Enhanced decolorization of Solar brilliant red 80 textile dye by an indigenous white rot fungus Schizophyllum commune IBL-06

An indigenously isolated white rot fungus, Schizophyllum commune IBL-06 was used to decolorize Solar brilliant red 80 direct dye in Kirk’s basal salts medium. In initial screening study, the maximum decolorization (84.8%) of Solar brilliant red 80 was achieved in 7 days shaking incubation period at pH 4.5 and 30 °C. Different physical and nutritional factors including pH, temperature and fungal inoculum density were statistically optimized through Completely Randomized Design (CRD), to enhance the efficiency of S. commune IBL-06 for maximum decolorization of Solar brilliant red 80 dye. The effects of inexpensive carbon and nitrogen sources were also investigated. Percent dye decolorization was determined by a reduction in optical density at the wavelength of maximum absorbance (λ_max, 590 nm). Under optimum conditions, the S. commune IBL-06 completely decolorized (100%) the Solar brilliant red 80 dye using maltose and ammonium sulfate as inexpensive carbon and nitrogen sources, respectively in 3 days. S. commune IBL-06 produced the three major ligninolytic enzymes lignin peroxidase (LiP), manganase peroxidase (MnP) and lacaase (Lac) during the decolorization of Solar brilliant red 80. LiP was the major enzyme (944 U/mL) secreted by S. commune IBL-06 along with comparatively lower activities of MnP and Laccase.     

Decolorization of textile azo dyes by aerobic bacterial consortium

The decolorization potential of two bacterial consortia developed from a textile wastewater treatment plant showed that among the two mixed bacterial culture SKB-II was the most efficient in decolorizing individual as well as mixture of dyes. At 1.3 g L^?1 starch supplementation in the basal medium by the end of 120 h decolorization of 80–96% of four out of the six individual azo dyes Congo red, Bordeaux, Ranocid Fast Blue and Blue BCC (10 mg L^?1) was noted. The culture exhibited good potential ability in decolorizing 50–60% of all the dyes (Congo red, Bordeaux, Ranocid Fast Blue and Blue BCC) when present as a mixture at 10 mg L^?1. The consortium SKB-II consisted of five different bacterial types identified by 16S rDNA sequence alignment as Bacillus vallismortis, Bacillus pumilus, Bacillus cereus, Bacillus subtilis and Bacillus megaterium which were further tested to decolorize dyes. The efficient ability of this developed consortium SKB-II to decolorize individual dyes and textile effluent using packed bed reactors is being carried out.     

Antioxidant response of a novel Streptomyces sp. M3004 isolated from legume rhizosphere to H2O2 and paraquat

This paper aims to investigate the effect of H₂O₂ and paraquat on the activities of superoxide dismutase (SOD) and catalase (CAT), and membrane lipid peroxidation (LPO) levels in newly isolated Streptomyces sp. M3004. SOD activities of Streptomyces sp. M3004, grown in 10 mM and 30 mM H₂O₂, were significantly lower than the control cultures. On the other hand, as an antioxidant enzyme, CAT activity in both H₂O₂ treatment conditions increased significantly compared with the control. These activity values in 10 mM and 30 mM H₂O₂ treatment on the 48th hour of incubation were 3.8- and 6.6-fold higher than the control, respectively. SOD activity decreased significantly with respect to paraquat concentration, which was added at the start of the incubation. CAT activities increased significantly in 1.0 mM and 3.0 mM paraquat treatments compared to control. As an indicative marker of membrane damage, LPO levels of the novel isolate Streptomyces sp. M3004 treated with H₂O₂, and paraquat stress conditions were significantly higher than the control. Nevertheless, compared with the 30 mM H₂O₂ in both treatment conditions, LPO levels in 10 mM H₂O₂ were significantly higher. The decreases in SOD activities in paraquat and H₂O₂ treatment conditions resulted in the increases in the LPO levels although it increases in CAT activities.     

Enzymatic decolorization of sulfonphthalein dyes

The white rot fungus (WRF) Pleurotus ostreatus produced manganese peroxidase (MnP) and manganese-independent peroxidase (MIP) activities during solid state fermentation of wheat straw, a natural lignocellulosic substrate. Most of the sulfonphthalein (SP) dyes were decolorized by MnP at pH 4.0. The higher K_m for meta-cresol purple (40 μM) and lower K_m for ortho-cresol red (26 μM) for MnP activities explained the preference for the position of methyl group at ortho than at meta on chromophore. Bromophenol blue decolorizing activity was higher at pH 3.5 and decreased as the concentration of Mn^II was increased. SP-decolorizing activity was associated not only with MnP but also with MIP. Additional bromine group along with the methyl group on SP chromophores decreases the rate of decolorization. Bromination of sulfonphthalein chromophore makes them the poorer substrate for MnP. This is evident from the higher K_m for bromocresol green (117 μM) when compared to bromocresol purple (36 μM) and bromophenol blue (78 μM). The order of preference for the SP dyes as substrate for the MnP-catalyzed decolorizing activity is phenol red > ortho-cresol red > meta-cresol purple > bromophenol red > bromocresol purple > bromophenol blue > bromocresol green and the order of preference for the SP dyes as substrate for the MIP-catalyzed decolorizing activity is bromocresol green > bromophenol blue > bromocresol purple > bromophenol red > meta-cresol purple > ortho-cresol red > phenol red. Inhibition of PR decolorizing activity by NaN₃ provided the evidence of decolorizing activity as an oxidative process.     

Hydrothermal synthesis and structural characterization of an organic–inorganic hybrid material: (H2tptz)2[δ-Mo8O26]·2H2O (tptz=2,4,6-tripyridyltriazine)

The reaction of MoO₃ and 2,4,6-tripyridyltriazine (tptz) in water at 180°C for 48 h and pH 5.5 produces (H₂tptz)₂[Mo₈O₂₆]·2H₂O in 70% yield. The structure is constructed from δ-Mo₈O₂₆ ⁴⁻ clusters, H₂tptz²⁺ and H₃O⁺ cations linked through hydrogen bonding into a network. Crystal data: C₁₈H₁₆Mo₄N₆O₁₄; monoclinic P2₁/n; a=10.2225(5) Å, b=14.0072(6) Å, c=18.1154(8) Å, β=93.896(1)°, V=2587.9(2) Å³, Z=4, D_calc=2.372 g cm⁻³; R₁=0.0271 based on 3212 reflections.     

Purification and characterization of an azoreductase from Escherichia coli CD-2 possessing quinone reductase activity

An oxygen-insensitive intracellular enzyme that is responsible for the decolorization of azo dyes was purified from Escherichia coli CD-2. The molecular weight of the purified enzyme was estimated as 27,000 ± 500 Da. Protein identification indicated that the enzyme had high sequence homology with E. coli K12 quinone reductase, and the enzyme was proved to have both azoreductase and quinone reductase activity. With methyl red as substrate, the optimal pH value and temperature were 6.5 and 37 °C, respectively. The enzyme was stable under different physiochemical conditions. The azoreductase activity was restrained by SDS and was almost completely inhibited by Co²⁺ and Hg²⁺. K_m and V_max values were 0.18 mM and 8.12 U mg^?1 of protein for NADH and 0.05 mM and 6.46 U mg^?1 of protein for methyl red, respectively. The purified enzyme could efficiently decolorize methyl red with both NADH and NADPH as electron donors.     

A new fungal peroxidase with alkaline-tolerant,chloride-enhancing activity and dye decolorization capacity

A new fungal peroxidase (Pspd) from Perenniporia subacida was purified by ammonium sulfate precipitation, DEAE-cellulose DE52 anionic exchange and Sepharose GL-6B chromatography, resulting in a high specific activity of 9.138 U mg⁻¹, 3.622-fold higher than that of crude enzyme at the same level. Polyacrylamide gel electrophoresis and UV–vis adsorption spectrum analysis showed that the purified enzyme is a heme-containing monomer with a molecular mass of 43.0 kDa. Optimal peroxidase activity was obtained at pH 5.5 and 30 °C when using 100.0 mM n-propanol as substrate, and under these conditions, the catalytic efficiency (k_cat/K_m) is 1.57 s⁻¹ μM⁻¹. Pspd was inhibited by l-cysteine, dithiothreitol, EDTA and sodium azide, but stimulated by Mn²⁺, Na⁺, Mg²⁺ and K⁺. The enzyme is stable over a broad pH range of 7.0–8.5 after incubation for 72 h, which indicated that the enzyme is lasting alkaline-tolerant. It was worth noting that the chloride at relatively low concentrations can enhance the peroxidase activity, with concomitant increase in substrate affinity. Additionally, Pspd performed high decolorization capability toward structurally various dyes and the capability was independent of the oxidizing mediators, with 75.31% of Neutral Red (50.0 mg L⁻¹) being decolorized by 1.5 U mL⁻¹ pure enzyme after incubation for 72 h. These properties demonstrated that Pspd has potentials for textile dyes decolorization applications.     

Tuning magnetic properties using targeted structural distortion: New additions to a family of Mn6 single-molecule magnets

We report the synthesis and magnetic properties of three hexametallic Mn clusters: [Mn₆O₂(Et-sao)₆(O₂C-Naphth)₂(EtOH)₄(H₂O)₂] (1) (HO₂C-Naphth = 1-naphthoic acid, Et-saoH₂ = 2-hydroxyphenylpropanone oxime), [Mn₆O₂(Et-sao)₆(O₂C-Anthra)₂(EtOH)₄(H₂O)₂] · 0.66EtOH · 0.33H₂O (HO₂C-Anthra = anthracene-9-carboxylic acid) (2 · 0.66EtOH · 0.33H₂O) and [Mn₆O₂(Et-sao)₆(O₂CPhCCH)₂(EtOH)₄(H₂O)₂] · 1.7EtOH · 0.3H₂O (HO₂CPhC  CH = 4-ethynylbenzoic acid) (3 · 1.7EtOH · 0.3H₂O). Clusters 1–3 exhibit ferromagnetic exchange between all six Mn^III centres resulting in S = 12 ground spin states. Ac magnetic susceptibility and single crystal micro-SQUID measurements on 1–3 confirm SMM behaviour with barriers to magnetisation reversal of 60.12 (1), 60.10 (2) and 66.79 (3) K.     

Effects of salt stress on ion content,antioxidant enzymes and protein profile in different tissues of Broussonetia papyrifera

Although some plant responses to salinity have been characterized, the precise mechanisms by which salt stress damages plants are still poorly understood especially in woody plants. In the present study, the physiological and biochemical responses of Broussonetia papyrifera, a tree species of the family, Moraceae, to salinity were studied. In vitro-produced plantlets of B. papyrifera were treated with varying levels of NaCl (0, 50, 100 and 150 mM) in hydroponic culture. Changes in ion contents, accumulation of H₂O₂, as well as the activities and isoform profiles of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in the leaves, stems and roots were investigated. Under salt stress, there was higher Na⁺ accumulation in roots than in stems and leaves, and Ca^2 +, Mg^2 + and P^3 + content, as well as K⁺/Na⁺ ratio were affected. NaCl treatment induced an increase in H₂O₂ contents in the tissues of B. papyrifera. The work demonstrated that activities of antioxidant defense enzymes changed in parallel with the increased H₂O₂ and salinity appeared to be associated with differential regulation of distinct SOD and POD isoenzymes. Moreover, SDS-PAGE analysis of total proteins extracted from leaves and roots of control and NaCl-treated plantlets revealed that in the leaves salt stress was associated with decrease or disappearance of some protein bands, and induction of a new protein band after exposure to 100 and 150 mM NaCl. In contrast, NaCl stress had little effect on the protein pattern in the roots. In summary, these findings may provide insight into the mechanisms of the response of woody plants to salt stress.     

Kinetic analysis for suicide-substrate inactivation of microperoxidase-11: A modified model for bisubstrate enzymes in the presence of reversible inhibitors

Kinetics of microperoxidase-11 (MP-11) as a heme–peptide enzyme model in oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied in the presence of amino acids, taking into account the inactivation of MP-11 during reaction by its suicide substrate, H₂O₂. Reliability of the kinetic equation was evaluated by non-linear mathematical fitting. Fitting of experimental data into a new integrated kinetic relation showed a close match between the kinetic model and the experimental data. Indeed, it was found that the mechanism of suicide-peroxide inactivation of MP-11 in the presence of amino acids is different from MP-11 and/or horseradish peroxidase. In this mechanism, amino acids compete with hydrogen peroxide for the sixth co-ordination position of iron atom in the heme group through a competitive inhibition mechanism.The proposed model can successfully determine the kinetic parameters including inactivation by hydrogen peroxide as well as the inhibitory rate constants by the amino acid inhibitor.Kinetic parameters of inactivation including the initial activity of MP-11, α₀, the apparent inactivation rate constant, k_i and the apparent inhibition rate constant for cysteine, k_I were obtained 0.282 ± 0.006 min^?1, 0.497 ± 0.013 min^?1 and 1.374 ± 0.007 min^?1 at [H₂O₂] = 1.0 mM, 27 °C, phosphate buffer 5.0 mM, pH 7.0. Results showed that inactivation and inhibition of microperoxidase as a peroxidase model enzyme occurred simultaneously even at low concentrations of hydrogen peroxide (0.4 mM). This kinetic analysis based on the suicide-substrate inactivation of microperoxidase-11, provides a tool and model for studying peroxidase models in the presence of reversible inhibitors. The introduced inhibition procedure can be used in designing activity tunable and specific protected enzyme models in the hidden and reversibly inhibited forms, which do not undergo inactivation.     

A comparative study of the use of inorganic carbon resources by Chara aspera and Potamogeton pectinatus

We studied the potential role of dissolved inorganic carbon (DIC) in determining vegetation dominance of Potamogeton pectinatus L. and Chara aspera Deth. ex Willd. by monitoring the seasonal dynamics of DIC in a shallow lake and comparing the use of DIC of the two species. The HCO₃⁻-concentration in summer dropped from 2.5 to <0.5 mM with seasonally increasing Chara biomass, whereas outside the vegetation concentrations remained at 2.5 mM. Inside Potamogeton spp. vegetation DIC decreased from 2.5 to ca. 0.75 mM HCO₃⁻. A growth experiment showed ash-free biomass for P. pectinatus was nearly two times as high as for C. aspera at 3 mM HCO₃⁻, but almost two times lower at 0.5 mM than at 3.0. In a separate experiment, P. pectinatus precultured at a relatively low HCO₃⁻-level had a lower net photosynthetic rate (P_max, 0.1 mmol O₂ g⁻¹ DW h⁻¹) than C. aspera (P_max, 0.1 mmol O₂ g⁻¹ DW h⁻¹) over the range of HCO₃⁻-concentrations tested (P_max, 0.14 mmol O₂ g⁻¹ DW h⁻¹). In response to CO₂ no significant differences between the compensation points (P. pectinatus, 28 mM; C. aspera 66 mM), were observed, but the photosynthetic rate increased faster than for C. aspera than for P. pectinatus. Under field conditions, the use of CO₂ is not important since inside vegetation CO₂-concentrations were below 10 μM, and thus, not available for photosynthesis of either species during the main part of the growth season. It is suggested that C. aspera may be a better competitor for HCO₃⁻ than P. pectinatus in conditions with a low HCO₃⁻ supply. As HCO₃⁻ is a strong limiting factor for growth inside the vegetation and probably the only carbon source available, the superior ability of C. aspera to use HCO₃⁻ may be an important factor explaining its present dominance in Veluwemeer.     

Production of β-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of β-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of β-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular β-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 °C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 °C. β-Fructofuranosidase activity was slightly activated by Cu²⁺, Mn²⁺, Mg²⁺, and Na⁺ at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K_d values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively.     

Purification and characterization of a thermostable α-galactosidase with transglycosylation activity from Aspergillus parasiticus MTCC-2796

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 (K_m = 0.83 mM), melibiose (K_m = 2.48 mM) and raffinose (K_m = 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.     

Thalassia testudinum response to the interactive stressors hypersalinity,sulfide and hypoxia

A large-scale mesocosm (sixteen 500 L tanks) experiment was conducted to investigate the effects of hypersalinity (45–65 psu), porewater sulfide (2–6 mM) and nighttime water column hypoxia (5–3 mg L⁻¹) on the tropical seagrass Thalassia testudinum Banks ex König. We examined stressor effects on growth, shoot survival, tissue sulfur (S⁰, TS, δ³⁴S) and leaf quantum efficiencies, as well as, porewater sulfides (∑TS_pw) and mesocosm water column O₂ dynamics. Sulfide was injected into intact seagrass cores of T. testudinum exposing below-ground tissues to 2, 4, and 6 mM S²⁻, but rapid oxidation resulted in ∑TS_pw < 1.5 mM. Hypersalinity at 65 psu lowered sulfide oxidation and significantly affected plant growth rates and quantum efficiencies (F_v/F_m < 0.70). The most depleted rhizome δ³⁴S signatures were also observed at 65 psu, suggesting increased sulfide exposure. Hypoxia did not influence ∑TS_pw and plant growth, but strengthened the hypersalinity response and decreased rhizome S⁰, indicating less efficient oxidation of ∑TS_pw. Following nighttime hypoxia treatments, ecosystem level metabolism responded to salinity treatments. When O₂ levels were reduced to 5 and 4 mg L⁻¹, daytime O₂ levels recovered to approximately 6 mg L⁻¹; however, this recovery was more limited when O₂ levels were lowered to 3 mg L⁻¹. Subsequent to O₂ reductions to 3 mg O₂ L⁻¹, nighttime O₂ levels rose in the 35 and 45 psu tanks, stayed the same in the 55 psu tanks, and declined in the 65 psu tanks. Thus, hypersalinity at 65 psu affects T. testudinum's oxidizing capacity and places subtle demands on the positive O₂ balance at an ecosystem level. This O₂ demand may influence T. testudinum die-off events, particularly after periods of high temperature and salinity. We hypothesize that the interaction between hypersalinity and sulfide toxicity in T. testudinum is their synergistic effect on the critical O₂ balance of the plant.     

Oxidation of the diphosphoinositol polyphosphate phosphohydrolase-like Nudix hydrolase Aps from Drosophila melanogaster induces thermolability—A possible regulatory switch?

Unlike mammalian cells, Drosophila melanogaster contains only a single member of the diphosphoinositol polyphosphate phosphohydrolase subfamily of the Nudix hydrolases, suggesting that functional specialisation has not occurred in this organism. In order to evaluate its function, Aps was cloned and characterized. It hydrolyses a range of (di)nucleoside polyphosphates, the most efficient being guanosine 5′-tetraphosphate (K_m = 11 μM, k_cat = 0.79 s^?1). However, it shows a 5-fold preference for the hydrolysis of diphosphoinositol pentakisphosphate (PP-InsP₅, K_m = 0.07 μM, k_cat = 0.024 s^?1). Assayed at 26 °C, Aps had an alkaline pH optimum and required a divalent ion: Mg²⁺ (10–20 mM) or Mn²⁺ (1 mM) were preferred for nucleotide hydrolysis and Mg²⁺ (0.5–1 mM) or Co²⁺ (1–100 μM) for PP-InsP₅ hydrolysis. GFP-fusions showed that Aps was predominantly cytoplasmic, with some nuclear localization. In the absence of dithiothreitol Aps was heat labile, rapidly losing activity even at 36 °C, while in the presence of dithiothreitol, Aps was heat stable, surviving for 5 min at 76 °C. Heat lability was restored by H₂O₂ and mass spectrometric analysis suggested that this was due to reversible dimerisation involving two inter-molecular disulphides between Cys23 and Cys25. Aps expression was highest in embryos and declined throughout development. The ratio of PP-InsP₅ to inositol hexakisphosphate also decreased throughout development, with the highest level of PP-InsP₅ found in embryos. These data suggest that the redox state of Aps may play a role in controlling its activity by altering its stability, something that could be important for regulating PP-InsP₅ during development.     

Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg²⁺ is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca²⁺ (alone) only supports DNA binding. To investigate the role of Mg²⁺ in DNA cleavage by restriction endonucleases, we have studied the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20 mM while no activity was detected in the presence of Mn²⁺ and in the presence of Ca²⁺ cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn²⁺ than in the presence of Mg²⁺ and can be activated by Ca²⁺. Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K_m value for pTrcHisB DNA of 6.15 nM and a V_max of 1.79 × 10^?2 nM min^?1, while EhoI had a K_m for pUC19 plasmid of 8.66 nM and a V_max of 2 × 10^?2 nM min^?1.     

Oxidative stress caused by pyocyanin impairs CFTR Cl− transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H₂O₂ threefold above the endogenous H₂O₂ production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 μM) oxidized the cytosol from a resting value of − 318 ± 5 mV by 48.0 ± 4.6 mV within 2 h; a comparable oxidation was induced by 100 μM H₂O₂. Whereas resting Cl⁻ secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl⁻ secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for ΔF508 CFTR failed to secrete Cl⁻ in response to pyocyanin or H₂O₂, indicating that these oxidants specifically target the CFTR and not other Cl⁻ conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H₂O₂, depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

Purification and characterization of a new carbonyl reductase from Leifsonia xyli HS0904 involved in stereoselective reduction of 3,5-bis(trifluoromethyl) acetophenone

Leifsonia xyli HS0904 can stereoselectively catalyze the bioreduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to its corresponding alcohol, which is a valuable chiral intermediate in the pharmaceuticals. In this study, a new carbonyl reductase derived from L. xyli HS0904 was purified and its biochemical properties were determined in detail. The carbonyl reductase was purified by 530-fold with a specific activity of 13.2 U mg⁻¹ and found to be a homodimer with a molecular mass of 49 kDa, in which the subunit molecular-weight was about 24 kDa. The purified enzyme exhibited a maximum enzyme activity at 34 °C and pH 7.2, and retained over 90% of its initial activity at 4 °C and pH 7.0 for 24 h. The addition of various additives, such as Ca²⁺, Mg²⁺, Mn²⁺, l-cysteine, l-glutathione, urea, PEG 1000 and PEG 4000, could enhance the enzyme activity. The maximal reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) of the purified carbonyl reductase for BTAP and NADH were confirmed as 33.9 U mg⁻¹, 0.383 mM and 69.9 U mg⁻¹, 0.412 mM, respectively. Furthermore, this enzyme was found to have a broad spectrum of substrate specificity and can asymmetrically catalyze the reduction of a variety of ketones and keto esters.     

Arginase from Bacillus thuringiensis SK 20.001: Purification,characteristics, and implications for l-ornithine biosynthesis

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 K_m and V_max 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.     

Quantitative analysis on inactivation and reactivation of recombinant glycerol dehydratase from Klebsiella pneumoniae XJPD-Li

The genes encoding glycerol dehydratase were cloned and characterized by genomic DNA from Klebsiella pneumoniae XJPD-Li, and the assigned accession number EF634063 was available from the GenBank database. The DNA sequence analysis showed that the clone included three ORFs (dhaB, dhaC and dhaE, encoding α, β and γ subunit of glycerol dehydratase, respectively). Among three subunits of glycerol dehydratase, amino acid residues H₁₃, S₁₉₃, N₃₅₉, E₄₀₇, and M₅₁₅ of α subunit, N₄₇, L₁₅₀, V₁₈₉ of β subunit are different with what had been reported. Subsequently, the expression vector was constructed and transformed into E. coli BL21, and the colony carried genes of glycerol dehydratase were selected. SDS-PAGE examination showed that the three subunits were well expressed. The specific activity of recombined glycerol dehydratase reached to 0.299 U mg^?1, which was about 3 times comparing with that of the wild strain. The research also displayed that both glycerol and O₂ could inactive the glycerol dehydratase expressed in E. coli quickly in 10 min. The inactivated glycerol dehydratase could be effectively reactivated under the system as follows: the concentration of ATP, Mg²⁺ and coenzyme B₁₂ were 50 mM, 10 mM and 3 μM, respectively, when the ratio (W/W) of glycerol dehydratase to reactivation factor was 4:1. The O₂-inactivated and glycerol-inactivated dehydratase could be reactivated to 97.3% and 98.9% of initial activity in 10 min in above-mentioned conditions, respectively. The reactivation factor together with ATP was considered as the “ON/OFF” reactivating condition.