Secondary Alcohol Dehydrogenase Research Articles

Description and genome analysis of a novel archaeon isolated from a syntrophic pyrite-forming enrichment culture and reclassification of Methanospirillum hungatei strains GP1 and SK as Methanospirillum purgamenti sp. nov.

The archaeal isolate J.3.6.1-F.2.7.3T was obtained from an anaerobic enrichment culture, where it may play an important role in methane production during pyrite formation. The new isolate formed a species-level clade with Methanospirillum hungatei strains GP1 and SK, which is separate from the type strain JF-1T. Cultivation-independent surveys indicate the occurrence of this phylogenetic group in sediments and anaerobic digesters. The abundance of this clade appears to be negatively affected by high nitrogen loads, indicating a sensitivity to certain nitrogen compounds that is not known in M. hungatei JF-1T. The relatively large core genome of this Methanospirillum clade is indicative of niche specialization and efficient control of horizontal gene transfer. Genes for nitrogenase and F420-dependent secondary alcohol dehydrogenase contribute to the metabolic versatility of this lineage. Characteristics of the new isolate such as the ability to utilize 2-propanol as an electron donor or the requirement for acetate as a carbon source are found also in the strains GP1 and SK, but not in the type strain M. hungatei JF-1T. Based on the genomic differences to related species, a new species within the genus Methanospirillum is proposed with the name M. purgamenti sp. nov. The determined phenotypic characteristics support this proposal and indicate a metabolic adaptation to a separate ecological niche.

Enantiocomplementary Asymmetric Reduction of 2-Haloacetophenones Using TeSADH: Synthesis of Enantiopure 2-Halo-1-arylethanols.

Enantiopure 2-halo-1-arylethanols are essential precursors for the synthesis of pharmaceuticals, agrochemicals, and fine chemicals. This study investigates the asymmetric reduction of 2-haloacetophenones and their substituted analogs to obtain their corresponding optically active 2-halo-1-arylethanols using secondary alcohol dehydrogenase from Thermoanaerobacter pseudethanolicus (TeSADH) mutants. Specifically, the ΔP84/A85G and P84S/A85G TeSADH mutants were evaluated for the asymmetric reduction of 2-haloacetophenones, generating their corresponding optically active halohydrins with high enantioselectivities. The asymmetric reduction of 2-haloacetophenones and their substituted analogs using the ΔP84/A85G TeSADH mutant yielded their corresponding (S)-2-halo-1-arylethanols with high enantiopurity in accordance with the anti-Prelog's rule. Conversely, the P84S/A85G TeSADH mutant produced (R)-alcohols when reducing 2-chloro-4'-chloroacetophenone, 2-chloro-4'-bromoacetophenone, and 2-bromo-4'-chloroacetophenone, while generating the (S)-configured halohydrin from 2-chloro-4'-fluoroacetophenone. Asymmetric reduction of the unsubstituted 2-bromoacetophenone, 2-chloroacetophenone, and 2,2,2-trifluoroacetophenone resulted in production of their (S)-halohydrins with the tested mutants, which reflects the importance of the nature of the substituent on the substrate's ring in controlling the stereopreference of these TeSADH-catalyzed reduction reactions. These findings contribute to the understanding and application of TeSADH in synthesizing optically active compounds and aid in the design of further mutants with the desired stereopreference.

Image Sensing of Gaseous Acetone Using Secondary Alcohol Dehydrogenase-Immobilized Mesh for Exhaled Air.

Human-borne acetone is a potent marker of lipid metabolism. Here, an enzyme immobilization method for secondary alcohol dehydrogenase (S-ADH), which is suitable for highly sensitive and selective biosensing of acetone, was developed, and then its applicability was demonstrated for spatiotemporal imaging of concentration distribution. After various investigations, S-ADH-immobilized meshes could be prepared with less than 5% variation by cross-linking S-ADH with glutaraldehyde on a cotton mesh at 40 °C for 15 min. Furthermore, high activity was obtained by adjusting the concentration of the coenzyme nicotinamide adenine dinucleotide (NADH) solution added to the S-ADH-immobilized mesh to 500 μM and the solvent to a potassium phosphate buffer solution at pH 6.5. The gas imaging system using the S-ADH-immobilized mesh was able to image the decrease in NADH fluorescence (ex 340 nm, fl 490 nm) caused by the catalytic reaction of S-ADH and the acetone distribution in the concentration range of 0.1-10 ppm-v, including the breath concentration of healthy people at rest. The exhaled breath of two healthy subjects at 6 h of fasting was quantified as 377 and 673 ppb-v, which were consistent with the values quantified by gas chromatography-mass spectrometry.

Enhanced catalytic efficiency for chiral alcohol pharmaceutical intermediate production using polyacrylic acid (PAA)-based nano-bienzyme conjugates at the organic-buffer biphasic interface

Nano-bienzyme conjugates (nano-BECs) for catalysing one-pot reactions can simplify the reaction process and improve the efficiency of biocatalytic reactions. In this work glucose dehydrogenase (GDH) and Candida parapsilosis secondary alcohol dehydrogenase (CpSADH) were immobilised with polyacrylic acid (PAA) to form two types of PAA-based nano-BECs (2-S, 2-P), which achieved in situ regeneration of NADH for the production of chiral alcohols in oil-water biphasic systems, such as the key chiral pharmaceutical intermediate of (S)-1-(3-trifluoromethylphenyl)ethanol ((S)-TMPE). At a substrate concentration of 100 mM 3-trifluoromethylacetophenone (TMAP), the conversion catalyzed by nano BEC was 98.2 % in dipentyl phthalate-phosphate buffer biphasic system. In contrast, the conversion was only 39.5 % and 38.9 % in the PB buffer and 8.4 % and 7.7 % in dipentyl phthalate system. The experiments on kinetic constants demonstrated that the catalytic efficiencies (Kcat/Km) of nano-BECs (2-S, 2-P) were enhanced by 6.7-fold and 7-fold, respectively, compared to free CpSADH. Additionally, nano-BECs showed stronger coenzyme regeneration ability. Under optimal reaction conditions, nano-BECs achieved 96.2 % conversion of TMAP to (S)-TMPE in an organic-buffer system with e.e. > 99.6 % at 300 mM substrate concentration. This research expands the potential applications of polymers and proteins, and offers valuable insights into the synergistic effects of PAA on bienzyme catalytic systems in biocatalysis.

Isopropanol production via the thermophilic bioconversion of sugars and syngas using metabolically engineered Moorella thermoacetica

BackgroundIsopropanol (IPA) is a commodity chemical used as a solvent or raw material for polymeric products, such as plastics. Currently, IPA production depends largely on high-CO2-emission petrochemical methods that are not sustainable. Therefore, alternative low-CO2 emission methods are required. IPA bioproduction using biomass or waste gas is a promising method.ResultsMoorella thermoacetica, a thermophilic acetogenic microorganism, was genetically engineered to produce IPA. A metabolic pathway related to acetone reduction was selected, and acetone conversion to IPA was achieved via the heterologous expression of secondary alcohol dehydrogenase (sadh) in the thermophilic bacterium. sadh-expressing strains were combined with acetone-producing strains, to obtain an IPA-producing strain. The strain produced IPA as a major product using hexose and pentose sugars as substrates (81% mol-IPA/mol-sugar). Furthermore, IPA was produced from CO, whereas acetate was an abundant byproduct. Fermentation using syngas containing both CO and H2 resulted in higher IPA production at the specific rate of 0.03 h−1. The supply of reducing power for acetone conversion from the gaseous substrates was examined by supplementing acetone to the culture, and the continuous and rapid conversion of acetone to IPA showed a sufficient supply of NADPH for Sadh.ConclusionsThe successful engineering of M. thermoacetica resulted in high IPA production from sugars. M. thermoacetica metabolism showed a high capacity for acetone conversion to IPA in the gaseous substrates, indicating acetone production as the bottleneck in IPA production for further improving the strain. This study provides a platform for IPA production via the metabolic engineering of thermophilic acetogens.

Heterologous Production of Isopropanol Using Metabolically Engineered Acetobacterium woodii Strains.

The depletion of fossil fuel resources and the CO2 emissions coupled with petroleum-based industrial processes present a relevant issue for the whole of society. An alternative to the fossil-based production of chemicals is microbial fermentation using acetogens. Acetogenic bacteria are able to metabolize CO or CO2 (+H2) via the Wood-Ljungdahl pathway. As isopropanol is widely used in a variety of industrial branches, it is advantageous to find a fossil-independent production process. In this study, Acetobacterium woodii was employed to produce isopropanol via plasmid-based expression of the enzymes thiolase A, CoA-transferase, acetoacetate decarboxylase and secondary alcohol dehydrogenase. An examination of the enzymes originating from different organisms led to a maximum isopropanol production of 5.64 ± 1.08 mM using CO2 + H2 as the carbon and energy source. To this end, the genes thlA (encoding thiolase A) and ctfA/ctfB (encoding CoA-transferase) of Clostridium scatologenes, adc (encoding acetoacetate decarboxylase) originating from C. acetobutylicum and sadH (encoding secondary alcohol dehydrogenase) of C. beijerinckii DSM 6423 were employed. Since bottlenecks in the isopropanol production pathway are known, optimization of the strain was investigated, resulting in a 2.5-fold increase in isopropanol concentration.

Two Cascade Reactions with Oleate Hydratases for the Sustainable Biosynthesis of Fatty Acid-Derived Fine Chemicals

Oleate hydratases (OHs) are of significant industrial interest for the sustainable generation of valuable fine chemicals. When combined with other enzymes in multi-step cascades, the direct formation of fatty acid congeners can be accomplished with minimal processing steps. In this study, two cascade reactions are presented, which can be applied in one-pot approaches. The first cascade was placed “upstream” of an OH derived from Rhodococcus erythropolis (OhyRe), where a lipase from Candida rugosa was applied to hydrolyze triglycerides into free fatty acids, a crucial step for OH conversion. Further, we tested the lipase–OhyRe cascade with various types of renewable triglycerides of plant and microbial origin. In this context, the most efficient conversion was observed for microbial oil from Cutaneotrichosporon oleaginosus leading the way toward its industrial application. In contrast, the second cascade was placed “downstream” of OhyRe, where a novel secondary alcohol dehydrogenase (secADH) was applied to oxidize the hydroxylated fatty acid into a fatty acid ketone. Optimal reaction parameters for the cascade with the secADH were established, which allows this to be applied to high-throughput screens. Moreover, we describe a light-dependent route, thereby extending the catalytic efficiency of the OH enzyme system.

How Medium Affects the Activity and Stereopreference of Thermoanaerobacter ethanolicus Secondary Alcohol Dehydrogenase Catalyzed Reduction of Ketones

Abstract: The activity and selectivity of W110G Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase were altered by cosubstrate and cosolvent at varying temperatures. A sharp drop in the enantiomeric excess (ee) was observed at 60 oC in the first 3 h, suggesting increased selectivity mistakes in the reduction of 4-phenyl-2-butanone to the expected (S)-4-phenyl-2-butanol using 5% v/v of 2-propanol as a cosubstrate. The ee increased exponentially with cosubstrate concentration, reaching ≥94% with 30-70% v/v 2-propanol. However, a decrease in enzyme activity was noticed at ≥30% v/v 2-propanol by a sharp drop in conversion. The lowest ee (<3%) was registered using 5% v/v 2- propanol at 30-40 oC, which prolonged enzyme life that allowed reversible redox reaction with selectivity mistakes to give the R-alcohol compared to ≥18% ee at 50-60 oC, where faster reaction rates promoted selectivity mistake, but enzyme life was shortened by protein denaturation at the elevated temperatures. Water-miscible and immiscible organic cosolvents (25% v/v) increased enzyme selectivity. For methanol, ethanol, ethylene glycol and tert-butanol, the activity/conversion decreased with an increase in pKa and log P while the stereoselectivity/ee increased.

Efficient isopropanol-butanol-ethanol (IBE) fermentation by a gene-modified solventogenic Clostridium species under the co-utilization of Fe(III) and butyrate

To elevate the efficiency of acetone-butanol-ethanol (ABE) fermentation by the wild-type strain WK, an optimal co-utilization system (20 mM Fe3+ and 5 g/L butyrate) was established to bring about a 22.22% increment in the yield of ABE mixtures with a significantly enhanced productivity (0.32 g/L/h). With the heterologous introduction of the secondary alcohol dehydrogenase encoded gene (adh), more than 95% of acetone was eliminated to convert 4.5 g/L isopropanol with corresponding increased butanol and ethanol production by 21.08% and 65.45% in the modified strain WK::adh. Under the optimal condition, strain WK::adh was capable of producing a total of 25.46 g/L IBE biosolvents with an enhanced productivity of 0.35 g/L/h by 45.83% over the original conditions. This work for the first time successfully established a synergetic system of co-utilizing Fe(III) and butyrate to demonstrate a feasible and efficient manner for generating the value-added biofuels through the metabolically engineered solventogenic clostridial strain.

Biological production of 2-propanol from propane using a metabolically engineered type I methanotrophic bacterium

2-Propanol is a widely used industrial solvents. Herein, we employed a unique feature of type I methanotrophic bacterium Methylotuvimicrobium alcaliphilum 20Z possessing only particulate methane monooxygenase (pMMO) for one-step direct production of pure 2-propanol from propane. By maintaining cell growth on glycerol, and after deletion of both Ca2+-dependent and La3+-dependent methanol dehydrogenases, propane was converted to 2-propanol by pMMO. Although most of the 2-propanol produced was further oxidized to acetone, deletion of active alcohol dehydrogenase, concomitant with synchronous overexpression of secondary alcohol dehydrogenase, significantly inhibited such undesirable oxidation. As a result, a remarkable enhancement (263 mg/L) of 2-propanol was achieved for 120 h by increasing cell growth with a supply of 50% (v/v) propane in headspace. This is the first demonstration to develop an engineered methanotrophic strain for the one-step direct production of pure 2-propanol from propane using one-phase cultivation without the supply of chemical inhibitors or additional reducing-power sources.

Insights into Evolutionary, Genomic, and Biogeographic Characterizations of Chryseobacterium nepalense Represented by a Polyvinyl Alcohol-Degrading Bacterium, AC3.

ABSTRACTChryseobacterium spp. are Gram-negative rods found ubiquitously in the environment, with certain species being reported as having unusual degrading properties. Polyvinyl alcohol (PVA) is used widely in industry but causes serious global environmental pollution. Here, we report the complete genome sequence of a novel bacterium, AC3, that efficiently degrades PVA. As the representative genome of Chryseobacterium nepalense, key genomic characteristics (e.g., mobile genetic elements, horizontal genes, genome-scale metabolic network, secondary metabolite biosynthesis gene clusters, and carbohydrate-active enzymes) were comprehensively investigated to reveal the potential genetic features of this species. Core genome phylogenetic analysis in combination with average nucleotide identity, average amino acid identity, and in silico DNA-DNA hybridization values provided an accurate taxonomic position of C. nepalense in the genus Chryseobacterium. Comparative genomic analysis of AC3 with closely related species suggested evolutionary dynamics characterized by a species-specific genetic repertoire, dramatic rearrangements, and evolutionary constraints driven by selective pressure, which facilitated the speciation and adaptative evolution of C. nepalense. Biogeographic characterization indicated that this species is ubiquitously distributed not only in soil habitats but also in a variety of other source niches. Bioinformatic analysis revealed the potential genetic basis of PVA degradation in AC3, which included six putative genes associated with the synthesis of PVA dehydrogenase, cytochrome c, oxidized PVA hydrolase, and secondary alcohol dehydrogenase. Our study reports the first complete genome of C. nepalense with PVA-degrading properties, providing comprehensive insights into the genomic characteristics of this species and increasing our understanding of the microbial degradation of PVA.IMPORTANCE Although PVA is a biodegradable polymer, the widespread use of PVA in global industrialization has resulted in serious environmental problems. To date, knowledge of effective and applicable PVA-degrading bacteria is limited, and thus, the discovery of novel PVA biodegraders is pertinent. Here, we isolated a novel bacterial strain, AC3, which efficiently degraded PVA. The complete genome of AC3 was sequenced as the first genome sequence of the species C. nepalense. Comparative genomic analysis was performed to comprehensively investigate the phylogenetic relationships, genome-scale metabolic network, key genomic characteristics associated with genomic evolution, evolutionary dynamics between AC3 and its close relatives, and biogeographic characterization of C. nepalense, particularly regarding the potential genetic basis of PVA degradation. These findings could advance our understanding of the genomic characteristics of C. nepalense and PVA bioremediation.

Crystallographic snapshots of ternary complexes of thermophilic secondary alcohol dehydrogenase from Thermoanaerobacter pseudoethanolicus reveal the dynamics of ligand exchange and the proton relay network.

Three-dimensional structures of I86A and C295A mutant secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter pseudoethanolicus were determined by x-ray crystallography. The tetrameric structure of C295A-SADH soaked with NADP+ and dimethyl sulfoxide (DMSO) was determined to 1.85 Å with an Rfree of 0.225. DMSO is bound to the tetrahedral zinc in each subunit, with ligands from SG of Cys-37, NE2 of His-59, and OD2 of Asp-150. The nicotinamide ring of NADP is hydrogen-bonded to the N of Ala-295 and the O of Val-265 and Gly-293. The O of DMSO is connected to a network of hydrogen bonds with OG of Ser-39, the 3'-OH of NADP, and ND1 of His-42. The structure of I86A-SADH soaked with 2-pentanol and NADP+ contains (R)-2-pentanol bound in each subunit, ligated to the tetrahedral zinc, and connected to the proton relay network. The structure of I86A-SADH soaked with 3-methylcyclohexanol and NADP+ has alcohol bound in three subunits. Two of the sites have the alcohol ligated to the zinc in an axial position, with OE2 of Glu-60 in the other axial position of a trigonal bipyramidal complex. One site has 3-methylcyclohexanol bound noncovalently, with the zinc in an inverted tetrahedral geometry with Glu-60. The fourth site also has the zinc in a trigonal bipyramidal complex with axial Glu-60 and water ligands. These structures demonstrate that ligand exchange of SADH involves pentacoordinate and inverted zinc complexes with Glu-60. Furthermore, we see a network of hydrogen bonds connecting the substrate oxygen to the external solvent that is likely to play a role in the mechanism of SADH.

Engineering Acetobacterium woodii for the production of isopropanol and acetone from carbon dioxide and hydrogen

The capability of four genetically modified Acetobacterium woodii strains for improved production of acetone from CO2 and hydrogen was tested. The acetone biosynthesis pathway was constructed by combining genes from Clostridium acetobutylicum and Clostridium aceticum. Expression of acetone production genes was demonstrated in all strains. In bioreactors with continuous gas supply, all produced acetic acid, acetone, and, surprisingly, isopropanol. The production of isopropanol was caused by an endogenous secondary alcohol dehydrogenase (SADH) activity at low gas-feeding rate. Although high amounts of the natural end product acetic acid of A. woodii were formed,14.5mM isopropanol and 7.6mM acetone were also detected, showing that this is a promising approach for the production of new solvents from C1 gases. The highest acetic acid, acetone, and isopropanol production was detected in the recombinant A. woodii [pJIR750_ac1t1] strain, with final concentrations of 438mM acetic acid, 7.6mM acetone, and 14.5mM isopropanol. The engineered strain A. woodii [pJIR750_ac1t1] was found to be the most promising strain for acetone production from a gas mixture of CO2 and H2 and the formation of isopropanol in A. woodii was shown for the first time.

Racemization of Enantiopure Alcohols Using Two Mutants of Thermoanaerobacter pseudoethanolicus Secondary Alcohol Dehydrogenase

AbstractRacemization of enantiopure alcohols is the key step to accomplish a dynamic kinetic resolution, and thus overcome the intrinsic drawback of 50 % yield with high enantioselectivity that is associated with kinetic resolution. Herein, the concurrent use of I86A and W110G mutants of Thermoanaerobacter pseudoethanolicus secondary alcohol dehydrogenase (TeSADH) in racemization of enantiopure phenyl‐ring‐containing alcohols is reported. The efficiency of this bienzymatic racemization approach is also compared with that reported previously using W110G TeSADH and is marginally more efficient.

Portable Smartphone Platform Based on a Single Dual-Emissive Ratiometric Fluorescent Probe for Visual Detection of Isopropanol in Exhaled Breath.

The components in the exhaled breath have been confirmed to be related to certain diseases, especially studies have shown that isopropanol (IPA) might be closely associated with illnesses such as lung cancer, and are considered as a biomarker. Herein, we designed a portable smartphone platform based on a chemically synthesized ratiometric fluorescent probe for real-time/on-site, sensitive, and quantitative visual detection of IPA in exhaled breath. The fluorescent probe was fabricated by a nicotinamide adenine dinucleotide (NAD+) functional modified onto fluorescent internal standard red carbon dots (RCDs). Whereas, IPA can convert NAD+ into reduced nicotinamide adenine dinucleotide (NADH) through an enzymatic reaction of secondary alcohol dehydrogenase (S-ADH). The electron transfer from IPA to NAD+ emitted a blue emission of NADH, which displayed consecutive color changes from red to light blue. Under optimum conditions, the fluorescent probe shows sensitive responses to IPA with a detection limit as low as 4.45 nM. Moreover, combined with the smartphone color recognizer application (APP), the ratio of fluorescence intensity response was recorded on a blue channel (B)/red channel (R), which has been employed for the visual quantitative determination of IPA with a detection limit of 8.34 nM and a recovery rate of 90.65-110.09% (RSD ≤ 4.83). The method reported here provides a convenient pathway for real-time/on-site and visual detection of IPA in exhaled air and is expected to extend the application of investigation of potential volatile biomarkers for preliminary monitoring and clinical diagnosis.

Efficient 1-Hydroxy-2-Butanone Production from 1,2-Butanediol by Whole Cells of Engineered E. coli

1-Hydroxy-2-butanone (HB) is a key intermediate for anti-tuberculosis pharmaceutical ethambutol. Commercially available HB is primarily obtained by the oxidation of 1,2-butanediol (1,2-BD) using chemical catalysts. In present study, seven enzymes including diol dehydrogenases, secondary alcohol dehydrogenases and glycerol dehydrogenase were chosen to evaluate their abilities in the conversion of 1,2-BD to HB. The results showed that (2R, 3R)- and (2S, 3S)-butanediol dehydrogenase (BDH) from Serratia sp. T241 could efficiently transform (R)- and (S)-1,2-BD into HB respectively. Furthermore, two biocatalysts co-expressing (2R, 3R)-/(2S, 3S)-BDH, NADH oxidase and hemoglobin protein in Escherichia coli were developed to convert 1,2-BD mixture into HB, and the transformation conditions were optimized. Maximum HB yield of 341.35 and 188.80 mM could be achieved from 440 mM (R)-1,2-BD and 360 mM (S)-1,2-BD by E. coli (pET-rrbdh-nox-vgb) and E. coli (pET-ssbdh-nox-vgb) under the optimized conditions. In addition, two biocatalysts showed the ability in chiral resolution of 1,2-BD isomers, and 135.68 mM (S)-1,2-BD and 112.43 mM (R)-1,2-BD with the purity of 100% could be obtained from 300 and 200 mM 1,2-BD mixture by E. coli (pET-rrbdh-nox-vgb) and E. coli (pET-ssbdh-nox-vgb), respectively. These results provided potential application for HB production from 1,2-BD mixture and chiral resolution of (R)-1,2-BD and (S)-1,2-BD.

A Bio-Fluorometric Acetone Gas Imaging System for the Dynamic Analysis of Lipid Metabolism in Human Breath

We constructed an imaging system to measure the concentration of acetone gas by acetone reduction using secondary alcohol dehydrogenase (S-ADH). Reduced nicotinamide adenine dinucleotide (NADH) was used as an electron donor, and acetone was imaged by fluorescence detection of the decrease in the autofluorescence of NADH. In this system, S-ADH–immobilized membranes wetted with buffer solution containing NADH were placed in a dark box, and UV-LED excitation sheets and a high-sensitivity camera were installed on both sides of the optical axis to enable loading of acetone gas. A hydrophilic polytetrafluoroethylene (H-PTFE) membrane with low autofluorescence was used as a substrate, and honeycomb-like through-hole structures were fabricated using a CO2 laser device. After loading the enzyme membrane with acetone gas standards, a decrease in fluorescence intensity was observed in accordance with the concentration of acetone gas. The degree of decrease in fluorescence intensity was calculated using image analysis software; it was possible to quantify acetone gas at concentrations of 50–2000 ppb, a range that includes the exhaled breath concentration of acetone in healthy subjects. We applied this imaging system to measure the acetone gas in the air exhaled by a healthy individual during fasting.

(Invited) Bio-Sniffers & Sniff-Cam: Biofluorometirc Gas Sensor and Imaging System for Human Volatile Chemicals

Introduction Various volatile organic compounds exist, such as those the transpired by humans, breath, body odor and smell of the living environment. The human body emits various volatile molecules, depending on a person’s genetics, disease and metabolic condition. In this contribution, novel gas-phase biosensors (bio-sniffer and sniff-cam) using bio-fluorometric techniques for human volatiles will be introduced as a future biosensing approach for preemptive medicine and healthcare. Bio-sniffer for breath acetone as early screening approach of diabetes mellitus. Secondary alcohol dehydrogenase (S-ADH) catalyzes the reduction of acetone and the oxidation of nicotinamide adenine dinucleotide (NADH to NAD+) in a weak acid environment (Fig.1). NADH is excited by 340 nm excitation lights and subsequently emit 490 nm fluorescence. Therefore, acetone can be measured by the decrease in NADH fluorescence intensity. A bio-fluorometric acetone sniffer was constructed by incorporating an optic-probe into a gas/liquid flow cell with S-ADH immobilized diaphragm (Fig.2) [1,2]. The developed biosniffer show rapid response, highly sensitivity and selectivity to acetone. The breath acetone analysis in healthy subjects shows that the mean values were 750.0 ± 434.4 ppb. The breath acetone did not show a statistical difference among different genders and ages. The breath acetone analysis for diabetic patients under medical treatment shows a mean value of 1207.7 ± 689.5 ppb, which was higher than that of healthy subjects (p < 1 × 10−6). In particularly, type-1 diabetic (T1D) patients exhaled a much higher concentration of acetone than type-2 diabetic (T2D) patients (p < 0.01) (Fig.3). These findings are worthwhile in the study of breath biomarkers for early screening of diabetes mellitus. Additionally, the developed bio-sniffers provide a new technique for volatolomics research. Sniff-cam with biofluorometric technique for spatiotemporal gas imaging A decade ago, a chemiluminescence sniff-cam system was developed and applied successfully for distribution imaging of high concentration ethanol vapor (ppm-level) such as breath ethanol after drinking (Fig.4) [3] and wine aroma from wine glass (Fig.5) [4]. In order to improve the performance, we applied the bio-fluorometric technique for highly sensitive sniff-cam system (ppb-level). The sniff-cam with ADH (alcohol dehydrogenase) redox reactions can show a spatiotemporal change of gaseous ethanol (EtOH) or acetaldehyde (AcH) in real-time [5,6]. The sniff-cam was composed of a highly sensitive camera, a UV-LED array sheet, and an ADH-immobilized mesh. The both redox reactions of ADH were employed for detection of gaseous EtOH and AcH where a relationship between fluorescence intensity from nicotinamide adenine dinucleotide. Moreover, the image differentiation method that calculated a fluorescence change rate was employed to visualize a real-time change in the concentration distribution of EtOH and AcH. The dynamic ranges of the sniff-cam for EtOH and AcH were 20ppb-300ppm and 100 ppb-10 ppm. As the physiological application, the sniff-cam successfully achieved the imaging of the concentration distribution of EtOH and AcH in breath and skin gas (Fig. 6). Future potential of the gas-phase biosensors (bio-sniffer and sniff-cam) The future potential of the bio-sniffer and sniff-cam for body volatiles includes the early disease screening, the identification of those locations, the real time evaluation of metabolic condition as a Bio-IoT monitoring.

Identification of New Microbial Isolates Capable of Transforming Oleic Acid to Solely 10‐Hydroxystearic Acid

Hydroxy fatty acids have a wide range of industrial applications including production of lubricants, plastics, waxes, cosmetics, and antimicrobial agents. Many plant oils contain a small amount of hydroxy fatty acids but are rich in oleic acid (OA) and linoleic acids. These fatty acids can be converted to hydroxy fatty acids by microorganisms with low to moderate yields. In some cases, OA can be converted to a mixture of 10-hydroxystearic acid (10-HSA) and 10-ketostearic acid (10-KSA). Oleate hydratase and secondary alcohol dehydrogenase (2o-ADH) are responsible for converting OA to 10-HSA and then 10-KSA, respectively. We are interested in obtaining microorganisms that can convert OA to solely 10-HSA without 10-KSA to eliminate downstream separation and purification of 10-HSA. While using CRISPR/Cas9 genome editing technology to knockout the 2o-ADH gene of Nocardia cholesterolicum NRRL5767 to obtain mutants that convert OA solely to 10-HSA (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230915), we also isolated three new microorganisms from soil of corn and soybean fields that convert OA solely to 10-HSA in high yield. Here we report isolation and identification of three new Isolates capable of transforming OA to solely 10-HSA. These microbial isolates were identified by Gram Stain and 16S rRNA gene sequencing. We have successfully amplified a ~1500 bp region in the 16S rRNA using 27F and 1492R primer pair from the genome of these isolates by PCR. The sequence of the entire fragment was determined on both strands by Sanger's Chain Termination method. The resulting nucleotide sequence was then used to search similar sequences in rRNA/ITS databases using nucleotide BLAST. We were able to identify the genus of these three microbes confidently.

Secondary Alcohol Dehydrogenases from Thermoanaerobacter pseudoethanolicus and Thermoanaerobacter brockii as Robust Catalysts.

Alcohol dehydrogenases (ADHs) are an important type of enzyme that have significant applications as biocatalysts. Secondary ADHs from Thermoanaerobacter pseudoethanolicus (TeSADH) and Thermoanaerobacter brockii (TbSADH) are well-known as robust catalysts. However, like most other ADHs, these enzymes suffer from their high substrate specificities (i. e., limited substrate scope), which to some extent restricts their use as biocatalysts. This minireview discusses recent efforts to expand the substrate scope and tune the enantioselectivity of TeSADH and TbSADH by using site-directed mutagenesis and directed evolution. Various examples of asymmetric synthesis of optically active alcohols using both enzymes are highlighted. Moreover, the unique thermal stability and organic solvent tolerance of these enzymes is illustrated by their concurrent inclusion with other interesting reactions to synthesize optically active alcohols and amines. For instance, TeSADH has been used in quantitative non-stereoselective oxidation of alcohols to deracemize alcohols via cyclic deracemization and in the racemization of enantiopure alcohols to accomplish a bienzymatic dynamic kinetic resolution.