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1.
Bacillus sp. X-b, a biocontrol agent against certain plant pathogenic fungi, secretes a complex of hydrolytic enzymes, composed of chitinase, chitosanase, laminarinase, lipase and protease. Homogenized mycelium of basidiomycete Macrolepiota procera induced activities of these enzymes more effectively than colloidal chitin or partially purified cell walls of another basidiomycete Polyporus squamosus. Subjected to a multi-step purification, the specific activity of chitinase increased 36-fold, chitosanase 69-fold, lipase 44-fold and laminarinase 15-fold. Partially purified chitinase showed two major bands with molecular masses of 46 000 and 35 000 on sodium dodecyl sulfate–polyacrylamide gel electrophoresis while chitosanase and lipase appeared as single bands with molecular masses of 27 000 and 62 000, respectively.  相似文献   

2.
Energy-converting hydrogenases (Ech) are ancient, membrane-bound enzymes that use reduced ferredoxin (Fd) as an electron donor to reduce protons to molecular H2. Experiments with whole cells, membranes and vesicle-fractions suggest that proton reduction is coupled to proton translocation across the cytoplasmatic membrane, but this has never been demonstrated with a purified enzyme. To this end, we produced a His-tagged Ech complex in the thermophilic and anaerobic bacterium Thermoanaerobacter kivui. The enzyme could be purified by affinity chromatography from solubilized membranes with full retention of its eight subunits, as well as full retention of physiological activities, i.e., H2-dependent Fd reduction and Fd2--dependent H2 production. We found the purified enzyme contained 34.2 ± 12.2 mol of iron/mol of protein, in accordance with seven predicted [4Fe-4S]-clusters and one [Ni-Fe]-center. The pH and temperature optima were at 7 to 8 and 66 °C, respectively. Notably, we found that the enzymatic activity was inhibited by N,N′-dicyclohexylcarbodiimide, an agent known to bind ion-translocating glutamates or aspartates buried in the cytoplasmic membrane and thereby inhibiting ion transport. To demonstrate the function of the Ech complex in ion transport, we further established a procedure to incorporate the enzyme complex into liposomes in an active state. We show the enzyme did not require Na+ for activity and did not translocate 22Na+ into the proteoliposomal lumen. In contrast, Ech activity led to the generation of a pH gradient and membrane potential across the proteoliposomal membrane, demonstrating that the Ech complex of T. kivui is a H+-translocating, H+-reducing enzyme.  相似文献   

3.
The expression of the gene encoding a thermostable α-amylase (EC 3.2.1.1) (optimal activity at 100°C) from the hyperthermophilic archaeon Pyrococcus woesei in the mesophilic hosts Escherichia coli and Halomonas elongata resulted in the formation of insoluble aggregates. More than 85% of the recombinant enzyme was present within the cells as insoluble but catalytically active aggregates. The recombinant α-amylase was purified to homogeneity in a single step by hydrophobic interaction chromatography on a phenyl superose column after solubilization of the enzyme under nondenaturing conditions. The enzyme was purified 258-fold with a final yield of 54%.  相似文献   

4.
F420-nonreactive and F420-reactive hydrogenases have been partially purified from Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a submarine hydrothermal vent. The molecular weights of both hydrogenases were determined by native gradient electrophoresis in 5 to 27% polyacrylamide gels. The F420-nonreactive hydrogenase produced one major band (475 kilodaltons), whereas the F420-reactive hydrogenase produced two major bands (990 and 115 kilodaltons). The F420-nonreactive hydrogenase consisted of two subunits (43 and 31 kilodaltons), and the F420-reactive hydrogenase contained three subunits (48, 32, and 25 kilodaltons). Each hydrogenase was active at very high temperatures. Methyl viologen-reducing activity of the F420-nonreactive hydrogenase was maximal at 80°C but was still detectable at 103°C. The maximum activities of F420-reactive hydrogenase for F420 and methyl viologen were measured at 80 and 90°C, respectively. Low but measureable activity toward methyl viologen was repeatedly observed at 103°C. Moreover, the half-life of the F420-nonreactive hydrogenase at 70°C was over 9 h, and that of the F420-reactive enzyme was over 3 h.  相似文献   

5.
Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (β) and light chain (α) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, under reducing conditions, with a Mr (molecular mass) of about 42 000 and 14 000 for heavy (β) and light chains (α), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine Mr 42 000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine Mr 42 000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin.  相似文献   

6.
A cardenolide-hydrolysing β-D-glucosidase was isolated from young leaves of Digitalis lanata. Since this enzyme differs from the cardenolide glucohydrolase (CGH) described and characterised previously, it was termed cardenolide glucohydrolase II (CGH II). CGH II was detected in various Digitalis tissue cultures as well as in young leaves of D. lanata. The latter source was used as the starting material for the isolation and purification of CGH II. The specific enzyme activity reached about 15 pkat·mg–1 protein in buffered leaf extracts. Optimal CGH II activity was seen at around pH 6.0 and 50 °C. CGH II was purified about 600-fold by anion exchange chromatography, size exclusion chromatography and hydroxyapatite chromatography. The apparent molecular mass of CGH II was 65 kDa as determined by SDS-PAGE. CGH II exhibited a high substrate specificity towards cardenolide disaccharides, especially to those with a 1-4-β-linked glucose-digitoxose moiety such as glucoevatromonoside. The Km- and Vmax-values for this particular substrate were calculated to be 101 μM and 19.8 nkat·mg–1 protein, respectively.  相似文献   

7.
The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 μm in diameter. The new isolate grew at temperatures between 60 and 95°C (optimum, 85°C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120°C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100°C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110°C. The enzyme formed mainly α-cyclodextrin with small amounts of β- and γ-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.  相似文献   

8.
A total of two different hemolymph proteins (designated P-I and P-II) of the Japanese oak silkworm, Antheraea yamamai, were purified from the hemolymph of the fifth instar larvae using four chromatographic steps: (a) hydrophobic interaction chromatography; (b) ion exchange chromatography; (c) gel-filtration; and (d) reverse-phase high performance liquid chromatography (HPLC). These two proteins were separated by TSKgel Phenyl-5PW RP column chromatography. P-I has an apparent molecular weight of 31 000 or 35 000, as determined by gel-filtration and SDS-PAGE, respectively. P-II shows a molecular weight of 22 000 or 25 000, by gel-filtration and SDS-PAGE, respectively. The molecular weight of P-I and P-II were determined to be 31 076 and 21 500 by MALDI-TOF MS, respectively. These results suggest that both P-I and P-II are monomers. The N-terminal sequence analysis suggests that P-I is closely related to the ommochrome-binding protein (OBP) from the hemolymph of Manduca sexta, with 40% identity in the first 30 residues, while P-II is similar to the biliproteins (BPs) from other lepidopteran insects (50% identity). Spectroscopic analysis shows that the blue chromophore of A. yamamai BP is not biliverdin IX, which is present in the biliproteins of most insects.  相似文献   

9.
We report on the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of the proteins Pabp41 and Pabp46, which show sequence similarities to the p49 and p58 subunits, respectively, of the eukaryotic polymerase α–primase complex. Both subunits were expressed, purified, and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribonucleotides and deoxyribonucleotides, and its activity was dependent on the presence of Mg2+ and Mn2+. Interestingly, Pab DNA primase also displayed DNA polymerase, gap-filling, and strand-displacement activities. Genetic analyses undertaken in Haloferax volcanii suggested that the eukaryotic-type heterodimeric primase is essential for survival in archaeal cells. Our results are in favor of a multifunctional archaeal primase involved in priming and repair.  相似文献   

10.
β-Turmerin from turmeric (Curcuma longa) waste grits obtained after extraction of curcumin was purified by successive gel permeation chromatography. Homogeneity of β-turmerin was confirmed by its movement as single band both in SDS-PAGE and as well as in native (basic) PAGE. The apparent molecular mass is 34 kDa by SDS-PAGE. It is more hydrophobic protein and showed sharp single peak in RP-HPLC with retention time of 62.17 min. It is a glycoprotein as it shows the presence of amino sugars up to 0.021 gm%. In three different model systems i.e., linolenic acid micelles, erythrocyte membrane systems and liposomes, β-turmerin at 0.125 μM offered 70%, 64%, and 60% inhibition of lipid peroxidation, which is 3200 times more efficient than the standard antioxidants BHA (400 μM) and α-tocopherol (400 μM). β-turmerin inhibited diene–triene and tetraene conjugation up to 54%, 72% and 47%, respectively. β-turmerin also effectively scavenges hydroxyl radicals when compared to BHA and α-tocopherol. β-turmerin (2.5 μM) further inhibited the activation of PMNL mediated by fMLP up to the extent of 75%, where as standards BHA (400 μM) and mannitol (10 μM) inhibited the same to 65% and 55%, respectively. At 0.125 μM dose β-turmerin prevented t-BOOH induced cell death at all time intervals. In addition to the above properties, it is non-toxic to lymphocytes as it did not affect the viability of cells. The mechanism of antioxidant action of β-turmerin could probably be by counteracting/quenching of reactive oxygen species (ROS). We report the purification and characterization of β-turmerin (34 kDa), a potent antioxidant protein from turmeric waste grits.  相似文献   

11.
The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na+/H+ antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na+ ions.  相似文献   

12.
The Rhodococcus erythropolis strain (N′4) possesses the ability to convert 4-chloro-3-hydroxybutyronitrile into the corresponding acid. This conversion was determined to be performed by its nitrile hydratase and amidase. Ammonium sulfate fractionation, DEAE ion exchange chromatography, and phenyl chromatography were used to partially purify nitrile hydratase from cell-free extract. A SDS-PAGE showed that the partially purified enzyme had two subunits and gel filtration chromatography showed that it consisted of four subunits of α2β2. The purified enzyme had a high specific activity of 860 U mg−1 toward methacrylonitrile. The enzyme was found to have high activity at low temperature range, with a maximum activity occurring at 25 °C and be stable in the presence of organic acids at higher temperatures. The enzyme exhibited a preference for aliphatic saturated nitrile substrates over aliphatic unsaturated or aromatic ones. It was inhibited by sulfhydryl, oxidizing, and serine protease inhibitors, thus indicating that essential cysteine and serine residues can be found in the active site.The purified nitrile hydratase was able to convert 4-chloro-3-hydroxybutyronitrile into the corresponding amide at 15 °C. GC analysis showed that the initial conversion rate of the reaction was 215 mg substrate consumed min−1 mg−1. This demonstrated that this enzyme could be used in conjunction with a stereoselective amidase to synthesize ethyl (S)-4-chloro-3-hydroxybutyrate, an intermediate for a hypercholesterolemia drug, Atorvastatin.  相似文献   

13.
Thermoplasma acidophilum is a thermophilic archaeon that uses both non-phosphorylative Entner-Doudoroff (ED) pathway and Embden-Meyerhof-Parnas (EMP) pathway for glucose degradation. While triosephosphate isomerase (TPI), a well-known glycolytic enzyme, is not involved in the ED pathway in T. acidophilum, it has been considered to play an important role in the EMP pathway. Here, we report crystal structures of apo- and glycerol-3-phosphate-bound TPI from T. acidophilum (TaTPI). TaTPI adopts the canonical TIM-barrel fold with eight α-helices and parallel eight β-strands. Although TaTPI shares ~30% sequence identity to other TPIs from thermophilic species that adopt tetrameric conformation for enzymatic activity in their harsh physiological environments, TaTPI exists as a dimer in solution. We confirmed the dimeric conformation of TaTPI by analytical ultracentrifugation and size-exclusion chromatography. Helix 5 as well as helix 4 of thermostable tetrameric TPIs have been known to play crucial roles in oligomerization, forming a hydrophobic interface. However, TaTPI contains unique charged-amino acid residues in the helix 5 and adopts dimer conformation. TaTPI exhibits the apparent Td value of 74.6°C and maintains its overall structure with some changes in the secondary structure contents at extremely acidic conditions (pH 1–2). Based on our structural and biophysical analyses of TaTPI, more compact structure of the protomer with reduced length of loops and certain patches on the surface could account for the robust nature of Thermoplasma acidophilum TPI.  相似文献   

14.
The protein subunit of RNase P from a thermophilic bacterium, Thermotoga maritima, was overexpressed in and purified from Escherichia coli. The cloned protein was reconstituted with the RNA subunit transcribed in vitro. The temperature optimum of the holoenzyme is near 50°C, with no enzymatic activity at 65°C or above. This finding is in sharp contrast to the optimal growth temperature of T.maritima, which is near 80°C. However, in heterologous reconstitution experiments in vitro with RNase P subunits from other species, we found that the protein subunit from T.maritima was responsible for the comparative thermal stability of such complexes.  相似文献   

15.
Two thermostable lipases were isolated and characterized from Thermosyntropha lipolytica DSM 11003, an anaerobic, thermophilic, alkali-tolerant bacterium which grows syntrophically with methanogens on lipids such as olive oil, utilizing only the liberated fatty acid moieties but not the glycerol. Lipases LipA and LipB were purified from culture supernatants to gel electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic interaction column chromatography. The apparent molecular masses of LipA and LipB determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 50 and 57 kDa, respectively. The temperature for maximal activity of LipA and LipB was around 96°C, which is, so far as is known, the highest temperature for maximal activity among lipases, and the pH optima for growth determined at 25°C (pH25°C optima) were 9.4 and 9.6, respectively. LipA and LipB at 100°C and pH25°C 8.0 retained 50% activity after 6 and 2 h of incubation, respectively. Both enzymes exhibited high activity with long-chain fatty acid glycerides, yielding maximum activity with trioleate (C18:1) and, among the p-nitrophenyl esters, with p-nitrophenyl laurate. Hydrolysis of glycerol ester bonds occurred at positions 1 and 3. The activities of both lipases were totally inhibited by 10 mM phenylmethylsulfonyl fluoride and 10 mM EDTA. Metal analysis indicated that both LipA and LipB contain 1 Ca2+ and one Mn2+ ion per monomeric enzyme unit. The addition of 1 mM MnCl2 to dialyzed enzyme preparations enhanced the activities at 96°C of both LipA and LipB by threefold and increased the durations of their thermal stability at 60°C and 75°C, respectively, by 4 h.  相似文献   

16.
An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg2+, Co2+, and Mn2+ were required for maximum activity of the enzyme. The Km values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while Kcat values were 2.3 × 102 s-1 and 0.5 × 102 s-1, respectively.  相似文献   

17.
The synthesis and characterization of the binary complex of copper(II) with the antiepileptic drug valproic acid sodium salt (Valp) and the related ternary complex with 1,10-phenanthroline (phen) are reported, as well as the anticonvulsant properties of the latter. The characterization was carried out by means of elemental analyses, infrared (IR), UV–visible (UV–vis) spectrophotometry and Electron Paramagnetic Resonance (EPR). The X-ray crystal structure of the mononuclear complex bis(2-propylpentanoate)(1,10-phenanthroline)copper(II) [Cu(Valp)2phen] is showed for the first time. It crystallized in C2/c space group with unit cell dimensions of a = 14.939(1) Å, b = 19.280(1) Å, c = 9.726(1) Å, β = 97.27(2)°, V = 2778.8(4) Å 3 and Z = 8. The carboxylates bond in an asymmetric chelating mode and the copper atom adopts a highly distorted octahedral coordination, characterized by the sum of the angles of 365.9° around Cu(II) and its nearest atoms in the CuN2O2 + O2 chromophore instead of the expected 360° for a basal square planar geometry found in most Cu(II) complexes. Molecules assemble three by three through slipped π–π stacking of the aromatic phen with respectively 3.519 and 3.527 Å distances, in a head-to-tail arrangement. Studies of the anticonvulsant properties of this bioligand chelate evidenced its lack of efficacy in preventing MES-induced seizures. Interestingly, complex 4 protected mice against the Minimal Clonic seizures at doses that do not cause Rotorod toxicity, with an ED50 documenting very potent anticonvulsant activity in this model of seizure, a particularly useful pharmacological profile of activity for the treatment of Petit Mal seizures.  相似文献   

18.
A recJ homolog was cloned from the extremely thermophilic bacterium Thermus themophilus HB8. It encodes a 527 amino acid protein that has 33% identity to Escherichia coli RecJ protein and includes the characteristic motifs conserved among RecJ homologs. Although T.thermophilus RecJ protein (ttRecJ) was expressed as an inclusion body, it was purified in soluble form through denaturation with urea and subsequent refolding steps. Limited proteolysis showed that ttRecJ has a protease-resistant core domain, which includes all the conserved motifs. We constructed a truncated ttRecJ gene that corresponds to the core domain (cd-ttRecJ). cd-ttRecJ was overexpressed in soluble form and purified. ttRecJ and cd-ttRecJ were stable up to 60°C. Size exclusion chromatography indicated that ttRecJ exists in several oligomeric states, whereas cd-ttRecJ is monomeric in solution. Both proteins have 5′→3′ exonuclease activity, which was enhanced by increasing the temperature to 50°C. Mg2+, Mn2+ or Co2+ ions were required to activate both proteins, whereas Ca2+ and Zn2+ had no effects.  相似文献   

19.
The method presented can easily produce milligram amounts of glycodelin from pregnancy endometrium, with a 19% yield. It involves anion-exchange chromatography, gel permeation and chromatofocusing; it results in one stainable band at Mr 28 000 after sodium dodecyl sulphate–polyacrylamide electrophoresis, as well as after immunoblot analysis, performed using an affinity-purified IgG fraction from an antiserum against glycodelin. In spite of this, the corresponding gel isoelectric focusing pattern gives four stainable bands with pI values between 4.55 and 5.2. Western immunoblot analysis of tissue extracts indicates the presence of glycodelin epitopes associated with materials heavier than the native protein. Circular dichroism spectra of the highly purified protein in water solutions indicate a large amount of β-sheet conformation, whereas those obtained with different proportions of 2-propanol in water, show an increased proportion of α-helix conformation.  相似文献   

20.
The Na+-pumping V-ATPase complex of the thermophilic bacterium Caloramator fervidus was purified and dissociated under controlled conditions. The structure of purified V1-ATPase subcomplexes differing in subunit composition was analyzed by electron microscopy and single particle analysis of 50 000 projections. Difference mapping of subcomplex projections revealed the presence and position of two subunits in the central stalk. A density with an elongated shape similar to the γ subunit of F-ATPases is partly located within V1 and corresponds, most likely, to subunit E. Subunit E is connected to the membrane-bound part V0 via subunit C, a spherical density that is connected to the center of V0. The presence of subunit C makes the central stalk substantially longer in comparison to the F-ATPases, in which the γ subunit connects directly to F0.  相似文献   

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