH production in rat liver is known to be repressed by thyroid hormone, these findings suggest that human ADH production may also be subject to thyroid hormone repression and that the mechanism involves an interference with retinoic acid induction.

Colloidal bismuth subcitrate and omeprazole inhibit alcohol dehydrogenase mediated acetaldehyde production by Helicobacter pylori

We have recently shown that Helicobacter pylori possesses marked alcohol dehydrogenase (ADH) activity and is capable - when incubated with an ethanol containing solution in vitro - of producing large amounts of acetaldehyde. In the present study we report that some drugs commonly used for the eradication of H. pylori and for the treatment of gastroduodenal diseases are potent ADH inhibitors and, consequently, effectively prevent bacterial oxidation of ethanol to acetaldehyde. Colloidal bismuth subcitrate (CBS), already at a concentration of 0.01 mM, inhibited H. pylori ADH by 93% at 0.5 M ethanol and decreased oxidation of 22 mM ethanol to acetaldehyde to 82% of control. At concentrations above 5 mM, CBS almost totally inhibited acetaldehyde formation. Omeprazole, a drug also known to suppress growth of H. pylori, also inhibited H. pylori ADH and suppressed bacterial acetaldehyde formation significantly to 69% of control at a drug concentration of 0.1 mM. By contrast, the H 2-receptor antagonists ranitidine and famotidine showed only modest effect on bacterial ADH and acetaldehyde production. We suggest that inhibition of bacterial ADH and a consequent suppression of acetaldehyde production from endogenous or exogenous ethanol may be a novel mechanism by which CBS and omeprazole exert their effect both on the growth of H. pylori as well as on H. pylori associated gastric injury.

Escherichia coli mutants with altered control of alcohol dehydrogenase and nitrate reductase

Mutants of Escherichia coli which overproduce alcohol dehydrogenase were obtained by selection for the ability to use ethanol as an acetate source in a strain auxotrophic for acetate. A mutant having a 20-fold overproduction of alcohol dehydrogenase was able to use ethanol only to fulfill its acetate requirement, whereas two mutants with a 60-fold overproduction were able to use ethanol as a sole carbon source. The latter two mutants produced only 25% of the wild-type level of nitrate reductase, when grown under anaerobic conditions. Alcohol dehydrogenase production was largely unaffected by catabolite repression but was repressed by nitrate under both aerobic and anaerobic conditions. The genetic locus responsible for alcohol dehydrogenase overproduction was located at min 27 on the E. coli genetic map; the gene order, as determined by transduction, was trp tonB adh chlC hemA. The possible relationship of alcohol dehydrogenase to anaerobic redox systems such as formate-nitrate reductase is discussed.

Dissociation-recombination of intergenic sunflower alcohol dehydrogenase isozymes and relative isozyme activities.

Two unlinked genes, Adh1 and Adh2, control of the production of alcohol dehydrogenase (ADH) in seeds of the animal sunflower (Helianthus annuus). Each gene is polymorphic, having F and S alleles. Starch gel electrophoretic zymograms of the four possible double homozygotes have three bands, representing two homodimers and an intermediately migrating intergenic isozyme. Zymograms of double heterozygotes consist of nine bands produced by ten isozymes: six intragenics and four intergenics, two of which are coincident. Results of dissociation-recombination (D-R) experiments are reported which demonstrate the subunit composition of the intergenic isozymes, thus supporting the relationships suggested by genetic studies. Densitometric tracings of the zymogram of a cleared gel and measurements of activities of homodimer isozymes eluted from gels follwoing D-R of an intergenic isozyme showed that the Adh2 isozymes were more that twice as active as those of Adh1. Measurements of activities of crude extracts from the four possible double homozygous genotypes indicated that the seeds of the genotype Adh1F/Adh2F, Adh2S/Adh2S produced more activity than the other three. This genotype is the most common one found in wild and cultivated stocks. Isozymes eluted following electrophoresis of the same extracts had averages of 19%, 70%, and 11% of total activity contributed by the Adh1, Adh2, and intergeneic isozymes, respectively. A simple but efficient method of isozyme elution from starch gels is described which resulted in nearly full expected recovery (approximately 46%) of the ADH activity in the applied sample.

EVIDENCE FOR TRIPLICATE GENES FOR ALCOHOL DEHYDROGENASE IN HEXAPLOID WHEAT

The alcohol dehydrogenase zymogram phenotypes of 40 wheat strains, each possessing a distinctive chromosomal constitution, were determined. Genes involved in the production of alcohol dehydrogenase were located on the beta arm of chromosome 4A and on chromosomes 4B and 4D. The results support the hypothesis that the active alcohol dehydrogenase isozymes are dimers composed of the six possible combinations of subunits coded by triplicate structural genes.