Regulation of the NADH supply by nuoE deletion and pncB overexpression to enhance hydrogen production in Enterobacter aerogenes

In this study, seven mutants from E. aerogenes IAM1183 wildtype were constructed via different strategies including deletion of lactate dehydrogenase, disruption of NADH dehydrogenase gene nuoE, overexpression of pncB and a combination of both to regulate of the NADH supply to enhance hydrogen production. Compared with the parental strain, the hydrogen yields of the strains IAM1183-E, IAM1183-L and IAM1183-EL increased by 23.3, 81.7 and 97.9%, respectively. When the pncB gene was overexpressed, the hydrogen yield of IAM1183/P, IAM1183-E/P, IAM1183-L/P and IAM1183-EL/P increased by 39.0, 6.5, 5.9, and 5.1% compared with the respective original knockout strains. Among them, the total hydrogen yield of strain IAM1183-EL/P with highest production efficiency was 58% higher than IAM1183. Further metabolite analysis indicated that the knockout of nuoE and ldhA, combined with the overexpression of pncB, resulted in a redistribution of the metabolic fluxes in E. aerogenes, which led to an improvement of the hydrogen yield.     

Kinetic behaviour of muscle LDH immobilized in nylon tubing

Immobilization was carried out of the lactate dehydrogenase (LDH) from rabbit muscle (EC 1.1.1.27), cross-linked through the bifunctional reactive glutar-aldehyde on to nylon tubing (1 m long, 53cm² internal surface area). Immobilized LDH inactivation kinetics are of first order (t_1/2 = 3·6 years, k = 5·4,e^?4 day^?1 to 5°C). The smaller effect of pH on activity than in the case of LDH in solution can be explained on the basis of limitation to proton diffusion towards the support. A limiting effect to free external diffusion of the substrate towards and products from the support was also observed, an effect which seems to determine the effective kinetic behaviour of immobilized LDH. The apparent optimum temperature is centred around 40°C, observing a clear inactivation (thermal denaturation) above this temperature. In the temperature range studied (10–40°C), the co-existence was seen of a kinetic control accompanied by another control, involving diffusional transport of substrates and products, on the global activity of the immobilized enzyme. This makes the Arrhenius profiles curvilinear. Both graphic and statistical non-linear regression analysis of the kinetic data—rate, v, versus substrate concentration [S]—carried out under conditions in which the diffusional limitations can be considered negligible (high recirculation flow rate), permitted investigation of the intrinsic kinetic behaviour of immobilized LDH. In this sense, it can be deduced that the rate equation to which these data seem to be fitted is of the polynomial quotient type in [S] of minimum degree 2:2. Although the diffusional limitations have a marked effect on the type of global kinetics shown by immobilized LDH, temperature was not found to affect its v[S] behaviour. The experimental evidence obtained thus indicates that the rate equation in the 10-40°C temperature range continues to be a rational equation of at least degree 2:2 in [S].     

A new amperometric biosensor for fructose determination based on epoxy-graphite-TTF-TCNQ-FDH-biocomposite

In this work a novel amperometric biosensor for fructose determination in solutions was developed. The device was constructed by the incorporation of a tetrathiofulvalene-tetracyanoquinodimethane organic conducting salt and fructose dehydrogenase enzyme, include in a polymeric matrix of epoxy resin and graphite powder. Because of the electrocatalytic function of the salt, the direct transfer of the electron between the reduced prosthetic group (PQQH₂) of the enzyme and the transducing material, was verified at a low working potential (150 mV vs. Ag/AgCl), where the interfering reactions were minimized. The response time at 90% of the steady state value was less than 20 s. The current response was directly proportional to the D-fructose concentration from 0.01 to 0.3 mmol/l with a detection limit of 0.005 mmol/l (signal/noise of 3) and a sensitivity of 1.9985 μA/mmol. The biosensor sensitivity diminishes when its surface is not polished between successive determinations, and remains constant (rsd=1.85, n=10) when the surface is polished between determinations. The effects of temperature and pH on the biosensor response were studied and analyzed; also the properties of the enzyme (Km _ap, I _max, Q₁₀) were determinate in this work. The biosensor was used to determine fructose in high fructose syrups and there were not significant differences between these results and those obtained by HPLC (p≤0.05). During 4 months, in intermittent determinations the biosensor kept 100% of its original sensitivity and after 18 months stored at 4°C, it only lost 32% of its sensitivity. The simplicity, low working potential, high stability and good performance of this biosensor shows a great potential for its use in the fructose determination.     

The Mystery of Extramitochondrial Proteins Lysine Succinylation

Lysine succinylation is a post-translational modification which alters protein function in both physiological and pathological processes. Mindful that it requires succinyl-CoA, a metabolite formed within the mitochondrial matrix that cannot permeate the inner mitochondrial membrane, the question arises as to how there can be succinylation of proteins outside mitochondria. The present mini-review examines pathways participating in peroxisomal fatty acid oxidation that lead to succinyl-CoA production, potentially supporting succinylation of extramitochondrial proteins. Furthermore, the influence of the mitochondrial status on cytosolic NAD⁺ availability affecting the activity of cytosolic SIRT5 iso1 and iso4—in turn regulating cytosolic protein lysine succinylations—is presented. Finally, the discovery that glia in the adult human brain lack subunits of both alpha-ketoglutarate dehydrogenase complex and succinate-CoA ligase—thus being unable to produce succinyl-CoA in the matrix—and yet exhibit robust pancellular lysine succinylation, is highlighted.     

Effect of coumarin and xanthotoxin on mitochondrial structure,oxygen uptake,and succinate dehydrogenase activity in onion root cells

At concentrations in which they occur on the plant surface and retard mitosis, coumarin and xanthotoxin lowered uptake of oxygen (by 60 and 30%, respectively) by meristematic cells ofAllium cepa root tips. They caused changes in the structure of the mitochondrial matrix to become dense, and protrusions of mitochondrial membranes were visible parallelling their hypertrophy, indicating alteration in the structure and physiology of these organelles. Coumarin and, to a lesser extent, xanthotoxin increased succinate dehydrogenase production in mitochondria and also in the cytoplasm, indicating changes in membrane permeability. Changes in oxygen uptake and mitochondrial structure, in addition to the retardation of mitosis, may be the reason these compounds act as allelochemicals after they have been removed from the plant surface and reach the root meristem.     

丙酮酸脱氢酶复合酶系研究进展

对丙酮酸脱氢酶复合酶系的组成、功能及特性进行了简要的综述.丙酸酸脱氢酶系是1个定位在线粒体中的多酶复合体.它是由3种酶:丙酮酸脱氢酶(E1)、二氢硫辛酸乙酰转移酶(E2)、二氢硫辛酸脱氢酶(E3)和调节它的活性的丙酮酸脱氢酶激酶、一种丙酮酸脱氢酶磷酸酶以及功能未知的蛋白X,还有一些辅助因子组成.在能量代谢调控过程中,丙酮酸脱氢酶系是1个主要的酶系.丙酸脱氢酶转化来自于碳水化合物和一些氨基酸的丙酮酸成为乙酰辅酶A.乙酰辅酶A是三羧酸循环的底物.丙酮酸脱氢酶系活性的调节可以通过PDH的磷酸化和脱磷酸化来调节平衡在许多组织中的源于碳水化合物和脂肪酸的乙酰辅酶A.丙酮酸脱氢酶发生突变后而不能行使正常功能,乳酸集结引起乳酸中毒.     

Effect of diet on phosphine toxicity,rate of development and reproduction of the rice weevil Sitophilus oryzae (Linnaeus)

One of the loci responsible for strong phosphine resistance encodes dihydrolipoamide dehydrogenase (DLD). The strong co-incidence of enzyme complexes that contain DLD, and enzymes that require thiamine as a cofactor, motivated us to test whether the thiamine deficiency of polished white rice could influence the efficacy of phosphine fumigation against insect pests of stored grain. Three strains of Sitophilus oryzae (susceptible, weak and strong resistance) were cultured on white rice (thiamine deficient), brown rice or whole wheat. As thiamine is an essential nutrient, we firstly evaluated the effect of white rice on developmental rate and fecundity and found that both were detrimentally affected by this diet. The mean time to reach adult stage for the three strains ranged from 40 to 43 days on brown rice and 50–52 days on white rice. The mean number of offspring for the three strains ranged from 7.7 to 10.3 per female over a three day period on brown rice and 2.1 to 2.6 on white rice. Growth and reproduction on wheat was similar to that on brown rice except that the strongly resistant strain showed a tendency toward reduced fecundity on wheat. The susceptible strain exhibited a modest increase in tolerance to phosphine on white rice as expected if thiamine deficiency could mimic the effect of the dld resistance mutation at the rph2 locus. The strongly resistant strain did not respond to thiamine deficiency, but this was expected as these insects are already strongly resistant. We failed, however, to observe the expected synergistic increase in resistance due to combining thiamine deficiency with the weakly resistant strain. The lack of interaction between thiamine content of the diet and the resistance genotype in determining the phosphine resistance phenotype suggests that the mode of inhibition of the complexes is a critical determinant of resistance.