Molecular cloning and characterization of a laccase gene from the basidiomycete<Emphasis Type="Italic"> Fome lignosus</Emphasis> and expression in<Emphasis Type="Italic"> Pichia pastoris</Emphasis>

A cDNA encoding for a laccase was isolated from the white-rot fungus Fome lignosus by RT-PCR. It contained an open reading frame of 1,557 bp. The deduced mature protein consisted of 497 amino acids and was preceded by a signal peptide of 21 amino acids. The genomic DNA of the laccase, containing 11 introns, was cloned by PCR. The cDNA was cloned into the vectors pGAPZA and pGAPZA, and expressed in the Pichia pastoris GS115. Laccase-secreting transformants were selected by their ability to oxidize the substrate 22-azinobis-(3-ethylbenzthiaoline-6-sufonic acid) (ABTS). The laccase activity obtained with the native signal peptide was found to be fivefold higher than that obtained with the -factor secretion signal peptide. The presence of 0.4 mM copper was necessary for optimal activity of the enzyme. The highest activity value reached 9.03 U ml^–1, and the optimal secreting time was 2~3 days at 20°C. The crude laccase was stable in a pH range from 6.0 to 10.0 and at temperatures lower than 30°C in pH4.5 for 24 h. The molecular mass of the enzyme was estimated to be 66.5 kDa by SDS-PAGE. The optimum pH and temperature were 2.4 and 55°C. The K_m and V_max values for ABTS were 177 M and 23.54 mol min^–1 respectively. The extent of glycosylation of the purified enzyme was 58.6%.     

Purification and characterization of a truncatedBacillus subtilis α-amylase produced byEscherichia coli

ABacillus subtilis amylase gene was inserted into a plasmid which transferred toEscherichia coli. During cloning, a 3 region encoding 171 carboxyterminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein. The transformed cells produced substantial amylolytic activity. The active protein was purified to apparent homogeneity. Its molecular mass (48 kDa), as estimated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was lower than the molecular mass values calculated from the derived amino acid sequences of theB. subtilis complete -amylase (57.7 kDa) and the truncated protein (54.1 kDa). This truncated enzyme form hydrolysed starch with aK _m of 3.845 mg/ml. Activity was optimal at pH 6.5 and 50°C, and the purified enzyme was stable at temperatures up to 50°C. While Hg²⁺, Fe³⁺ and Al³⁺ were effective in inhibiting the truncated enzyme Mn²⁺ and Co²⁺ considerably enhanced the activity.     

Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterization

The medicinal mushroom Agaricus blazei produced high amounts of laccase (up to 5,000 units l^–1) in a complex, agitated liquid medium based on tomato juice, while only traces of the enzyme (<100 units l^–1) were detected in synthetic glucose-based medium. Purification of the enzyme required three chromatographic steps, including anion and cation exchanging. A. blazei laccase was expressed as a single protein with a molecular mass of 66 kDa and an isoelectric point of 4.0. Spectroscopic analysis of the purified enzyme confirmed that it belongs to the blue copper oxidases. The enzymes pH optimum for 2,6-dimethoxyphenol (DMP) and syringaldazine was pH 5.5; but for 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) no distinct pH optimum was observed (highest activity at the lowest pH tested). Purified laccase was stable at 20°C, pH 7.0 and pH 3.0, but rapidly lost its activity at 40°C or pH 10. Sodium chloride strongly inhibited the enzyme activity, although the inhibition was completely reversible. The following kinetic constants were determined (K_m, k_cat): 63 M, 21 s^–1 for ABTS, 4 M, 5 s^–1 for syringaldazine, 1,026 M, 15 s^–1 for DMP and 4307 M, 159 s^–1 for guaiacol. The results show that—in addition to the wood-colonizing white-rot fungi—the typical litter-decomposing basidiomycetes can also produce high titers of laccase in suitable liquid media.     

Purification and characterization of laccase from the white-rot fungus<Emphasis Type="Italic"> Daedalea quercina</Emphasis> and decolorization of synthetic dyes by the enzyme

The white-rot fungus Daedalea quercina produced the ligninolytic enzymes laccase and Mn-dependent peroxidase. Laccase was purified using anionexchange and size-exclusion chromatographies. SDS-PAGE showed the purified laccase to be a monomeric protein of 69 kDa (71 kDa using gel filtration) with an isoelectric point near 3.0. The optimum pH for activity was bellow 2.0 for 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (K_m=38 M), 4.0 for 2,6-dimethoxyphenol (K_m=48 M), 4.5 for guaiacol (K_m=93 M) and 7.0 for syringaldazine (K_m=131 M). The temperature optimum was between 60 and 70 °C depending on the pH and buffer used. The enzyme was stable up to 45 °C, and stability was higher at alkaline pH. Enzyme activity was increased by the addition of Cu²⁺ and inhibited by Mn²⁺, sodium azide, dithiothreitol, and cysteine. Laccase from Daedalea quercina was able to decolorize the synthetic dyes Chicago sky blue, poly B-411, remazol brilliant blue R, trypan blue and reactive blue 2.     

Cloning and characterization of an exoinulinase from Bacillus polymyxa

A gene encoding an exoinulinase (inu) from Bacillus polymyxa MGL21 was cloned and sequenced. It is composed of 1455 nucleotides, encoding a protein (485 amino acids) with a molecular mass of 55522 Da. Inu was expressed in Escherichia coli and the His-tagged exoinulinase was purified. The purified enzyme hydrolyzed sucrose, levan and raffinose, in addition to inulin, with a sucrose/inulin ratio of 2. Inulinase activity was optimal at 35°C and pH 7, was completely inactivated by 1 mM Ag⁺ or Hg²⁺. The K _m and V _max values for inulin hydrolysis were 0.7 mM and 2500 M min^–1 mg^–1 protein. The enzyme acted on inulin via an exo-attack to produce fructose mainly.     

Hyperthermophilic α-L-arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression, and characterization of the recombinant protein

A putative -L-arabinofuranosidase (AFase) gene belonging to family 51 of glycosyl hydrolases of a hyperthermophilic bacterium Thermotoga maritima MSB8 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein (Tm-AFase) was purified to apparent homogeneity by heat treatment (80°C, 30 min), followed by hydrophobic interaction, anion-exchange, and gel permeation column chromatography. Tm-AFase had a molecular mass of 55,284 Da on matrix assisted laser desorption ionization time-of-flight mass spectrometry and ~332 kDa on gel permeation column chromatography. Therefore, Tm-AFase comprised six identical subunits as in the case of homologous AFase from Geobacillus stearothermophilus. Regarding substrate specificity, Tm-AFase was active with p-nitrophenyl -L-arabinofuranoside but not with p-nitrophenyl -L-arabinopyranoside. Regarding polysaccharides, Tm-AFase hydrolyzed arabinan and debranched arabinan but not arabinoxylan, arabinogalactan, and carboxymethyl cellulose. Tm-AFase was extremely thermophilic, displaying an optimal reaction temperature of 90°C in a 10 min assay. When Tm-AFase was heated at 90°C, no loss of activity was observed for at least 24 h. At 100°C, the activity dropped to ~50% in 20 min; thereafter, inactivation occurred very slowly exhibiting a half-life of ~2.7 h, characterizing the enzyme to be the most thermophilic AFase reported thus far.     

Genetic analysis of functional connectivity between substrate recognition domains ofEscherichia coli glutaminyl-tRNA synthetase

It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype onEscherichia coli. Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination. The three-dimensional structure of the tRNA^Gln: GlnRS:ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3 end of tRNA^Gln. The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischargesupF tRNA^Tyr considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not. These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguishsupE tRNA _CUA ^Gln from tRNA ₂ ^Gln considerably more efficiently than GlnRS E222K. Furthermore, GlnRS-E222K was found to have a 15-fold higher K_m for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase. These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.     

Production of a thermostable metal-tolerant laccase from Trametes versicolor and its application in dye decolorization

Production of laccase using a submerged culture of Trametes versicolor sdu-4 was optimized using a central composite design of the Response Surface Methodology. Optimized conditions gave a laccase yield of 4,213 U/L which was approximately three times of that in basal medium. The laccase was purified to homogeneity using a three-step process. The overall yield of the purification was 58%, with a purification fold of 11.4 and a specific activity of 1320.7 U/mg protein. The molecular mass of the laccase was 60 kDa. The optimum pH values of the enzyme were 2.2, 3.7, and 7 for the oxidations of ABTS, DMP, and syringaldazine, respectively. The enzyme had adaptability to a broad pH range and high temperature and wsa stable at pH 3.0 ∼ 10.0. The half-life of this laccase at 70°C was 2.2 h. Methyl red, 2-bromophenol, and 4-bromophenol were oxidized by the purified laccase in the absence of mediators. Purified laccase was effective in the decolorization of several dyes and was not inhibited by Cu²⁺, Mn²⁺, Zn²⁺, Na⁺, K⁺, Mg²⁺, Ba²⁺, and Ca²⁺ at 5 mM. These excellent characteristics made it a highly attractive candidate for industrial use.     

Purification and characterization of β-mannanase from Bacillus licheniformis for industrial use

An easily scaled-up technique has been designed to purify -mannanase from Bacillus licheniformis. Using flocculation, ultrafiltration and ion-exchange chromatography, the enzyme was purified 33-fold with a final recovery of 47% and a specific activity of 4341 U mg^–1protein. The enzyme had maximum activity at 60 °C and pH 7.0. It was stable at 50 °C and pH 6.0 for 6 h, but lost all of its activity when held at 70 °C and pH 6.0 for 1 h.     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Die Phenoloxydasen des Ascomyceten Podospora anserina

Summary Laccase of the wild strain of Podospora anserina was purified by subsequent treatment with protamine sulfate, precipitation with ammonium sulfate and column chromatography on DEAE-Sephadex and hydroxylapatite. The purity was confirmed by sedimentation and electrophoresis (molecular weight about 361 000, isoeletric point at pH 5.1).The blue-coloured pure laccase has its absorption-maxima at 280 and 605 m. The substrate specifity of the enzyme corresponds to results which have been earlier obtained with unpurified preparations (Esser 1963 b). Laccase is very temperature sensitive. It loses its activity after both freezing and heat treatment (half-life time at 60°C about 6 min). The Michaelis-constants as determined with Dopa, potassium ferrocyanide and catechol are in the range of 2 to 5·10^-3 Mol/l. The appropriate value for ascorbic acid is about 10^-2 less. The laccase contains about 12% carbohydrate and about 7,5% nitrogen. According to its copper content of 0.123% the laccase carries seven atoms of copper per molecule.

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Teil I erschien in Arch. Mikrobiol. 46, 217–226 (1963).     

Purification and characterization of a thermally stable yellow laccase from <Emphasis Type="Italic">Daedalea flavida</Emphasis> MTCC-145 with higher catalytic performance towards selective synthesis of substituted benzaldehydes

A laccase from the culture filtrate of white rot fungus Daedalea flavida MTCC-145 has been purified and characterized. The method involved concentration of the culture filtrate by ultrafiltration and an anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (native PAGE) both gave single protein bands indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 75.0 kDa. Purification fold was 21.5 while recovery of the enzyme activity was 11.52%. Using 2,6-dimethoxyphenol, diammonium salt of 2,2'-[azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)] and 3,5-dimethoxy-4-hydroxybenzaldehyde azine as substrates, the Km, kcat, and k _cat/K _m values of the laccase were found to be 440 µM, 6.45 s^–1, 1.47 × 10⁴ M^–1 s^–1; 366 µM, 6.45 s^–1, 1.76 × 10⁴ M^–1 s^–1; and 226 µM, 6.45 s^–1, 2.85 × 10⁴ M^–1 s^–1, respectively. The pH and temperature optima were 4.5 and 50°C, respectively. The enzyme was most stable at pH 5.0 when exposed for 1 h. The purified laccase has yellow color and shows no absorption band around 610 nm characteristic of blue laccases. The enzyme transforms toluene and substituted toluenes to corresponding benzaldehyde and substituted benzaldehydes in the absence of mediator molecules with higher catalytic efficiency as compared to other known laccases.     

Enzymatic properties and nucleotide and amino acid sequences of a thermostable β-agarase from a novel species of deep-sea<Emphasis Type="Italic"> Microbulbifer</Emphasis>

An agar-degrading bacterium, strain JAMB-A7, was isolated from the sediment in Sagami Bay, Japan, at a depth of 1,174 m and identified as a novel species of the genus Microbulbifer. The gene for a novel -agarase from the isolate was cloned and sequenced. It encodes a protein of 441 amino acids with a calculated molecular mass of 48,989 Da. The deduced amino acid sequence showed similarity to those of known -agarases in glycoside hydrolase family 16, with only 34–55% identity. A sequence similar to a carbohydrate-binding module was found in the C-terminal region of the enzyme. The recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host, and the enzyme purified to homogeneity had a specific activity of 398 U (mg protein)^–1 at pH 7.0 and 50°C. It was thermostable, with a half-life of 502 min at 50°C. The optimal pH and temperature for activity were around 7 and 50°C, respectively. The pattern of agarose hydrolysis showed that the enzyme was an endo-type -agarase, and the final main product was neoagarotetraose. The activity was not inhibited by NaCl, EDTA, and various surfactants at high concentrations. In particular, sodium dodecyl sulfate had no inhibitory effect up to 2%.     

Divergence of glutamate and glutamine aminoacylation pathways: Providing the evolutionary rationale for mischarging

Aminoacyl-tRNA for protein synthesis is produced through the action of a family of enzymes called aminoacyl-tRNA synthetases. A general rule is that there is one aminoacyl-tRNA synthetase for each of the standard 20 amino acids found in all cells. This is not universal, however, as a majority of prokaryotic organisms and eukaryotic organelles lack the enzyme glutaminyl-tRNA synthetase, which is responsible for forming Gln-tRNA^Gln in eukaryotes and in Gram-negative eubacteria. Instead, in organisms lacking glutaminyl-tRNA synthetase, Gln-tRNA^Gln is provided by misacylation of tRNA^Gln with glutamate by glutamyl-tRNA synthetase, followed by the conversion of tRNA-bound glutamate to glutamine by the enzyme Glu-tRNA^Gln amidotransferase. The fact that two different pathways exist for charging glutamine tRNA indicates that ancestral prokaryotic and eukaryotic organisms evolved different cellular mechanisms for incorporating glutamine into proteins. Here, we explore the basis for diverging pathways for aminoacylation of glutamine tRNA. We propose that stable retention of glutaminyl-tRNA synthetase in prokaryotic organisms following a horizontal gene transfer event from eukaryotic organisms (Lamour et al. 1994) was dependent on the evolving pool of glutamate and glutamine tRNAs in the organisms that acquired glutaminyl-tRNA synthetase by this mechanism. This model also addresses several unusual aspects of aminoacylation by glutamyl- and glutaminyl-tRNA synthetases that have been observed.Based on a presentation made at a workshop—Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: D. Söll     

Purification and characterization of α-glucosidase from Aspergillus carbonarious

Summary An -glucosidase was purified from Aspergillus carbonarious CCRC 30414 over 20 fold with 37 % recovery. Its molecular mass was estimated to be 328 kDa by gel filtration with an optimum pH from 4.2 to 5.0, and pI=5.0. The optimum temperature is at 60°C over 40 min. The enzyme was partially inhibited by 5 mM Ag⁺, Hg²⁺, Ba²⁺, Pb²⁺, and Aso₄ ⁺.     

Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov

One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn²⁺, Hg²⁺, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K _m and V _max of the enzyme for CMC were 3.4 mg ml^–1 and 44.7 mol min^–1 (mg protein)^–1, respectively.     

Purification and Characterization of S-Adenosyl-L-Methionine Nicotinic Acid-N-Methyltransferase from Leaves of Glycine max

Trigonelline (TRG), which act as a cell cycle regulator and a compatible solute in response to salinity and water-stress, is the N-methyl conjugate of nicotinic acid the formation of which is catalyzed by S-adenosyl-L-methionine nicotinic acid-N-methyltransferase. The enzyme was purified 2650-fold from soybean (Glycine max L.) leaves with a recovery of 4 %. The purification procedure included ammonium sulfate (45 – 60 %) precipitation, linear gradient DEAE-Sepharose chromatography, adenosine-agarose affinity chromatography, hydroxyapatite chromatography and gel filtration (Sephacryl-S-200). The purified enzyme preparation showed a major band with a molecular mass of 41.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that is related to the enzyme activity. The native enzyme had a molecular mass of about 85 kDa as estimated by gel filtration. The K_m values for S-adenosyl-L-methionine and nicotinic acid were 31 and 12.5 M, respectively. The purified enzyme showed optimum activity at pH 6.5 and temperature of 40 – 45 °C. High concentration of dithiothreitol (10 mM) and glycerol (20 %) stabilize the enzyme during purification and storage. Hg²⁺ strongly inhibits enzyme activity.     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

Purification and properties of laccase from Ganoderma lucidum

Summary The enzyme laccase has been partially purified from the culture fluid of Ganoderma lucidum by acetone precipitation, ammonium sulphate fractionation and adsorption on alumina C gel. The enzyme has been shown to be specific for ortho and para hydroxyphenolic compounds, having Km values of 5.5×10^-5 M and 2.86×10^-5 M for catechol and hydroquinone respectively. The optimum pH for the oxidation of catechol and hydroquinone are 5.4 and 5.0 respectively. The enzyme is inactivated above 60°C and is inhibited by enzyme inhibitors and metal chelating agents like azide, cyanide etc.     

Gln54Leu of the conserved polar residue inthe interfacial coiled coil position (d) results in significant stabilization of the original structure of the COMP pentamerization domain

The assembly domain of cartilage oligomeric matrixprotein (COMP) forms an -helical coiled coilhomopentamer with a conserved polar glutamine in theinterior (d) position. We substituted Gln⁵⁴ forapolar Leu in the recombinant fragment of the rat COMPdomain. Biochemical studies and circular dichroism(CD) spectroscopy showed that the mutant, similarly tothe wild-type (w.t.) peptide, forms spontaneously an-helical pentamer. Thermal transitions of thew.t. and mutant pentamers were analyzed by CDspectroscopy and differential scanning calorimetry.The Gln⁵⁴Leu mutation increased the thermal stabilityof the pentamer with reduced disulfide bonds from73 °C to 104 °C. The denaturation of thedisulfide bonded w.t. pentamer was observed at108 °C while the mutant pentamer cannot bedenatured up to 120 °C (the apparatus limit).Thus, by Gln⁵⁴Leu mutation we found a way tosignificantly stabilize the coiled coil pentamer,making this peptide even more attractive as an oligomerization tool for various biotechnological applications.