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1.
N,N'-dimethyl-4,4'-azopyridinium methyl sulfate (MAZP) was characterized as an electron transfer mediator for oxidation reactions catalyzed by NAD+- and pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases. The bimolecular rate constant of NADH reactivity with MAZP was defined as (2.2 ± 0.1) × 105 M−1 s−1, whereas the bimolecular rate constant of reactivity of the reduced form of PQQ-dependent alcohol dehydrogenase with MAZP was determined to be (4.7 ± 0.1) × 104 M−1 s−1. The use of MAZP for the regeneration of the cofactors was investigated by applying the electrochemical oxidation of the mediator. The total turnover numbers of mediator MAZP and cofactor NADH for ethanol oxidation catalyzed by NAD+-dependent alcohol dehydrogenase depended on the concentration of the substrate and the duration of the electrolysis, and the yield of the reaction was limited by the enzyme inactivation and the electrochemical process. The PQQ-dependent alcohol dehydrogenase was more stable, and the turnover number of the enzyme reached a value of 2.3 × 103. In addition, oxidation of 1,2-propanediol catalyzed by the PQQ-dependent alcohol dehydrogenase proceeded enantioselectively to yield l-lactic acid. 相似文献
2.
Xiaoju Chen Shaotong Jiang Zhi Zheng Lijun Pan Shuizhong Luo 《Process Biochemistry》2012,47(8):1250-1255
During mixed-acid fermentation by Corynebacterium crenatum under anaerobic conditions, two moles of NADH are required to synthesize 1 mol of succinic acid. In this work, four controlled culture redox potentials and different carbon sources with different oxidation states were used to investigate the possibility of enhancing the succinic acid production by increasing the availability of NADH. When the culture redox potential was ?300 mV, the yield of succinic acid was 0.31 g/g, representing a 72% increase compared with the yield when the culture redox potential was ?40 mV. Meanwhile, the molar ratio of succinic acid/lactic acid increased from 0.27 to 0.48. When 0.1% neutral red was added to the acid production medium, the yield of succinic acid was 0.25 g/g, and the molar ratio of succinic acid/lactic acid was 0.38. Both values were higher than those obtained from glucose only (0.19 g/g, 0.26) or gluconate (0.05 g/g, 0.18). A higher NADH/NAD+ ratio and increased enzymatic activity could be achieved to enhance the succinic acid production by manipulating the culture to a more reductive environment. 相似文献
3.
In wild-type Escherichia coli, 1 mol of CO2 was fixated in 1 mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD+ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD+ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72 h, 14.5 g L−1 of glucose was consumed to generate 12.08 g L−1 of succinic acid. Furthermore, under optimized condition of CO2 supply, the succinic acid productivity and the CO2 fixation rate reached 223.88 mg L−1 h−1 and 83.48 mg L−1 h−1, respectively. 相似文献
4.
Bo Peng Chunrong Liu Zhen Li Jacob J. Day Yun Lu David J. Lefer Ming Xian 《Bioorganic & medicinal chemistry letters》2017,27(3):542-545
Here we report the model studies of the reactions between NADH models (using HEH and BNAH) and sulfane sulfurs (using polysulfides). Such reactions could lead to the oxidation of NADH models and the production of hydrogen sulfide (H2S). Kinetics of the reaction between BNAH and elemental sulfur S8 were determined in ethanol and the second-order rate constant was found to be 0.074 M?1 min?1 (at 37 °C) suggesting this is a slow process. 相似文献
5.
《Journal of Molecular Catalysis .B, Enzymatic》2011,72(3-4):159-165
Active pharmaceutical ingredients (APIs) such as l-sugars and keto acids are favorably accessed through selective oxidation of sugar alcohols and amino acids, respectively, catalyzed by NAD(P)-dependent dehydrogenases. Cofactor regeneration from NAD(P)H conveniently is achieved via water-forming NAD(P)H oxidases (nox2), which only need molecular oxygen as co-substrate. Turnover-dependent overoxidation of the conserved cysteine residue in the active site of water-forming NADH oxidases is the presumed cause of the limited nox2 stability.We present a novel NAD(P)H oxidase, NoxV from Lactobacillus plantarum, with specific activity of 167 U/mg and apparent kinetic constants at air saturation and 25 °C of kcat,app = 212 s−1 and KM,app = 50.2 μM in the broad pH optimum from 5.5 to 8.0. The enzyme features a higher stability than other NAD(P)H oxidases against overoxidation, as is evidenced by a higher total turnover number, in the presence (168,000) and, most importantly, also in the absence (128,000) of exogenously added reducing agents. While the native enzyme shows exclusively activity on NADH, we engineered the substrate binding pocket to generate variants, G178K,R and L179K,R,H that accommodate and oxidize both NADH and NADPH as substrates. 相似文献
6.
《Process Biochemistry》2010,45(6):980-985
Carbon sources with different oxidation states were used to investigate the possibility increasing the availability of NADH and the NADH/NAD+ ratio and to determine the effect of this manipulation on the distribution of metabolites in Actinobacillus succinogenes NJ113. The sugars glucose, sorbitol and gluconate were each used at an initial concentration of 40 g/L.The yield of succinic acid (0.75) and the ratio of succinic acid to acetic acid (5.06) were both higher for sorbitol than the values obtained with glucose (0.66 and 2.68, respectively). In contrast, with gluconate as the carbon source the yield of succinic acid was 0.54 and the ratio of succinic acid to acetic acid was only 1.70. This work showed that different levels of NADH availability and the NADH/NAD+ ratio can be achieved by using carbon sources that have different oxidation states.Highly reduced sorbitol was examined as a possible carbon substrate for maximizing the redox potential during the production of succinic acid. 相似文献
7.
Peter S. Dragovich Benjamin P. Fauber Laura B. Corson Charles Z. Ding Charles Eigenbrot HongXiu Ge Anthony M. Giannetti Thomas Hunsaker Sharada Labadie Yichin Liu Shiva Malek Borlan Pan David Peterson Keith Pitts Hans E. Purkey Steve Sideris Mark Ultsch Erica VanderPorten Xuying Zhang 《Bioorganic & medicinal chemistry letters》2013,23(11):3186-3194
A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH) was identified by high-throughput screening (IC50 = 8.1 μM). Biochemical, surface plasmon resonance, and saturation transfer difference NMR experiments indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50 = 0.48 μM). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure–activity relationships. 相似文献
8.
Previous studies have made many exciting achievements on pushing the functional reversal of beta-oxidation cycle (r-BOX) to more widespread adoption for synthesis of a wide variety of fuels and chemicals. However, the redox cofactor requirement for the efficient operation of r-BOX remains unclear. In this work, the metabolic efficiency of r-BOX for medium-chain fatty acid (C6-C10, MCFA) production was optimized by redox cofactor engineering. Stoichiometric analysis of the r-BOX pathway and further experimental examination identified NADH as a crucial determinant of r-BOX process yield. Furthermore, the introduction of formate dehydrogenase from Candida boidinii using fermentative inhibitor byproduct formate as a redox NADH sink improved MCFA titer from initial 1.2 g/L to 3.1 g/L. Moreover, coupling of increasing the supply of acetyl-CoA with NADH to achieve fermentative redox balance enabled product synthesis at maximum titers. To this end, the acetate re-assimilation pathway was further optimized to increase acetyl-CoA availability associated with the new supply of NADH. It was found that the acetyl-CoA synthetase activity and intracellular ATP levels constrained the activity of acetate re-assimilation pathway, and 4.7 g/L of MCFA titer was finally achieved after alleviating these two limiting factors. To the best of our knowledge, this represented the highest titer reported to date. These results demonstrated that the key constraint of r-BOX was redox imbalance and redox engineering could further unleash the lipogenic potential of this cycle. The redox engineering strategies could be applied to acetyl-CoA-derived products or other bio-products requiring multiple redox cofactors for biosynthesis. 相似文献
9.
《Journal of Molecular Catalysis .B, Enzymatic》2007,44(1):27-31
We evaluated the photochemical and enzymatic synthesis of methanol from formaldehyde with alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae and NAD+ photoreduction by the visible-light sensitization of zinc tetraphenylporphyrin tetrasulfonate (ZnTPPS) in the presence of methylviologen (MV2+), diaphorase, and triethanolamine (TEOA). When the sample solution containing ZnTPPS, MV2+, NAD+, diaphorase, and TEOA in potassium phosphate buffer solution was irradiated, the NADH produced increased with the irradiation time. After irradiation for 180 min, the conversion yield of NAD+ to NADH was about 60% under 0.1 mM NAD+ condition. The methanol production also depended on the conversion yield of NAD+ to NADH. After irradiation for 180 min, 0.38 μM of methanol was produced from formaldehyde (16 μM). The conversion ratio of formaldehyde to methanol was about 2.3%. This result indicates that a system for the photochemical synthesis of methanol from formaldehyde was developed with ADH and the NADH produced by the photosensitization of ZnTPPS in water media. 相似文献
10.
Reducing equivalents are an important cofactor for efficient synthesis of target products. During metabolic evolution to improve succinate production in Escherichia coli strains, two reducing equivalent-conserving pathways were activated to increase succinate yield. The sensitivity of pyruvate dehydrogenase to NADH inhibition was eliminated by three nucleotide mutations in the lpdA gene. Pyruvate dehydrogenase activity increased under anaerobic conditions, which provided additional NADH. The pentose phosphate pathway and transhydrogenase were activated by increased activities of transketolase and soluble transhydrogenase SthA. These data suggest that more carbon flux went through the pentose phosphate pathway, thus leading to production of more reducing equivalent in the form of NADPH, which was then converted to NADH through soluble transhydrogenase for succinate production. Reverse metabolic engineering was further performed in a parent strain, which was not metabolically evolved, to verify the effects of activating these two reducing equivalent-conserving pathways for improving succinate yield. Activating pyruvate dehydrogenase increased succinate yield from 1.12 to 1.31 mol/mol, whereas activating the pentose phosphate pathway and transhydrogenase increased succinate yield from 1.12 to 1.33 mol/mol. Activating these two pathways in combination led to a succinate yield of 1.5 mol/mol (88% of theoretical maximum), suggesting that they exhibited a synergistic effect for improving succinate yield. 相似文献
11.
《Process Biochemistry》2014,49(5):740-744
The effects of oxido-reduction potential (ORP) control on succinic acid production have been investigated in Escherichia coli LL016. In LL016, two CO2 fixation pathways were achieved and NAD+ supply was enhanced by co-expression of heterologous pyruvate carboxylase (PYC) and nicotinic acid phosphoribosyltransferase (NAPRTase). During anaerobic fermentation, cell growth and metabolite distribution were changed with redox potential levels in the range of −200 to −400 mV. From the results, the ORP level of −400 mV was preferable, which resulted in the high succinic acid concentration (28.6 g/L) and high succinic acid productivity (0.33 g/L/h). Meanwhile, the yield of succinic acid at the ORP level of −400 mV was 39% higher than that at the ORP level of −200 mV. In addition, a higher NADH/NAD+ ratio and increased enzyme activities were also achieved by regulating the culture to a more reductive environment, which further enhanced the succinic acid production. 相似文献
12.
《Process Biochemistry》2014,49(12):2039-2043
Bacitracin fermentation by Bacillus licheniformis in this work showed three characteristics: (1) the extracellular propionate, butyrate, acetoin and 2,3-butanediol accumulates under conditions of low dissolved oxygen (zero after 4 h cultivation), reaching a total content of approximately 11.1 g/L; (2) cell growth occurs quickly subsequent to cell autolysis and the second growth; and (3) there is a low content of 2,3-butanediol, a reduced product of acetoin catalyzed by acetoin reductase, in the culture process. In this study, addition of MnCl2 (0.3 mg/L) to the production medium increased the acetoin reductase activity, redirected the NADH oxidation partly from the propionate- and butyrate-production pathways to the 2,3-butanediol synthesis pathway, reduced the intracellular NADH/NAD+ ratio, and facilitated cell growth, ultimately achieving a 11.6% increase in bacitracin production (1076 U/mL) versus the control. The results provide useful information regarding large-scale bacitracin production by B. licheniformis. 相似文献
13.
Exopolysaccharides (EPS) are important food and drug additives with beneficial antioxidant, anticancer, and immune-related effects on human health. However, the EPS is limited by low yields and the need for complex culture conditions in fermentation. Here, we report that hydrogen peroxide and calcium stimulated probiotic activity and production of crude exopolysaccharide (c-EPS) by Lactobacillus rhamnosus ZY. Accordingly, supplementation with 3 mM H2O2 allowed c-EPS biosynthesis to reach 567 mg/L after 24 h. Addition of both CaCl2 and H2O2 resulted in a c-EPS yield of 2498 mg/L after 12 h, over 9-fold higher than that of an anaerobic culture. We observed that exposure to calcium and hydrogen peroxide made the cells more hydrophobic and led to the over-expression of GroEL, NADH peroxidase, and glyceraldehyde 3-phosphate dehydrogenase, thus increasing energy storage and EPS production. Chromatographic analysis revealed c-EPS was composed mainly of mannose (5.1%), galactose (15.3%), glucose (20–30%), and rhamnose (50–60%). Preliminary in vitro tests revealed that H2O2 and CaCl2 enhanced the 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radical scavenging capacities, resulting in a notable protective effect against oxidative damage in NIH/3T3 cells. Our study provides a simple and cost-effective approach for achieving high yields of good quality EPS using Lactobacillus rhamnosus. 相似文献
14.
Continuous anaerobic fermentations were performed in a novel external-recycle, biofilm reactor using d-glucose and CO2 as carbon substrates. Succinic acid (SA) yields were found to be an increasing function of glucose consumption with the succinic acid to acetic acid ratio increasing from 2.4 g g−1 at a glucose consumption of 10 g L−1, to 5.7 g g−1 at a glucose consumption of 50 g L−1. The formic acid to acetic acid ratio decreased from an equimolar value (0.77 g g−1) at a glucose consumption of 10 g L−1 to a value close to zero at 50 g L−1. The highest SA yield on glucose and highest SA titre obtained were 0.91 g g−1 and 48.5 g L−1 respectively. Metabolic flux analysis based on the established C3 and C4 metabolic pathways of Actinobacillus succinogenes revealed that the increase in the succinate to acetate ratio could not be attributed to the decrease in formic acid and that an additional source of NADH was present. The fraction of unaccounted NADH increased with glucose consumption, suggesting that additional reducing power is present in the medium or is provided by the activation of an alternative metabolic pathway. 相似文献
15.
Binghai Zhao Yanhui Chu Dianne O. Hardy Xiao-kun Li Ren-Shan Ge 《The Journal of steroid biochemistry and molecular biology》2010,118(1-2):13-17
Perfluorooctane acid (PFOA) is classified as a persistent organic pollutant and as an endocrine disruptor. The mechanism by which PFOA causes reduced testosterone production in males is not known. We tested our hypothesis that PFOA interferes with Leydig cell steroidogenic enzymes by measuring its effect on 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) activities in rat testis microsomes and Leydig cells. The IC50s of PFOA and mode of inhibition were assayed. PFOA inhibited microsomal 3β-HSD with an IC50 of 53.2 ± 25.9 μM and 17β-HSD3 with an IC50 17.7 ± 6.8 μM. PFOA inhibited intact Leydig cell 3β-HSD with an IC50 of 146.1 ± 0.9 μM and 17β-HSD3 with an IC50 of 194.8 ± 1.0 μM. The inhibitions of 3β-HSD and 17β-HSD3 by PFOA were competitive for the substrates. In conclusion, PFOA inhibits 3β-HSD and 17β-HSD3 in rat Leydig cells. 相似文献
16.
Najeeb Kaid Nasser Al-Shorgani Mohd Sahaid Kalil Wan Mohtar Wan Yusoff Aidil Abdul Hamid 《Saudi Journal of Biological Sciences》2018,25(2):339-348
The effect of pH and butyric acid supplementation on the production of butanol by a new local isolate of Clostridium acetobutylicum YM1 during batch culture fermentation was investigated. The results showed that pH had a significant effect on bacterial growth and butanol yield and productivity. The optimal initial pH that maximized butanol production was pH 6.0 ± 0.2. Controlled pH was found to be unsuitable for butanol production in strain YM1, while the uncontrolled pH condition with an initial pH of 6.0 ± 0.2 was suitable for bacterial growth, butanol yield and productivity. The maximum butanol concentration of 13.5 ± 1.42 g/L was obtained from cultures grown under the uncontrolled pH condition, resulting in a butanol yield (YP/S) and productivity of 0.27 g/g and 0.188 g/L h, respectively. Supplementation of the pH-controlled cultures with 4.0 g/L butyric acid did not improve butanol production; however, supplementation of the uncontrolled pH cultures resulted in high butanol concentrations, yield and productivity (16.50 ± 0.8 g/L, 0.345 g/g and 0.163 g/L h, respectively). pH influenced the activity of NADH-dependent butanol dehydrogenase, with the highest activity obtained under the uncontrolled pH condition. This study revealed that pH is a very important factor in butanol fermentation by C. acetobutylicum YM1. 相似文献
17.
《Process Biochemistry》2014,49(1):25-32
The compound 1,2,4-butanetriol (BT) is a valuable chemical used in the production of plasticizers, polymers, cationic lipids and other medical applications, and is conventionally produced via hydrogenation of malate. In this report, BT is biosynthesized by an engineered Escherichia coli from d-xylose. The pathway: d-xylose → d-xylonate → 2-keto-3-deoxy-d-xylonate → 3,4-dihydroxybutanal → BT, was constructed in E. coli by recruiting a xylose dehydrogenase and a keto acid decarboxylase from Caulobacter crescentus and Pseudomonas putida, respectively. Authentic BT was detected from cultures of the engineered strain. Further improvement on the strain was performed by blocking the native d-xylose and d-xylonate metabolic pathways which involves disruption of xylAB, yjhH and yagE genes in the host chromosome. The final construct produced 0.88 g L−1 BT from 10 g L−1 d-xylose with a molar yield of 12.82%. By far, this is the first report on the direct production of BT from d-xylose by a single microbial host. This may serve as a starting point for further metabolic engineering works to increase the titer of BT toward industrial scale viability. 相似文献
18.
《Bioorganic & medicinal chemistry letters》2014,24(16):3764-3771
A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50 = 1.7 μM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50 = 0.18 μM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure–activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F = 45%). 相似文献
19.
Chanha Jun Young Seung Sa Sol-A Gu Jeong Chan Joo Seil Kim Kyung-Jin Kim Yong Hwan Kim 《Process Biochemistry》2013,48(1):109-117
The demand on thermostable d-lactate dehydrogenases (d-LDH) has been increased for d-lactic acid production but thermostable d-DLHs with industrially applicable activity were not much explored. To identify a thermostable d-LDH, three d-LDHs from different Lactobacillus jensenii strains were screened by genome mining and then expressed in Escherichia coli. One of the three d-LDHs (d-LDH3) exhibited higher optimal reaction temperature (50 °C) than the others. The T5010 value of this thermostable d-LDH3 was 48.3 °C, much higher than the T5010 values of the others (42.7 and 42.9 °C) and that of a commercial d-lactate dehydrogenase (41.2 °C). The Tm values were 48.6, 45.7 and 55.7 °C for the three d-LDHs, respectively. In addition, kinetic parameter (kcat/Km) of d-LDH3 for pyruvate reduction was estimated to be almost 150 times higher than that for lactate oxidation at pH 8.0 and 25 °C, implying that d-lactate production from pyruvate is highly favored. These superior thermal and kinetic features would make the d-LDH3 characterized in this study a good candidate for the microbial production of d-lactate at high temperature from glucose if it is genetically introduced to lactate producing microbial. 相似文献
20.
Caldicellulosiruptor saccharolyticus displays superior H2 yields on a wide range of carbon sources provided that lactate formation is avoided. Nevertheless, a low lactate flux is initiated as the growth rate declined in the transition to the stationary phase, which coincides with a drastic decrease in the glucose consumption and acetate production fluxes. In addition, the decrease in growth rate was accompanied by a sudden increase and then decrease in NADH levels. The V′MAX of the lactate dehydrogenase (LDH) doubled when the cells entered the stationary phase. Kinetic analysis revealed that at the metabolic level LDH activity is regulated through (i) competitive inhibition by pyrophosphate (PPi, ki=1.7 mM) and NAD (ki=0.43 mM) and (ii) allosteric activation by FBP (300%), ATP (160%) and ADP (140%). From these data a MWC-based model was derived. Simulations with this model could explain the observed lactate shift by displaying how the sensitivity of LDH activity to NADH/NAD ratio varied with different PPi concentrations. Moreover, the activation of LDH by ATP indicates that C. saccharolyticus uses LDH as a means to adjusts its flux of ATP and NADH production. To our knowledge, this is the first time PPi is observed as an effector of LDH. 相似文献