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.

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.

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.

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.

Isolation and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus.

A protocol for the rapid purification of the glycerol dehydrogenase (glycerol: NAD+ 2-oxidoreductase, EC 1.1.1.6) from the thermophile Bacillus stearothermophilus has been developed using a combination of chromatographic techniques including affinity chromatography on a Sepharose-immobilised triazine dye (Procion red, HE3B, ICI). Substrate specificity has been examined and Km values determined. The protein has been shown to have an oligomeric Mr of approx. 180,000 and consists of four identical subunits of Mr 42,000. Exposure to chelating agents (e.g., EDTA) leads to total loss of activity; the EDTA-inactivated enzyme can be reactivated by Zn2+ and requires 1 mol equivalent of zinc per subunit for full catalytic activity. Other divalent cations such as Cd2+ and Co2+ will reactivate the apo-enzyme but yields an enzyme of lower specific activity. The enzyme binds 1 equivalent of NADH per subunit and during catalysis transfers the 4-pro-R hydride from the nicotinamide ring of the reduced-coenzyme to the substrate. Glycerol increases the dissociation constant for the interaction between NADH and Zn-metallo-glycerol dehydrogenase (ZnGDH) but has no effect on the equilibrium between NADH and metal-depleted enzyme.

Immunohistochemical localization of aldose reductase. I. Enzyme purification and antibody preparation--localization in peripheral nerve, artery, and testis.

Aldose reductase (AR) was purified from rat and bovine seminal vesicles using DEAE-cellulose, hydroxylapatite, and Sephadex-gel column chromatography. The purification resulted in the obtention of an AR pool and a contaminating pool. Antibodies were raised in rabbits against both enzymes by subcutaneous injection of the AR pool. The antisera was judged to be specific for AR by immunoprecipitation of AR activity and by Ouchterlony double immunodiffusion and immunoelectrophoretic methods. Antibodies against rat AR were used in the unlabeled antibody-enzyme (PAP) technique to demonstrate the cellular location of the enzyme in a number of tissues known to be sites of diabetic lesions. Antibodies against bovine AR were not cross reactive with the rat enzyme, as determined by Ouchterlony and competitive protein-binding studies. AR was localized in rat tissues to the Schwann cell sheath of peripheral nerve, arterial endothelium, and the sustentacular (Sertoli) cells and mature spermatids of the testis.

Immunohistochemical localization of aldose reductase. II. Rat eye and kidney.

Aldose reductase (AR) was purified from rat seminal vesicles. Specific antibodies to this enzyme were prepared in rabbits and were used in the unlabeled antibody-enzyme (PAP) technique to localize AR in a number of tissues known to be sites of diabetic lesions. AR was localized in the following structures in the eye: lens epithelial lining and cortical lenticular fibers; corneal endothelium; the inner, nonpigmented layer of ciliary body epithelium and its extension as the posterior surface of the iris; and neuroglial (Müller) cells in the retina. Retinal capillary endothelium did not contain immunoreactive AR. In the kidney, staining was intense in the inner medulla. Specific structures included thin limbs of the loop of Henle, collecting tubules deep in the inner medulla, and transitional epithelial cells lining the pelvic space: structures in the cortex including glomerular podocytes and distal convoluted tubules. Collecting tubules in the outer medulla and cortex, as well as proximal convoluted tubules and glomerular capillary endothelium did not contain immunoreactive AR.

Localization of aldose reductase in the human eye.

The presence of the enzyme aldose reductase is increasingly being linked to diabetic complications. The distribution of this enzyme in human cornea, lens, retina, and optic nerve has been studied using specific antibodies against purified human placental aldose reductase raised in both rabbit and goat. The antisera from both animals gave equal, specific reactions. In frozen sections of ocular tissues, significant aldose reductase localization was reproducibly demonstrated in the endothelium and epithelium of the cornea and in the basal cell layers of the conjunctiva. In the lens, staining was observed in the epithelium and superficial lens fibers. In retinal sections, the presence of aldose reductase was demonstrated in the Mueller's cells, especially near the inner limiting membrane. It was also found in some ganglion and cone cells. In the optic nerve, positive staining was observed in the axon. All other cells of the tissues examined revealed only weak, nonspecific staining.

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.

Aldose reductase and p-crystallin belong to the same protein superfamily as aldehyde reductase.

Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH-dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40-50% homology with p-crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and p-crystallin are members of a superfamily of related proteins.

Acetyl-blocked N-terminal structures of sorbitol and aldehyde dehydrogenases.

Two new dehydrogenase structures, the 354-residue polypeptide chain of sorbitol dehydrogenase (from sheep liver) and the 500-residue polypeptide chain of cytoplasmic aldehyde dehydrogenase (from human liver), have blocked N-termini. The N-terminal peptides were purified by reverse-phase high-performance liquid chromatography and submitted to mass spectrometry after derivatization. They were also analyzed by dipeptidyl carboxypeptidase digestion, utilizing gas chromatography-mass spectrometry for dipeptide identifications. Results are consistent and establish that sorbitol dehydrogenase has N-terminal acetylalanine and aldehyde dehydrogenase N-terminal acetylserine in amino acid sequences that are compatible with estimates from chemical analyses. The two N-terminal residues found are typical of acetylated proteins in general, extend the group of known acetylated dehydrogenases, and show that these intracellular proteins are frequently N-terminally acetylated.

Aldose reductase localization in human retinal mural cells.

A hallmark of early diabetic retinopathy is the selective loss of the retinal mural cells (pericytes) from vessels. Using antibodies prepared against purified human placental aldose reductase, the presence of the enzyme aldose reductase can be demonstrated immunohistochemically in the cytoplasm of retinal mural cells of trypsin-digested human retinal vessels. This enzyme, which reduces various hexose sugars to their sugar alcohols, has been implicated in the pathogenesis of several diabetic complications.

The sorbitol pathway in the human lens: aldose reductase and polyol dehydrogenase.

The sorbitol pathway in human lenses is evaluated on the enzymic level. Adult lenses, normal and nondiabetic as well as diabetic cataracts, are found to contain limited levels of aldose reductase (AR) and high levels of polyol dehydrogenase (PD) relative to the animal lens. AR is confined primarily to the lens epithelium and is two to three times higher in juvenile lenses than in the adult lens. The level of AR in the epithelium of juvenile lenses is sufficient to cause significant osmotic stress. The Km of glucose of AR is roughly 200 mM, whereas the Km for NADPH is 0.06 mM. NADP inhibits human lens AR noncompetitively and has a Ki equivalent to the Km for NADPH. PD occurs in both the lens epithelium and cortex, remains persistently high with age, and decreases with increased cortical involvement. The Km of sorbitol for PD is 1.4 mM and for NAD is 0.06 mM. NADH (Ki 0.002 mM) competitively inhibits PD in the forward direction. PD purified 100-fold from diabetic and nondiabetic cataracts and normal lenses exhibit similar kinetic constants. PD has an extremely high Vmax in the fructose-to-sorbitol direction. The Km of fructose is 40 mM and for NADH is 0.02 mM. At high enough concentration, alrestatin also inhibits PD. The added activities of AR and PD in producing sorbitol and fructose in combination with decreased hexokinase with age may account for diabetic cataract formation in human lenses exposed to a high glucose stress. Nucleotide levels are reported for senile cataractous lenses.

Purification and properties of N-acyl-D-mannosamine dehydrogenase from Flavobacterium sp. 141-8.

A new enzyme, N-acyl-D-mannosamine dehydrogenase, was purified to apparent homogeneity from a cell-free extract of Flavobacterium sp. 141-8 and some of its properties were investigated. The enzyme showed optimum activity at pH 8.0-9.5. N-Acetyl- and N-glycolyl-D-mannosamine were oxidized but other commonly existing sugars, such as N-acetylglucosamine, N-acetylgalactosamine, amino sugars, neutral hexoses, and pentoses, were not oxidized. NAD+ was specifically utilized as an effective hydrogen acceptor. The apparent Km values for N-acetyl- and N-glycolyl-D-mannosamine, and NAD+ were 1.0, 13.3, and 0.41 mM, respectively. The stoichiometry data showed that 1 mol each of N-acetyl-D-mannosamine and NAD+ were converted to 1 mol each of N-acetyl-D-mannosaminic acid and NADH, respectively. Although the formation of lactone was detected in the enzyme reaction mixture, the reverse reaction of the enzyme, the reduction of N-acetyl-D-mannosamino-lactone, was not observed. The enzyme activity was strongly inhibited by Hg2+ and SDS, but metal-chelating reagents and sulfhydryl-group-blocking reagents had almost no effect. The molecular weight of the enzyme was estimated to be 120,000 on gel filtration and 29,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was at pH 4.8. On trial application of the enzyme, it was indicated that N-acetylneuraminic acid can be determined quantitatively with the combined enzyme system involving the new enzyme and N-acetylneuraminic acid aldolase.

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.

Microdetermination of aldose and aldehyde reductases from human tissues.

Microfluorometric method has been described for the determination of aldose reductase and aldehyde reductase II activities in human erythrocyte, brain, and lens. The enzyme activity determined by the microfluorometric method was compared with the activity determined spectrophotometrically by following the oxidation of NADPH and fluorometrically by the formation of sorbitol. The activity of aldose reductase in homogenous preparations from human lens, brain, and erythrocyte was identical when determined by NADPH oxidation, NADP formation, and sorbitol formation using glucose as substrate and NADPH as co-factor. This indicated that NADPH oxidation by aldose reductase is not due to a non-specific oxidation by oxidants generated as a result of interaction of aldose reductase and glucose. Similarly, the activity of aldehyde reductase II obtained by NADPH oxidation and that by NADP formation were in good agreement. The microfluorescent method is convenient and accurate and can be used for the determination of aldose reductase in human tissues using glucose as substrate even when the sample size is small.

[On the localization of sorbitol dehydrogenase in the rat kidney (author's transl)]. / Zur Lokalisation der Sorbitdehydrogenase in der Rattenniere.

We have investigated the localization and demonstration of sorbitol dehydrogenase activity in the rat kidney by comparative histochemical-electrophoretical technique. Under histochemical conditions an adequate demonstration of the activity of the sorbitol dehydrogenase in native sections is possible by membrane incubating technique in presence of PMS and KCN. In the glomerulum the strong and uniform reaction with regard to the specifity is not clear. In the glomerula of semithin sections from glutaraldehyde fixed, incubated and Epon-embedded cryostate sections the reaction product is localized predominantly in the maesangium cells. By micropolyacrylamidgel gradient electrophoresis of the supernatant from the kidney cortex and of isolated glomerula 2 sorbitol dehydrogenase fractions were found. The results are discussed in connection with the specifity of the reaction.

[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.

[Physico-chemical characteristics of sorbitol dehydrogenase from the cytoplasm of bovine liver cells]. / Fiziko-khimicheskie kharakteristiki sorbitoldegidrogenazy iz tsitoplazmy kletok pecheni byka.

Partially purified preparation of sorbitol dehydrogenase, isolated from hepatocytes of bovine liver tissue, was active at a wide range of pH exhibiting the maximal activity at pH 9.0 in presence of NAD but not of NADP. The high rate of sorbitol and xylitol dehydration was observed, whereas the enzyme dehydrated ribitol at the 4-fold lower rate. Disc electrophoresis of the preparation in polyacrylamide gel, where sorbitol, xylitol and ribitol were used as substrates for colorimetric detection of the enzyme activity, exhibited six enzymatic zones with Rf 0.387, 0.266, 0.338, 0.193, 0.129 and 0.064. Optimal conditions were developed for storage of the active enzyme.