The first in-depth exploration of the genome of the engineered bacterium, Gluconobacter thailandicus.

Glycerol is an abundant byproduct of biodiesel production that has significant industrial value and can be converted into dihydroxyacetone (DHA). DHA is widely used for the production of various chemicals, pharmaceuticals, and food additives. Gluconobacter can convert glycerol to DHA through two different pathways, including membrane-bound dehydrogenases with pyrroloquinoline quinone (PQQ) and NAD(P)+ -dependent enzymes. Previous work has indicated that membrane-bound dehydrogenases are present in Gluconobacter oxydans and Gluconobacter frateurii, but the metabolic mechanism of Gluconobacter thailandicus's glycerol conversion is still not clear. Through in-depth analysis of the G. thailandicus genome and annotation of its metabolic pathways, we revealed the existence of both PQQ and NAD(P)+ -dependent enzymes in G. thailandicus. In addition, this study provides important information related to the tricarboxylic acid cycle, glycerol dehydrogenase level, and phylogenetic relationships of this important species.

GDH and toxin immunoassay for the diagnosis of Clostridioides (Clostridium) difficile infection is not a 'one size fit all' screening test.

Diagnosing Clostridioides (Clostridium) difficile infection is challenged by lack of a clear gold standard. We sought to determine if the two-step algorithm (screening GDH and toxin lateral flow assay followed by tcdB PCR) would have adequate clinical performance at a tertiary care center. Of 486 patients, 310 (63.8%) were immunocompromised. Of 150 PCR-positive specimens, 52 (34.7%) were toxin-positive and 126 (84.0%) were GDH positive. Positive GDH or toxin results corresponded to lower PCR cycle threshold values (P < 0.01). PCR-positive patients had more frequently documented antibiotic usage (78.4% vs 66.9%, P = 0.05) and diarrhea (91.0% vs. 79.4%, P < 0.01) and less frequent alternate etiologies of diarrhea (27.3% vs. 41.1%, P = 0.004) or laxative use (24.6% vs 36.1%, P = 0.02). Toxin positivity was associated with antibiotic use (P < 0.01), but not with neutropenia, diarrhea, malignancy, or chemotherapy (P > 0.05). The application of the 2-step algorithm should be thoroughly evaluated in immunocompromised patient populations before implementation.

Preparation of Expoxy-Functionalized Magnetic Nanoparticles for Immobilization of Glycerol Dehydrogenase.

Immobilization of glycerol dehydrogenase (GDH) from Serratia marcescens H30 onto epoxy functional magnetic nanoparticles by covalent attachment was carried out. The optimal immobilization conditions were obtained as follows: enzyme/support 6.08 mg/g, temperature 25 °C, pH 7.0 and time 8 h. Under these conditions, a high immobilization yield above 90% was obtained. The characterization of the immobilized GDH indicated that enhanced pH and thermal stability were achieved. Kinetic parameters Km of free and immobilized GDH were determined as 10.35 mM and 15.76 mM, respectively. The immobilized GDH retained about 85% initial activity after ten cycles. These results suggested that GDH immobilized onto magnetic nanoparticles is a simple and efficient way for preparation of stable enzyme. And the immobilized GDH has potential applications in the production of DHA.

Clostridium difficile infection.

Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis - the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota.

Evaluation of inoculum of Candida guilliermondii grown in presence of glucose on xylose reductase and xylitol dehydrogenase activities and xylitol production during batch fermentation of sugarcane bagasse hydrolysate.

The effect of glucose on xylose-xylitol metabolism in fermentation medium consisting of sugarcane bagasse hydrolysate was evaluated by employing an inoculum of Candida guilliermondii grown in synthetic media containing, as carbon sources, glucose (30 g/L), xylose (30 g/L), or a mixture of glucose (2 g/L) and xylose (30 g/L). The inoculum medium containing glucose promoted a 2.5-fold increase in xylose reductase activity (0.582 IU/mgprot) and a 2-fold increase in xylitol dehydrogenase activity (0.203 IU/mgprot) when compared with an inoculum-grown medium containing only xylose. The improvement in enzyme activities resulted in higher values of xylitol yield (0.56 g/g) and productivity (0.46 g/[L.h]) after 48 h of fermentation.

Modification of analytical procedures for determining vitamin C enzyme (L-gulonolactone oxidase) activity in swine liver.

Modifications of the analytical method to determine L-gulono-gamma-lactone oxidase (EC 1.1.8) enzyme activity were conducted in pig liver by evaluating the concentration of added substrate (L-gulono-gamma-lactone), glutathione, and various tissue sample-to-buffer ratios in the incubation mixture. Sampling different liver sites (lobes), the effect of different cooling temperatures of the liver immediately after collection, and the effect of tissue storage length on subsequent enzyme activity were evaluated. Our results demonstrated that 10 mM of substrate added to the reaction media maximized L-gulono-gamma-lactone oxidase enzyme activity, whereas increasing levels of glutathione did not greatly affect enzyme activity. High sample-to-buffer ratios resulted in higher L-gulono-gamma-lactone oxidase activities but sample analytical variations and background interferences were greater. A 1:4 tissue sample to buffer ratio (weight:weight) resulted in repeatable values, but the importance of maintaining the same ratio of the two components seems to be critical within an experiment. Expressing L-gulono-gamma-lactone oxidase enzyme activity on a liver protein rather than on a liver weight basis also resulted in more consistent results. No difference in liver L-gulono-gamma-lactone oxidase enzyme activities or ascorbic acid concentrations occurred between liver lobes. L-gulono-gamma-lactone oxidase enzyme activity from 0 to 90 day of storage was not affected when tissue samples were immediately frozen in liquid nitrogen, or placed on crushed ice. During a 90-day storage the oxidized form of ascorbic acid (dehydroascorbic acid) decreased (P < 0.01), the reduced (ascorbic acid) form increased (P < 0.01), while total ascorbic acid concentration remained constant.

Efeito do hidrolisado hemicelulósico de bagaço de cana-de-açúcar submetido a diferentes tratamentos sobre a atividade da xilose redutase de Candida guilliermondii / Effect of the sugarcane bagasse hemicellulose hydrolysate submited to different treatments on xylose reductase of Candida guilliermondii activity

No presente trabalho, foi avaliada a influência da concentração e de diferentes conbinações de tratamentos do hidrolisado hemicelulósico ácido de bagaço de cana-de-açúcar, sobre atividade da enzima xilose redutase (XR) de Candida guilliermondii. Em paralelo, determinou-se a atividade da enzima xilitol deshidrogenase (XD) presente nas células, já que o nível de atividade desta enzima pode interferir no rendimento em xilitol, quando a bioconversão é feita por via fermentativa. As células foram cultivadas nos hidrolisados provinientes das diferentes combinaçõoes de tratamentos pela alteração do pH utilizando bases e ácidos e adsorção com carvão ativo. Amostras foram retiradas para a medição da atividade emzimática em extrato...

Enzymatic method for branched chain alpha-ketoacid determination: application to rapid analysis of urine and plasma samples from maple syrup urine disease patients.

A new method for the determination of branched-chain alpha-ketoacid concentration using lactate dehydrogenase (E C 1.1.1.27) isozyme C4 (LDH C4) from mouse testes is proposed. The assay is performed on urine and plasma without previous treatment. Alpha-ketoglutarate and pyruvate are determined on the same sample using glutamate dehydrogenase (GDH, EC 1.4.1.2) and lactate dehydrogenase isozyme A4 (LDH5) respectively and subtracted from the total alpha-ketoacid concentration obtained with LDH C4. This value corresponds to the branched chain alpha-ketoacid. Results were linear within the concentration range 8 to 170 mumoles/L. Detection limit was 8 mumoles/L. Analytical recovery was higher than 91%. For microplate assays, recoveries were higher than 84% and the detection limit was 20 mumoles/L. Determinations performed with GDH, LDH A4 and LDH C4 allow differentiation of E3 deficiency from other clinical phenotypes of maple syrup urine disease. The method is simple and fast, and adaptation to microplates would allow screening of newborns.

Enzymatic method for branched chain alpha-ketoacid determination: application to rapid analysis of urine and plasma samples from maple syrup urine disease patients

A new method for the determination of branched-chain alpha-ketoacid concentration using lactate dehydrogenase (E C 1.1.1.27) isozyme C4 (LDH) C4) from mouse testes is proposed. The assay is performed on urine and plasma without previous treatment. Alpha-ketoglutarate and pyruvate are determined on the same sample using glutamate dehydrogenase (GDH,EC 1.4.1.2) and lactate dehydrogenase isozyme A4 (LDH5) respectively and subtracted from the total alpha-ketoacid concentration obtained with LDH C4. This value corresponds to the branched chain alpha-ketoacid. Results were linear within the concentration range 8 to 170 mumoles/L. Detection limit was 8 mumoles/L. Analytical recovery was higher than 91 per cent. For microplate assays, recoveries were higher than 84 per cent and the detection limit was 20 mumoles/L. Determinations performed with GDH, LDH A4 and LDH C4 allow differentiation of E3 deficiency from other clinical phenotypes of maple syrup urine disease. The method is simple and fast, and adaptation to microplates would allow screening of newborns. (AU)

Enzymatic method for branched chain alpha-ketoacid determination: application to rapid analysis of urine and plasma samples from maple syrup urine disease patients

A new method for the determination of branched-chain alpha-ketoacid concentration using lactate dehydrogenase (E C 1.1.1.27) isozyme C4 (LDH) C4) from mouse testes is proposed. The assay is performed on urine and plasma without previous treatment. Alpha-ketoglutarate and pyruvate are determined on the same sample using glutamate dehydrogenase (GDH,EC 1.4.1.2) and lactate dehydrogenase isozyme A4 (LDH5) respectively and subtracted from the total alpha-ketoacid concentration obtained with LDH C4. This value corresponds to the branched chain alpha-ketoacid. Results were linear within the concentration range 8 to 170 mumoles/L. Detection limit was 8 mumoles/L. Analytical recovery was higher than 91 per cent. For microplate assays, recoveries were higher than 84 per cent and the detection limit was 20 mumoles/L. Determinations performed with GDH, LDH A4 and LDH C4 allow differentiation of E3 deficiency from other clinical phenotypes of maple syrup urine disease. The method is simple and fast, and adaptation to microplates would allow screening of newborns.

Loss of protein kinase-catalyzed phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, by mutation of the ptsH gene confers catabolite repression resistance to several catabolic genes of Bacillus subtilis.

In gram-positive bacteria, HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), is phosphorylated by an ATP-dependent, metabolite-activated protein kinase on seryl residue 46. In a Bacillus subtilis mutant strain in which Ser-46 of HPr was replaced with a nonphosphorylatable alanyl residue (ptsH1 mutation), synthesis of gluconate kinase, glucitol dehydrogenase, mannitol-1-P dehydrogenase and the mannitol-specific PTS permease was completely relieved from repression by glucose, fructose, or mannitol, whereas synthesis of inositol dehydrogenase was partially relieved from catabolite repression and synthesis of alpha-glucosidase and glycerol kinase was still subject to catabolite repression. When the S46A mutation in HPr was reverted to give S46 wild-type HPr, expression of gluconate kinase and glucitol dehydrogenase regained full sensitivity to repression by PTS sugars. These results suggest that phosphorylation of HPr at Ser-46 is directly or indirectly involved in catabolite repression. A strain deleted for the ptsGHI genes was transformed with plasmids expressing either the wild-type ptsH gene or various S46 mutant ptsH genes (S46A or S46D). Expression of the gene encoding S46D HPr, having a structure similar to that of P-ser-HPr according to nuclear magnetic resonance data, caused significant reduction of gluconate kinase activity, whereas expression of the genes encoding wild-type or S46A HPr had no effect on this enzyme activity. When the promoterless lacZ gene was put under the control of the gnt promoter and was subsequently incorporated into the amyE gene on the B. subtilis chromosome, expression of beta-galactosidase was inducible by gluconate and repressed by glucose. However, we observed no repression of beta-galactosidase activity in a strain carrying the ptsH1 mutation. Additionally, we investigated a ccpA mutant strain and observed that all of the enzymes which we found to be relieved from carbon catabolite repression in the ptsH1 mutant strain were also insensitive to catabolite repression in the ccpA mutant. Enzymes that were repressed in the ptsH1 mutant were also repressed in the ccpA mutant.

[The possible effect of amino acids forming a loop in the surface layer of subunits on the electrophoretic mobility of enzymes in the alcohol/polyol dehydrogenase family]. / Vozmozhnoe vliianie aminokislot, obrazuiushchikh petliu v poverkhnostnom sloe sub''edinits, na élektroforeticheskuiu podvizhnost' fermentov semeistva alkogol'/polioldegidrogenaz.

Relative electrophoretic mobility (REM) of alcohol dehydrogenases from equine hepatocyte cytoplasm was low probably due to the presence of a loop which consisted of 21 amino acid residues in the surface layer of the enzyme subunits. The REM of multiple molecular forms of alcohol dehydrogenases from yeast cell cytoplasm was higher as consistent with the absence of this loop in the surface layer of the enzyme subunits. Possible role of amino acid residues comprising the loop, in the formation of total charge and their effect on REM values of enzymes from the alcohol/polyol dehydrogenase family are discussed.

Micro-determination of L-gulono-gamma-lactone oxidase activity.

Highly sensitive assay method of L-gulono-gamma-lactone oxidase (GLO) was constructed. In this method, L-ascorbic acid formed in the enzymatic reaction was converted to its bis(dinitrophenyl)hydrazone derivative, and the amount of the latter was determined by high-performance liquid chromatography. Twenty picomoles of ascorbic acid was detected, which makes this method 25 times more sensitive than the previously used dipyridyl one. By the present method, a minute activity of GLO in liver microsomes prepared from rats of the Osteogenic Disorder Shionogi strain (ODS-od/od) could be measured.

Isolation and characterization of Escherichia coli mutants able to utilize the novel pentose L-ribose.

Wild-type strains of Escherichia coli were unable to utilize L-ribose for growth. However, L-ribose-positive mutants could be isolated from strains of E. coli K-12 which contained a ribitol operon. L-ribose-positive strains of E. coli, isolated after 15 to 20 days, had a growth rate of 0.22 generation per h on L-ribose. Growth on L-ribose was found to induce the enzymes of the L-arabinose and ribitol pathways, but only ribitol-negative mutants derived from strains originally L-ribose positive lost the ability to grow on L-ribose, showing that a functional ribitol pathway was required. One of the mutations permitting growth on L-ribose enabled the mutants to produce constitutively an NADPH-linked reductase which converted L-ribose to ribitol. L-ribose is not metabolized by an isomerization to L-ribulose, as would be predicted on the basis of other pentose pathways in enteric bacteria. Instead, L-ribose was metabolized by the reduction of L-ribose to ribitol, followed by the conversion to D-ribulose by enzymes of the ribitol pathway.

Purification and properties of NADP(+)-dependent glycerol dehydrogenases from Aspergillus nidulans and A. niger.

Glycerol dehydrogenase, NADP(+)-specific (EC 1.1.1.72), was purified from mycelium of Aspergillus nidulans and Aspergillus niger using different purification procedures. Both enzymes had an Mr of approximately 38,000 and were immunologically cross-reactive, but had different amino acid compositions and isoelectric points. For both enzymes, the substrate specificity was limited to glycerol and erythritol for the oxidative reaction and to dihydroxyacetone (DHA), diacetyl, methylglyoxal, erythrose and D-glyceraldehyde for the reductive reaction. The A. nidulans enzyme had a turnover number twice that of the A. niger enzyme at pH 6.0, whereas inhibition by NADP+ was less (Ki = 45 microM vs 13 microM). It is proposed that both enzymes catalyse in vivo the reduction of DHA to glycerol and that they are regulated by the anabolic reduction charge.

Gulonolactone oxidase is missing in teleost fish. The direct spectrophotometric assay.

Data in the literature imply that some fish species evolved with the capacity to synthesize ascorbic acid. Gulonolactone oxidase activity has been reported in kidney and/or liver tissues. However, it is shown here that this microsomal enzyme activity is missing in common carp hepatopancreas and kidney, whereas high activity was confirmed in pigeon kidney, rat liver, bovine liver and amphibian (Xenopus) kidney tissues. A new assay using either the whole tissue homogenate or microsomes solubilized by sodium deoxycholate was developed to directly measure the formation of ascorbic acid spectrophotometrically. Identical values were found using this assay as well as the assay in which formed ascorbate was determined by the dinitrophenyl hydrazine (DNPH) method. In some experiments, these results were confirmed by polarographically measured oxygen consumption.

[The detection of sorbitol dehydrogenase in the cytoplasm of liver cells in birds and its relation to alcohol dehydrogenase and glucose-6-phosphate dehydrogenase]. / Obnaruzhenie sorbitoldegidrogenazy v tsitoplazme kletok pecheni nekotorykh ptits i ee sviaz' s alkogol'degidrogenazoi i gliukozo-6-fosfatdegidrogenazoi.

Comparative studies have been made in the specific activity of sorbitol dehydrogenase, glucose-6-phosphate and alcohol dehydrogenases in the cytoplasm from the liver of wild and domestic ducks, hen and pheasant. High activity of all the three enzymes was found in ducks indicating the effective sorbitol (polyol) metabolism of glucose. The activity of glucose-6-phosphate dehydrogenase is an order lower as compared with the activity of sorbitol and alcohol dehydrogenases in the cytoplasm of hen liver. The same relationship was found for the activity of sorbitol dehydrogenase in the cytoplasm of pheasant liver.

[The thiobarbituric method for analyzing sugars. III. A method for studying sorbitol dehydrogenase activity]. / Tiobarbiturov metod za analiz na zakhari. III. Metod za izsledvane na sorbitoldekhidrogenaznata aktivnost.

A method for studying of sorbitol dehydrogenase activity of Acetobacter suboxydans has been worked out which can be applied to other microbe cells too. The enzyme transformation of D-sorbitol into L-sorbose is determined through the reaction between ketohexose and 2-thiobarbituric acid under heating in 1 M oxalic acid (pH = 0.64) medium for 15 or 30 min. The yellow-coloured product was measured at 400 nm. The method is characterized with its high specificity, good reproducibility and accuracy, which allows for its application for quantitative analysis of sorbitol in dietetic foods. For this purpose a sorbitol dehydrogenase preparation isolated from bacterial cells is needed. The main purpose of the spectrophotometric method is to use it as a control in the process of microbial transformation of sorbitol into sorbose both under laboratory and industrial conditions.

[The heterogeneity of sorbitol dehydrogenase from the cytoplasm of mammalian brain cells]. / Geterogennost' sorbitoldegidrogenazy iz tsitoplazmy kletok mozga mlekopitaiushchikh.

The multiple molecular forms of sorbitoldehydrogenase in cytoplasm of brain cells of bull, ground squirrel, guinea-pig, rat, hamster and mouse have been found using the method of electrophoresis in polyacrylamide gel and the subsequent specific dyeing for the fermentative activity. All revealed zones of activity are related to the slowly migrating ones. A set of multiple molecular forms from different sources is various. A form with relative electrophoretic activity 0.385 is found in all analyzed animals. The conditions for obtaining of distinct zones of activity on zymograms are chosen.