Cis-epoxysuccinic Acid Research Articles

FRI025 Succinate Receptor Activation Increases Respiration In Human But Not Murine Brown Adipocytes

Abstract Disclosure: B.T. McNeill: None. A. Kelman: None. L.E. Ramage: None. K.J. Suchacki: None. S.J. Wakelin: None. N.M. Morton: None. R.H. Stimson: None. Brown adipose tissue (BAT) is a thermogenic tissue that generates heat to maintain body temperature. BAT activation is a potential therapeutic target for treating obesity-related cardiometabolic disease. We performed RNA sequencing of human primary brown and white adipocytes and identified SUCNR1, which encodes the succinate receptor, as highly differentially expressed (20-fold) in brown versus white adipocytes. Succinate is known to increase BAT thermogenesis, while SUCNR1 activation inhibits lipolysis in white adipose tissue (WAT). We hypothesized that SUCNR1 activation increases BAT thermogenesis in humans and in mice. To test this, we investigated the effect of succinate and the selective SUCNR1 agonist cis-epoxysuccinic acid (C-ESA) in human and murine primary brown/ beige adipocytes. In addition, we determined the metabolic effects of disruption of Sucnr1 in mice either fed control or a high fat (HFD) for 12 weeks and measured energy expenditure when housed at standard room temperature (21°C) and during cold exposure (4°C). In human brown and white adipocytes, succinate increased basal and noradrenaline-stimulated respiration, while C-ESA increased basal respiration only in human brown adipocytes without altering UCP1 mRNA levels. SUCNR1 activation reduced glycerol release only in human white adipocytes. Male (but not female) Sucnr1-/- mice exhibited impaired glucose tolerance versus Sucnr1+/+ littermates after 2 weeks of HFD, and greater fat mass was also observed in the Sucnr1-/- mice following chronic HFD. However, energy expenditure, locomotor activity and tail vein temperature were unchanged in male Sucnr1-/- mice either at 21°C or 4°C. In addition, expression of Ucp1 and Pgc1α in BAT and inguinal WAT were similar between genotypes. Sucnr1 deletion had no effect on fat mass or glucose tolerance in mice fed a control diet. In vitro, differentiated brown adipocytes from Sucnr1-/- mice demonstrated comparable cellular respiration and Ucp1 mRNA levels both basally and during noradrenaline stimulation to Sucnr1+/+ adipocytes. Succinate and C-ESA did not stimulate respiration in murine brown or white adipocytes. These data reveal that SUCNR1 activation enhances brown adipocyte respiration in human but not murine brown adipocytes, highlighting a novel pathway regulating BAT function in addition to differences between species. These data also reveal novel sex-specific differences in the metabolic effects of SUCNR1, although the mechanisms through which female mice are protected from the adverse metabolic effects of Sucnr1 deletion are unclear. SUCNR1 activation may be a novel therapeutic target to treat obesity and associated metabolic disease, but requires further research in humans to determine the specific mechanisms through which SUCNR1 regulates BAT thermogenesis. Presentation: Friday, June 16, 2023

Role of the succinate and GPR91 pathway in atrial fibrillation mechanisms

Atrial fibrillation (AF) is the most common arrhythmia and is associated with increased morbidity and mortality. The mechanisms underlying the transition from paroxysmal to persistent AF are still a matter of debate. Recent studies indicate a potential role for metabolic remodelling in the AF stabilization process and increased levels of a specific metabolic substrate, succinate, have been found in blood plasma of AF patients. While succinate is known to increase reactive oxygen species (ROS) production, the role of the succinate receptor (GPR91) in atrial function is unknown. To assess the impact of the succinate pathway on AF vulnerability in a sheep model. GPR91 expression levels were quantified by Western Blot (WB) in a previously described sheep model of persistent AF induced by burst-pacing. Optical experiments were subsequently performed in ex vivo right atria (RA) from control sheep (n = 5). RA were perfused by Tyrode solution, then supplemented with the cis-epoxysuccinic acid (cESA), a specific activator of GPR91 (150 μM) to study its involvement in atrial electrophysiology and AF. Action potential duration at 80% of repolarization (APD80) were assessed from 2 to 5 Hz pacing frequency. We used an S1S2 pacing protocol to determine effective refractory period (ERP) and a burst pacing protocol (30 Hz) to assess ex vivo AF vulnerability. Finally, we investigated these properties in a human RA from an AF patient. WB experiments revealed an increase in GPR91 expression in AF sheep compared to sham sheep. Interestingly, in resistant sheep (no AF after 90 days of burst pacing), expression levels were decreased compared to sham. During cESA perfusion in control sheep RA, sinus rhythm (119 vs. 95 bpm; P = 0.03) and ERP (202 vs. 178 ms; P = 0.36) were decreased when compared to baseline. We also observed a decrease in APD80 (199 vs. 177 ms; P = 0.009) and in CV (127 vs. 115 cm/sec; P = 0.01). These electrophysiological perturbations led to an increase in AF vulnerability by burst pacing (0% vs. 60%) and an increase in spontaneous arrhythmias. These results were corroborated in the human RA were cESA decreased APD80 (265 vs. 241 ms), ERP (240 vs. 220 ms), and CV (119 vs. 94 cm/sec) and increased arrhythmia vulnerability. For the first time, we demonstrated that GPR91 is expressed in atrial tissue and that its activation through succinate can play a major role in AF mechanisms.

PO-01-159 ROLE OF THE SUCCINATE PATHWAY IN ATRIAL FIBRILLATION MECHANISMS

Atrial fibrillation (AF) is the most common arrhythmia and is associated with increased morbidity and mortality. The mechanisms underlying the transition from paroxysmal to persistent AF are still a matter of debate. Recent studies indicate a potential role for metabolic remodelling in the AF stabilization process and increased levels of a specific metabolic substrate, succinate, have been found in blood plasma of AF patients. While succinate is known to increase reactive oxygen species (ROS) production, the role of the succinate receptor (GPR91) in atrial function is unknown. To assess the impact of the succinate pathway on AF vulnerability in a sheep model. GPR91 expression levels were quantified by Western Blot (WB) in a previously described sheep model of persistent AF induced by burst-pacing. Optical experiments were subsequently performed in ex vivo right atria (RA) from control sheep (N=5). RA were perfused by Tyrode solution, then supplemented with the cis-epoxysuccinic acid (cESA), a specific activator of GPR91 (150μM) to study its involvement in atrial electrophysiology and AF. Action potential duration at 80% of repolarization (APD80) were assessed from 2 to 5Hz pacing frequency. We used an S1S2 pacing protocol to determine effective refractory period (ERP) and a burst pacing protocol (30Hz) to assess ex vivo AF vulnerability. Finally, an organ donation program allowed us to investigate these properties in a human RA from an AF patient. WB experiments revealed an increase in GPR91 expression in AF sheep compared to sham sheep. Interestingly, in resistant sheep (no AF after 90 days of burst pacing), expression levels were decreased compared to sham. During cESA perfusion in control sheep RA, sinus rhythm (119 vs 95 bpm; p=0,03) and ERP (202 vs 178 ms; p=0,36) were decreased when compared to baseline. We also observed a decrease in APD80 (199 vs 177 ms; p=0.009) and in CV (127 vs 115 cm/sec; p=0.01). These electrophysiological perturbations led to an increase in AF vulnerability by burst pacing (0% vs 60%) and an increase in spontaneous arrhythmias. These results were corroborated in the human RA were cESA decreased APD80 (265 vs 241 ms), ERP (240 vs 220 ms), and CV (119 vs 94 cm/sec) and increased arrhythmia vulnerability. For the first time, we demonstrated that GPR91 is expressed in atrial tissue and that its activation through succinate can play a major role in AF mechanisms.

Development of highly efficient whole-cell catalysts of cis-epoxysuccinic acid hydrolase by surface display

Bacterial cis-epoxysuccinic acid hydrolases (CESHs) are intracellular enzymes used in the industrial production of enantiomeric tartaric acids. The enzymes are mainly used as whole-cell catalysts because of the low stability of purified CESHs. However, the low cell permeability is the major drawback of the whole-cell catalyst. To overcome this problem, we developed whole-cell catalysts using various surface display systems for CESH[L] which produces L(+)-tartaric acid. Considering that the display efficiency depends on both the carrier and the passenger, we screened five different anchoring motifs in Escherichia coli. Display efficiencies are significantly different among these five systems and the InaPbN-CESH[L] system has the highest whole-cell enzymatic activity. Conditions for InaPbN-CESH[L] production were optimized and a maturation step was discovered which can increase the whole-cell activity several times. After optimization, the total activity of the InaPbN-CESH[L] surface display system is higher than the total lysate activity of an intracellular CESH[L] overexpression system, indicating a very high CESH[L] display level. Furthermore, the whole-cell InaPbN-CESH[L] biocatalyst exhibited good storage stability at 4 °C and considerable reusability. Thereby, an efficient whole-cell CESH[L] biocatalyst was developed in this study, which solves the cell permeability problem and provides a valuable system for industrial L(+)-tartaric acid production.Graphical

Role of the succinate pathway in the electrophysiological properties of right atria in a persistent atrial fibrillation sheep model

Atrial fibrillation (AF) is the most sustained arrhythmia and increases morbidity and mortality. However, development and maintenance of AF is a still poorly understood process. Metabolic remodelling could induce transition from paroxysmal to persistent AF. Succinate levels are increased in AF patients indicating a potential role for metabolic remodelling. To assess the impact of the succinate pathway on the electrophysiological properties of right atria (RA) in persistent AF sheep model. Optical experiments were performed in ex vivo RA from a burst pacing AF sheep model. RA were perfused by Tyrode solution with glucose (5.6 mM), subsequently replaced by succinate (10 mM) to increase mitochondrial reactive oxygen species (ROS) production which are known to modulate excitation-contraction coupling (ECC) and study metabolic remodelling. An activator of the succinate β-adrenergic receptor (GPR91), cis-epoxysuccinic acid (300 μM), was used to study the GPR91 involvement in atrial electrophysiology and AF. Action potential duration at 80% of repolarization (APD80) were assessed from 2 to 5 Hz pacing frequency and during sinus rhythm (SR). We used an S1S2 pacing protocol to determine effective refractory period (ERP) and a burst pacing protocol (30 Hz) to assess ex vivo AF vulnerability. Finally, an organ donation program allowed us to investigate these properties in a human RA from an AF patient. During succinate perfusion SR is decreased in Sham (1,5 vs 1,2 Hz; P = 0,03), AF (1,5 vs 1 Hz; P = 0,003) and resistant (1,3 vs 1 Hz; P = 0,01) sheep, ERP is increased in AF sheep (184 vs 344 ms; P = 0,0007) and APD80 is increased in Sham (217 vs 264 ms; P = 0,0004) and AF sheep (195 vs 275 ms; P = 0,003). We also observed an increase in amplitude alternans which can induce re-entries and therefore be pro-arrhythmic. These results seems to be confirmed on human AF RA by increasing APD80 (257 vs 309 ms), ERP (260 vs 330 ms) and spontaneous arrhythmias. GPR91 activation led to a slowing of SR (1,7 vs 1,3 Hz; P = 0,03), a shortening of APD80 (200 vs 170 ms; P = 0,007) and an increase in spontaneous arrhythmias. A dysregulation of ECC could explain our results consistently with the ROS involvement and metabolic remodelling in AF. For the first time, we also demonstrated that GPR91 pathway is involved in atrial electrophysiology and could be involved in AF mechanisms.

BS-526-02 ROLE OF THE SUCCINATE PATHWAY IN THE ELECTROPHYSIOLOGICAL PROPERTIES OF RIGHT ATRIA IN A PERSISTENT ATRIAL FIBRILLATION SHEEP MODEL

The progression from paroxysmal to persistent atrial fibrillation (AF) is still a poorly understood process. Succinate levels are increased in AF patients indicating a potential role for metabolic remodeling in this transition process. To assess the impact of the succinate pathway on the electrophysiological properties of right atria (RA) in a persistent AF sheep model. Optical mapping was performed on ex vivo RA from a burst pacing AF sheep model, including 8 sham, 7 persistent AF, and 3 resistant animals. RA were perfused by Tyrode solution with glucose (5.6mM), subsequently replaced by succinate (10mM), known to increase mitochondrial Reactive Oxygen Species (ROS). An activator of the succinate β-adrenergic receptor (GPR91), cis-epoxysuccinic acid (300μM), was also used to study the GPR91 involvement in atrial electrophysiology and AF (N=6). Action potential duration at 80% of repolarization (APD80) were assessed from 2 to 5Hz pacing frequency and during sinus rhythm (SR). We used an S1S2 pacing protocol to determine effective refractory period (ERP) and a burst pacing protocol (30Hz) to assess ex vivo AF vulnerability. Finally, an organ donation program allowed us to investigate these properties in a human RA from an AF patient. During succinate perfusion SR is decreased in Sham (1,5 vs 1,2 Hz ; p=0,03), AF (1,5 vs 1 Hz ; p=0,003) and resistant (1,3 vs 1 Hz ; p=0,01) sheep, ERP is increased in AF sheep (184 vs 344 ms ; p=0,0007) and APD80 is increased in Sham (217 vs 264 ms ; p=0,0004) and AF sheep (195 vs 275 ms ; p=0,003). We also observed an increase in amplitude alternans and decrease in frequencies at which alternans was observed. These results appear to be confirmed in a human AF RA where succinate increased APD80 (257 vs 309 ms), ERP (260 vs 330 ms) and the occurrence of spontaneous arrhythmias. Finally, in sheep, GPR91 activation led to a slowing of SR (1,7 vs 1,3 Hz ; p=0,03), a shortening of APD80 (200 vs 170 ms ; p=0,007) and an increase in spontaneous arrhythmias. Succinate induces significant electrophysiological modifications, especially in persistent AF, and increases vulnerability to AF. We have shown that the GPR91 pathway is involved in atrial electrophysiology and succinate-induced arrhythmogenesis, most likely in combination with increased ROS production.

Isolation of Penicillium expansum WH-3 for the production of L(+)-tartaric acid.

The L(+)-form of tartaric acid (L(+)-TA) exists extensively in nature, and is widely used in the food, chemical, textile, building, and pharmaceutical industries (Su et al., 2001). The main method for L(+)-TA production is microbial transformation by cis-epoxysuccinate hydrolase (CESH), which can catalyze the asymmetric hydrolysis of cis-epoxysuccinic acid or its salts to TA or tartrate (Bao et al., 2019). Seventeen species containing CESH have been isolated so far. However, most species for L(+)-TA production have been reported from bacteria (Xuan and Feng, 2019). The only fungus isolated from soil by our lab recently, that could be used as catalyst for the process under acidic condition, is Aspergillus niger WH-2 (Bao et al., 2020). In order to find strains with new characteristics, this study attempted to isolate a new CESH source from fungi and investigate its application value.

L-TARTARIC ACID: PRODUCTION TECHNOLOGY, ECONOMIC GROWTH AND QUALITY CONTROL

Natural substances are the basis of many types of industries and represent a growing market. The study of these products and the development of analytical methods should accompany this growth to ensure quality and provenance to consumers. An example to be discussed is the L(+)-Tartaric acid, an organic compound of molecular formula C4H6O6. This organic acid is widely applied in wine, food and pharmaceutical industry. It is obtained naturally through the fermentation of fruits, especially grape and tamarind. Synthetically, there are two ways of obtaining L(+)-tartaric acid on industrial scale. It can be synthesized by the reaction of maleic anhydride with hydrogen peroxide, which is derived from petroleum by-products. And by biotechnological synthesis, in which cis-epoxy succinic acid, also derived from petroleum, is converted into L(+)-tartaric acid by hydrolase enzyme. The market for tartaric acid is growing and is considered promising. Currently, there is a lack of legislation and specific rules that allow classification of tartaric acid according to its origin. This legal vacuum precludes quality assurance for the consumer. This lack of safety is a matter of great concern as applications of tartaric acid come directly to final consumer.

Identification and pharmacological characterization of succinate receptor agonists.

The succinate receptor (formerly GPR91 or SUCNR1) is described as a metabolic sensor that may be involved in homeostasis. Notwithstanding its implication in important (patho)physiological processes, the function of succinate receptors has remained ill-defined because no pharmacological tools were available. We report on the discovery of the first family of potent synthetic agonists. We screened a library of succinate analogues and analysed their activity on succinate receptors. Also, we modelled a pharmacophore and a binding site for this receptor. New agonists were identified based on the information provided by these two approaches. Their activity was studied in various bioassays, including measurement of cAMP levels, [Ca2+ ]i mobilization, TGF-α shedding and recruitment of arrestin 3. The in vivo effects of activating succinate receptors with these new agonists was evaluated on rat BP. We identified cis-epoxysuccinic acid and cis-1,2-cyclopropanedicarboxylic acid as agonists with an efficacy similar to that of succinic acid. Interestingly, cis-epoxysuccinic acid was 10- to 20-fold more potent than succinic acid on succinate receptors. For example, cis-epoxysuccinic acid reduced cAMP levels with a pEC50 =5.57±0.02 (EC50 =2.7μM), compared with succinate pEC50 =4.54±0.08 (EC50 =29μM). The rank order of potency of the three agonists was the same in all in vitro assays. Both cis-epoxysuccinic and cis-1,2-cyclopropanedicarboxylic acid were as potent as succinate in increasing rat BP. We describe new agonists at succinate receptors that should facilitate further research on this understudied receptor.

Improvement of the Production Efficiency of l-(+)-Tartaric Acid by Heterogeneous Whole-Cell Bioconversion

An Escherichia coli-engineered bacterium with cis-epoxysuccinate hydrolase (ESH) activity was used to catalyze the stereospecific hydrolysis of cis-epoxysuccinic acid to L-(+)-tartaric acid. The effect of the substrate composition on the production efficiency of L-(+)-tartaric acid was investigated. Based on the sodium-type homogeneous substrate system, a heterogeneous substrate system, composed of 1.2 M sodium-type substrate and 1.8 M calcium-type substrate, was designed to improve ESH catalytic efficiency. After process optimization, a catalytic efficiency of 9.37 × 10(-3) g U(-1) h(-1) was obtained with fed-batch mode in the heterogeneous substrate system, about a twofold increase compared to the traditional bioconversion process with Nocardia tartaricans cells. The scale-up tests were carried out in a 15-m(3) stirred tank reactor, which indicated that the heterogeneous substrate system had great application prospect for the L-(+)-tartaric acid industrial production.

Efficiency and Stability Enhancement of Cis-epoxysuccinic Acid Hydrolase by Fusion with a Carbohydrate Binding Module and Immobilization onto Cellulose

Cis-epoxysuccinic acid hydrolase (CESH) is an enzyme that catalyzes cis-epoxysuccinic acid to produce enantiomeric L(+)-tartaric acid. The production of tartaric acid by using CESH would be valuable in the chemical industry because of its high yield and selectivity, but the low stability of CESH hampers its application. To improve the stability of CESH, we fused five different carbohydrate-binding modules (CBMs) to CESH and immobilized the chimeric enzymes on cellulose. The effects of the fusion and immobilization on the activity, kinetics, and stability of CESH were compared. Activity measurements demonstrated that the fusion with CBMs and the immobilization on cellulose increased the pH and temperature adaptability of CESH. The chimeric enzymes showed significantly different enzyme kinetics parameters, among which the immobilized CBM30-CESH exhibited twofold catalytic efficiency compared with the native CESH. The half-life measurements indicated that the stability of the enzyme in its free form was slightly increased by the fusion with CBMs, whereas the immobilization on cellulose significantly increased the stability of the enzyme. The immobilized CBM30-CESH showed the longest half-life, which is more than five times the free native CESH half-life at 30 °C. Therefore, most CBMs can improve enzymatic properties, and CBM30 is the best fusion partner for CESH to improve both its enzymatic efficiency and its stability.

High Yield Recombinant Expression, Characterization and Homology Modeling of Two Types of Cis-epoxysuccinic Acid Hydrolases

The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form D: (-)-tartaric acid or L: (+)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[D: ] and CESH[L: ], producing D: (-)- and L: (+)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver-Burk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[L: ] showed much higher catalytic efficiency than CESH[D: ], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[L: ] and CESH[D: ] have different structural folds and potential active site residues. CESH[L: ] adopted a typical α/β-hydrolase fold with a cap domain and a core domain, whereas CESH[D: ] possessed a unique TIM barrel fold composed of 8 α-helices and 8 β-strands, and 2 extra short α-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[D: ], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs.

Purification and characterization of a cis-epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli

A highly enantioselective cis-epoxysuccinic acid hydrolase from Nocardia tartaricans was purified to electrophoretic homogeneity. The enzyme was purified 184-fold with a yield of 18.8%. The purified cis-epoxysuccinic acid hydrolase had a monomeric molecular weight of 28kDa, and its optimum conditions were 37°C and pH 7-9. With sodium cis-epoxysuccinate as the substrate, Michaelis-Menten enzyme kinetics analysis gave a Km value of 35.71mM and a Vmax of 2.65mMmin(-1). The enzyme was activated by Ni(2+) and Al(3+), while strongly inhibited by Fe(3+), Fe(2+), Cu(2+), and Ag(+). The cis-epoxysuccinic acid hydrolase gene was cloned, and its open reading frame sequence predicted a protein composed of 253 amino acids. A pET11a expression plasmid carrying the gene under the control of the T7 promoter was introduced into Escherichia coli, and the cis-epoxysuccinic acid hydrolase gene was successfully expressed in the recombinant strains.

From Infinite One-Dimensional Helix to Discrete CuII15 Cluster along with in Situ SN2 Ring-Cleavage of cis-Epoxysuccinic Acid: pH-Controlled Assemblies, Crystal Structures, and Properties

Two Cu(II) coordination complexes {[Cu(ces)(H(2)O)(2)](H(2)O)(0.5)}(n) (1) and [Cu(15)(dhs)(6)(OH)(6)(H(2)O)(10)](H(2)O)(20) (2) have been synthesized from cis-epoxysuccinic acid (cis-H(2)ces) and Cu(II) perchlorate under different pH conditions (ces = cis-epoxysuccinate and dhs = 2,3-dihydroxysuccinate), and fully characterized by IR spectra, elemental analyses, as well as single crystal and powder X-ray diffraction techniques. Notably, when the reaction was performed at pH above about 7.4, a one-dimensional (1-D) helical chain complex 1 is formed, whereas a neutral isolated Cu(15) nanocluster 2 is generated when the pH value is decreased to the range of about 6.6-7.3, being concomitant with in situ S(N)2 ring-cleavage reaction of cis-H(2)ces to form (2S,3S)- and (2R,3R)-H(4)dhs. Further, extended supramolecular architectures are constructed via secondary interactions in both structures. The magnetic properties of 1 and 2 have also been studied in detail, showing that 1 is an antiferromagnetic helical chain of S = 1/2 spins and 2 possesses an S = 3/2 spin ground state.

Purification and characterization of a cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3

Purification of a cis-epoxysuccinic acid hydrolase was achieved by ammonium sulfate precipitation, ionic exchange chromatography, hydrophobic interaction chromatography followed by size-exclusion chromatography. The enzyme was purified 177-fold with a yield of 14.4%. The apparent molecular mass of the enzyme was determined to be 33 kDa under denaturing conditions. The optimum pH for enzyme activity was 7.0, and the enzyme exhibited maximum activity at about 45 °C in 50 mM sodium phosphate buffer (pH 7.5). EDTA and o-phenanthrolin inhibited the enzyme activity remarkably, suggesting that the enzyme needs some metal cation to maintain its activity. Results of inductively coupled plasma mass spectrometry analysis indicated that the cis-epoxysuccinic acid hydrolase needs Zn 2+ as a cofactor. Eight amino acids sequenced from the N-terminal region of the cis-epoxysuccinic acid hydrolase showed the same sequence as the N-terminal region of the beta subunit of the cis-epoxysuccinic acid hydrolase obtained from Alcaligenes sp.

Optimization of culture conditions for production of cis-epoxysuccinic acid hydrolase using response surface methodology

Response surface methodology, which allows for rapid identification of important factors and optimization of them to enhance enzyme production, was employed here to optimize culture conditions for the production of cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3. In the first step, a Plackett–Burman design was used to evaluate the effects of nine variables (yeast extract, cis-epoxysuccinic acid, KH 2PO 4, K 2HPO 4 · 3H 2O, MgSO 4 · 7H 2O, trace minerals solution, culture volume, initial pH and incubation time) on the enzyme production. Yeast extract, cis-epoxysuccinic acid and KH 2PO 4 had significant influences on cis-epoxysuccinic acid hydrolase production and their concentrations were further optimized using central composite design and response surface analysis. A combination of adjusting the concentration of yeast extract to 7.8 g/l, cis-epoxysuccinic acid to 9.8 g/l, and KH 2PO 4 to 1.12 g/l would favor maximum cis-epoxysuccinic acid hydrolase production. An enhancement of cis-epoxysuccinic acid hydrolase production from 5.6 U/ml to 9.27 U/ml was gained after optimization.

Isolation and identification of a novel Rhodococcus sp. ML-0004 producing epoxide hydrolase and optimization of enzyme production

Rhodococcus sp. ML-0004, a novel strain for producing epoxide hydrolase, was isolated from soil in this study. The epoxide hydrolase can catalyze the stereo-specific hydrolysis of cis-epoxysuccinic acid to generate l(+)-tartaric acid. By examining physiological, biochemical characteristics and comparing its 16S rDNA gene sequence, it was identified as Rhodococcus opacus, and named R. opacus ML-0004. The optimal conditions for epoxide hydrolase production from R. opacus ML-0004 were also investigated. Propanediol and (NH 4) 2SO 4 were selected as carbon source and nitrogen source, respectively, for the production of R. opacus ML-0004 epoxide hydrolase. The optimal conditions for epoxide hydrolase production were fermentation temperature = 28 °C, pH 7.0, and cultivation time = 26 h. Under these conditions, the maximum epoxide hydrolase activity reached 10.5 U mL −1.

Isolation and characterization of a new bacterium capable of biotransformingcis-epoxysuccinic acid to d(−)-tartaric acid

A bacterial strain 1-3 capable of enantioselectively hydrolyzing cis-epoxysuccinic acid to D(-)-tartaric acid was isolated from soil. Phenotypic characteristics, Biolog GN test and phylogenetic analysis of strain 1-3 indicated that it belongs to the genus Bordetella. cis-Epoxysuccinic acid could be used as a carbon source and inducer for production of cis-epoxysuccinic acid hydrolase. After the purification of the enzymatic biotransformed product, a colorless and scentless crystal was obtained, which was confirmed to be the tartaric acid with laevorotatory optical rotation by the (1)H nuclear magnetic resonance spectrum and optical rotation analysis.

Kinetics and mechanism of homogeneous catalytic hydroxylation of maleic acid by hydrogen peroxide: III. Kinetics of the hydrolysis ofcis-epoxysuccinic acid

The objective of this work was to study the hydrolysis kinetics and also the character of the involvement of the epoxidation catalyst (Na2WO4 – sodium tungstate) on the hydrolysis of cis-epoxysuccinic acid (the initial product in the hydroxylation reaction of maleic acid by hydrogen peroxide). The results obtained at 65 °C clearly revealed that the hydrolysis reaction exhibits a considerably low rate in the absence of a catalyst whilst the rate is significantly enhanced by the introduction of catalytic quantities of Na2WO4. The phenomenon of end-product inhibition was observed in this study and the results obtained permitted the development of a kinetic model consistent with experimental observations. Analysis of the kinetic model shows that the reaction is first order with respect to the concentrations of the catalyst and the epoxide. However, tartaric acid has a strong inhibitive influence on the overall reaction rate. © 1999 Society of Chemical Industry

Influence of molybdate and tungstate ions on activity of cis-epoxysuccinate hydrolase in Nocardia tartaricans

L-Tartaric acid was produced by transformation of cis-epoxysuccinic acid using Nocardia tartaricans ATCC 31191 in a molar yield of 100%. cis-Epoxysuccinic acid was prepared by epoxidation of maleic acid in the presence of molybdate or tungstate ions. The effect of these ions on the subsequent activity of the cis-epoxysuccinate hydrolase was determined in both intact cells and cell-free extracts of Nocardia tartaricans. Based on kinetic studies, the tungstate ions behaved as a strong inhibitor while the molybdate ions as a stimulator of the enzyme. © Rapid Science Ltd. 1998