Efficiency of superoxide anions in the inactivation of selected dehydrogenases

The most ubiquitous of the primary reactive oxygen species, formed in all aerobes, is the superoxide free radical. It is believed that the superoxide anion radical shows low reactivity and in oxidative stress it is regarded mainly as an initiator of more reactive species such as OH and ONOO^‐.In this paper, the effectiveness of inactivation of selected enzymes by radiation-generated superoxide radicals in comparison with the effectiveness of the other products of water radiolysis is examined. We investigate three enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH).We show that the direct contribution of the superoxide anion radical to GAPDH and ADH inactivation is significant. The effectiveness of the superoxide anion in the inactivation of GAPDH and ADG was only 2.4 and 2.8 times smaller, respectively, in comparison with hydroxyl radical. LDH was practically not inactivated by the superoxide anion.Despite the fact that the studied dehydrogenases belong to the same class of enzymes (oxidoreductases), all have a similar molecular weight and are tetramers, their susceptibility to free-radical damage varies. The differences in the radiosensitivity of the enzymes are not determined by the basic structural parameters analyzed. A significant role in inactivation susceptibility is played by the type of amino acid residues and their localization within enzyme molecules.     

Enzymatic method for the determination of inorganic and organic inhibitors of alcohol dehydrogenase

The inhibiting effect of heavy metal ions, organic nitrogen-containing heterocyclic compounds, and amino acids on the catalytic activity of yeast alcohol dehydrogenase (ADH) in the reaction of ethanol oxidation by nicotinamide adenine dinucleotide was detected. Sensitive procedures were developed for the determination of the most effective ADH inhibitors [mercury (II), silver(I), zinc(II), copper(II), 1,10-phenanthroline, 2,2′-dipyridyl, 8-hydroxyquinoline, quinoline, 3,4-dimethylimidazole, benzimidazole, 1,2,3-benzotriazole, histidine, tryptophan, proline, and histamine] with c_L = 5x 10^-10-1 x 10^-4M (RSD = 1–12%).     

A Study on the Stability and Enzymatic Activity of Yeast Alcohol Dehydrogenase in Presence of the Self-Assembling Block Copolymer Poloxamer 407

Yeast alcohol dehydrogenase (ADH) is an enzyme widely studied for biotechnological applications due to its involvement in fermentation industry, and various attempts to improve its catalytic properties and its thermal stability have been carried out. In this paper, the influence of a block copolymer (Poloxamer 407) on ADH enzymatic activity and thermal behaviour has been studied in order to get new insights about the use of poloxamers in formulation of sustained release systems for therapeutic proteins. Poloxamer 407 has the ability to form micelles and gel due to its self-assembling and thermoresponsive properties. The effect of the copolymer towards thermal stress and pH changes, which often reduce enzymes activity it has been investigated by means of enzymatic assays and differential scanning calorimetry. Results showed that at pH?9.1 and 7.3, the Poloxamer in the form of unimeric, micellar and gel state is able to effectively preserve the enzyme from thermoinactivation. In addition by calorimetric data Poloxamer 407 has showed an effect in preserving ADH from aggregation at pH?7.3. In conclusion, Poloxamer 407 seems to be very effective in protecting ADH from stress related events, like alkaline inactivation and aggregation.     

Computational studies of human class V alcohol dehydrogenase - the odd sibling

Background

All known attempts to isolate and characterize mammalian class V alcohol dehydrogenase (class V ADH), a member of the large ADH protein family, at the protein level have failed. This indicates that the class V ADH protein is not stable in a non-cellular environment, which is in contrast to all other human ADH enzymes. In this report we present evidence, supported with results from computational analyses performed in combination with earlier in vitro studies, why this ADH behaves in an atypical way.

Results

Using a combination of structural calculations and sequence analyses, we were able to identify local structural differences between human class V ADH and other human ADHs, including an elongated β-strands and a labile α-helix at the subunit interface region of each chain that probably disturb it. Several amino acid residues are strictly conserved in class I–IV, but altered in class V ADH. This includes a for class V ADH unique and conserved Lys51, a position directly involved in the catalytic mechanism in other ADHs, and nine other class V ADH-specific residues.

Conclusions

In this study we show that there are pronounced structural changes in class V ADH as compared to other ADH enzymes. Furthermore, there is an evolutionary pressure among the mammalian class V ADHs, which for most proteins indicate that they fulfill a physiological function. We assume that class V ADH is expressed, but unable to form active dimers in a non-cellular environment, and is an atypical mammalian ADH. This is compatible with previous experimental characterization and present structural modelling. It can be considered the odd sibling of the ADH protein family and so far seems to be a pseudoenzyme with another hitherto unknown physiological function.

     

Preparation of poly(ε-caprolactone)@attapulgite nanocomposites via a combination of controlled ring-opening polymerization and click chemistry

In this work, by a combination of controlled ring-opening polymerization (CROP) and click chemistry, we report a facile and useful method to synthesize linear poly(ε-caprolactone)@attapulgite nanocomposites with well-defined structures. For this, first, the chlorine-terminated attapulgite was prepared by the self-assembly of 3-chloropropyltrimethoxysilane from the surfaces of attapulgite. And then, the terminal chlorines of modified attapulgite were substituted with azido groups. As the second step, linear propargyl-terminated poly(ε-caprolactone) (PCLs) with different molecular weights were synthesized by the CROP of ε-CL monomer in toluene with stannous octoate as a catalyst and propargyl alcohol as an initiator. The structural characteristics of the obtained linear PCLs have been determined by ¹H NMR and gel permeation chromatography analysis. Finally, the azido-terminated attapulgite was reacted with propargyl-terminated PCLs via the click reaction.     

Mutation of Tyr-218 to Phe in <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> Secondary Alcohol Dehydrogenase: Effects on Bioelectronic Interface Performance

Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP⁺), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD⁺). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD⁺ by mutating Tyr²¹⁸ to Phe (Y218F 2° ADH). This mutation increased the K _m(app) for NADP⁺ 200-fold while decreasing the K _m(app) for NAD⁺ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD⁺, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD⁺. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD⁺ was electrically recycled 6.7 × 10⁴ times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.     

A Rigidoporus Vinctus Alcohol Dehydrogenase and Its Characterization

Alcohol dehydrogenases (ADHs; E.C. 1.1.1.1) are widely distributed enzymes found in many microorganisms. ADHs are oxidoreductases that catalyze the NAD(P)⁺‐dependent conversion of alcohols to aldehydes or ketones as well as the reverse reaction. The ADH cloned from Rigidoporus vinctus (RvADH) was 1035 bp that encodes a protein of 344 amino acid residues with calculated molecular mass of 38.39 kDa. This ADH is belonging to the medium‐chain family (medium‐chain dehydrogenase/reductase (MDR) and has the highly conserved GXXGXXG sequence found in the MDR family which found as the coenzyme‐binding pocket. To characterize the ADH protein, the coding region was subcloned into an expression vector pET‐20b(+) and transformed into E. coli Rosetta (DE3). The recombinant His6‐tagged ADH was overexpressed and purified by Ni²⁺‐nitrilotriacetic acid Sepharose. The purified enzyme showed one band on 12 % sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The Michaelis constant (K_M) value of the recombinant enzyme for ethanol was 0.79 mM. In substrates specificity analysis showed that RvADH had great oxidative activity toward primary alcohols. However, the less activtiy toward secondary alcohols and alcohol derivatives were compared with ethanol. Regarding the reductase activity showed low or even no activity to aldehydes and ketone.     

Generation of Oxidoreductases with Dual Alcohol Dehydrogenase and Amine Dehydrogenase Activity

The l -lysine-ϵ-dehydrogenase (LysEDH) from Geobacillus stearothermophilus naturally catalyzes the oxidative deamination of the ϵ-amino group of l -lysine. We previously engineered this enzyme to create amine dehydrogenase (AmDH) variants that possess a new hydrophobic cavity in their active site such that aromatic ketones can bind and be converted into α-chiral amines with excellent enantioselectivity. We also recently observed that LysEDH was capable of reducing aromatic aldehydes into primary alcohols. Herein, we harnessed the promiscuous alcohol dehydrogenase (ADH) activity of LysEDH to create new variants that exhibited enhanced catalytic activity for the reduction of substituted benzaldehydes and arylaliphatic aldehydes to primary alcohols. Notably, these novel engineered dehydrogenases also catalyzed the reductive amination of a variety of aldehydes and ketones with excellent enantioselectivity, thus exhibiting a dual AmDH/ADH activity. We envisioned that the catalytic bi-functionality of these enzymes could be applied for the direct conversion of alcohols into amines. As a proof-of-principle, we performed an unprecedented one-pot “hydrogen-borrowing” cascade to convert benzyl alcohol to benzylamine using a single enzyme. Conducting the same biocatalytic cascade in the presence of cofactor recycling enzymes (i.e., NADH-oxidase and formate dehydrogenase) increased the reaction yields. In summary, this work provides the first examples of enzymes showing “alcohol aminase” activity.     

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.     

Detection of NADH and ethanol at a graphite electrode modified with titania sol-gel/Meldola’s Blue/MWCNT/Nafion nanocomposite film

For electrocatalytic determination of NADH, a graphite electrode modified with titania sol-gel/Meldola’s Blue/MWCNT/Nafion nanocomposite was proposed. The composition of the matrix film was optimised in terms of the content of carbon nanotubes and Nafion. Incorporation of a redox mediator, Meldola’s Blue, into the nanocomposite film enabled electrocatalytic determination of NADH at a low potential, −50 mV. For determination of ethanol, alcohol dehydrogenase (ADH) was immobilized into the matrix layer. Experimental conditions affecting the biosensor response were examined, including enzyme loading, temperature of measurement and pH of background electrolyte. Assessments of the analytical characteristics of the biosensor were performed with respect to sensitivity, limit of detection, operational stability, repeatability and reproducibility. The proposed biosensor showed electrocatalytic activity toward oxidation of ethanol with sensitivity of 2.24 μA L mmol⁻¹, linear range from 0.05 to 1.1 mmol L⁻¹, and limit of detection of 25 μmol L⁻¹. The apparent Michaelis-Menten constant was 1.24 mmol L⁻¹, indicating a high biological affinity of ADH/titania sol-gel/Meldola’s Blue/MWCNT/Nafion electrode for ethanol. The developed biosensor was tested in determinations of ethanol content in alcoholic beverages.     

Amperometric Ethanol Biosensors Based on Chitosan‐NAD+‐Alcohol Dehydrogenase Films

The reagentless and oxygen‐independent biosensors for ethanol were developed based on the covalent immobilization of alcohol dehydrogenase (ADH) and its cofactor nicotinamide adenine dinucleotide (NAD⁺) on chitosan (CHIT) chains. The CHIT‐NAD⁺‐ADH structures were adsorbed onto carbon nanotubes (CNT) in order to provide a signal transduction based on the recycling of redox states of NAD cofactor at CNT (detection limit, 8–30 µM ethanol; dynamic range up to 20 mM). The CHIT‐NAD⁺‐dehydrogenase/CNT hybrid material represents a general approach to the development of dehydrogenases‐based electrochemical biosensors. Interestingly, the CHIT‐NAD⁺ solutions preserved their enzymatic activity even after five years of storage at 4 °C.     

Electrical communication between electrode and dehydrogenase by a ferrocene-labeled high molecular-weight cofactor derivative: application to a reagentless biosensor

A ferrocene-labeled high molecular weight cofactor derivative (PEI-Fc-NAD) was prepared by attaching both ferrocene and nicotinamide adenine dinucleotide (NAD⁺) to a water soluble polyelectrolyte, polyethylenimine (PEI). Approximately 9.8% and 2.9% of all the primary amino groups of PEI were coupled with ferrocene and the bioactive cofactor, respectively. The cyclic voltammograms of PEI-Fc-NAD exhibited a one-electron transfer process, and the difference between the anodic and cathodic peak potential was found to be 80 mV. The PEI-Fc-NAD was used together with NAD-dependent dehydrogenase to construct a reagentless biosensor. An NAD-dependent alcohol dehydrogenase (ADH) was selected as the model enzyme, and both ADH and PEI-Fc-NAD were retained onto the sensing area of gold electrodes by a dialysis membrane or immobilized by layer-by-layer adsorption method. In both cases, the modified electrodes showed current response to ethanol without the addition of native NAD⁺ to the system, which suggested that the electrical communication between ADH and the electrode was achieved through PEI-Fc-NAD. In summary, PEI-Fc-NAD provides a new way for immobilization of mediator and cofactor, and exhibits the potential as a platform for constructing reagentless NAD-dependent dehydrogenase biosensors.     

Effect of Different Variables on the Efficiency of the Baker's Yeast Cell Disruption Process to Obtain Alcohol Dehydrogenase Activity

Cell disruption process of dry baker's yeast was studied in this work to obtain maximum activity of alcohol dehydrogenase (ADH). Disruption by ultrasonication, glass beads, and combination of these two methods was compared. A 1.8-fold increase of ADH activity can be achieved by combining glass beads with ultrasonication in comparison to ultrasonication. To achieve maximum volume activity of ADH, the effect of different variables on the cell disruption process was investigated (time, glass bead diameter, mass of glass beads, and ultrasound amplitude). Using the Design-Expert© software, 2⁴ factorial experimental design was performed. Two ultrasound probes were tested: MS 73 and KE 76. Optimal conditions (process variables) for cell disruption process were obtained. Optimal ADH activities after cell disruption with MS 73 and KE 76 probes were 1,890.9 and 1,531.7 U cm^?3, respectively. Necessary ultrasonication time and ultrasound amplitude should be at the maximum values in the investigated variable range (30 min and 62 %). Bead size should be at maximum (4 mm) when using MS 73 probe and at minimum (0.3 mm) when using KE 76 probe. Partial purification of the enzyme was carried out and it was kinetically characterized using several oxidation and reduction systems.     

What to sacrifice? Fusions of cofactor regenerating enzymes with Baeyer-Villiger monooxygenases and alcohol dehydrogenases for self-sufficient redox biocatalysis

A collection of fusion biocatalysts has been generated that can be used for self-sufficient oxygenations or ketone reductions. These biocatalysts were created by fusing a Baeyer-Villiger monooxygenase (cyclohexanone monooxygenase from Thermocrispum municipale: TmCHMO) or an alcohol dehydrogenase (alcohol dehydrogenase from Lactobacillus brevis: LbADH) with three different cofactor regeneration enzymes (formate dehydrogenase from Burkholderia stabilis: BsFDH; glucose dehydrogenase from Sulfolobus tokodaii: StGDH, and phosphite dehydrogenase from Pseudomonas stutzeri: PsPTDH). Their tolerance against various organic solvents, including a deep eutectic solvent, and their activity and selectivity with a variety of substrates have been studied. Excellent conversions and enantioselectivities were obtained, demonstrating that these engineered fusion enzymes can be used as biocatalysts for the synthesis of (chiral) valuable compounds.     

4-Ferrocenylphenol as an electron transfer mediator in PQQ-dependent alcohol and glucose dehydrogenase-catalyzed reactions

Enzyme electrodes containing pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) as a biological component in combination with 4-ferrocenylphenol (1) as an electron transfer mediator between PQQ and a carbon electrode were constructed and used for measurements of ethanol and d-glucose. Analysis of the current response of the carbon electrodes modified with 1 at different pH and potentials demonstrated that 1 participates in the bioelectrocatalytic oxidation of d-glucose or ethanol. The biosensors showed the highest response at pH 5.5 and the working potentials of 0.3 and 0.4 V (versus Ag|AgCl) for ADH and GDH, respectively. The electrocatalytic processes under such conditions at these electrodes are characterized by the apparent values of the Michaelis constants K_M^app of 7.1 and 13 mM and the maximal current density j_max 40 and 26 μA cm⁻² for ethanol and d-glucose, respectively. No electrocatalysis was found when glucose oxidase from Aspergillus niger was used instead of GDH.     

On.column pre‐concentration of alcohol dehydrogenase in capillary electrophoresis

The analysis of alcohol dehydrogenase (ADH) at low concentration using capillary electrophoresis is described. Several simple and effective ways to improve detection limits and sensitivity are investigated. These include large volume sample stacking, head column field amplified sample stacking, and sweeping. Results indicate that by using a combination of head‐column field amplified sample stacking and sweeping, fluorescently labelled alcohol dehydrogenase can be pre‐concentrated online by dissolving samples in water or other low conductivity matrices, and injecting into a high conductivity micellar buffer. The abrupt changes in conductivity cause narrowing of the analyte length and thus enhance the detection sensitivity. Combination of this approach with laser induced fluorescence detection yields a limit of detection of 5×10^–13 M. Both qualitative and quantitative aspects of this method are investigated.     

Meldola’s Blue — doped titania sol-gel sensor for NADH determination

Titania layers obtained by a sol-gel technique doped with redox mediator, Meldola’s Blue, were employed for construction of a new NADH senor. Optimization of preparation process as well as experimental conditions affecting the response of the sensor were examined. Under optimal conditions NADH could be determined in the wide linear range from 90 to 2300 μM with detection limit 12 μM and a high sensitivity 12.5 nA μM⁻¹. The usefulness of developed sensor was preliminarily checked in determination of NADH forming during enzymatic oxidation of ethanol catalyzed by alcohol dehydrogenase (ADH).      

Coenzyme Regeneration in Hexanol Oxidation Catalyzed by Alcohol Dehydrogenase

The enzymatic ways of coenzyme regeneration include the addition of a second enzyme to the system or the addition of the co-substrate. In the present study, both methods of enzymatic coenzyme (NAD⁺) regeneration were studied and compared in the reaction of hexanol oxidation catalyzed by alcohol dehydrogenase (ADH). As a source of ADH, commercial isolated enzyme and the whole baker??s yeast cells were used. First, coenzyme regeneration was employed in the reaction of acetaldehyde reduction catalyzed by the same enzyme that catalyzed the main reaction, and then NAD⁺ regeneration was applied in the reaction of pyruvate reduction catalyzed by l-lactate dehydrogenase (l-LDH). Hexanal was obtained as the product of hexanol oxidation catalyzed by isolated ADH while hexaonic acid was detected as a product of the same reaction catalyzed by baker??s yeast cells. All of the used biocatalysts were kinetically characterized. The mass reactions were described by the mathematical models. All models were validated in the batch reactor. One hundred percent hexanol conversion was obtained using permeabilized yeast cells using both methods of cofactor regeneration. By using isolated enzyme ADH, the higher conversion was achieved in a system with cofactor regeneration catalyzed by l-LDH.     

A Simple Biosystem for the High-Yielding Cascade Conversion of Racemic Alcohols to Enantiopure Amines

The amination of racemic alcohols to produce enantiopure amines is an important green chemistry reaction for pharmaceutical manufacturing, requiring simple and efficient solutions. Herein, we report the development of a cascade biotransformation to aminate racemic alcohols. This cascade utilizes an ambidextrous alcohol dehydrogenase (ADH) to oxidize a racemic alcohol, an enantioselective transaminase (TA) to convert the ketone intermediate to chiral amine, and isopropylamine to recycle PMP and NAD⁺ cofactors via the reversed cascade reactions. The concept was proven by using an ambidextrous CpSADH-W286A engineered from (S)-enantioselective CpSADH as the first example of evolving ambidextrous ADHs, an enantioselective BmTA, and isopropylamine. A biosystem containing isopropylamine and E. coli (CpSADH-W286A/BmTA) expressing the two enzymes was developed for the amination of racemic alcohols to produce eight useful and high-value (S)-amines in 72–99 % yield and 98–99 % ee, providing with a simple and practical solution to this type of reaction.     

Manipulating Pyruvate Decarboxylase by Addition of Enzyme Regulators during Fermentation of Rhizopus oryzae to Enhance Lactic Acid Production

Ethanol was found as the major by-product in lactate fermentation by Rhizopus oryzae. Several methods have been conducted in order to limit ethanol formation, thus increasing the lactate yield. The direct way to suppress ethanol production can be done by inhibition of the responsible enzymes in the related pathway. Pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are responsible for ethanol production in R. oryzae. Shunting the ethanol production pathway by targeting at PDC was attempted in this study. Three compounds including 4-methylpyrazole, glyoxylic acid, and 3-hydroxypyruvate with the in vitro reversible inhibitory effect on PDC were selected from the literature and were used to regulate the living cell of R. oryzae during the fermentation. The results show that 0.1 mM 4-methylpyrazole of which the structure resembled a thiazolium ring in thiamine diphosphate, PDC cofactor, and 1.0 μm 3-hydroxypyruvate, pyruvate analog, effectively hampered ethanol production. Further observation on the enzyme expression indicated that these two regulators not only targeted PDC but also caused changes in ADH and lactate dehydrogenase (LDH) activities. This was perhaps due to the living cell of R. oryzae that responded to the presence of the regulators to balance the pyruvate flux and subsequently maintain its metabolic activities.