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.     

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.     

Expression and Characterization of Monomeric Recombinant Isocitrate Dehydrogenases from Corynebacterium glutamicum and Azotobacter vinelandii for NADPH Regeneration

The enzymatic transformation of various chemicals, especially using NADPH-dependent hydroxylase, into more soluble and/or high value-added products has steadily garnered increasing attention. However, the industrial application of these NADPH-dependent hydroxylases has been limited due to the high cost of the cofactor NADPH. As an alternative, enzymatic NADPH-regeneration systems have been developed and are frequently used in various fields. Here, we expressed and compared two recombinant isocitrate dehydrogenases (IDHs) from Corynebacterium glutamicum and Azotobacter vinelandii in Escherichia coli. Both enzymes were hyper-expressed in the soluble fraction of E. coli and were single-step purified to apparent homogeneity with yields of more than 850 mg/L. These enzymes also functioned well when paired with NADPH consumption systems. Specifically, NADPH was regenerated from NADP⁺ when an NADPH-consuming cytochrome P450 BM3 from Bacillus megaterium was incorporated. Therefore, both enzymes could be used as alternatives to the commonly used regeneration system for NADPH. These enzymes also have promising potential as genetic fusion partners with NADPH-dependent enzymes due to the monomeric nature of their quaternary structure, thereby resulting in self-sufficient biocatalysts via NADPH regeneration in a single polypeptide with NADPH-dependent activity.     

重组大肠杆菌表达17β-羟基类固醇脱氢酶全细胞催化合成宝丹酮的研究

宝丹酮作为一种重要的蛋白同化雄性激素类固醇,具有提升肌肉质量和耐力的功能。宝丹酮的传统合成方法是以1,4-雄烯二酮(ADD)为底物通过化学法合成,但过程复杂、污染严重。17β-羟基类固醇脱氢酶(17β-HSD)可催化甾体化合物C-17位点的氧化还原反应,实现ADD和宝丹酮的相互转化。本研究通过基因序列同源性分析,筛选到6种不同来源的17β-HSD基因并对其在大肠杆菌中进行异源表达。利用不同重组菌转化ADD合成宝丹酮,结果表明重组菌BL21/pET28a-HSDPy的ADD转化率最高,因此选择BL21/pET28a-HSDPy进行进一步研究。鉴定了重组菌的酶学性质并优化其全细胞转化条件。结果表明在生物量为36 g·L^-1、底物浓度为5.40 g·L^-1条件下,经过两次补料,获得了3.66g·L^-1宝丹酮,比优化前提高了4.1倍。而且在生物转化过程中未检测到副产物。为生物合成宝丹酮提供了可能。     

醇脱氢酶在聚乙烯膜表面的固定化研究

以聚丙烯酸(PAA)改性的聚乙烯(PE)膜为载体,研究了醇脱氢酶(ADH)的两种固定化路线,并以甲醛为底物考察了固定化酶的催化性能。路线1用聚乙烯亚胺(PEI)进一步改性,使用戊二醛(GA)固定化ADH。最优固定化pH为6.0,温度为5～15℃,酶浓度为1.0 mg/ml,GA浓度为0.01%(质量);固定化酶的最适反应pH为6.5,温度为15～30℃,反应速率最高为9.6μmol/(L·min);重复利用10次后可保持47.3%的活性。路线2以PAA-PE为载体,用1-(3-二甲氨基丙基)-2-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)为活化剂,固定化ADH。EDC和NHS最优摩尔比为1∶0.5,固定化时间为24 h;固定化酶的最适反应pH为6.5,温度为20～37℃,反应速率为15.58μmol/(L·min);重复利用10次后可保持53.8%的活性。     

Tracking W-Formate Dehydrogenase Structural Changes During Catalysis and Enzyme Reoxidation

Metal-dependent formate dehydrogenases (Fdh) catalyze the reversible conversion of CO₂ to formate, with unrivalled efficiency and selectivity. However, the key catalytic aspects of these enzymes remain unknown, preventing us from fully benefiting from their capabilities in terms of biotechnological applications. Here, we report a time-resolved characterization by X-ray crystallography of the Desulfovibrio vulgaris Hildenborough SeCys/W-Fdh during formate oxidation. The results allowed us to model five different intermediate structures and to chronologically map the changes occurring during enzyme reduction. Formate molecules were assigned for the first time to populate the catalytic pocket of a Fdh. Finally, the redox reversibility of DvFdhAB in crystals was confirmed by reduction and reoxidation structural studies.     

Long-Chain and Medium-Chain Fatty Acids in Energy Metabolism of Murine Kidney Mitochondria

Scientists have long established that fatty acids are the primary substrates for kidney mitochondria. However, to date we still do not know how long-chain and middle-chain fatty acids are oxidized at the mitochondrial level. Our previous research has shown that mitochondria from the heart, brain, and kidney oxidize palmitoylcarnitine at a high rate only in the presence of succinate, glutamate, or pyruvate. In this paper, we report properties of the isolated kidney mitochondria and how malate and succinate affect the oxidation of C16 and C8 acylcarnitines. The isolated kidney mitochondria contain very few endogenous substrates and require malate to oxidize pyruvate, glutamate, and C16 or C8 acylcarnitines. We discovered that with 10 µM of C16 or C8 acylcarnitines, low concentrations of malate (0.2 mM) or succinate (0.5 mM) enhance the States 4 and 3 respiratory rates several times. The highest respiration rates were observed with C16 or C8 acylcarnitines and 5 mM succinate mixtures. Results show that kidney mitochondria, unlike the heart and brain mitochondria, lack the intrinsic inhibition of succinate dehydrogenase. Additionally, results show that the oxidation of fatty acid by the small respirasome’s supercomplex generates a high level of CoQH2, and this makes SDH in the presence of succinate reverse the flow of electrons from CoQH2 to reduce fumarate to succinate. Finally, we report evidence that succinate dehydrogenase is a key mitochondrial enzyme that allows fast oxidation of fatty acids and turns the TCA cycle function from the catabolic to the anabolic and anaplerotic metabolic pathways.