Purification, crystallisation and preliminary X-ray diffraction characterisation of methanol dehydrogenase from Methylosinus trichosporium OB3b

Methanol dehydrogenase was purified from the obligate methanotroph, Methylosinus trichosporium OB3b, in two steps from disrupted biomass by aqueous two-phase partition and ion-exchange chromatography. Copartitioning of a cytochrome c was dependent upon the pH at which aqueous partition was carried out. The native enzyme has a Mr of 120,000, as determined by gel filtration chromatography, and consists of two identical subunits. The purified enzyme contained four electrophoretically distinct isoenzymes, with pI values of 6.3, 6.58, 6.63 and 6.88. The native enzyme has been crystallised in a form suitable for high-resolution X-ray crystallographic studies. The crystals diffract to better than 0.19 nm spacing and are relatively stable to irradiation with X-rays. The space group is P6(1)22 (or P6(5)22) with cell dimensions a = b = 10.21 nm, c = 29.32 nm and the crystal probably contains a single monomer in the asymmetric unit.     

Purification, crystallisation and preliminary X-ray study of haemoglobin from Crocodilis palustris and Crocodilis porosus

The unsolved three-dimensional structure of crocodile haemoglobin and its prospects as a blood substitute have led us to initiate the purification and crystallisation of haemoglobin molecules from crocodile species (Crocodilis palustris or mugger and Crocodilis porosus or salt water crocodile). The work has resulted in the prevention of polymerisation of naked haemoglobin molecules using N-ethylmaleimide or iodoacetamide. The purified monomeric haemoglobin molecule of C. porosus was crystallised in two different forms and X-ray diffraction data were collected up to 2 A resolution for both forms. Form I: a=53.62, b=53.55, c=103.77 A; beta=93.35 degrees, space group P2(1), Z=2. Form II: a=71.30, b=54.70, c=80.00 A; beta=106.4 degrees, space group P2(1), Z=2. Structure solution and rigid body refinement of form I data resulted in a model with R(free)=0.42 and R=0.35.     

Separation and purification of laccases from two different fungi using aqueous two-phase extraction

Selective purification still poses a challenge in the downstream processing of biomolecules such as proteins and especially enzymes. In this study a polyethylene glycol 3000 (PEG 3000)–phosphate aqueous two-phase system at 25 °C and pH 7 was successfully used for laccase purification and separation. Initially, the effect of phase forming components on enzyme activities in homogenous systems was studied. In the course of the extraction experiments tie lines, enzyme source, initial enzyme activities, phase ratio and sodium chloride concentrations were varied and their influence on the activity partitioning was determined. Partitioning results were validated using clear-native-PAGE and isoelectric focusing. Based on these results, the separation of laccases from Trametes versicolor and Pleurotus sapidus was investigated using the principle of superposition. Sodium chloride was used to adjust laccase partitioning in the applied aqueous two-phase system (ATPS). Finally, two modes of operation are proposed depending on the aim of the purification task. One mode with 0.133 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and without sodium chloride separates P. sapidus laccases from T. versicolor laccases with clearance factors of 5.23 and 6.45, respectively. The other mode of operation with 0.124 g g⁻¹ of PEG3000, 0.063 g g⁻¹ of phosphate and 0.013 g g⁻¹ of sodium chloride enables a partitioning of both laccases into the bottom phase of the ATPS resulting in a purification factor of 2.74 and 96% activity recovery.     

Purification and characterization of two laccase isoenzymes from a ligninolytic fungus Trametes gallica

Constant laccase activities were detected in culture supernatant of newly isolated basidiomycete Trametes gallica. Tryptone and glucose have great effects on the production of laccase. Two laccase isoenzymes (Lac I and Lac II) produced by T. gallica were purified to homogeneity (51- and 50-fold, respectively) by gel filtration chromatography, anion exchange chromatography, and improved native PAGE, with an overall yield of 24.8%. Lac I and Lac II from this fungus are glycoproteins with 3.6% and 4% carbohydrate content, the same molecular masses (by SDS-PAGE) of 60 kDa, and the pI of 3.1 and 3.0, respectively. Native gel electrophoresis indicates that the two laccases have different migration ratios. Lac I and Lac II have the same optimal pH of 3.0 on 2,6-dimethoxyphenol (DMP), pH 2.2 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and of pH 4.0 on guaiacol. The highest rate of ABTS oxidation for both laccases was reached at 70 degrees C. Both laccases are stable from pH 6 to 9, retaining 88-90% activity after 24 hr incubation, and show good stability when incubated at temperatures lower than 40 degrees C. The Km values of Lac I for ABTS, DMP, and guaiacol are 0.118 x 10(-2), 0.420, and 0.405 mM, respectively; the Km values of Lac II for ABTS, DMP, and guaiacol are 0.086 x 10(-2), 0.41, and 0.40 mM, respectively. Their N-terminal sequences are determined and show strong similarity with those from other basidiomycetes. Graphite-furnace atomic absorption analysis revealed that both laccases have four copper atoms per protein molecule, but they have no type I copper signal at around 600 nm and a type III copper signal near 330 nm. Cyanide, azide, and halides completely inhibit the enzyme activity, whereas EDTA has less inhibition.     

Purification, crystallization, and X-ray diffraction studies of lactotransferrin from buffalo colostrum.

Lactotransferrin is an iron-binding protein. It has been purified from buffalo colostrum. The purified lactotransferrin has been crystallized in 10% ethanol solution. The crystals are orthorhombic and the space group is P2(1)2(1)2(1) with unit cell dimensions a = 161.70 A, b = 155.75 A, c = 113.48 A. The asymmetric unit contains three molecules of the protein with a solvent content of about 59%. The crystals were stable in the X-ray beam and diffract beyond 3.5 A resolution. The native data have been collected and the structure determination is in progress.     

Purification, partial characterization, and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17

Two isozymes of laccase were obtained from an induced liquid culture of Marasmius quercophilus with p-hydroxybenzoic acid as the inducer. Both the constitutive and the induced isozyme have a molecular mass of 60 kDa as determined by polyacrylamide gel electrophoresis. Using isoelectric focusing, we found three isozymes with the constitutive enzyme (pI 4, 4.2, 4.4) and four of the induced form (pI 4.75, 4.85, 4.95, 5.1). We observed certain differences between these two isozymes; the specific activity of the induced isozyme was twice as high, and two optimum pH levels (5 and 6) were observed with the induced isozyme (only one, pH 5, for the constitutive isozyme). However, both of these enzymes have the same thermal stability and the same temperature for their highest activity (80 degrees C). Furthermore, the reactivity of both these enzymes with aromatic compounds was similar. The use of mediators extended the oxidized substrate range of the laccases studied. Various products of degradation were observed, depending on the mediator used. When laccase was used alone, the decrease of the signal corresponding to the aromatic cycle, without any formations of other peaks at different wavelengths, suggested polymerisation of aromatic compounds.     

Purification,crystallization, and preliminary X-ray diffraction studies of tRNA-guanine transglycosylase from Zymomonas mobilis

The tRNA modifying enzyme tRNA–guanine transglycosylase (Tgt) catalyzes the exchange of guanine in the first position of the anticodon with the queuine precursor 7-aminomethyl-7-deazaguanine. Tgt from Zymomonas mobilis has been purified by crystallization and further recrystallized to obtain single crystals suitable for x-ray diffraction studies. Crystals were grown by vapor diffusion/gel crystallization methods using PEG 8,000 as precipitant. Macroseeding techniques were employed to produce large single crystals. The crystals of Tgt belong to the monoclinic space group C2 with cell constants a = 92.1 Å, b = 65.1 Å, c = 71.9 Å, and β = 97.5°, and contain one molecule per asymmetric unit. A complete diffraction data set from one native crystal has been obtained at 1.85 Å resolution.     

High redox potential laccases from the ligninolytic fungi Pycnoporus coccineus and Pycnoporus sanguineus suitable for white biotechnology: from gene cloning to enzyme characterization and applications

Aims: Exploitation of natural biodiversity in species Pycnoporus coccineus and Pycnoporus sanguineus to screen for a new generation of laccases with properties suitable for the lignin‐processing sector. Methods and Results: Thirty strains originating from subtropical and tropical environments, mainly isolated from fresh specimens collected in situ, were screened for laccase activity. On the basis of levels of enzyme activity and percentage of similarity between protein sequences, the laccases from strains BRFM 938, BRFM 66 and BRFM 902 were selected for purification and characterization. Each BRFM 938, BRFM 66 and BRFM 902 laccase gene encoded a predicted protein of 518 amino acids; the three deduced proteins showed 68·7–97·5% similarity with other Polyporale laccases. The three laccases (59·5–62·9 kDa with 7–10% carbohydrate content) had high redox potentials (0·72–0·75 V vs normal hydrogen electrode at pH 6), remained highly stable up to 75–78°C and at pH 5–7 mixtures, and were resistant to methyl and ethyl alcohols, acetonitrile and dimethylsulfoxide at concentrations as high as 50% (v/v). The best laccase‐1‐hydroxybenzotriazole systems permitted almost 100% of various polyphenolic dye decolourization and oxidation of adlerol and veratryl alcohol. Conclusions: The three laccases showed complementary biochemical features. BRFM 938 laccase had the highest thermo‐ and pH stability, catalytic efficiency towards 2,2′‐azino‐bis‐[3‐ethylthiazoline‐6‐sulfonate] and resistance to alcoholic solvents. BRFM 66 laccase had the highest rates of dye decolourization and oxidation of nonphenolic compounds. Significance and Impact of the Study: This study identified P. coccineus and P. sanguineus as outstanding producers of high redox potential laccases, easy to purify and scale‐up for industrial production. Three new laccases proved to be suitable models for white biotechnology processes and for further molecular breeding to create a new generation of tailor‐made enzymes.     

Purification, crystallization, and preliminary X-ray diffraction analysis of the Tricorn protease hexamer from Thermoplasma acidophilum

Tricorn protease from Thermoplasma acidophilum is a hexameric enzyme; in vivo the hexamers assemble further to form large icosahedral capsids of 14.6 MDa. Recombinant Tricorn protease was purified as an enzymatically active hexamer of 0.72 MDa that formed crystals of octahedral morphology under low-ionic-strength conditions. These crystals belong to space group C2 with unit cell dimensions a = 307.5 A, b = 163.2 A, c = 220.9 A, beta = 105.5 degrees and diffract to 2.2-A resolution using high-brilliance synchrotron radiation. Based on analysis of the self-rotation function and the presence of a pseudo-origin peak in the native Patterson map, a packing model was derived for the complex, comprising 1.5 hexamers per asymmetric unit with a solvent content of 43%. Due to the ninefold noncrystallographic symmetry the Tricorn crystals represent an interesting case for phasing X-ray crystallographic data by electron microscopic phase information.     

X-ray diffraction evidence for fully interdigitated bilayers of 1-stearoyllysophosphatidylcholine

X-ray diffraction experiments have been performed on 1-stearoyllysophosphatidylcholine or C(18):C(0)PC as a function of hydration at temperatures below the order/disorder transition (Tm = 26.2 degrees C). At these temperatures, hydrated C(18):C(0)PC forms lamellae. The bilayer thickness, as determined by the saturation hydration method and electron-density profile, is 35-36 A, and the average area per C(18):C(0)PC molecule at the lipid/water interface is 45.5 A2. The packing geometry of C(18):C(0)PC in the lamella is proposed to adopt a fully interdigitated model in which the long C(18) acyl chain extends across the entire hydrocarbon width of the bilayer. Thus far, three different types of interdigitated bilayers are known for phosphatidylcholines. These various types of chain interdigitation are discussed in terms of the chain length difference between the sn-1 and sn-2 acyl chains.     

Purification, crystallisation and characterization of quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa

Pseudomonas aeruginosa ATCC 17933 when grown on ethanol produces high levels of a quinoprotein ethanol dehydrogenase, which amounts to 7% of the soluble protein. The enzyme has been purified to homogeneity and it crystallizes readily in the presence of polyethylene glycol 1550 or 6000. The ethanol dehydrogenase (Km(ethanol) = 14 microM) resembles the dye-dependent quinoprotein methanol dehydrogenases of methylotrophic bacteria, but has a low affinity for methanol (Km (methanol) = 94mM). In addition the enzyme oxidizes secondary alcohols. With its catalytic properties the ethanol dehydrogenase is similar to the enzyme isolated from P. aeruginosa LMD 80.53 (Groen, B., Frank, J. Jzn. & Duine, J.A. (1984) Biochem. J. 223, 921-924). In contrast to this enzyme from P. aeruginosa LMD 80.53, which is a monomer, the ethanol dehydrogenase isolated from P. aeruginosa ATCC 17933 is a dimer of identical subunits of relative molecular mass 60,000. The N-terminal amino acid is lysine. Inactivation with cyclopropanone ethylhemiketal reveals one molecule of pyrroloquinoline quinone per subunit. As shown by active enzyme sedimentation, the dimer is the enzymatically active form.     

Production,partial purification and characterization of ligninolytic enzymes from selected basidiomycetes mushroom fungi

In recent years, many research on the quantity of lignocellulosic waste have been developed. The production, partial purification, and characterisation of ligninolytic enzymes from various fungi are described in this work. On the 21st day of incubation in Potato Dextrose (PD) broth, Hypsizygus ulmarius developed the most laccase (14.83 × 10⁻⁶ IU/ml) and manganese peroxidase (24.11 × 10⁻⁶ IU/ml), while Pleurotus florida produced the most lignin peroxidase (19.56 × ⁻⁶ IU/ml). Laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), all generated by selected basidiomycetes mushroom fungi, were largely isolated using ammonium sulphate precipitation followed by dialysis. Laccase, lignin peroxidase, and manganese peroxidase purification findings indicated 1.83, 2.13, and 1.77 fold purity enhancements, respectively. Specific activity of purified laccase enzyme preparations ranged from 305.80 to 376.85 IU/mg, purified lignin peroxidase from 258.51 to 336.95 IU/mg, and purified manganese peroxidase from 253.45 to 529.34 IU/mg. H. ulmarius laccase (376.85 IU/mg) with 1.83 fold purification had the highest specific activity of all the ligninolytic enzymes studied, followed by 2.13 fold purification in lignin peroxidase (350.57 IU/mg) and manganese peroxidase (529.34 IU/mg) with 1.77-fold purification. Three notable bands with molecular weights ranging from 43 to 68 kDa and a single prominent band with a molecular weight of 97.4 kDa were identified on a Native PAGE gel from mycelial proteins of selected mushroom fungus. The SDS PAGE profiles of the mycelial proteins from the selected mushroom fungus were similar to the native PAGE. All three partially purified ligninolytic isozymes display three bands in native gel electrophoresis, with only one prominent band in enzyme activity staining. The 43 kDa, 55 kDa, and 68 kDa protein bands correspond to laccase, lignin peroxidase, and manganese peroxidase, respectively.     

Purification, crystallization and preliminary X-ray diffraction studies of the PvuII endonuclease.

The PvuII endonuclease (PvuIIR) is a restriction enzyme from a type II restriction-modification system of Proteus vulgaris coded on plasmid pPvu1. The protein recognizes the DNA sequence 5' CAG'CTG 3' and shows no sequence homology to other restriction enzymes. This makes PvuIIR an interesting subject for structural determination. A purification procedure was developed that yields milligram quantities of the PvuIIR from plasmids expressed in the Escherichia coli strain HB101. The protein was crystallized using ammonium sulphate as precipitant. The crystals are orthorhombic, space group P2(1)2(1)2 with cell dimensions: a = 84.2 A, b = 106.2 A, c = 46.9 A. The asymmetric unit contains one PvuIIR dimer. Diffraction extends to 2.3 A, so the crystals may permit structural determination at atomic resolution.     

Degradation of lindane and endosulfan by fungi, fungal and bacterial laccases

The ability of two white-rot fungi (Trametes versicolor and Pleurotus ostreatus) and one brown-rot fungus (Gloeophyllum trabeum) to degrade two organochlorine insecticides, lindane and endosulfan, in liquid cultures was studied and dead fungal biomass was examined for adsorption of both insecticides from liquid medium. Lindane and endosulfan were also treated with fungal laccase and bacterial protein CotA, which has laccase activities. The amount of degraded lindane and endosulfan increased with their exposure period in the liquid cultures of both examined white-rot fungi. Endosulfan was transformed to endosulfan sulphate by T. versicolor and P. ostreatus. A small amount of endosulfan ether was also detected and its origin was examined. Degradation of lindane and endosulfan by a brown rot G. trabeum did not occur. Mycelial biomasses of all examined fungi have been found to adsorb lindane and endosulfan and adsorption onto fungal biomass should therefore be considered as a possible mechanism of pollutant removal when fungal degradation potentials are studied. Bacterial protein CotA performed more efficient degradation of lindane and endosulfan than fungal laccase and has shown potential for bioremediation of organic pollutants.     

Purification,crystallization, and preliminary X-ray diffraction analysis of even-skipped homeodomain complexed to DNA

Embryonic development in metazoa, to a significant extent, is directed by genes which contain a conserved sequence motif named the homeobox. This sequence encodes a polypeptide called the homeodomain which has sequence specific DNA-binding activity. We report the purification, crystallization, and preliminary diffraction analysis of the Drosophila Even-skipped homeodomain (Eve HD) bound to two different oligonucleotides. Crystals of Eve HD complexed with an AT-rich sequence belong to space group P2₁, a = 34.06, b = 61.61, c = 39.99 Å, b=90.0°. These crystals diffract to at least 2.0 Å and both native and derivative data sets have been collected. Crystals of Eve HD complexed with a GC-rich sequence belong to space group P6₃, a = b = 124.52, c = 66.78 Å and diffract to 3.5 Å resolution. A native data set has been collected. © 1995 Wiley-Liss, Inc.     

Purification and characterization of two thermostable laccases with high cold adapted characteristics from Pycnoporus sp. SYBC-L1

The white-rot fungus Pycnoporus sp. SYBC-L1 produced large amount of laccase in submerged fermentation. Two laccase isozymes (LacI and LacII) were purified using (NH₄)₂SO₄ fractionation, DEAE-cellulose and Sephadex G-100 column chromatography. The molecular masses of LacI and Lac II were 55.89 and 63.07 kDa, respectively by SDS-PAGE. Both the laccases showed acidic pH optima and high catalytic activities at low temperature for oxidations of 2,6-dimethoxyphenol (DMP), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonate acid) (ABTS), syringaldazine and guaiacol. LacI and LacII were not only with high cold adaptation, but also fairly stable at high temperature. The half-lives of LacI at 50, 60 and 70 °C were 69.31, 2.58 and 0.13 h, respectively, whereas LacII was more stable with half-lives of 256.72, 21.00 and 2.06 h respectively. The best substrates for the enzymes were both found to be ABTS, in which the K_m values of LacI and LacII were 0.0166 and 0.0435 mM and the catalytic efficiencies were 19640.36 and 31172.64 S⁻¹ mM⁻¹, respectively. EDTA and low concentration of Cu²⁺ and Mn²⁺ almost had non-inhibitions on their activities. LacII with syringaldehyde efficiently decolorized Remazol Brilliant Blue R. The high thermostabilities as well as cold adapted properties made Pycnoporus sp. SYBC-L1 laccases to be excellent candidates in harsh industry.     

Purification, characterization and preliminary X-ray study of fumarase from Saccharomyces cerevisiae.

Fumarase (fumarate hydratase, EC 4.2.1.2) from Saccharomyces cerevisiae has been purified to homogeneity by a method including acetone fractionation, DEAE ion-exchange and dye-sorbent affinity chromatography. The suggested method allows fumarase purification with a yield higher than 60% and may be used to obtain large enzyme quantities. The native protein consists of four subunits with a approximately 50 kDa molecular mass each and has an isoelectric point at pH 6.5 +/- 0.3. The equilibrium constant for fumarate hydration is about 4.3 (25 degrees C, pH 7.5), the Michaelis constants for fumarate and 1-malate are approximately 30 microM and approximately 250 microM, respectively. The enzyme is activated by substrates and multivalent anions, the activation seems to be of a non-competitive type. The fumarase complex with meso-tartaric acid has been crystallized by the vapor diffusion method. The unit cell parameters are a = 93.30, b = 94.05 and c = 106.07 A, space group P2(1)2(1)2(1). The unit cell contains 2 protein molecules. The crystals diffract to at least 2.6 A resolution and are suitable for X-ray structure analysis.