Dependent Secondary Alcohol Dehydrogenase Research Articles

Fluorometric Bio-Sniffer (Gas Phase Biosensor) for Breath Acetone as a Volatile Product of Lipid Metabolism

Breath analysis for healthcare and disease monitoring has become popular recently in global. There are hundreds of volatile organic compounds (VOCs) exist in human breath, and some of them can offer medical and biological information as the bloodstream. Particularly,acetone is one of the typical VOCs in exhaled breath, which is related to lipid metabolism in fasting, exercise and diabetes mellitus. The breath acetone concentrations have been previously shown to correlate strongly with blood acetone concentrations as well as with other ketones such as beta-hydroxybutyrate. Besides, there are many studies found the correlation between the breath acetone concentrations and blood glucose concentrations. Previous research indicated that the breath acetone concentration in healthy people shows the range from 200 to 900 ppb and higher concentration was found in breath of diabetic patients. Increase of the acetone concentration is usually a sign that cells cannot effectively use glucose as energy source, which often occurs in starvation, aerobic exercise, lack of insulin or resistance of insulin. Thus, acetone in exhaled breath has a potential to be a biomarker for non-invasive monitoring the progressive of diabetes. Typical quantitation of gaseous acetone usually uses gas chromatography with mass spectrometry (GC/MS) or proton transfer reaction mass spectrometry (PTR-MS). These methods are highly sensitivity but still have disadvantages in cost, measurement time and hard for continuity. Therefore, a rapid, convenient and highly selective method for acetone determination is strongly required. In this study, a highly sensitive acetone bio-sniffer (gas-phase biosensor) was developed and used to assess the acetone from exhale breath for evaluating the lipid metabolism. Nicotinamide adenine dinucleotide (NADH)-dependent secondary alcohol dehydrogenase (S-ADH) is a kind of enzyme that can reduce acetone to be isopropanol and meanwhile oxidase NADH to be NAD+ in acidic condition. It is known that NADH can be excited by UV (ultraviolet) light at central wavelength of 340 nm and release a fluorescent emission at peak of 490 nm simultaneously, while NAD+ do not have similar optical property. Thus, acetone concentration can be measured using the decreasing of NADH fluorescence intensity that is consumed by the enzymatic reaction of S-ADH. The fluorometric acetone bio-sniffer was composed of a NADH fluorescence measurement system, a flow-cell attaching on the fiber probe and an enzyme-immobilized membrane. The NADH measurement system consisted of a UV-LED as the excitation light source, band-pass filters ( λ = 340 ± 10 nm and λ = 490 ± 10 nm) for reducing noise, a photomultiplier tube (PMT) as fluorescence detector and bifurcated optical fibers for assemble. S-ADH was immobilized on the hydrophilic-PTFE membrane and equipped on the top of flow-cell by silicone O-ring. Phosphate buffer, which contained NADH, was circulated in the flow-cell to supply fresh co-enzyme and eliminate isopropanol and NAD+that produced by the enzyme membrane. When bio-sniffer contacted the gaseous acetone, enzymatic reaction that mentioned above occurred immediately and the change of fluorescence would detect by the PMT. This acetone bio-sniffer showed rapid reaction, highly sensitivity and selectivity. When the acetone vapors were supplied to the sensing region, fluorescent intensity decreased immediately and reached to a steady state. Then, the intensity would return to the initial value in short time when the acetone vapor supply was stopped. The relation between the concentration of acetone and the change of fluorescent intensity was investigated and the dynamic range was confirmed from 20 to 5300 ppb of acetone in the gas phase. This range encompasses the concentration of acetone vapors found in breath of healthy people and of those suffering from disorders of carbohydrate metabolism. The selectivity of the S-ADH was also characterized; several different VOCs were employed to compare the responses. The interference of 2-butanol and 2-pentanone were ignorable because they contained in healthy people in very low concentration. Finally, we applied the bio-sniffer to measure breath acetone for evaluation of lipid metabolism during long time fasting and an aerobic exercise test. The results showed that the mean breath acetone concentration of long time fasting healthy volunteers were higher than those who did not have fasting and display significant difference. Furthermore, the concentration of acetone in breath was observed significantly increase during aerobic exercise and it would recover to the initial value after long time rest. This highly sensitivity acetone bio-sniffer provides a new kind of analytical tool for non-invasive evaluation of human lipid metabolism and it is also expected to use for the clinical applications such as monitoring the progression of diabetes mellitus and diagnosis of other metabolism diseases.

An acetone bio-sniffer (gas phase biosensor) enabling assessment of lipid metabolism from exhaled breath.

Several volatile organic compounds (VOCs) are released from human breath or skin. Like chemical substances in blood or urine, some of these vapors can provide valuable information regarding the state of the human body. A highly sensitive acetone biochemical gas sensor (bio-sniffer) was developed and used to measure exhaled breath acetone concentration, and assess lipid metabolism based on breath acetone analysis. A fiber-optic biochemical gas sensing system was constructed by attaching a flow-cell with nicotinamide adenine dinucleotide (NADH)-dependent secondary alcohol dehydrogenase (S-ADH) immobilized membrane onto a fiber-optic NADH measurement system. The NADH measurement system utilizes an ultraviolet-light emitting diode with peak emission of 335 nm as an excitation light source. NADH is consumed by the enzymatic reaction of S-ADH, and the consumption is proportional to the concentration of acetone vapor. Phosphate buffer which contained NADH was circulated into the flow-cell to rinse products and the excessive substrates from the optode. The change of fluorescent emitted from NADH is analyzed by the PMT. Hence, fluorescence intensity decreased as the acetone concentration increased. The relationship between fluorescence intensity and acetone concentration was identified from 20 ppb to 5300 ppb. This interval included the concentration of acetone vapor in the breath of healthy people and those suffering from disorders of carbohydrate metabolism. Finally, the acetone bio-sniffer was used to measure breath acetone during an exercise stress test on an ergometer after a period of fasting. The concentration of acetone in breath was shown to significantly increase after exercise. This biosensor allows rapid, highly sensitive and selective measurement of lipid metabolism.

2-Butanol and butanone production in Saccharomyces cerevisiae through combination of a B12 dependent dehydratase and a secondary alcohol dehydrogenase using a TEV-based expression system.

2-Butanol and its chemical precursor butanone (methyl ethyl ketone – MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuterii), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions.

A bifunctional enzyme from Rhodococcus erythropolis exhibiting secondary alcohol dehydrogenase-catalase activities.

Alcohol dehydrogenases have long been recognized as potential biocatalyst for production of chiral fine and bulk chemicals. They are relevant for industry in enantiospecific production of chiral compounds. In this study, we identified and purified a nicotinamide adenine dinucleotide (NAD)-dependent secondary alcohol dehydrogenase (SdcA) from Rhodococcus erythropolis oxidizing γ-lactols into γ-lactones. SdcA showed broad substrate specificity on γ-lactols; secondary aliphatic alcohols with 8 and 10 carbon atoms were also substrates and oxidized with (2S)-stereospecificity. The enzyme exhibited moderate stability with a half-life of 5 h at 40 °C and 20 days at 4 °C. Mass spectrometric identification revealed high sequence coverage of SdcA amino acid sequence to a highly conserved catalase from R. erythropolis. The corresponding encoding gene was isolated from genomic DNA and subsequently overexpressed in Escherichia coli BL21 DE3 cells. In addition, the recombinant SdcA was purified and characterized in order to confirm that the secondary alcohol dehydrogenase and catalase activity correspond to the same enzyme.

Novel chiral tool, (R)-2-octanol dehydrogenase, from Pichia finlandica: purification, gene cloning, and application for optically active α-haloalcohols

A novel enantioselective alcohol dehydrogenase, (R)-2-octanol dehydrogenase (PfODH), was discovered among methylotrophic microorganisms. The enzyme was purified from Pichia finlandica and characterized. The molecular mass of the enzyme was estimated to be 83,000 and 30,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The enzyme was an NAD(+)-dependent secondary alcohol dehydrogenase and showed a strict enantioselectivity, very broad substrate specificity, and high tolerance to SH reagents. A gene-encoding PfODH was cloned and sequenced. The gene consisted of 765 nucleotides, coding polypeptides of 254 amino acids. The gene was singly expressed and coexpressed together with a formate dehydrogenase as an NADH regenerator in an Escherichia coli. Ethyl (S)-4-chloro-3-hydroxybutanoate and (S)-2-chloro-1-phenylethanol were synthesized using a whole-cell biocatalyst in more than 99% optical purity.

A novel secondary alcohol dehydrogenase from Micrococcus luteus WIUJH20: purification, cloning, and properties

A novel NAD+‐dependent secondary alcohol dehydrogenase (2o‐ADH) has been purified to homogeneity from Micrococcus luteus WIUJH20 (M. luteus WIUJH20). It has a molecular weight of ~ 140 kDa as determined by gel filtration chromatography, and a molecular weight of ~ 34 kDa as determined by SDS‐PAGE. Thus, the enzyme is a homotetramer. The gene encodes for this enzyme has been amplified by PCR from the genome of M. luteus WIUJH20, cloned into pGEX2T expression vector, and recombinant protein overexpressed as a GST fusion protein (rGST‐2o‐ADH). Based on sequence homology, the enzyme is a NAD+‐dependent L‐3‐hydroxyacyl‐CoA dehydrogenase. But based on its catalytic activity, the enzyme belongs to secondary alcohol dehydrogenase. The rGST‐2o‐ADH fusion protein, the recombinant 2o‐ADH (r2o‐ADH) with GST‐tag removed, and the 2o‐ADH isolated from M. Luteus WIUJH20 had broad substrate specificity. Enzyme kinetics analysis showed that the r2o‐ADH possesses a Km and Vmax of 3.25 x 10−6 M and 150.3 μmol/min/mg respectively, when 12‐hydroxyoctadecanoic acid was the substrate. This r2o‐ADH prefers to use linear secondary alcohols as substrates over the cyclic secondary alcohols. The enzyme prefers long‐chain alcohol over short‐chain alcohol as shown by the decreasing in Km from 4‐heptanol (7 mM) to 6‐dodecanol (8 μM). Primary alcohols (particularly ethanol) are inhibitors to the 2o‐ADH and the inhibition constant Ki increases as the hydrocarbon chain length decreases.The research was supported in part by grants from USDA from USDA CSREES 04‐35504‐14712 and 02‐015470, Western Illinois University Foundation, College of Arts and Sciences and the Honors College at Western Illinois University.

Propane monooxygenase and NAD+-dependent secondary alcohol dehydrogenase in propane metabolism by Gordonia sp. strain TY-5.

A new isolate, Gordonia sp. strain TY-5, is capable of growth on propane and n-alkanes with C(13) to C(22) carbon chains as the sole source of carbon. In whole-cell reactions, significant propane oxidation to 2-propanol was detected. A gene cluster designated prmABCD, which encodes the components of a putative dinuclear-iron-containing multicomponent monooxygenase, including the large and small subunits of the hydroxylase, an NADH-dependent acceptor oxidoreductase, and a coupling protein, was cloned and sequenced. A mutant with prmB disrupted (prmB::Kan(r)) lost the ability to grow on propane, and Northern blot analysis revealed that polycistronic transcription of the prm genes was induced during its growth on propane. These results indicate that the prmABCD gene products play an essential role in propane oxidation by the bacterium. Downstream of the prm genes, an open reading frame (adh1) encoding an NAD(+)-dependent secondary alcohol dehydrogenase was identified, and the protein was purified and characterized. The Northern blot analysis results and growth properties of a disrupted mutant (adh1::Kan(r)) indicate that Adh1 plays a major role in propane metabolism. Two additional NAD(+)-dependent secondary alcohol dehydrogenases (Adh2 and Adh3) were also found to be involved in 2-propanol oxidation. On the basis of these results, we conclude that Gordonia sp. strain TY-5 oxidizes propane by monooxygenase-mediated subterminal oxidation via 2-propanol.

Characterization of cold-active dehydrogenases for secondary alcohols and glycerol in psychrotolerant bacteria isolated from Antarctic soil

A NAD(P) +-dependent secondary alcohol dehydrogenase (2°ADH) and a NAD +-dependent glycerol dehydrogenase (GDH) have been identified in a psychrotolerant Micrococcus sp. and Arthrobacter sp., respectively, both isolated from Antarctic soil. In partially purified cell-free extracts (enriched 84- and 78-fold for 2°ADH and GDH, respectively), the enzymes displayed cold activity: the 2°ADH had optimum for activity at 33 °C and retained 32% of its maximum activity at 6 °C, the corresponding values for the GDH were 35 °C and 43%. The enzymes were inactivated within 10 min at 50 and 60 °C, for 2°ADH and GDH, respectively. The 2°ADH was active against a wide range of medium chain-length alcohols, preferring ( R)-stereoisomers; the enzyme was reversible with a higher affinity for the reduction of ketones. The GDH was active against glycerol and diols containing a secondary alcohol group. These properties make the enzymes candidates for low temperature biotransformations.

Thermophilic alcohol dehydrogenase from the mesophile Entamoeba histolytica: crystallization and preliminary X-ray characterization.

The tetrameric NADP(+)-dependent secondary alcohol dehydrogenase from Entamoeba histolytica has been crystallized in its apo form. The crystals belong to space group C222(1), with unit-cell parameters a = 76.89, b = 234.24, c = 96.24 A, and diffract to 1.9 A at liquid-nitrogen temperature. Analysis of the Patterson self-rotation function shows that the crystals contain one dimer per asymmetric unit.

Bioaffinity purification of NADP(+)-dependent dehydrogenases: studies with alcohol dehydrogenase from Thermoanaerobacter brockii.

This study describes the development and application of a bioaffinity chromatographic system for the one-step purification of an NADP(+)-dependent secondary alcohol dehydrogenase from the obligate anaerobe, Thermoanaerobacter brockii (TBADH, EC 1.1.1.2). The general approach is based upon improving the selectivity of immobilized cofactor derivatives (general ligand approach to bioaffinity chromatography) through using soluble enzyme-specific substrate analogues in irrigants to promote biospecific adsorption (the kinetic locking-on tactic). Specifically, the following is described: Evaluation of 8'-azo-linked, C(8)-linked, N(1)-linked, and N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of TBADH; evaluation of 2', 5'-ADP as a stripping ligand for TBADH bioaffinity purifications using an 8'-azo-linked immobilized NADP(+) derivative in the locking-on mode; and application of the developed bioaffinity chromatographic system to the purification of TBADH from a crude cellular extract. Surprizingly, of the four immobilized NADP(+) derivatives investigated, only the 8'-azo-linked immobilized NADP(+) derivative proved effective for TBADH affinity purification when used in conjunction with pyrazole (a competitive inhibitor of TBADH activity) as the locking-on ligand. This is in contrast to other NADP(+)-dependent dehydrogenases where the immobilized N(6)-linked cofactor proved to be suitable. While the one-step purification of TBADH to electrophoretic homogeneity is described in the present study (92% yield), results from the model chromatographic studies point to improvements that could be made to the immobilized cofactor derivative to improve its suitability for TBADH bioaffinity purification and to facilitate future large scale protein purification operations.

Secondary alcohol dehydrogenase from a vinyl alcohol oligomer-degrading Geotrichum fermentans; stabilization with Triton X-100 and activity toward polymers with polymerization degrees less than 20

An NAD+-dependent secondary alcohol dehydrogenase has been found in the cells of Geotrichum fermentans WF9101 grown on 2,4-pentanediol (the constitutional unit of vinyl alcohol polymers). Although the enzyme was unstable and lost its activity very rapidly, we found that it could be purified after stabilization in a buffer containing 0.01% Triton X-100. The molecular weight of the purified enzyme was 88,000 with subunit size of molecular weight 42,000. The enzyme, which was specific for the (S)-(+)-enantiomers, was active on various secondary mono-ols and diols, notably, 2-hexanol. The Km values at 30 °C, pH 8.0, were 240 μm for 2-hexanol and 48.7 μm for NAD+. This enzyme showed activity toward vinyl alcohol polymers with average degrees of polymerization of less than 20, and was assumed to contribute on the degradation of poly(vinyl alcohol) in synergism with other microorganisms.

Purification and characterization of (S)-1,3-butanediol dehydrogenase from Candida parapsilosis.

(S)-1,3-Butanediol (BDO) oxidizing enzyme was purified from Candida parapsilosis IFO 1396, which could produce (R)-1,3-BDO from the racemate. The purified enzyme was an NAD(+)-dependent secondary alcohol dehydrogenase that oxidized (S)-1,3-BDO to 4-hydroxy-2-butanone stereo-specifically.

Purification and characterisation of an NAD +-dependent secondary alcohol dehydrogenase from Pseudomonas maltophilia MB11L

A constitutive NAD +-linked alcohol dehydrogenase was purified 338-fold from cells of Pseudomonas maltophilia MB11L grown on glucose. Maximum activity was observed with cyclic and linear secondary alcohols, with little activity seen against primary or aromatic alcohols. Substrate oxidation activity was maximal at pH 10.0, while substrate reduction was optimal at pH 4.5. The K m values for propan-2-ol, NAD + and acetone were 87, 413 and 143 μM respectively. The enzyme is a tetramer with subunit M r of approximately 44 000. It has an isoelectric point of 4.75, and was inhibited by chelating agents, thiol reagents and certain metal ions.

Purification and characterisation of an NAD+-dependent secondary alcohol dehydrogenase fromPseudomonas maltophiliaMB11L

Purification and characterisation of an NAD⁺-dependent secondary alcohol dehydrogenase from<i>Pseudomonas maltophilia</i>MB11L

Purification and characterization of a NAD+-dependent secondary alcohol dehydrogenase from propane-grown Rhodococcus rhodochrous PNKb1

NAD+-linked primary and secondary alcohol dehydrogenase activity was detected in cell-free extracts of propane-grown Rhodococcus rhodochrous PNKb1. One enzyme was purified to homogeneity using a two-step procedure involving DEAE-cellulose and NAD-agarose chromatography and this exhibited both primary and secondary NAD+-linked alcohol dehydrogenase activity. The Mr of the enzyme was approximately 86,000 with subunits of Mr 42,000. The enzyme exhibited broad substrate specificity, oxidizing a range of short-chain primary and secondary alcohols (C2−C8) and representative cyclic and aromatic alcohols. The pH optimum was 10. At pH 6.5, in the presence of NADH, the enzyme catalysed the reduction of ketones to alcohols. The K m values for propan-1-ol, propan-2-ol and NAD were 12 mM, 18 mM and 0.057 mM respectively. The enzyme was inhibited by metal-complexing agents and iodoacetate. The properties of this enzyme were compared with similar enzymes in the current literature, and were found to be significantly different from those thus far described. It is likely that this enzyme plays a major role in the assimilation of propane by R. rhodochrous PNKb1.