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1.
Xylitol-2-dehydrogenase from Candida albicans was cloned and overexpressed in Escherichia coli. The purified recombinant XDH has an apparent molecular weight of 40 kDa which belongs to the medium chain alcohol dehydrogenase family and exclusively uses NAD+ as a cofactor. The recombinant caXDH has a KM of 8.8 mM and 37.7 μM using the substrate xylitol and NAD+, respectively, and its catalytic efficiency is 53,200 min?1 mM?1. Following site-directed mutagenesis, one of the engineered caXDHs with six mutations at Ser95Cys, Ser98Cys, Tyr101Cys, Asp206Ala, Ile207Arg, and Phe208Ser shifted its cofactor dependence from NAD+ to NADP+ in which the KM and kcat/KM towards NADP+ are 119 μM and 26,200 min?1 mM?1, respectively. 相似文献
2.
Higher energy content and hydrophobicity make bio-based n-butanol a preferred building block for chemical and biofuels manufacturing. Butanol is obtained by Clostridium sp. based ABE fermentation process. While the ABE process is well understood, the enzyme systems involved have not been elucidated in detail. The important enzyme ß-hydroxybutyryl CoA dehydrogenase from Clostridium acetobutylicum ATCC 824 (Hbd) was purified and characterized. Surprisingly, Hbd shows extremely high temperature (T > 60 °C), pH (4–11) and solvent (1-butanol, isobutanol, ethanol) stability. Hbd catalyzes acetoacetyl CoA hydration to ß-hydroxybutyryl CoA up to pH 9.5, where the reaction is reversed. Substrate (acacCoA, ß-hbCoA) and cofactor (NADH, NAD+, NADPH and NADP+) specificities were determined. We identified NAD+ as an uncompetitive inhibitor. Identification of process relevant enzymes such as Hbd is key to optimize butanol production via cellular or cell-free enzymatic systems. 相似文献
3.
《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
4.
Shinuo Cao Gabriel Oluga Aboge Mohamad Alaa Terkawi Mo Zhou Yuzi Luo Longzheng Yu Yan Li Younkyoung Goo Ketsarin Kamyingkird Tatsunori Masatani Hiroshi Suzuki Ikuo Igarashi Yoshifumi Nishikawa Xuenan Xuan 《Parasitology international》2013,62(2):87-94
The inosine monophosphate dehydrogenase (IMPDH) enzyme has been characterized and validated as a molecular drug target in other apicomplexans but not in the genus Babesia. Subsequently, we cloned and expressed a Babesia gibsoni IMPDH (BgIMPDH) cDNA in Escherichia coli. We also determined the inhibitory effect of mycophenolic acid (MPA) on recombinant BgIMPDH (rBgIMPDH) activity and the Babesia-growths in vitro. The translated BgIMPDH peptide contained thirteen amino acid residues responsible for substrate and cofactor binding in its catalytic domain with Gly374 in BgIMPDH being replaced by Ser388 in mammalian IMPDH. The native BgIMPDH enzyme in the parasite was approximately 54-kDa a mass similar to His-tag rBgIMPDH protein. The Km values of the rBgIMPDH were 8.18 ± 0.878 (mean ± standard error of the mean) μM and 360.80 ± 43.41 μM for IMP and NAD+, respectively. MPA inhibited the rBgIMPDH activity yielding a Ki value of 20.93 ± 1.83 μM with respect to NAD+. For Babesia growths, the IC50s were 0.95 ± 0.21 and 2.88 ± 0.49 μM for B. gibsoni and B. bovis, respectively. Therefore, our results suggest that MPA may inhibit the replication of Babesia parasites by targeting IMPDH enzyme of the purine pathway. 相似文献
5.
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
6.
《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
7.
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
8.
Design and development of new class of Mycobacterium tuberculosis l-alanine dehydrogenase inhibitors
《Bioorganic & medicinal chemistry》2016,24(18):4499-4508
Mycobacterium tuberculosis l-alanine dehydrogenase (MTB l-AlaDH) is one of the important drug targets for treating latent/persistent tuberculosis. In this study we used crystal structure of the MTB l-AlaDH bound with cofactor NAD+ as a structural framework for virtual screening of our in-house database to identified new classes of l-AlaDH inhibitor. We identified azetidine-2,4-dicarboxamide derivative as one of the potent inhibitor with IC50 of 9.22 ± 0.72 μM. Further lead optimization by synthesis leads to compound 1-(isonicotinamido)-N2,N4-bis(benzo[d]thiazol-2-yl)azetidine-2,4-dicarboxamide (18) with l-AlaDH IC50 of 3.83 ± 0.12 μM, 2.0 log reduction in nutrient starved dormant MTB model and MIC of 11.81 μM in actively replicative MTB. 相似文献
9.
α-Ketoglutarate (aKG) represents a central intermediate of cell metabolism. It is used for medical treatments and as a chemical building block. Enzymatic cascade reactions have the potential to sustainably synthesize this natural product. Here we report a systems biocatalysis approach for an in vitro reaction set-up to produce aKG from glucuronate using the oxidative pathway of uronic acids. Because of two dehydrations, a decarboxylation, and reaction conditions favoring oxidation, the pathway is driven thermodynamically towards complete product formation. The five enzymes (including one for cofactor recycling) were first investigated individually to define optimal reaction conditions for the cascade reaction. Then, the kinetic parameters were determined under these conditions and the inhibitory effects of substrate, intermediates, and product were evaluated. As cofactor supply is critical for the cascade reaction, various set-ups were tested: increasing concentrations of the recycling enzyme, different initial NAD+ concentrations, as well as the use of a bubble reactor for faster oxygen diffusion. Finally, we were able to convert 10 g L−1 glucuronate with 92% yield of aKG within 5 h. The maximum productivity of 2.8 g L−1 h−1 is the second highest reported in the biotechnological synthesis of aKG. 相似文献
10.
Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)+ and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD+ from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60 °C, the NAD+ concentration could be kept almost constant for 15 h. 相似文献
11.
Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c2 +) as the electron donor. In this study, the oxidation of horse cyt c2 + by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c2 + and cyt c3 +. This allowed an analysis of the effects of cyt c3 + on the steady-state kinetics between CcO and cyt c2 +. The results show that cyt c3 + exhibits product inhibition by two mechanisms: competition with cyt c2 + at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c2 + at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6 × His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). 相似文献
12.
Purushottam R. Lomate Vandana K. Hivrale 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2010,155(2):164-170
We report the partial purification to apparent homogeneity of a soluble aminopeptidase (EC 3.4.11.1) from midgut of Helicoverpa armigera larvae, which preferentially degraded Leucine p-nitroanilide (LpNA). After midgut isolation, extraction and precipitation of soluble proteins with acetone, proteins were purified in two consecutive steps including gel filtration and ion-exchange chromatographies. Aminopeptidase activity was increased 8.95 fold after gel filtration chromatography. The purified enzyme appeared as single band with a molecular mass of ~ 112 kDa in SDS-PAGE, with a pH optimum of 7.0. Zymogram analysis revealed two enzymatically active proteinases using LpNA as substrate. The optimal temperature of aminopeptidase activity was 50–60 °C. The enzyme was characterized as metalloprotease as it was strongly inhibited by 1,10 phenanthroline. Strong inhibition was also being observed using the specific aminopeptidase inhibitor bestatin. Heavy metal ions, EDTA and cysteine strongly inhibited the enzyme, while Ca+ 2, Mn+ 2 and Mg+ 2 somewhat stimulated aminopeptidase activity. Besides LpNA, the purified aminopeptidase also cleaved with decreasing activity ApNA, VpNA and BApNA. Study could be helpful to understand the mechanism of action of N-terminal degrading enzymes and also important is to further study the differential interaction of Bacillus thuringiensis cry insecticidal toxin with midgut receptor of insects. 相似文献
13.
In the present work, NAD+-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD+, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system. 相似文献
14.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2007,1774(12):1500-1507
Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl− salts) the effectiveness follows the series: Cu+2, Fe+2 > Zn+2 > Li+ > Ca+2 > Mg+2 > Cs+ > NH4+ > Rb+ > K+ > Na+ and for anions (Na+ salts) the series is: I− > ClO4− > −SCN > Br− ≈ NO3− > Cl− ≈ −OAc > F− ≈ SO4− 2. The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of kcat/Km for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pKas and not to an effect on the pH-independent second-order rate constant, (kcat/Km)lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pKa1 increases (from 3.7 to 4.9) and the apparent pKa2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (kcat/Km)lim (= 9 × 104 M− 1 s− 1) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pKas when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pKa shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pKa of 3.7 and dissociated from the group with a pKa of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two pKas become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (kcat/Km)lim, of the monoprotonated species. For other enzymes which may show such salt-induced pKa shifts, this provides a convenient test for the role of reverse protonation. 相似文献
15.
《Process Biochemistry》2010,45(7):1088-1093
An extracellular thermostable α-galactosidase from Aspergillus parasiticus MTCC-2796 was purified 16.59-fold by precipitation with acetone, followed by sequential column chromatography with DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found to be a monomeric protein with a molecular weight of about 67.5 kDa. The purified enzyme showed optimum activity against o-nitrophenyl-α-d-galactopyranoside (oNPG) at pH 5.0 and a temperature of 50 °C. The enzyme was thermostable, showing complete activity even after heating at 65 °C for 30 min. The enzyme showed strict substrate specificity for α-galactosides and hydrolyzed oNPG (Km = 0.83 mM), melibiose (Km = 2.48 mM) and raffinose (Km = 5.83 mM). Among metal ions and reagents tested, Ca2+ and K+ enhanced the enzymatic activity, but Mg2+, Mn2+, ethylenediaminetetraacetic acid (EDTA) and 2-mercaptoethanol showed no effect, while Ag+, Hg2+ and Co2+ strongly inhibited the activity of the enzyme. The enzyme catalyzed the transglycosylation reaction for the synthesis of melibiose. 相似文献
16.
《Bioorganic & medicinal chemistry letters》2014,24(3):911-916
The enoyl acyl-carrier protein reductase (ENR) enzyme of the apicomplexan parasite family has been intensely studied for antiparasitic drug design for over a decade, with the most potent inhibitors targeting the NAD+ bound form of the enzyme. However, the higher affinity for the NADH co-factor over NAD+ and its availability in the natural environment makes the NADH complex form of ENR an attractive target. Herein, we have examined a benzimidazole family of inhibitors which target the NADH form of Francisella ENR, but despite good efficacy against Toxoplasma gondii, the IC50 for T. gondii ENR is poor, with no inhibitory activity at 1 μM. Moreover similar benzimidazole scaffolds are potent against fungi which lack the ENR enzyme and as such we believe that there may be significant off target effects for this family of inhibitors. 相似文献
17.
NAD(P)-dependent oxidoreductases represent a great interest in the field of biotechnology and biotransformation. Although they have many advantages, the biggest drawback and limitation of oxidoreductase usage is the price of the coenzymes. In order to solve this problem, many in situ methods for regeneration of coenzymes have been studied and developed. Unfortunately, although results indicate that those methods are suitable for regeneration procedure, most of the processes need additional optimization to make them more sustainable. As an alternative, microreactor technology could be used as a new technique for coenzyme regeneration processes due to many advantages.In this study regeneration of coenzyme NAD+ was carried out in a microreactor by acetaldehyde reduction to ethanol using enzyme alcohol dehydrogenase (ADH). Suspended and immobilized whole permeabilized baker’s yeast cells were used as the source of the ADH enzyme. A 65.3% conversion of NADH was achieved with suspended permeabilized baker’s yeast cells for a residence time of τ = 36 s and equimolar concentration of substrates (ci,NADH = 5.5 mmol/dm3, ci,acetaldehyde = 5.5 mmol/dm3). When working with immobilized cells, conversion achieved for the same residence time was 10 fold lower. When permeabilized baker’s yeast cells were used for coenzyme regeneration process was stabile for 6 days of continuous operation which makes this system a good alternative for coenzyme regeneration. 相似文献
18.
LaaA,a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031T
Alpha-amylase is a kind of broadly used industrial enzymes, most of which have been exploited from terrestrial organism. Comparatively, alpha-amylase from marine environment was largely undeveloped. In this study, a novel alkalophilic alpha-amylase with high activity, Luteimonas abyssi alpha-amylase (LaaA), was cloned from deep-sea bacterium L. abyssi XH031T and expressed in Escherichia coli BL21. The gene has a length of 1428 bp and encodes 475 amino acids with a 35-residue signal peptide. The specific activity of LaaA reached 8881 U/mg at the optimum pH 9.0, which is obvious higher than other reported alpha-amylase. This enzyme can remain active at pH levels ranging from 6.0 to 11.0 and temperatures below 45 °C, retaining high activity even at low temperatures (almost 38% residual activity at 10 °C). In addition, 1 mM Na+, K+, and Mn2+ enhanced the activity of LaaA. To investigate the function of potential active sites, R227G, D229K, E256Q/H, H327V and D328V mutants were generated, and the results suggested that Arg227, Asp229, Glu256 and Asp328 were total conserved and essential for the activity of alpha-amylase LaaA. This study shows that the alpha-amylase LaaA is an alkali-tolerant and high-active amylase with strong potential for use in detergent industry. 相似文献
19.
l-Amino acid oxidases (LAAOs) are useful catalysts for the deracemisation of racemic amino acid substrates when combined with abiotic reductants. The gene nadB encoding the l-aspartate amino acid oxidase from Pseudomonas putida (PpLASPO) has been cloned and expressed in E. coli. The purified PpLASPO enzyme displayed a KM for l-aspartic acid of 2.26 mM and a kcat = 10.6 s−1, with lower activity also displayed towards l-asparagine, for which pronounced substrate inhibition was also observed. The pH optimum of the enzyme was recorded at pH 7.4. The enzyme was stable for 60 min at up to 40 °C, but rapid losses in activity were observed at 50 °C. A mutational analysis of the enzyme, based on its sequence homology with the LASPO from E. coli of known structure, appeared to confirm roles in substrate binding or catalysis for residues His244, His351, Arg386 and Arg290 and also for Thr259 and Gln242. The high activity of the enzyme, and its promiscuous acceptance of both l-asparagine and l-glutamate as substrates, if with low activity, suggests that PpLASPO may provide a good model enzyme for evolution studies towards AAOs of altered or improved properties in the future. 相似文献
20.
Tao Zhang Yujie Guo Hao Zhang Wanmeng Mu Ming Miao Bo Jiang 《Process Biochemistry》2013,48(4):663-668
An l-ornithine high producing strain Bacillus thuringiensis SK20.001 was screened by our laboratory. An intracellular arginase used to biosynthesize l-ornithine from the strain was purified and characterized. The final specific arginase activity was 589.2 units/mg, with 70.1 fold enrichment and 22.4% recovery. The molecular weight of the enzyme was approximately 33,000 Da as evaluated by SDS-PAGE and 191,000 Da as determined by gel filtration. The enzyme had an optimum pH of 10.0 and an optimum temperature of 40 °C. It was stable from pH 8.0–12.0 and <50 °C without Mn2+. The presence of Mn2+ and Ni2+ had strong effects on the enzyme activity, and Mn2+ significantly increased the thermal stability of the enzyme. The arginase was slightly inhibited by Ca2+, Fe2+ and Zn2+. Trp, Asp, Glu, Tyr, and Arg residues were directly involved in the arginase activity evaluated by chemical modifications. The Km and Vmax for l-arginine were estimated to be 15.6 mM and 538.9 μmol/min/mg. The biosynthesis yield of l-ornithine was 72.7 g/L with the enzyme. 相似文献