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Pyruvate:quinone oxidoreductase (PQO) is a flavin‐containing peripheral membrane enzyme catalyzing the decarboxylation of pyruvate to acetate and CO2 with quinone as an electron acceptor. Here, we investigate PQO activity in Corynebacterium glutamicum, examine purified PQO, and describe the crystal structure of the native enzyme and a truncated version. The specific PQO activity was highest in stationary phase cells grown in complex medium, lower in cells grown in complex medium containing glucose or acetate, and lowest in cells grown in minimal acetate‐medium. A similar pattern with about 30‐fold higher specific PQO activities was observed in C. glutamicum with plasmid‐bound pqo expression under the control of the tac promoter, indicating that the differences in PQO activity are likely due to post‐transcriptional control. Continuous cultivation of C. glutamicum at dilution rates between 0.05 and 0.4 h−1 revealed a negative correlation between PQO activity and growth rate. Kinetic analysis of PQO enzymes purified from cells grown in complex or in minimal acetate‐medium revealed substantial differences in specific activity (72.3 vs. 11.9 U·mg protein−1) and turnover number (kcat: 440 vs. 78 s−1, respectively), suggesting post‐translational modifications affecting PQO activity. Structural analysis of PQO revealed a homotetrameric arrangement very similar to the Escherichia coli pyruvate oxidase PoxB except for the C‐terminal membrane binding domain, which exhibited a conformation markedly different from its PoxB counterpart. A truncated PQO variant lacking 17 C‐terminal amino acids showed higher affinity to pyruvate and was independent of detergent activation, highlighting the importance of the C‐terminus for enzyme activation and lipid binding. [ABSTRACT FROM AUTHOR]

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Alanine amino transaminase (ALT) is an enzyme that can be used as a biomarker for liver injury and other diseases. In this work, we report the development of the first microelectrode based on a molecularly imprinted pyruvate oxidase enzyme to be applied as an electrochemical biosensor for ALT detection. The biosensor is based on pyruvate oxidase enzyme (POx), imprinted using 4-aminophenol (functional monomer-on-platinum microelectrode modified (PME)) with platinum nanoparticles and 4-aminoantypirine (4-AAP)/sodium pyruvate as an electrochemical indicator. The operational conditions of the biosensor were optimized and characterized morphologically using scanning electron microscopy (SEM) and electrochemically using electrochemical impedance spectroscopy (EIS). The biosensor was found to have a fast response towards ALT within a linear range of 25–700 U/L and a limit of detection of 2.97 U/L. The biosensor did not exhibit cross-reactivity towards other tested enzymes, including nicotinamide adenine dinucleotide (Beta-NAD), catalase (CAT), glutathione peroxidase (GPx), and L-glutathione reduced (GSH) enzymes. The biosensor was efficiently applied for the assay of ALT in plasma samples; with recovery values ranging from 99.80–103.82% and RSD of values 0.27–2.01% and these results were found to be comparable to those of the reference diagnostic kits, without any need for complicated procedures or protein extraction. In addition to being highly sensitive, low cost, and portable, the use of microelectrodes allows the application of the proposed sensor for point-of-care diagnostics of liver function and online monitoring of ALT levels in hospitalized patients without the need for withdrawing samples, which indicates the promising applicability of the presented ALT sensor for point-of-care diagnostics. [ABSTRACT FROM AUTHOR]

The study describes the effect of aerobic conditions on the proteome of homofermentative lactic acid bacterium Lacticaseibacillus rhamnosus CM MSU 529 grown in a batch culture. Aeration caused the induction of the biosynthesis of 43 proteins, while 14 proteins were downregulated as detected by label-free LC-MS/MS. Upregulated proteins are involved in oxygen consumption (Pox, LctO, pyridoxine 5'-phosphate oxidase), xylulose 5-phosphate conversion (Xfp), pyruvate metabolism (PdhD, AlsS, AlsD), reactive oxygen species (ROS) elimination (Tpx, TrxA, Npr), general stress response (GroES, PfpI, universal stress protein, YqiG), antioxidant production (CysK, DkgA), pyrimidine metabolism (CarA, CarB, PyrE, PyrC, PyrB, PyrR), oligopeptide transport and metabolism (OppA, PepO), and maturation and stability of ribosomal subunits (RbfA, VicX). Downregulated proteins participate in ROS defense (AhpC), citrate and pyruvate consumption (CitE, PflB), oxaloacetate production (AvtA), arginine synthesis (ArgG), amino acid transport (GlnQ), and deoxynucleoside biosynthesis (RtpR). The data obtained shed light on mechanisms providing O2-tolerance and adaptation to aerobic conditions in strain CM MSU 529. The biosynthesis of 39 from 57 differentially abundant proteins was shown to be O2-sensitive in lactic acid bacteria for the first time. To our knowledge this is the first study on the impact of aerobic cultivation on the proteome of L. rhamnosus. [ABSTRACT FROM AUTHOR]

Pyruvate:quinone oxidoreductase (PQO) is a flavin-containing peripheral membrane enzyme catalyzing the decarboxylation of pyruvate to acetate and CO 2 with quinone as an electron acceptor. Here, we investigate PQO activity in Corynebacterium glutamicum, examine purified PQO, and describe the crystal structure of the native enzyme and a truncated version. The specific PQO activity was highest in stationary phase cells grown in complex medium, lower in cells grown in complex medium containing glucose or acetate, and lowest in cells grown in minimal acetate-medium. A similar pattern with about 30-fold higher specific PQO activities was observed in C. glutamicum with plasmid-bound pqo expression under the control of the tac promoter, indicating that the differences in PQO activity are likely due to post-transcriptional control. Continuous cultivation of C. glutamicum at dilution rates between 0.05 and 0.4 h -1 revealed a negative correlation between PQO activity and growth rate. Kinetic analysis of PQO enzymes purified from cells grown in complex or in minimal acetate-medium revealed substantial differences in specific activity (72.3 vs. 11.9 U·mg protein -1 ) and turnover number (k cat : 440 vs. 78 s -1 , respectively), suggesting post-translational modifications affecting PQO activity. Structural analysis of PQO revealed a homotetrameric arrangement very similar to the Escherichia coli pyruvate oxidase PoxB except for the C-terminal membrane binding domain, which exhibited a conformation markedly different from its PoxB counterpart. A truncated PQO variant lacking 17 C-terminal amino acids showed higher affinity to pyruvate and was independent of detergent activation, highlighting the importance of the C-terminus for enzyme activation and lipid binding., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Alanine amino transaminase (ALT) is an enzyme that can be used as a biomarker for liver injury and other diseases. In this work, we report the development of the first microelectrode based on a molecularly imprinted pyruvate oxidase enzyme to be applied as an electrochemical biosensor for ALT detection. The biosensor is based on pyruvate oxidase enzyme (POx), imprinted using 4-aminophenol (functional monomer-on-platinum microelectrode modified (PME)) with platinum nanoparticles and 4-aminoantypirine (4-AAP)/sodium pyruvate as an electrochemical indicator. The operational conditions of the biosensor were optimized and characterized morphologically using scanning electron microscopy (SEM) and electrochemically using electrochemical impedance spectroscopy (EIS). The biosensor was found to have a fast response towards ALT within a linear range of 25–700 U/L and a limit of detection of 2.97 U/L. The biosensor did not exhibit cross-reactivity towards other tested enzymes, including nicotinamide adenine dinucleotide (Beta-NAD), catalase (CAT), glutathione peroxidase (GPx), and L-glutathione reduced (GSH) enzymes. The biosensor was efficiently applied for the assay of ALT in plasma samples; with recovery values ranging from 99.80–103.82% and RSD of values 0.27–2.01% and these results were found to be comparable to those of the reference diagnostic kits, without any need for complicated procedures or protein extraction. In addition to being highly sensitive, low cost, and portable, the use of microelectrodes allows the application of the proposed sensor for point-of-care diagnostics of liver function and online monitoring of ALT levels in hospitalized patients without the need for withdrawing samples, which indicates the promising applicability of the presented ALT sensor for point-of-care diagnostics.

A novel nanostructured platform for pyruvate oxidase biosensors comprises poly(neutral red) (PNR) prepared by electropolymerization of NR in ethaline deep eutectic solvent (DES) with acid dopant, on a multiwalled carbon nanotubes (MWCNT) modified glassy carbon electrode (GCE). Characterization was by cyclic voltammetry and electrochemical impedance spectroscopy and morphology was examined by scanning electron microscopy. Ascorbate and H2O2 gave a better response at PNRDES/GCE than at PNRaq/GCE. Biosensors for pyruvate and phosphate, immobilizing pyruvate oxidase onto PNRDES/MWCNT/GCE enabled selective determination of pyruvate and phosphate, with micromolar limits of detection. Pyruvate was determined in onion samples and phosphate in water samples. [ABSTRACT FROM AUTHOR]

The study describes the effect of aerobic conditions on the proteome of homofermentative lactic acid bacterium Lacticaseibacillus rhamnosus CM MSU 529 grown in a batch culture. Aeration caused the induction of the biosynthesis of 43 proteins, while 14 proteins were downregulated as detected by label-free LC-MS/MS. Upregulated proteins are involved in oxygen consumption (Pox, LctO, pyridoxine 5’-phosphate oxidase), xylulose 5-phosphate conversion (Xfp), pyruvate metabolism (PdhD, AlsS, AlsD), reactive oxygen species (ROS) elimination (Tpx, TrxA, Npr), general stress response (GroES, PfpI, universal stress protein, YqiG), antioxidant production (CysK, DkgA), pyrimidine metabolism (CarA, CarB, PyrE, PyrC, PyrB, PyrR), oligopeptide transport and metabolism (OppA, PepO), and maturation and stability of ribosomal subunits (RbfA, VicX). Downregulated proteins participate in ROS defense (AhpC), citrate and pyruvate consumption (CitE, PflB), oxaloacetate production (AvtA), arginine synthesis (ArgG), amino acid transport (GlnQ), and deoxynucleoside biosynthesis (RtpR). The data obtained shed light on mechanisms providing O2-tolerance and adaptation to aerobic conditions in strain CM MSU 529. The biosynthesis of 39 from 57 differentially abundant proteins was shown to be O2-sensitive in lactic acid bacteria for the first time. To our knowledge this is the first study on the impact of aerobic cultivation on the proteome of L. rhamnosus.

[Display omitted] • Alanine aminotransferase and Aspartate aminotransferase are important enzymes found in serum and various body tissues. • Alanine aminotransferase is an enzyme which catalyzes conversion of alanine and α-ketoglutarate to pyruvate and glutamate. • Aspartate aminotransferase determine reversible exchange of amino group between glutamate and aspartate. • Levels of these enzymes can be used as prognostic tool for diagnosis of damages to the liver and other body tissues. • For quantification of these enzymes a novel portable, cheap and fast detecting device could be fabricated. This review article describes the brief information of currently developed electrochemical biosensors for the analysis of Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) levels. Levels of these enzymes can be used as prognostic tool for diagnosis of damages to heart, liver and other body tissues. The normal levels of ALT and AST in blood serum are 5–35 U/L and 5–40 U/L respectively. There is increment in the level of ALT and AST in blood serum when there is damaged to the tissues or body organs. ALT biosensor are found to be run within 5−180 s with potential range of <50 mV to + 0.7 V in 7.0–7.5 pH ranges. ALT concentration range 5–140 U/L to 10–1000 U/L with LOD of 1.56 U/L to 25 U/L. AST biosensors are found to be worked in concentration range of 0–134 U/L to 30–240 U/L with limit of detection of 0.5268 U/L–20 U/L. For AST biosensors, potential range are found within − 0.1 V to + 0.4 V at pH range of 7.0–7.5. There is need to develop a portable, cost effective and very sensitive mini device for the measurement of ALT and AST. [ABSTRACT FROM AUTHOR]

Streptococcal pyruvate oxidase (SpxB) is a hydrogen peroxide‐generating enzyme and plays a critical role in Streptococcus sanguinis interspecies interactions, but less is known about its biochemistry. We examined SpxB subcellular localization using protein fractionation and microscopy and found SpxB to be primarily cytoplasmic, but a small portion is also membrane associated. Potential post‐translational modifications of SpxB were determined using coimmunoprecipitation and mass spectrometry. Two mutant strains were constructed to further validate the presence of predicted site‐specific post‐translational modifications. These site mutated SpxB proteins exhibited reduced solubility in vivo, which likely contributes to the observed phenotypic changes in colony morphology, bacterial growth, and H2O2 production. Overall, our data suggest that SpxB post‐translational modifications likely play a major role to regulate SpxB function in S. sanguinis. [ABSTRACT FROM AUTHOR]

Abstract Background Pyruvate oxidase (Pox) is an important enzyme in bacterial metabolism for increasing ATP production and providing a fitness advantage via hydrogen peroxide production. However, few Pox enzymes have been characterized from bacterial species. The tetrameric non-hydrogen-peroxide producing Pox from E. coli is activated by phospholipids, which is important for its function in vivo. Results We characterized the hydrogenperoxide-producing Pox from L. delbrueckii strain STYM1 and showed it is specifically activated by phosphotidylethanolamine (16:0–18:1), but not by phosphotidylcholine or phosphotidylglycerol. This activation is a mixture of K- and V-type activation as both km and enzyme turnover are altered. Furthermore, we demonstrated that the L. delbrueckii Pox forms pentamers and either decamers or dimers of pentamers in solution, which is different from other characterized Pox enzymes. Lastly, we generated a C-terminal truncation mutant that was only weakly activated by phosphotidylethanolamine, which suggests the C-terminus is important for lipid activation. Conclusions To our knowledge this is the first known hydrogenperoxide-producing Pox enzyme that is activated by phospholipids. Our results suggest that there are substantial differences between Pox enzymes from different bacterial species, which could be important for their role in biological systems as well as in the development of Pox-based biosensors.

Significance:Streptococcus pneumoniae (Spn), a facultative anaerobic Gram-positive human pathogen with increasing rates of penicillin and macrolide resistance, is a major cause of lower respiratory tract infections worldwide. Pneumococci are a primary agent of severe pneumonia in children younger than 5 years and of community-acquired pneumonia in adults. A major defense mechanism toward Spn is the generation of reactive oxygen species, including hydrogen peroxide (H2O2), during the oxidative burst of neutrophils and macrophages. Paradoxically, Spn produces high endogenous levels of H2O2 as a strategy to promote colonization. Recent Advances: Pneumococci, which express neither catalase nor common regulators of peroxide stress resistance, have developed unique mechanisms to protect themselves from H2O2. Spn generates high levels of H2O2 as a strategy to promote colonization. Production of H2O2 moreover constitutes an important virulence phenotype and its cellular activities overlap and complement those of other virulence factors, such as pneumolysin, in modulating host immune responses and promoting organ injury. Critical Issues: This review examines the dual role of H2O2 in pneumococcal pneumonia, from the viewpoint of both the pathogen (defense mechanisms, lytic activity toward competing pathogens, and virulence) and the resulting host–response (inflammasome activation, endoplasmic reticulum stress, and damage to the alveolar–capillary barrier in the lungs). Future Directions: An understanding of the complexity of H2O2-mediated host–pathogen interactions is necessary to develop novel strategies that target these processes to enhance lung function during severe pneumonia. [ABSTRACT FROM AUTHOR]

The pyruvate oxidase (SpxB)-dependent production of H2O2 is widely distributed among oral commensal streptococci. Several studies confirmed the ability of H2O2 to antagonize susceptible oral bacterial species, including caries-associated Streptococcus mutans as well as several periodontal pathobionts. Here we report a potential mechanism to bolster oral commensal streptococcal H2O2 production by magnesium (Mg2+) supplementation. Magnesium is a cofactor for SpxB catalytic activity, and supplementation increases the production of H2O2 in vitro. We demonstrate that Mg2+ affects spxB transcription and SpxB abundance in Streptococcus sanguinis and Streptococcus gordonii. The competitiveness of low-passage commensal streptococcal clinical isolates is positively influenced in antagonism assays against S. mutans. In growth conditions normally selective for S. mutans, Mg2+ supplementation is able to increase the abundance of S. sanguinis in dual-species biofilms. Using an in vivo biophotonic imaging platform, we further demonstrate that dietary Mg2+ supplementation significantly improves S. gordonii oral colonization in mice. In summary, our results support a role for Mg2+ supplementation as a potential prebiotic to promote establishment of oral health-associated commensal streptococci. [ABSTRACT FROM AUTHOR]

Background: Pyruvate oxidase (Pox) is an important enzyme in bacterial metabolism for increasing ATP production and providing a fitness advantage via hydrogen peroxide production. However, few Pox enzymes have been characterized from bacterial species. The tetrameric non-hydrogen-peroxide producing Pox from E. coli is activated by phospholipids, which is important for its function in vivo. Results: We characterized the hydrogenperoxide-producing Pox from L. delbrueckii strain STYM1 and showed it is specifically activated by phosphotidylethanolamine (16:0–18:1), but not by phosphotidylcholine or phosphotidylglycerol. This activation is a mixture of K- and V-type activation as both km and enzyme turnover are altered. Furthermore, we demonstrated that the L. delbrueckii Pox forms pentamers and either decamers or dimers of pentamers in solution, which is different from other characterized Pox enzymes. Lastly, we generated a C-terminal truncation mutant that was only weakly activated by phosphotidylethanolamine, which suggests the C-terminus is important for lipid activation. Conclusions: To our knowledge this is the first known hydrogenperoxide-producing Pox enzyme that is activated by phospholipids. Our results suggest that there are substantial differences between Pox enzymes from different bacterial species, which could be important for their role in biological systems as well as in the development of Pox-based biosensors. [ABSTRACT FROM AUTHOR]

Graphene@MXene composite nanomaterials were utilized to construct an electrochemical sensor for alanine aminotransferase (ALT) detection. The combination of graphene nanosheets with MXene avoids the self-stacking of MXene and graphene, and broadens the charge transfer channel. In addition, the composite nanomaterial provides increased loading sites for pyruvate oxidase. The principle of ALT detection is a two-step enzymatic reaction. L-Alanine was initially transferred to pyruvate catalyzed by ALT. The formed pyruvate was then oxidized by pyruvate oxidase, generating H 2 O 2 . Through the detection of the generated H 2 O 2 , ALT activity was measured. The linear range of the sensor to ALT was from 5 to 400 U·L -1 with a detection limit of 0.16 U·L -1 (S/N = 3). For real sample analysis, the spiked recovery test results of ALT in serum samples were between 96.89 and 103.93% with RSD < 5%, confirming the reliability of the sensor testing results and potential clinical application of the sensor., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

2-Oxobutyrate (2-OBA), as a toxic metabolic intermediate, generally arrests the cell growth of most microorganisms and blocks the biosynthesis of target metabolites. In this study, we demonstrated that using the acetate bypass to replace the pyruvate dehydrogenase complex (PDHc) in Escherichia coli could recharge the intracellular acetyl-CoA pool to alleviate the metabolic toxicity of 2-OBA. Furthermore, based on the crystal structure of pyruvate oxidase (PoxB), two candidate residues in the substrate-binding pocket of PoxB were predicted by computational simulation. Site-directed saturation mutagenesis was performed to attenuate 2-OBA-binding affinity, and one of the variants, PoxBF112W, exhibited a 20-fold activity ratio of pyruvate/2-OBA in substrate selectivity. PoxBF112W was employed to remodel the acetate bypass in E. coli, resulting in l-threonine (a precursor of 2-OBA) biosynthesis with minimal inhibition from 2-OBA. After metabolic detoxification of 2-OBA, the supplies of intracellular acetyl-CoA and NADPH (nicotinamide adenine dinucleotide phosphate) used for l-threonine biosynthesis were restored. Therefore, 2-OBA is the substitute for pyruvate to engage in enzymatic reactions and disturbs pyruvate metabolism. Our study makes a straightforward explanation of the 2-OBA toxicity mechanism and gives an effective approach for its metabolic detoxification.

BACKGROUND: Recombinant pyruvate oxidase (pod) is in high demand owing to its broad application in biochemical analysis and low yield from wild microbial strains. Recombinant Escherichia coli harboring pET28a‐pod is an efficient producer of pod, facilitating its production in high yield. Optimizing cultivation using shake flasks is necessary for high‐level soluble pod production. RESULTS: During recombinant E. coli cultivation, 96.9% of pod activity was lost, and 3.1% of residual pod activity remained in a 25 mL working volume in 250 mL flasks owing to accumulation of pod in insoluble inclusion bodies. pH stress was the factor responsible for this phenomenon based on investigating dissolved oxygen and carbon source addition. Residual pod activity was increased to 43.3, 88.0 and 61.5% by maintaining the pH at 5.0, 6.0 and 7.0 respectively during the induction phase, which decreased inclusion body formation and increased production of soluble pod. Glycerol addition increased the cell density and volumetric pod activity to 21 243.3 U L−1 at pH 6.0 after 64 h induction. CONCLUSION: Soluble pod is converted to inclusion bodies under pH stress conditions, and this can be avoided by pH maintenance. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Mediatorless bienzyme-type amperometric biosensors that couple a hydrogen peroxidase (H 2 O 2)-generating oxidase (OxD) reaction on the electrode surface with a direct electron transfer-type bioelectrocatalytic reduction of H 2 O 2 by horseradish peroxidase (HRP) allow the detection of the substrate of the OxD without any mediator. Constructed was an analytical model involving four steps: the diffusion of the substrate of an OxD, the diffusion of oxygen, the enzymatic reaction of the OxD, and the enzymatic reaction of HRP to understand the response of the mediatorless bienzyme-type biosensors. Pyruvate oxidase was used as a model OxD to detect pyruvate. The steady-state amperometric response of the pyruvate sensor in a rotating disk mode as functions of the pyruvate concentration and the rotating speed was well explained on the model to yield the kinetic parameters for the enzymatic reactions and the mass transfer in the biosensor. Based on the kinetic parameters, we constructed a mediatorless bienzyme-type microelectrode to detect pyruvate with a steady-state diffusion-controlled performance under quiescent conditions. Unlabelled Image • Bienzyme and mediatorless amperometric pyruvate sensors are constructed. • Kinetics of the oxidase and peroxidase type sensor is modeled. • The model explains the responses of the constructed pyruvate sensor for the pyruvate and H 2 O 2. • The rate determining step in the constructed sensor is predicted by the model. • The mass-transfer controlled microdisk-type pyruvate sensor is realized. [ABSTRACT FROM AUTHOR]

Abstract This review analyzes electrochemical biosensors for the determination of lactate (lactic acid) and pyruvate (pyruvic acid) concentrations in liquid samples, especially in the blood serum. The biosensor systems for the simultaneous determination of both substances and commercial variants of the biosensors are presented, and the biosensors for medical diagnostics are highlighted. The information concerning the necessity of separate and simultaneous determination of lactate and pyruvate, as well as lactate to pyruvate ratio, is given; the traditional methods for the determination of these substances are briefly described. Lactate dehydrogenase and lactate oxidase are shown to be most commonly used in the biosensors for lactate detection. Pyruvate oxidase and living cells are used in the biosensors for pyruvate detection. Different methods of the enzymes immobilization are presented, as well as strategies for enhancement of the biosensor sensitivity. An additional requirement for practical applications is the biosensor resistance to electroactive interferents, inhibitors, biofouling, and electrode passivation; thus, the variants of solving these problems in the biosensors for lactate and pyruvate detection are analyzed. Highlights • Necessity of the detection of lactate, pyruvate and their ratio in blood is shown. • Electrochemical biosensors for the lactate and pyruvate determination are compared. • The biosensors contain lactate dehydrogenase, lactate oxidase, or pyruvate oxidase. • Biosensor systems (arrays) and commercial variants of the biosensors are presented. • The importance of further development of such biosensors is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Pyruvate oxidase is an important enzyme used as a reagent in kits and biochemical analyses; however, the yield of pyruvate oxidase from wild microbial strains is low. In this study, high‐level expression of Aerococcus viridans pyruvate oxidase was achieved in recombinant Escherichia coli by optimizing the expression system and induction conditions. Three recombinant pET vectors were constructed for pyruvate oxidase expression in E. coli. The isopropyl‐β‐d‐thiogalactoside (IPTG) concentration and induction temperature were optimized, with the result that the highest pyruvate oxidase yield (4106·9 U l−1) of the recombinant E. colipET28a‐pod was obtained under conditions of 25°C, 0·5 mmol l−1 IPTG, 0·5 OD600, after 24 h of induction, which was 34·2 times the yield achieved with the wild‐type strain. The soluble pyruvate oxidase contributed 99·6% of the total pyruvate oxidase expressed. Significance and Impact of the Study: This study demonstrates that a highly soluble pyruvate oxidase can be obtained in recombinant Escherichia coli by optimizing vectors and induction conditions. The pyruvate oxidase yield achieved is the highest reported so far, which provides a convenient and cost‐saving way to produce pyruvate oxidase. This research promotes pyruvate oxidase application in the pharmaceutical and biochemical industries. [ABSTRACT FROM AUTHOR]

Pyruvate oxidase (PyOD) is a very powerful enzyme for clinical diagnostic applications and environmental monitoring. Influences of temperature on cell growth, plasmid stability, and PyOD expression during the PyOD fermentation process by recombinant Escherichia coli were investigated. Based on the influences of temperature on the physiological metabolism, a novel high-cell density fed-batch cultivation with gradient temperature decrease strategy for effective PyOD production was achieved, under which the biomass (OD600) of recombinant E. coli could reach to 71 and the highest PyOD activity in broth could reach to 3,307 U/L in 26 hr fermentation. [ABSTRACT FROM AUTHOR]

Lactobacillus brevis is an obligatory heterofermentative lactic acid bacterium that produces high levels of acetate, which improve the aerobic stability of silages against deterioration caused by yeasts and molds. However, the mechanism involved in acetate accumulation has yet to be elucidated. Here, experimental evidence indicated that aerobiosis resulted in the conversion of lactate to acetate after glucose exhaustion in L. brevis ATCC 367 (GenBank accession number NC_008497). To elucidate the conversion pathway, in silico analysis showed that lactate was first converted to pyruvate by the reverse catalytic reaction of lactate dehydrogenase (LDH); subsequently, pyruvate conversion to acetate might be mediated by pyruvate dehydrogenase (PDH) or pyruvate oxidase (POX). Transcriptional analysis indicated that the pdh and pox genes of L. brevis ATCC 367 were upregulated 37.92- and 18.32-fold, respectively, by oxygen and glucose exhaustion, corresponding to 5.32- and 2.35-fold increases in the respective enzyme activities. Compared with the wildtype strain, the transcription and enzymatic activity of PDH remained stable in the Δpox mutant, while those of POX increased significantly in the Δpdh mutant. More lactate but less acetate was produced in the Δpdh mutant than in the wild-type and Δpox mutant strains, and more H2O2 (a product of the POX pathway) was produced in the Δpdh mutant. We speculated that the high levels of aerobic acetate accumulation in L. brevis ATCC 367 originated mainly from the reuse of lactate to produce pyruvate, which was further converted to acetate by the predominant and secondary functions of PDH and POX, respectively. IMPORTANCE PDH and POX are two possible key enzymes involved in aerobic acetate accumulation in lactic acid bacteria (LAB). It is currently thought that POX plays the major role in aerobic growth in homofermentative LAB and some heterofermentative LAB, while the impact of PDH remains unclear. In this study, we reported that both PDH and POX worked in the aerobic conversion of lactate to acetate in L. brevis ATCC 367, in dominant and secondary roles, respectively. Our findings will further develop the theory of aerobic metabolism by LAB. [ABSTRACT FROM AUTHOR]

ABSTRACT The catalase-negative, facultative anaerobe Streptococcus pneumoniae D39 is naturally resistant to hydrogen peroxide (H2O2) produced endogenously by pyruvate oxidase (SpxB). Here, we investigate the adaptive response to endogenously produced H2O2. We show that lactate oxidase, which converts lactate to pyruvate, positively impacts pyruvate flux through SpxB and that ΔlctO mutants produce significantly lower H2O2. In addition, both the SpxB pathway and a candidate pyruvate dehydrogenase complex (PDHC) pathway contribute to acetyl coenzyme A (acetyl-CoA) production during aerobic growth, and the pyruvate format lyase (PFL) pathway is the major acetyl-CoA pathway during anaerobic growth. Microarray analysis of the D39 strain cultured under aerobic versus strict anaerobic conditions shows upregulation of spxB, a gene encoding a rhodanese-like protein (locus tag spd0091), tpxD, sodA, piuB, piuD, and an Fe-S protein biogenesis operon under H2O2-producing conditions. Proteome profiling of H2O2-induced sulfenylation reveals that sulfenylation levels correlate with cellular H2O2 production, with endogenous sulfenylation of ≈50 proteins. Deletion of tpxD increases cellular sulfenylation 5-fold and has an inhibitory effect on ATP generation. Two major targets of protein sulfenylation are glyceraldehyde-3-phosphate dehydrogenase (GapA) and SpxB itself, but targets also include pyruvate kinase, LctO, AdhE, and acetate kinase (AckA). Sulfenylation of GapA is inhibitory, while the effect on SpxB activity is negligible. Strikingly, four enzymes of capsular polysaccharide biosynthesis are sulfenylated, as are enzymes associated with nucleotide biosynthesis via ribulose-5-phosphate. We propose that LctO/SpxB-generated H2O2 functions as a signaling molecule to downregulate capsule production and drive altered flux through sugar utilization pathways. IMPORTANCE Adaptation to endogenous oxidative stress is an integral aspect of Streptococcus pneumoniae colonization and virulence. In this work, we identify key transcriptomic and proteomic features of the pneumococcal endogenous oxidative stress response. The thiol peroxidase TpxD plays a critical role in adaptation to endogenous H2O2 and serves to limit protein sulfenylation of glycolytic, capsule, and nucleotide biosynthesis enzymes in S. pneumoniae.

Long ripened cheeses, such as Grana Padano (GP), a Protected Designation of Origin (PDO) Italian cheese, harbor a viable microbiota mainly composed of non-starter lactic acid bacteria (NSLAB), which contribute to the final characteristics of cheese. The NSLAB species Lactobacillus rhamnosus, Lb. casei and Lb. paracasei are frequently found in GP, and form a closely related taxonomic group ( Lb. casei group), making it difficult to distinguish the three species through 16S rRNA sequencing. SpxB , a metabolic gene coding for pyruvate oxidase in Lb. casei group, was recently used to distinguish the species within this bacterial group, both in pure cultures and in cheese, where it could provide an alternative energy source through the conversion of pyruvate to acetate. The aim of this work was to study the evolution of the metabolically active microbiota during different stages of GP ripening, targeting 16S rRNA to describe the whole microbiota composition, and spxB gene to monitor the biodiversity within the Lb. casei group. Furthermore, activation of pyruvate oxidase pathway was measured directly in cheese by reverse transcription real time PCR (RT–qPCR). The results showed that Lb. casei group dominates throughout the ripening and high-throughput sequencing of spxB allowed to identify four clusters inside the Lb. casei group. The dynamics of the sequence types forming the clusters were followed during ripening. Pyruvate oxidase pathway was expressed in cheese, showing a decreasing trend over ripening time. This work highlights how the composition of the microbiota in the early manufacturing stages influences the microbial dynamics throughout ripening, and how targeting of a metabolic gene can provide an insight into the activity of strains relevant for dairy products. [ABSTRACT FROM AUTHOR]

A promising field-effect transistor (FET) biosensor has been fabricated based on pyruvate oxidase (PyO) functionalized ZnO nanorods (ZnO NRs) array grown on seeded SiO 2 /Si substrate. The direct and vertically grown ZnO NRs on the seeded SiO 2 /Si substrate offers high surface area for enhanced PyO immobilization, which further helps to detect phosphate with higher specificity. Under optimum conditions, the fabricated FET biosensor provided a convenient method for phosphate detection with high sensitivity (80.57 μA mM −1 cm −2 ) in a wide-linear range (0.1 µM–7.0 mM). Additionally, it also showed very low effect of electroactive species, stability and good reproducibility. Encouraging results suggest that this approach presents a promising method to be used for field measurements to detect phosphate. [ABSTRACT FROM AUTHOR]

A new L-lactate selective impedimetric biosensor based on a lactate dehydrogenase (LDH)/pyruvate oxidase (PyrOx) bioselective membrane is reported, fabricated using enzyme drop coating and cross-linking via glutaraldehyde vapours. The sensing strategy is based on the measurement of the change in impedance caused by the generation of charged ions (CH 3 COO − , H + and HCO 3 − ), from PyrOx catalysed pyruvate oxidation, the pyruvate resulting from L-lactate oxidation catalysed by LDH + nicotinamide adenine dinucleotide (NAD + ). The experimental conditions, pH and buffer concentration, were optimized for best biosensor response, and operational/storage stability and selectivity/specificity were investigated. The limit of detection for L-lactate, was 17 and 20 μM, considering changes in resistance and charge separation parameters at and close to the LDH-NAD + /PyrOx interface, respectively. The biosensor was successfully employed for the determination of L-lactate in yogurt samples. [ABSTRACT FROM AUTHOR]

This study focused on the spxB gene, which encodes for pyruvate oxidase. The presence of spxB in the genome and its transcription could be a way to produce energy and allow bacterial growth during carbohydrate starvation. In addition, the activity of pyruvate oxidase, which produces hydrogen peroxide, could be a mechanism for interspecies competition. Because this gene seems to provide advantages for the encoding species for adaptation in complex ecosystems, we studied spxB in a large set of cheese isolates belonging to the Lactobacillus casei group. Through this study, we demonstrated that this gene is widely found in the genomes of members of the L. casei group and shows variability useful for taxonomic studies. In particular, the HRM analysis method allowed for a specific discrimination between Lactobacillus rhamnosus , Lactobacillus paracasei and L. casei . Regarding the coding region, the spxB functionality in cheese was shown for the first time by real-time PCR, and by exploiting the heterogeneity between the L. casei group species, we identified the bacterial communities encoding the spxB gene in this ecosystem. This study allowed for monitoring of the active bacterial community involved in different stages of ripening by following the POX pathway. [ABSTRACT FROM AUTHOR]

The two-component regulatory system CiaRH of Streptococcus pneumoniae is implicated in competence, ß-lactam resistance, maintenance of cell integrity, bacteriocin production, host colonization, and virulence. Depending on the growth conditions, CiaR can be highly active in the absence of its cognate kinase CiaH, although phosphorylation of CiaR is required for DNA binding and gene regulation. To test the possibility that acetyl phosphate (AcP) could be the alternative phosphodonor, genes involved in pyruvate metabolism were disrupted to alter cellular levels of acetyl phosphate. Inactivating the genes of pyruvate oxidase SpxB, phosphotransacetylase Pta, and acetate kinase AckA, resulted in very low AcP levels and in strongly reduced CiaR-mediated gene expression in CiaH-deficient strains. Therefore, alternative phosphorylation of CiaR appears to proceed via AcP. The AcP effect on CiaR is not detected in strains with CiaH. Attempts to obtain elevated AcP by preventing its degradation by acetate kinase AckA, were not successful in CiaH-deficient strains with a functional SpxB, the most important enzyme for AcP production in S. pneumoniae. The ciaH-spxB-ackA mutant producing intermediate amounts of AcP could be constructed and showed a promoter activation, which was much higher than expected. Since activation was dependent on AcP, it can apparently be used more efficiently for CiaR phosphorylation in the absence of AckA. Therefore, high AcP levels in the absence of CiaH and AckA may cause extreme overexpression of the CiaR regulon leading to synthetic lethality. AckA is also involved in a regulatory response, in which is mediated by CiaH. Addition of acetate to the growth medium switch CiaH from kinase to phosphatase. This switch is lost in the absence of AckA indicating metabolism of acetate is required, which starts with the production of AcP by AckA. Therefore, AckA plays a special regulatory role in the control of the CiaRH two-component regulatory system.