6-Aminoquinoline as a fluorogenic leaving group in peptide cleavage reactions: A new fluorogenic substrate for chymotrypsin

A new fluorogenic substrate capable of measuring the amidolytic activity of chymotrypsin and based upon the enzyme-catalyzed release of a highly fluorescent aromatic amine, 6-aminoquinoline, was prepared. The substrate, 6-(N-glutaryl-l-phenylalanylamido)quinoline, was found to have at pH 8.0 and 25°C K_m = 1.77 mm and k_cat = 1.4 × 10^?1 s^?1. The aminoquinoline is a unique leaving group in that its appearance can be measured fluorometrically at its excitation and emission maxima, while, under these conditions, fluorescence associated with unhydrolyzed substrate is negligible.     

Laccase isoforms with unusual properties from the basidiomycete Steccherinum ochraceum strain 1833

Aims: To isolate and characterize the laccase isoforms from S. ochraceum 1833 – a new active producer of high extracellular laccase activity. Methods and Results: Three laccase isoforms (laccases I, II and III) with 57·5, 59·5 and 63 kDa molecular masses respectively were purified from S. ochraceum 1833 and in contrast to the known laccases had strongly pronounced absorption at 611 nm with molar extinction coefficients ranging from 7170 to 7830 mol^?1 l cm^?1. All isoforms showed maximal activity with ABTS at low pH (≤2) and temperatures in the range 70–80°C, were stable for long time of incubation at high temperature (60–80°C) and at pH values ranging from 2 to 6. Laccase II showed a higher activity and wider substrate specificity. N‐terminal amino acid sequence analysis of the purified laccase II (VQIGPVTDLH) showed 80% identity with the N‐terminal amino acid sequence of laccase from Lentinula edodes [Appl Microbiol Biotechnol 60 (2002) 327]. Conclusions: Elevated temperature optima, high thermo‐ and pH‐stabilities, the broad substrate specificity of the isoforms make the laccases from S. ochraceum 1833 a suitable model for biotechnological processes proceeding at high temperatures. Significance and Impact of the Study: For the first time, new basidiomycete strain S. ochraceum was reported as a producer of novel thermostable, pH stable, acidophilic laccases with unusual spectral properties.     

Action of a human plasma fraction on tetradecapeptide,angiotensin I and angiotensin II

A protein fraction designated PF70 was isolated from human plasma and partially purified on Sephadex G-100. PF70 proteins, molecular weight 37, 000 to 41, 500, formed angiotensin I (AI) and angiotensin II (AII) from ¹⁴C-tetradecapeptide renin substrate (TDP) at 37 C. Hydrolysis was maximal at pH 6.9 but there was no change in the relative quantity of AI and AII formed at different pH values. Data indicate that AI was formed first and at a faster rate than AII, but typical converting enzyme activity was not detected. Radiolabeled AII was converted to Des-Asp¹-angiotensin II (angiotensin III); [³H]AI was degraded to a single tritiated product, possibly the nonapeptide. These aspartyl hydrolase reactions were apparently inhibited by TDP and were not involved in AI or AII generation from TDP. It is concluded that these enzymic activities represent two or more enzymes that are associated with the renin-angiotensin system.     

Peptide inhibitors of converting enzyme.

Human plasma and atypical lung converting enzyme, and porcine plasma converting enzyme are substantially inhibited by other components of the renin-angiotensin system, and by angiotensin II and its analogues. Des-Asp¹ angiotensin II (angiotensin III) 0.1 mM and tridecapeptide renin substrate 0.1 mM are both effective inhibitors of human lung, plasma and porcine plasma converting enzymes. Des-Asp¹-Arg² angiotensin II also was an effective inhibitor of plasma enzymes. Bradykininase activity (kininase II) of the converting enzymes was also inhibited by angiotensin I, angiotensin III, tetradecapeptide renin substrate and tridecapeptide renin substrate. The substantial kininase and converting enzyme inhibitory effects of components of the renin-angiotensin system, suggest a potential close physiologic relationship between the kallikrein-kinin system and the renin-angiotensin system.     

[DES-ASP1] angiotensin II in the sheep: Blood levels and its effect on plasma renin concentration

The present study describes an improved method for measuring angiotensin III in arterial blood. This was accomplished by SE-sephadex column to separate angiotensin II from angiotensin III prior to radioimmunoassay. The arterial concentration of angiotensin III measured before and after 24 to 48 hours sodium depletion by acute cannulation of parotid gland was 12.4 ± 1.7 fmol/ml (SEM, n=7) and 49.8 ± 10.3 fmol/ml (SEM, n=7) respectively. The arterial concentration of Val⁴-angiotensin III obtained from continuous infusion of Val⁴-angiotensin III at rates of 24 and 48 nmol/h in sodium deficient sheep were 245 ± 32.5 fmol/ml (n=6) and 330 ± 11.4 fmol/ ml (n=7) respectively. The clearance rate of exogenous Val⁴-angiotensin III in sodium deficient sheep after correction for endogenous level was calculated to be 140 ± 13.6 L/h (SEM, n=13). This was in the same order as Ile⁵-angiotensin II and Ile⁴-angiotensin III reported earlier in sodium replete sheep. Prolonged intravenous infusion of Val⁴-angiotensin III at a rate of 48 nmol/h in sodium- deficient sheep suppressed plasma renin concentration to the same extent as equimolar infusions of angiotensin II. This suggests that angiotensin III may inhibit renin secretion by a similar mechanism to angiotensin II.     

Identification of serine proteinases with tonin-like activity in the rat submandibular and prostate glands.

Two enzymes with tonin-like activity, designated rSMT3 and rSMT4, were purified from rat submandibular glands and another, rPT1, was obtained from the prostate. The three enzyme fractions hydrolysed angiotensin I, angiotensinogen (AG) and synthetic AG(1-14) to form angiotensin II. With angiotensin I as substrate, pH optima were 6.5 for rSMT3, 6.8 for rSMT4 and 7.5 for rPT1. With AG(1-14), the three enzymes had optimal activity at pH 7.5. The three enzymes had negligible activity upon a kallikrein substrate, Ac-Phe-Arg-Nan. The enzymes were inhibited by aprotinin, soybean trypsin inhibitor and phenylmethanesulfonyl fluoride but not by two angiotensin converting enzyme inhibitors, ethylenediaminetetracetic acid or enalaprilat. N-tosyl-L-phenylalanine chloromethyl ketone (1 mM) inhibited rPT1 and rSMT4 but not rSMT3. Molecular weights (SDS-PAGE) were 31,700 for rSMT3, 29,800 for rSMT4 and 28,100 for rPT1. Total activity in the prostate is 150-times lower than in the submandibular gland, where 92% of the tonin activity is related to rSMT4. Physical and chemical properties suggest that rSMT4 is tonin, whereas rSMT3 and rPT1 are tonin-like enzymes which can generate angiotensin II from different substrates.     

Reduction of blood clearance rate of [Val5] angiotensin II by [Sar1, ILE8] angiotensin II in sheep

The present study examines the effect of [Sar¹, Ile⁸] angiotensin II ([Sar¹, Ile⁸] ANG II) on the blood clearance rate of [Val⁵] angiotensin II ([Val⁵] ANG II) in conscious, sodium-replete sheep. Animals were infused simultaneously with [Val⁵] ANG II and [Sar¹, Ile⁸] ANG II at a rate of 42 nmol/h and 6 μmol/h respectively. Blood [Val⁵] ANG II was quantitatively determined with care taken in separating [Val⁵] ANG II from [Sar¹, Ile⁸] ANG II prior to radioimmunoassay. The blood clearance rate of [Val⁵] ANG II calculated from infusion rate/blood concentration was significantly different before and during [Sar¹, Ile⁸] ANG II infusion, being 141 ± 13 L/h (n = 12) and 95 ± 10 L/h (n = 12) respectively. Plasma renin concentration remained suppressed after the commencement of [Sar¹, Ile⁸] ANG II infusion. $\text{In-vitro}$ studies showed no significant decrease in the rate of degradation of [Val⁵] ANG II in blood in the presence of [Sar¹, Ile⁸] ANG II. Possible interpretation of this reduction of blood clearance rate of [Val⁵] ANG II by 45 ± 15 L/h (n = 6) was discussed.     

The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties

Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l⁻¹) were produced in response to Cu²⁺. Highest laccase levels (19,000 U l⁻¹) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had M _w of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability. Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60°C, and at 5°C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any activity during 6 months storage at 4°C. Kinetic constants (K _m, k _cat) were determined for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine.     

Studies of monoamine oxidases: properties of the enzyme in bovine and rabbit brain mitochondria

Brain mitochondria were prepared from rabbit and bovine cerebral cortex and the purity and intactness of the preparation assessed through the use of enzyme markers and electron microscopy. Enzymatic properties of monoamine oxidase were studied in the purified mitochondrial preparations which were essentially devoid of major contamination by other organelles, especially microsomes. Five substrates were used for characterization of the enzyme: dopamine, kynuramine, serotonin, tryptamine and tyramine. It was found that there was considerable substrate variation in the properties, but in general, the two species showed similar characteristics. The more pertinent findings were: (1) apparent K_m values ranged from 1.1 ± 10^?5m for tryptamine to 2.5 ± 10^?4m for dopamine; (2) substrate specificity from V_max values in decreasing order was tyramine > dopamine > kynuramine > serotonin > tryptamine for the bovine enzyme and tyramine > kynuramine > dopamine > serotonin > tryptamine for rabbit; (3) there appeared to be three distinct pH optima according to substrate: pH 7.5 for phenylethylamines, pH 8.2–8.5 for the indolylamines and pH 9.1 for kynuramine; and (4) the activity with tyramine was highly sensitive to increased oxygen tension while kynuramine showed no sensitivity. It is proposed that the properties of monoamine oxidase, a membrane-bound enzyme, might be influenced by the microenvironment and results are also discussed in terms of multiple forms or multiple activity sites on a single form.     

Properties of spinach chloroplast fructose-1,6-diphosphatase

Photosynthetic fructose-1,6-diphosphatase (FDPase) fractions I and II, earlier purified from spinach leaves, show a similar amino acid composition, with the exception of a higher glutamic acid content in the latter. In both fractions glutamic and aspartic acids are the main amino acids. pH activity profiles of fractions I and II are similar, with optima at 8·65–8·70, both showing a high specificity for fructose- 1,6-diphosphate. These two fractions are Mg²⁺-dependent for activity, with an Optimum Mg²⁺ concentration of 10 mM in standard conditions, which shifts to 5 mM when the MG²⁺/EDTA ratio is increased to 10; Mn²⁺ and Co²⁺ are slightly active. EDTA enhances FDPase activity slightly, with an optimum at 0·4–0·8 mM. Cysteine has no activating effect, and acts as an inhibitor above 10 mM. Both I and II have an optimum substrate concentration of 4 mM, and the substrate inhibits at concns above this value. Kinetic velocity curves are sigmoidal, with the concave zone located in the range of physiological substrate concns. (Hill coefficient 1·75 for both). This suggests a strong regulatory role of fructose-1,6-diphosphate. K_m values are 1·4 × 10⁻³ M (fraction I) and 1·1 × 10⁻³ M (fraction II). The highest activity rate occurs at 60°, in accordance with the high thermostability of both fractions; the activation energies are 14·3 kcal/mol (fraction I) and 13·0 kcal/mol (fraction II).     

Synthesis,characterization, and biological activity of Nα-(N-fluoresceinthiocarbamoyl)-(Asp1, Ile5)-angiotensin II

A fluorescent analog of angiotensin II was synthesized by reacting fluorescein 5′-isothiocyanate with (Asp¹, Ile⁵)-angiotensin II. N^α-(N-Fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II was purified by chromatography on DEAE-cellulose and Sephadex G-25. Analysis of the analog by thin-layer chromatography, thin-layer electrophoresis, and reversed-phase high-performance liquid chromatography indicated that the analog was free of angiotensin II and fluorescein 5′-isothiocyanate. N-Terminal sequence analysis demonstrated that fluorescein 5′-isothiocyanate reacted with the N-terminal aspartic acid residue of angiotensin II. N^α-(N-Fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II has an absorption maximum at 492 nm, and the value of the molar extinction coefficient, ?, is 7.7 × 10⁴m^?1 cm^?1. The fluorescence emission maximum occurs at 520 nm. Infusion of the analog (0.69 μg/min/kg body wt) directly into the renal artery of an anesthetized rat reduced the blood flow by 12 to 27% within 2 min. Infusion of angiotensin II (0.48 μg/min/kg body wt) reduced renal arterial blood flow by 35 to 53% within 2 min. Saralasin, a partial agonist and antagonist of angiotensin II, inhibited the biologic effect of the fluorescent analog and angiotensin II by 75 and 70%, respectively. The purity, spectral properties, and in vivo biologic activity of N^α-(N-fluoresceinthiocarbamoyl)-(Asp¹, Ile⁵)-angiotensin II indicate that this analog should facilitate characterization of angiotensin II receptors.     

Estrogen modulation of angiotensin-stimulated phosphoinositide hydrolysis in slices of rat anterior pituitary

In this study, slices of rat anterior pituitary were prelabeled with [³H]myo-inositol and the ability of angiotensins II and III to stimulate [³H]phosphoinositide hydrolysis was characterized. When using tissue derived from ovariectomized rats, dose-response experiments revealed that angiotensin II significantly increases [³H]inositol monophosphate formation (in the presence of 10 mM LiCI) at concentrations of 10 nM and above. Maximal stimulation by angiotensin II was observed at 1 μM (228% of basal) and 50% maximal stimulation was at 10.8 ± 2.7 nM. Angiotensin III was less potent when compared to angiotensin II (maximal stimulation at 10 μM; 220% of basal: 50% maximal stimulation, 475 ± 159 nM). When using normal female rats, significant stimulation by angiotensin II was not observed until 1 μM angiotensin II. When ovariectomized rats were treated for 7 days with 17β-estradiol, increases in [³H]inositol monophosphate induced by 1 μM angiotensin II were significantly reduced when compared to sesame oil vehicle controls.This study shows that estrogen down-regulates angiotensin receptor coupling in the anterior pituitary. Moreover, it illustrates the influence of the hormonal state of the animal on the regulation of the effects of angiotensins in this tissue.     

Identification and characterization of angiotensin II receptors in cardiac sarcolemma

We have used [¹²⁵I] angiotensin II to investigate the presence of specific angiotensin II receptors in beef heart sarcolemmal membranes. The observed binding is saturable, reversible and specific. The apparent equilibrium dissociation constant is 2.23 ± 0.15 ($\text{x}$ ± SEM) and the maximal number of binding sites per mg membrane protein is 32.8 ± 5.4 fmol ($\text{x}$ ± SEM). The specific binding is 80–100% of the total [¹²⁵I] angiotensin II bound and is directly proportional to membrane protein concentration over the range of 33–173 μg protein per ml. Angiotensin II and its antagonists competed for binding in a potency order of (agent, K_i): angiotensin II, 0.9nM > Sar¹ Ala³, 7 nM > Sar¹-Ile³, 51 nM > Sar¹-Leu³, 427nM > angiotensin I, 1709 nM. The ability to characterize and quantify these receptors should now provide a method for investigating the mechanisms underlying the effects of angiotensin II on myocardial tissues.     

Conformation and activity in angiotensin II peptides

Angiotensin II and its competitive inhibitor [Sar¹, Ile⁸]-angiotensin II, as well as several analogs of these two compounds specifically chosen for their well-defined pharmacological properties, were studied by circular dichroism and nuclear magnetic resonance methods at various pH values in aqueous solution and in d₆-dimethylsulfoxide. The results were compared with their biological activities. This allowed us to establish relationships between conformation and pressor activity, explaining most of the properties of angiotensin II, its inhibitor, and the analogs successively substituted in positions 3 and 5.     

Absolute quantification of endogenous angiotensin II levels in human plasma using ESI-LC-MS/MS

Background

Angiotensin II acts as a peptide hormone and component of renin-angiotensin- system (RAS) regulating the blood pressure, and seems to be involved in renal and vascular disorders. There is no reliable quantification method for angiotensin II available until now and the angiotensin II plasma levels described in the literature are correspondingly strongly divergent. Therefore, we developed and validated a sensitive, selective and reliable LC-ESI-MS/MS method for absolute quantification of angiotensin II concentration in human plasma based on the AQUA strategy.

Methods

Plasma samples were extracted using MAX Oasis cartridges and were subjected to a further immunoaffinity-purification using immobilized anti-angiotensin II antibodies in order to isolate endogenous angiotensin II. Stable isotope (¹³C- and ¹⁵ N-) labeled angiotensin II was used as an internal standard. The fractionated samples were analysed using LC-ESI-MS/MS.

Results

The calibration curve was established in plasma in the concentration range 6–240 pM (r² > 0.999). The developed and validated method was successfully applied for quantification of endogenous angiotensin II in human plasma of healthy volunteers and chronic kidney disease (CKD-5D) patients. The mean plasma angiotensin II levels were found to be 18.4 ± 3.3 pM in healthy subjects and 64.5 ± 32.4 pM in CKD-5D patients (each n =9).

Conclusion

The LC-ESI-MS/MS-based method for quantification of angiotensin II levels in human plasma was successfully evaluated within the study. This method is applicable for clinical applications aiming at the validation of the impact of highly physiologically and pathophysiologically active angiotensin II.     

Angiotensin II Promotes Leptin Production in Cultured Human Fat Cells by an ERK1/2‐dependent Pathway

Objective: The fat cell hormone leptin is known to be implicated in the pathogenesis of hypertension and cardiovascular disease. Here we tested whether angiotensin (Ang) II is involved in the control of leptin release from human adipocytes. Research Methods and Procedures: Leptin secretion was assessed from in vitro differentiated human adipocytes by radioimmunoassay. Western blot experiments were used to test for the signaling pathway activated by Ang II. Results: Ang II increased leptin secretion into the culture medium in a dose‐ and time‐dependent fashion. At 10^?5 M Ang II, the leptin concentration in the medium was increased at 24 hours by 500 ± 222% compared with control cultures (p < 0.05). This effect was also seen at the mRNA level. Similar effects were seen after exposure of fat cells to Ang III and Ang IV. Preincubation of fat cells with candesartan, an angiotensin II type 1 receptor antagonist, or the extracellular‐signal‐regulated kinases 1 and 2 inhibitor UO126 completely abolished the effect of Ang II on leptin production. The peroxisome proliferator‐activated receptor‐gamma agonist troglitazone modestly attenuated leptin release. Discussion: In conclusion, Ang II and its metabolites stimulated leptin production in human adipocytes. This effect is mediated through an extracellular‐signal‐regulated kinases 1 and 2‐dependent pathway and includes the angiotensin II type 1 receptor subtype.     

Effects of angiotensin II antagonists on the contractile and hydrosmotic effect of AT II and AT III in the toad (Bufo arenarum)

The effects of peptide and non-peptide angiotensin II receptor antagonists on the responses to angiotensin II were examined using aortic rings and skin isolated from the toad. The contractile responses of aortic rings to (Ala-Pro-Gly) angiotensin II were inhibited by the angiotensin II analogue Leu⁸ angiotensin II, with a pA₂ value of 7.6. Similarly, the concentration response curve for (Ala-Pro-Gly) angiotensin II was displaced to the right by the specific angiotensin receptor subtype antagonist DuP 753, with a pA₂ value of 6.0. In contrast, the angiotensin receptor subtype 2 antagonists PD 123177 and CGP 42112A did not modify the contractile response to (Ala-Pro-Gly) angiotensin II. None of the antagonists was able to alter the contractile response to norepinephrine. Both Leu⁸ angiotensin II (10^-8 mol·l^-1) and DuP 753 (10^-6 mol·l^-1) partially inhibited angiotensin III-induced contractions in toad aorta. Angiotensin III, in turn, exhibited lower activity than [Asn¹-Val⁵] angiotensin II in this preparation, its molar potency ratio being 0.293. Previous work from this laboratory reported that osmotic water permeability in the skin of the toad Bufo arenarum was increased by angiotensin II, the effect being blocked by the peptide antagonist Leu⁸ angiotensin II. The hydrosmotic response to [Asn¹-Val⁵] angiotensin II (10^-7 mol·l^-1) was significantly inhibited by DuP 753 (10^-6 and 5×10^-6 mol·l^-1), whereas the response was not inhibited by a tenfold higher concentration of either PD 123177 or CGP 42112A. DuP 753 (10^-6 mol·l^-1) also inhibited the hydrosmotic response to angiotensin III (10^-7 mol·l^-1). These results suggest that receptors for angiotensin II present in isolated toad aorta and skin exhibit pharmacological features similar to those characterized as angiotensin subtype 1 in mammalian tissues.Abbreviations AT ₁ angiotensin receptor subtype 1 - AT ₂ angiotensin receptor subtype 2 - AT II angiotensin II - AT III angiotensin III - CDRC cumulative doseresponse curve(s) - NE norepinephrine - SCC short-circuit current     

Purification and Properties of Hypoxanthine Phosphoribosyltransferase from Streptomyces cyanogenus

Hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) of a strain of Streptomyces cyanogenus was purified 1,900-fold to an apparent homogenity from cell-free extracts. The enzyme had a molecular weight of 150,000 and consisted of eight identical subunits with a molecular weight of 18,000. The isoelectric point was at pH 4.4. The enzyme required Mg²⁺ or Ma²⁺ for activity and had a pH optimum at 8.5. Hypoxanthine and guanine were good substrates for the enzyme. Xanthine was a very poor substrate and adenine was not a substrate. Apparent Km values of the enzyme for hypoxanthine, guanine and 5-phosphoribose-1-pyro-phosphate were 1.6 × 10^?8, 2.7 × 10^?6 and 6.3 × 10^?5 m, respectively. All purine nucleotides tested inhibited the activity significantly, apparently by competing with 5-phosphoribose-1-pyrophosphate.     

An improved method for measuring angiotensin I converting enzyme activity using a highly sensitive angiotensin II radioimmunoassay

A highly sensitive assay for angiotensin I converting enzyme has been developed by using angiotensin I as a substrate. Angiotensin II generated in the reaction mixture was measured by a newly developed specific radioimmunoassay. To protect against angiotensin II destruction, bestatin, an inhibitor of renin, was also used to inhibit plasma renin activity. The reaction was stopped by adding EDTA and MK-521, inhibitors of angiotensin I converting enzyme. The specificity of the antiserum used for the angiotensin II radioimmunoassay was very high. The cross reactivity with angiotensin I was less than 0.5% and none of the proteolytic enzyme inhibitors crossreacted in the assay. The inhibitory effect of pepstatin on plasma renin activity was very high (more than 80%) under the standard assay conditions employed. Serum angiotensinase activity was completely inhibited by the addition of bestatin. An excellent correlation was obtained between this new method and the spectrophotometric method using a synthetic substrate, Hip-His-Leu. The generation of as little as 12 pM of Angiotensin II can be detected. Such low concentration have not been measurable with the usual spectrophotometric method. This new method will facilitate clinical and experimental studies on this unique enzyme, since very low levels of activity can be determined by this highly sensitive radioimmunoassay for angiotensin II.     

Characterization of a thermally stable and organic solvent-adaptative NAD+ -dependent formate dehydrogenase from Bacillus sp. F1

Aims: To characterize a robust NAD⁺‐dependent formate dehydrogenase firstly obtained from a nonmethylotroph, Bacillus sp. F1. Methods and Results: The Bacillus sp. F1 NAD⁺‐dependent formate dehydrogenase (BacFDH) gene was cloned by TAIL‐PCR and heterologous expressed in Escherichia coli. BacFDH was stable at temperatures below 55°C, and the half‐life at 60°C was determined as 52·9 min. This enzyme also showed a broad pH stability and retained more than 80% of the activities after incubating in buffers with different pH ranging from 4·5 to 10·5 for 1 h. The activity of BacFDH was significantly enhanced by some metal ions. Moreover, BacFDH exhibited high tolerance to 20% dimethyl sulfoxide, 60% acetone, 10% methanol, 20% ethanol, 60% isopropanol and 20% n‐hexane. Like other FDHs, BacFDH displayed strict substrate specificity for formate. Conclusion: We isolated a robust formate dehydrogenase, designated as BacFDH, which showed excellent thermal stability, organic solvent stability and a broad pH stability. Significance and Impact of the Study: The multi‐aspect stability makes BacFDH a competitive candidate for coenzyme regeneration in practical applications of chiral chemicals and pharmaceuticals synthesis with a relatively low cost, especially for the catalysis performed in extreme pH conditions and organic solvents.