Purification and properties of a citrate synthase from Nitrosomonas sp. TK794 and a comparison with the enzymes of another nitrifying bacteria

Citrate(si)-synthase (citrate oxaloacetate-lyasem EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from an ammonia-oxidizing chemoautotrophic bacterium, Nitrosomonas sp. TK794. The molecular mass of the native enzyme was estimated to be about 287 kDa by gel filtration, whereas SDS-PAGE produced one band with M_r values of 44.7 kDa, suggesting that the enzyme is a hexamer consisting of identical subunits. The isoelectric point of the enzyme was 5.0. The pH and temperature optima for citrate synthase (CS) activity was about 7.5–8.0 and 40°C, respectively. The citrate synthase was stable over a pH range of 6.0–8.5 and up to 40°C. The apparent K_m values for oxaloacetate and acetyl-CoA were about 11 μM and 247 μM, respectively. The activity of the citrate synthase was not inhibited by ATP, NADH or 2-oxoglutarate at 5mM, and was activated by potassium chloride at 0.1–100 mM. The N-terminal amino acid sequence of the enzyme protein was PPQDVATLSPGENKKTIELPILG.     

l-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea

The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. l-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP⁺-dependent enzymes from chloroplasts and was separated from the NAD⁺-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis–Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K _m for oxaloacetate was 20 μM; the K _m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD⁺ increased with pH but there was very little activity with NADP⁺. At pH 7.0, the K _m for l-malate was 5 mM and the K _m for NAD⁺ was 24 μM. The reductive activity was quite insensitive to inhibition by l-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10–20 times higher than the oxidative activity. These results indicate that the l-malate dehydrogenase in N. europaea is similar to other NAD⁺-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.     

Purification and Characterization of a Novel (<Emphasis Type="Italic">R</Emphasis>)-1-Phenylethanol Dehydrogenase from <Emphasis Type="Italic">Lysinibacillus</Emphasis> sp. NUST506

A novel (R)-1-phenylethanol dehydrogenase was successfully purified from Lysinibacillus sp. NUST506 by preparative polyacrylamide gel electrophoresis. The enzyme is a NAD+-dependent oxidoreductase. The molecular weight of the (R)-1-phenylethanol dehydrogenase measured by SDS-PAGE was about 28 kDa. Furthermore, the optimal reaction conditions for the oxidative reaction were 70°C and pH 9.5 and for the reductive reaction were 65°C and pH 6.5. Under the optimal conditions, the K_M and k_cat values with (R)-1-phenylethanol as a substrate were found to be 0.78 mM and 123 s^–1 and with acetophenone they were 0.56 mM and 125 s^–1, respectively. The (R)-1-phenylethanol dehydrogenase became more stable at pH 9.5 in comparison with pH 5.0 and high stability was noticed at 4 and 37°C. Properties of the enzyme place it as a promising candidate for industrial applications.     

Purification and some properties of citrate synthase from a nitrite-oxidizing chemoautotroph,Nitrobacter agilis ATCC 14123

Citrate (si)-synthase (citrate oxaloacetate-lyase, EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from a nitrite-oxidizing chemoautotrophic bacterium, Nitrobacter agilis ATCC 14123. The molecular mass (M_r) of the native enzyme was estimated to be about 250,000 by gel filtration, whereas SDS-PAGE gave two bands with M_r values of 45,000 and 80,000, respectively, suggesting that the enzyme is a tetramer consisting of two different subunits (α: 45,000, β: 80,000). The isoelectric point of the enzyme was 5.4. The pH and temperature optima on the citrate synthase activity were about 7.5–8.0 and 30–35°C, respectively. The citrate synthase was stable in the pH range of 6.0–9.0 and up to 55°C. The apparent K_m values for oxaloacetate and acetyl-CoA were about 27 μM and 410 μM, respectively. The activity of citrate synthase was not inhibited by ATP (1 mM), NADH (1 mM) or 2-oxoglutarate (10 mM), but was strongly inhibited by SDS (1 mM). Activation by metal ions was not observed.     

Expression,purification, and characterization of recombinant mangrove glutamine synthetase

To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was ~40 kDa. Optimal conditions for biosynthetic activity were found to be 35 °C at pH 7.5. The Mg²⁺-dependent biosynthetic activity was strongly inhibited by Ni²⁺, Zn²⁺, and Al³⁺, whereas was enhanced by Co²⁺. The apparent K _m values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, and 2.54 mM for ATP, 2.80 mM for NH₄ ⁺ respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions.     

Purification and characterization of malate dehydrogenase from the phototrophic bacterium,Rhodopseudomonas capsulata

Malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37) has been purified about 480-fold from crude extract of the facultative phototrophic bacterium, Rhodopseudomonas capsulata by only two purification steps, involving Red-Sepharose affinity chromatography. The enzyme has a molecular mass of about 80 kDa and consists of two subunits with identical molecular mass (35 kDa). The enzyme is susceptible to heat inactivation and loses its activity completely upon incubation at 40°C for 10 min. Addition of NAD⁺, NADH and oxaloacetate, but not l-malate, to the enzyme solution stabilized the enzyme. The enzyme catalyzes exclusively the oxidation of l-malate, and the reduction of oxaloacetate and ketomalonate in the presence of NAD⁺ and NADH, respectively, as the coenzyme. The pH optima are around 9.5 for the l-malate oxidation, and 7.75–8.5 and 4.3–7.0 for the reduction of oxaloacetate and ketomalonate, respectively. The K_m values were determined to be 2.1 mM for l-malate, 48 μM for NAD⁺, 85 μM for oxaloacetate, 25 μM for NADH and 2.2 mM for ketomalonate. Initial velocity and product inhibition patterns of the enzyme reactions indicate a random binding of the substrates, NAD⁺ and l-malate, to the enzyme and a sequential release of the products: NADH is the last product released from the enzyme in the l-malate oxidation.     

Purification and characterization of L-amino acid oxidase from the solid-state grown cultures of Aspergillus oryzae ASH

L-Amino acid oxidase (L-AAO) was purified from the solid state-grown cultures of A. oryzae ASH (JX006239.1) by fractional salting out, followed by ion exchange and gel filtration chromatography, to its molecular homogeneity, displaying 3.38-fold purification in comparison with the crude enzyme. SDS-PAGE revealed the enzyme to be a homo-dimer with ～55-kDa subunits, with approximate molecular weight on native PAGE of 105–110 kDa. Two absorption maxima, at 280 nm and 341 nm, for the apoproteinic and FMN prosthetic group of the enzyme, respectively, were observed, with no detected surface glycosyl residues. The enzyme had maximum activity at pH 7.8–8.0, with ionic structural stability within pH range 7.2–7.6 and pH precipitation point (pI) 4.1–5.0. L-AAO exhibited the highest activity at 55°C, with plausible thermal stability below 40°C. The enzyme had T _1/2 values of 21.2, 8.3, 3.6, 3.1, 2.6 h at 30, 35, 40, 50, 60°C with Tm 61.3°C. Kinetically, A. oryzae L-AAO displayed a broad oxidative activity for tested amino acids as substrates. However, the enzyme had a higher affinity towards basic amino acid L-lysine (K _m 3.3 mM, K _cat 0.04 s^?1) followed by aromatic amino acids L-tyrosine (K _m 5.3 mM, K _cat 0.036 s^?1) and L-phenylalanine (K _m 6.6 mM), with 1ow affinity for the S-amino acid L-methionine (K _m 15.6 mM). The higher specificity of A. oryzae L-AAO to L-lysine as substrate seems to be a unique property comparing to this enzyme from other microbes. The enzyme was significantly inhibited by hydroxylamine and SDS, with slight inhibition by EDTA. The enzyme had a little effect on AST and ALT, with no effect on platelet aggregation and blood hemolysis in vivo with an obvious cytotoxic effect towards HepG2 (IC₅₀ 832.2 μg/mL) and MCF-7 (IC₅₀, 370.6 μg/mL) tumor cells in vitro.     

Purification and characterization of piceid-β-d-glucosidase from Aspergillus oryzae

The piceid-β-d-glucosidase that hydrolyzes the β-d-glucopyranoside bond of piceid to release resveratrol was isolated from Aspergillus oryzae sp.100 strain, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 77 kDa. The optimum temperature of the piceid-β-d-glucosidase was 60 °C, and the optimum pH was 5.0. The piceid-β-d-glucosidase was stable at less than 60 °C, and pH 4.0–5.0. Ca²⁺, Mg²⁺ and Zn²⁺ ions have no significant effect on enzyme activity, but Cu²⁺ ion inhibits enzyme activity strongly. The K_m value was 0.74 mM and the V_max value was 323 nkat mg⁻¹ for piceid.     

Fatty Acid Synthesis in Leucoplasts Isolated from Developing Seeds of Brassica campestris

Fatty acid synthesis from Na (1-¹⁴C) acetate in leucoplasts isolated from developing seeds of Brassica compestris was found to be maximum when leucoplasts were supplied with 0.8 mM acetate, 20 mM NaHCO₃, 8 mM ATP, 8 mM MgCl₂, 4 mM MnCl₂, 0.6 mM CoA, 1 mM NADH, 1 mM NADPH and 0.2 M sorbitol and incubated at 30°C for 2 h. The rate of fatty acid synthesis was highest at pH 8.5 In presence of 0.4 M Bistris-propane buffer and linear for upto 4 h at 30°C with 80–110 μg plastid protein. Sorbitol was an essential requirement as it prevented the rupturing of leucoplasts by osmosis. ATP and divalent cations were almost absolute requirements, whereas nucleotides, CoA and bicarbonate improved the rate of fatty acid synthesis by two to ten folds. Mg²⁺ and NADH were the preferred cation and nucleotide, respectively. High concentration of dithiothreltol inhibited the incorporation of (¹⁴C) acetate Into fatty acids. The system developed as above could be used for in vitro studies.     

Transhydroxylase of Pelobacter acidigallici: a molybdoenzyme catalyzing the conversion of pyrogallol to phloroglucinol

Trihydroxybenzenes are degraded anaerobically through the phloroglucinol pathway. In Pelobacter acidigallici as well as in Pelobacter massiliensis, pyrogallol is converted to phloroglucinol in the presence of 1,2,3,5-tetrahydroxybenzene by intermolecular hydroxyl transfer. The enzyme catalyzing this reaction was purified to chromatographic and electrophoretic homogeneity. Gel filtration and electrophoresis revealed a heterodimer structure with an apparent molecular mass of 127 kDa for the native enzyme and 86 kDa and 38 kDa, respectively, for the subunits. The enzyme was not sensitive to oxygen. HgCl₂, p-chloromercuribenzoic acid, and CuCl₂ inhibited strongly the reaction indicating an essential function of SH-groups. Transhydroxylase had a pH-optimum of 7.0 and a pI of 4.1. The apparent temperature optimum was in the range of 53°C to 58°C. The activation energy for the conversion of pyrogallol and 1,2,3,5-tetrahydroxybenzene to phloroglucinol and tetrahydroxybenzene was 31.4 kJ per mol. Purified enzyme exhibited a specific activity of 3.1 mol. m⁻¹ mg⁻¹ protein and an apparent K_m for pyrogallol and 1,2,3,5-tetrahydroxybenzene of 0.70 mM and 0.71 mM, respectively. The enzyme was found to contain per mol heterodimer 1.1 mol molybdenum, 12.1 mol iron and 14.5 mol acid-labile sulfur. Requirement for molybdenum for transhydroxylating enzyme activity was proven also by cultivation experiments. No hints for the presence of flavins were obtained. The results presented here support the hypothesis that a redox reaction is involved in this intermolecular hydroxyl transfer.     

Purification and characterization of SDG-β-d-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Aspergillus oryzae

The SDG-β-d-glucosidase that hydrolyzes the glucopyranoside bond of secoisolariciresinol diglucoside (SDG) to release secoisolariciresinol (SECO) was isolated from Aspergillus oryzae 39 strain and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 64.9 kDa. The optimum temperature of the SDG-β-d-glucosidase was 40 °C, and the optimum pH was 5.0. The SDG-β-d-glucosidase was stable at less than 65 °C, and pH 4.0–6.0. Ca²⁺, K⁺, Mg²⁺ and Na⁺ ions have no significant effect on enzyme activity, Zn²⁺ and Cu²⁺ ions have weakly effect on enzyme activity, but Fe³⁺ ion inhibits enzyme activity strongly. The K_m value of SDG-β-d-glucosidase was 0.14 mM for SDG.     

Cytochrome a1 of Nitrosomonas europaea resembles aa3-type cytochrome c oxidase in many respects

Cytochrome c oxidase of Nitrosomonas europaea has been called cytochrome a₁ by Erickson et al. (Erickson, R.H., Hooper, A.B. and Terry, K.R. (1972) Biochim. Biophys. Acta 283, 155–166) because the reduced form of their preparation had the α peak at 595 nm. In the present studies, the enzyme was purified to an electrophoretically almost homogeneous state and some of its properties were studied. The enzyme much resembled cytochrome aa₃-type oxidase although its reduced form showed the α peak at 597 nm. (1) The absorption spectra of the CO compound of the reduced enzyme and CN⁻ compounds of the oxidized and reduced enzyme were similar to those of the respective compounds of cytochrome aa₃, as well as the absorption spectrum of the intact enzyme resembled that of the cytochrome. (2) The enzyme possessed two molecules of haem a and 1–2 atoms of copper in the molecule. (3) The enzyme molecule was composed of two kinds of subunits of M_r 50000 and 33000, respectively, as are other bacterial cytochromes aa₃. Although the enzyme resembled other bacterial cytochromes aa₃ in many properties, it differed greatly in two properties; its CO compound was easily dissociated into the oxidized enzyme and CO in air, and 50% inhibition of its activity by CN⁻ required approx. 100 μM of the reagent. The enzyme oxidized 0.57, 1.6 and 1.8 mol horse, Candida krusei and N. europaea ferrocytochromes c per s per mol haem a, respectively, in 10 mM phosphate buffer, pH 6.0. The turnover numbers with eukaryotic ferrocytochromes c were increased to 32 and 14, respectively, by addition of cardiolipin (14 μ · ml⁻¹).     

S-Adenosylmethionine synthetase in bloodstream Trypanosoma brucei

S-adenosylmethionine synthetase was studied from bloodstream forms of Trypanosoma brucei brucei, the agent of African sleeping sickness. Two isoforms of the enzyme were evident from Eadie Hofstee and Hanes-Woolf plots of varying ATP or methionine concentrations. In the range 10–250 μM the K_m for methionine was 20 μM, and this changed to 200 μM for the range 0.5–5.0 mM. In the range 10–250 μM the K_m for ATP was 53 μM, and this changed to 1.75 mM for the range 0.5–5.0 mM. The trypanosome enzyme had a molecular weight of 145 kDa determined by agarose gel filtration. Methionine analogs including selenomethionine, L-2-amino-4-methoxy-cis but-3-enoic acid and ethionine acted as competitive inhibitors of methionine and as weak substrates when tested in the absence of methionine with [¹⁴C]ATP. The enzyme was not inducible in procyclic trypomastigotes in vitro, and the enzyme half-life was > 6 h. T. b. brucei AdoMet synthetase was inhibited by AdoMet (K_i 240 μM). The relative insensitivity of the trypanosome enzyme to control by product inhibition indicates it is markedly different from mammalian isoforms of the enzyme which are highly sensitive to AdoMet. Since trypanosomes treated with the ornithine decarboxylase antagonist DL-α-difluoromethylornithine accumulate AdoMet and dcAdoMet (final concentration ≈ 5 mM), this enzyme may be the critical drug target linking inhibition of polyamine synthesis to disruption of AdoMet metabolism.     

Purification and characterization of a novel (−) gamma-lactamase from<Emphasis Type="Italic">Microbacterium hydrocarbonoxydans</Emphasis>

A (?) gamma-lactamase fromMicrobacterium hydrocarbonoxydans was purified to homogeneity by chromatography methods. SDS-PAGE showed the molecular weight of the enzyme was about 31 kDa. The purified enzyme had a specific activity of 61.3±2.5 U mg^?1 for 2-azabicyclo [2.2.1] hept-5-en-3-one [(?) gamma-lactam]. The enantioselectivity factor (E) of the purified enzyme was 9.5±0.8 for unreacted (+) gamma-lactam. TheK _m andV _max value were 2.3±0.2 mM and 80.0±15.4 U mg^?1 respectively. The highest activity was found at 30 °C and pH 8.0. ESIMS mass spectrometry analysis results and N-terminal sequence indicated the (?) gamma-lactamase might be a new enzyme.     

Characterization flavanone 3β-hydroxylase expressed from <Emphasis Type="Italic">Populus euphratica</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in dihydroflavonol production

Flavanone 3β-hydroxylase plays very important role in the biosynthesis of flavonoids. A putative flavanone 3β-hydroxylase gene (Pef3h) from Populus euphratica was cloned and over-expressed in Escherichia coli. Induction performed with 0.1 mM IPTG at 20°C led to localization of PeF3H in the soluble fraction. Recombinant enzyme was purified by Ni-NTA affinity. The optimal activity of PeF3H was revealed at pH 7.6 and 35°C. The purified enzyme was stable over pH range of 7.6–8.8 and had a half-life of 1 h at 50°C. The activity of PeF3H was significantly enhanced in the presence of Fe²⁺ and Fe³⁺. The K _M and V _max for the enzyme using naringenin as substrate were 0.23 mM and 0.069 μmoles mg–1min-1, respectively. The K _m and V _max for eriodictyol were 0.18 mM and 0.013 μmoles mg^–1min^–1, respectively. The optimal conditions for naringenin bioconversion in dihydrokaempferol were obtained: OD₆₀₀ of 3.5 for cell concentration, 0.1 mM IPTG, 5 mM α-ketoglutaric acid and 20°C. Under the optimal conditions, naringenin (0.2 g/L) was transformed into 0.18 g/L dihydrokaempferol within 24 h by the recombinant E. coli with a corresponding molar conversion of 88%. Thus, this study provides a promising flavanone 3β-hydroxylase that may be used in biosynthetic applications.     

Cloning, expression and characterization of a novel esterase from Bacillus pumilus

The gene encoding esterase (CE1) from Bacillus pumilus ARA with a calculated molecular weight of 28.4 kDa was cloned, sequenced and efficiently expressed in Escherichia coli. The open reading frame of 747 nucleotides encoded a protein, which was classified as a carboxylesterase with an identity of 87 % to esterase from Bacillus subtilis 168. Recombinant CE1 was purified in a single step to electrophoretic homogeneity by IMAC (Ni²⁺). The enzyme displayed maximum activity toward p-nitrophenyl (pNP) acetate at 37–40 °C and pH?6.5–7.0. It was stable in the pH range from 6.5 to 8.0, and at temperature from 25 to 40 °C. Among four p-nitrophenyl esters tested, the best substrate was pNP acetate with K _m and k _cat values of 0.33 mM and 4.07 s^?1, respectively. Amounts of 2 mM Ca²⁺ and Co²⁺ significantly increased the esterase activity to 190 and 121 %, respectively. These results suggest that CE1 has very attractive applications of increasing feed digestibility in animal nutrition in this moderate temperature range.     

Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite

Nitrosomonas europaea, an obligate ammonia-oxidizing bacterium, lost an increasing amount of ammonia oxidation activity upon exposure to increasing concentrations of nitrite, the primary product of ammonia-oxidizing metabolism. The loss of activity was specific to the ammonia monooxygenase (AMO) enzyme, as confirmed by a decreased rate of NH₄⁺-dependent O₂ consumption, some loss of active AMO molecules observed by polypeptide labeling with ¹⁴C₂H₂, the protection of activity by substrates of AMO, and the requirement for copper. The loss of AMO activity via nitrite occurred under both aerobic and anaerobic conditions, and more activity was lost under alkaline than under acidic conditions except in the presence of large concentrations (20 mM) of nitrite. These results indicate that nitrite toxicity in N. europaea is mediated by a unique mechanism that is specific for AMO.     

Structural and biochemical properties of lichenase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affinity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-configured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu²⁺ ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu²⁺ (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni²⁺, Co²⁺ and Zn²⁺ did not affect the activity of the enzyme at low concentrations (0–10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (β/α)₈-fold a common fold among many glycoside hydrolase families. A cleft was identified that represented the probable location of active site.     

Purification and properties of acetate kinase from Clostridium thermoaceticum

Acetate kinase (ATP: acetate phosphotransferase EC 2.7.2.1) has been purified from Clostridium thermoaceticum. The enzyme of a specific activity of 282 μmoles min^-1 mg^-1 appeared homogeneous as judged from Sephadex chromatography and sedimentation velocity. Polyacrylamide gel electrophoretic patterns at pH 9.0 and 9.5 showed heterogeneity. Velocity curves obtained with varying amount of acetate were of the Michaelis-Menten type with an apparent K _m of 0.135 M. With varying amounts of ATP sigmoidal kinetic was observed (S_0.5=1.64 mM), suggesting cooperative binding of this substrate. The enzyme had only moderate thermal stability with a temperature optimum of about 60°C and exhibited a broken line in an Arrhenius graph. From gel filtration a molecular weight of about 60 000 daltons was estimated for the enzyme. The S_20w value was 6.0 S.