A novel membrane-bound flavocytochrome c sulfide dehydrogenase from the colourless sulfur bacterium Thiobacillus sp. W5

A novel membrane-bound sulfide-oxidizing enzyme was purified 102-fold from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5 by means of a six-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 40 and 11 kDa. The smaller subunit contains covalently bound haem c, as was shown by haem staining. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide-oxidizing heterodimer contains one molecule of haem c and one molecule of flavin. It appeared that the sulfide-oxidizing enzyme is a member of a small class of redox proteins, the flavocytochromes c, and is structurally most related to the flavocytochrome c sulfide dehydrogenase of the green sulfur bacterium Chlorobium limicola. The pH optimum of the enzyme is 8.6. At pH 9, the V _max was 2.1 ± 0.1 μmol cytochrome c (mg protein)^–1 min^–1, and the K _m values for sulfide and cytochrome c were 1.7 ± 0.4 μM and 3.8 ± 0.8 μM, respectively. Cyanide inhibited the enzyme by the formation of an N-5 adduct with the flavin moiety of the protein. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product. Received: 15 October 1996 / Accepted: 7 January 1997     

Purification and characterization of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes sp. nov.

Summary Membrane-bound alcohol dehydrogenase (ADH) was purified from the membrane fraction of an industrial-vinegar-producing strain, A. polyoxogenes sp. nov. NBI1028 by solubilization using Triton X-100 and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneous on polyacrylamide disc gel electrophoresis (PAGE). Upon sodium dodecyl sulphate-PAGE, the enzyme showed the presence of two subunits with a molecular mass of 72 000 daltons and 44 000 daltons, respectively. The small subunit was identified as cytochrome c. In addition, absorption and fluorescence spectra showed the the presence of pyrroloquinoline quinone in the purified ADH. The ADH preferentially oxidized aliphatic alcohols with a straight carbon chain except for methanol. Formaldehyde and acetaldehyde were also oxidizable substrates. The apparent K _m for ethanol was 1.2 mM. The optimum pH and temperature were 5.0–6.0 and 40°C, respectively. p-Chloromercuribenzoic acid and heavy metals such as CuSO₄ were inhibitory to the enzyme activity. Ferricyanide was effective as an electron acceptor.Offprint requests to: M. Fukaya     

Purification and characterization of alcohol oxidase from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> isolated as a formaldehyde-resistant fungus

Paecilomyces variotii IRI017 was isolated as a formaldehyde-resistant fungus from wastewater containing formaldehyde. The fungus grew in a medium containing 0.5% formaldehyde and had consumed formaldehyde completely after 5 days. Alcohol oxidase was purified from the fungus grown on methanol. A 20-fold purification was achieved with a yield of 44%. The molecular mass of the purified enzyme was estimated to be 73 and 450 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively, suggesting that the enzyme consists of six identical subunits. The N-terminal amino acid sequence of the subunit was TIPDEVDIII. The enzyme showed an absorption spectrum typical of a flavoprotein and had a noncovalently bound flavin different from FAD, FMN, and riboflavin. The pH optimum of the enzyme activity was pH 6–10. The enzyme was stable in the pH range of pH 5–10. The enzyme retained full activity after incubation at 50°C for 30 min. The enzyme oxidized not only methanol but also lower primary alcohols and formaldehyde. The K _m values for methanol, ethanol, and formaldehyde were 1.9, 3.8, and 4.9 mmol l⁻¹, respectively.     

Electrochemical study on cytochrome c peroxidase from Paracoccus denitrificans: a shifting pattern of structural and thermodynamic properties as the enzyme is activated

 The di-haem cytochrome c peroxidase of Paracoccus denitrificans is a calcium binding dimer of 37.5 kDa subunits. It is responsible for reduction of H₂O₂ to H₂O with oxidation of cytochrome c ₅₅₀ and is isolated in a fully oxidised state (inactive) in which one haem (centre I) is in a high-spin/low-spin equilibrium and high potential and the other (centre II) is low-spin and low potential. The enzyme undergoes direct electron transfer (without the need for mediators) with a 4,4′-dithiodipyridine-modified gold electrode and the response of both haem groups can be observed. By combination of the cyclic and pulse voltammetric data with the established spectroscopic information, it was demonstrated that entry of one electron to the high potential haem leads (in a mechanism involving strong haem-haem interactions) to a complex change of spin states and redox potentials of both haems in order to attain a "ready state" for binding, reduction and cleavage of the hydrogen peroxide. In the absence of endogenous calcium, haem communication can be completely disconnected and is recovered only when Ca²⁺ is added, an essential step for the formation of the peroxidatic site. The intricate electrochemical behaviour of this enzyme was interpreted as a mechanism involving, both reduction and oxidation of the high potential haem, an interfacial electron transfer coupled to a homogenous chemical reaction (EC mechanism). We discuss two different models for the sequence of events leading to the appearance of the active pentacoordinated peroxidatic haem. Received: 29 April 1998 / Accepted: 3 September 1998     

Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721^T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl, Na₂SO₄, NaBr, NaNO₃, CH₃COONa, LiCl, NH₄NO₃, and NH₄Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe²⁺ and Hg²⁺ ions), regardless whether NaCl was present or not. * The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession no. AB211544.     

Purification and characterization of an endoxylanase from the culture broth of <Emphasis Type="Italic">Bacillus cereus</Emphasis> BSA1

An extracellular xylanase from the fermented broth of Bacillus cereus BSA1 was purified and characterized. The enzyme was purified to 3.43 fold through ammonium sulphate precipitation, DEAE cellulose chromatography and followed by gel filtration through Sephadex-G-100 column. The molecular mass of the purified xylanse was about 33 kDa. The enzyme was an endoxylanase as it initially degraded xylan to xylooligomers. The purified enzyme showed optimum activity at 55°C and at pH 7.0 and remained reasonably stable in a wide range of pH (5.0–8.0) and temperature (40–65°C). The K _m and V _max values were found to be 8.2 mg/ml and 181.8 μmol/(min mg), respectively. The enzyme had no apparent requirement of cofactors, and its activity was strongly inhibited by Cu²⁺, Hg²⁺. It was also a salt tolerant enzyme and stable upto 2.5 M of NaCl and retained its 85% activity at 3.0 M. For stability and substrate binding, the enzyme needed hydrophobic interaction that revealed when most surfactants inhibited xylanase activity. Since the enzyme was active over wide range of pH, temperature and remained active in higher salt concentration, it could find potential uses in biobleaching process in paper industries.     

Purification and characterization of a sulfite:cytochrome c oxidoreductase from Thiobacillus acidophilus

Cell-free extracts of Thiobacillus acidophilus prepared at neutral pH showed oxidation of sulfite to sulfate with ferricyanide as electron acceptor. Horse heart cytochrome c could be used as alternative electron acceptor; however, the observed activity was only 0.1% of that found for ferricyanide. The enzyme responsible for the oxidation of sulfite was purified to homogeneity. The purified enzyme was a monomer of 42 kDa and contained one haem c per monomer. Electron paramagnetic resonance (EPR) spectroscopical analysis of the sulfite:cytochrome c oxidoreductase showed the presence of molybdenum (V), only after reduction of the enzyme with sulfite. The pH optimum for the enzymatic reaction was 7.5 and the temperature optimum 40°C. Enzymatic activity was strongly reduced in the presence of the anions: chloride, phosphate and nitrate. In contrast to other enzymes involved in sulfur metabolism and previously isolated from T. acidophilus, sulfite:cytochrome c oxidoreductase activity is not stimulated by the presence of sulfate ions.     

Heterologous Expression and Characterization of an Alcohol Dehydrogenase from the Archeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Analysis of the Thermoplasma acidophilum DSM 1728 genome identified two putative alcohol dehydrogenase (ADH) open reading frames showing 50.4% identity against each other. The corresponding genes Ta0841 and Ta1316 encode proteins of 336 and 328 amino acids with molecular masses of 36.48 and 36.01 kDa, respectively. The genes were expressed in Escherichia coli and the recombinant enzymes were functionally assessed for activity. Throughout the study only Ta1316 ADH resulted active in the oxidative reaction in the pH range 2–8 (optimal pH 5.0) and temperatures from 25 to 90°C (optimal 75°C). This ADH catalyzes the oxidation of several alcohols such as ethanol, methanol, 2-propanol, butanol, and pentanol during the reduction of the cofactor NAD⁺. The highest activity was found in the presence of ethanol producing optically pure acetaldehyde. The specific enzyme activity of the purified Ta1316 ADH with ethanol as a substrate in the optimal conditions was 628.7 U/mg.     

Purification and characterization of a novel β-galactosidase from Bacillus sp MTCC 3088

An extracellular β-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was harvested from the late stationary-phase of Bacillus sp MTCC 3088. The enzyme was purified 36.2-fold by ZnCl₂ precipitation, ion exchange, hydrophobic interaction and gel filtration chromatography with an overall recovery of 12.7%. The molecular mass of the purified enzyme was estimated to be about 484 kDa by gel filtration on a Sephadex G-200 packed column and the molecular masses of the subunits were estimated to be 115, 86.5, 72.5, 45.7 and 41.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the native enzyme, determined by polyacrylamide gel electrofocusing, was 6.2. The optimum pH and temperature were 8 and 60°C, respectively. The Michaelis–Menten constants determined with respect to o-NO₂-phenyl-β-D-galactopyranoside and lactose were 6.34 and 6.18 mM, respectively. The enzyme activity was strongly inhibited (68%) by galactose, the end product of lactose hydrolysis reaction. The β-galactosidase was specific for β-D anomeric linkages. Enzyme activity was significantly inhibited by metal ions (Hg²⁺, Cu²⁺ and Ag⁺) in the 1–2.5 mM range. Mg²⁺ was a good activator. Catalytic activity was not affected by the chelating agent EDTA. Journal of Industrial Microbiology & Biotechnology (2000) 24, 58–63. Received 09 February 1999/ Accepted in revised form 24 September 1999     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Expression, Purification and Characterization of a Recombinant β-Glucosidase from Volvariella Volvacea

For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a K_m of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside.     

Co-expression of α and β subunits of the 3-methylcrotonyl-coenzyme A carboxylase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa is a versatile bacterium that can grow using citronellol or leucine as sole carbon source. For both compounds the degradation pathways converge at the key enzyme 3-methylcrotonyl coenzyme-A carboxylase (MCCase). This enzyme is a complex formed by two subunits (α and β), encoded by the liuD and liuB genes, respectively; both are essential for enzyme function. Previously, both subunits had been separately expressed and then the complex re-constituted, however this methodology is laborious and produces low yield of active enzyme. In this work, the MCCase subunits were co-expressed in the same plasmid and purified in one step by affinity chromatography using the LiuD-His tag protein, interacting with the LiuB-S tag recombinant protein. The purified enzyme lost most of the activity within few hours of storage. The co-expressed subunits formed an (αβ)₄ complex that suffered a modification of its oligomerization state after storage, which probably contributed to the loss on activity observed. The recombinant MCCase enzyme presented optimum pH and temperature values of 9.0 and 30o C, respectively. Functionally, MCCase showed Michaelian kinetics behavior with a K_m for its substrate and V_max of 168 μM and 430 nmoles mg⁻¹min⁻¹, respectively. The results suggest that the co-expression and co-purification of the subunits is a suitable procedure to obtain the active complex of the MCCase from Pseudomonas aeruginosa in a single step.     

Purification and Properties of Alcohol Oxidase from Candida methanosorbosa M-2003

Alcohol oxidase from Candida methanosorbosa was purified about sixfold with a yield of 37.6% from the culture broth of Candida methanosorbosa M-2003. The enzyme preparation was homogeneous on slab gel electrophoresis. The purified enzyme had an optimal pH from 6.0 to 9.0 and was stable in the range 6.0–8.5. Its optimal temperature of reaction was 50°C, and it was stable below 50°C. In the presence of NaN₃, the enzyme retained its initial activity at 30°C for 35 days, indicating stability for a long term, so far. The isoelectric point was pH 4.3. Its molecular weight was 620,000 by gel filtration chromatography and 80,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. These results indicate that the enzyme consists of 8 subunits. Received: 1 October 1996 / Accepted: 12 December 1996     

A novel thermostable and halostable carboxymethylcellulase from marine bacterium <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>AU01

A novel thermostable, halostable carboxymethylcellulase (CMCase) from a marine bacterium Bacillus licheniformisAU01 was purified 10.4-fold with 18% yield with a specific activity of 88.43 U/mg and the molecular weight was estimated as 37 kDa. The enzyme was optimally active at pH 9–10 and temperature 50–60°C and it was most stable up to pH 12 and 20–30% of NaCl concentration. The enzyme activity was reduced when treated with Hg²⁺, Fe²⁺ and EDTA and stimulated by Co²⁺, Mn²⁺, Mg²⁺ and Ca²⁺. Various cationic, anionic detergents and commercial detergents were not much affected CMCase activity.     

Purification and characterization of pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an αβγδ-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80° C. The pH optimum was 7.6–7.8. The apparent K _m values for pyruvate and coenzyme A at 70° C were 3.45 mM and 54 μM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t _50%) at 70° C was approximately 8 h. Received: 9 September 1996 / Accepted: 27 December 1996     

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development. Received: 24 February 1998 / Accepted: 25 May 1998     

Purification and biological characterization of a halophilic thermostable protease from <Emphasis Type="Italic">Haloferax lucentensis</Emphasis> VKMM 007

An extremely halophilic archaeon Haloferax lucentensis VKMM 007, isolated from a solar saltern, was found to produce a protease. This extracellular enzyme consisted of a single polypeptide chain of 57.8 kDa as determined by SDS–PAGE and was purified by a combination of ultrafiltration, bacitracin–Sepharose affinity chromatography and Sephadex G-100 gel filtration. The purified protein was stable in a wide range of temperatures (20–70°C), NaCl concentrations (0.85–5.13 M) and pH (5.0–9.0) with maximal activity observed at 60°C, 4.3 M NaCl and pH 8.0. Proteolytic activity was enhanced by Ca²⁺, K⁺, Mg²⁺, Na⁺, and Fe²⁺ ions and the protein was classified as a trypsin-like serine protease. Further assays indicated highest degree of specificity when hemoglobin was used as an enzyme substrate. Most importantly, the proteolytic activity remained stable or only marginally inhibited in the presence of various polar and non-polar solvents, surfactants and reducing agents thus emphasizing the biotechnological potential of this novel halophilic protease.     

Purification and Characterization of an Extracellular β-Glucosidase from the Wood-Grown Fungus Xylaria regalis

Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces β-glucosidase (EC 3.2.1.21) extracellularly. The β-glucosidase was purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. With p-nitrophenyl β-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the K _m was 1.72 mM and V _max was 326 μmol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0 at temperatures up to 50°C. The purified β-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl β-D-galactopyranoside. The activity of β-glucosidase was stimulated by Ca²⁺, Mg²⁺, Mn²⁺, Cd²⁺ and β-mercaptoethanol, and inhibited by Ag⁺, Hg²⁺, SDS, and p-chloromercuribenzoate (PCMB). Received: 30 March 1996 / Accepted: 3 May 1996     

Purification and characterization of an extracellular acid trehalase fromLentinula edodes

Trehalase from the culture filtrate ofLentinula edodes was purified and characterized. Molecular masses were estimated to be 158 kDa and 79–91 kDa by gel filtration and SDS-PAGE under the reduced condition, respectively. The enzyme was composed of two identical subunits and contained carbohydrate molecules. The optimum temperature and pH were obtained at around 40°C and pH 5.0, respectively. The enzyme was stable up to 40°C and in a range pH of 4–10 at 30°C. It cleaved α-1,1 linkages of trehalose, but not α-1,4, α-1,6 or β-1,4 glycosyl linkages, and was defined as an acid trehalase.     

Detection of β-glucosidase as saprotrophic ability from an ectomycorrhizal mushroom, Tricholoma matsutake

We investigated extracellular carbohydrase production in the medium of an ectomycorrhizal fungus, Tricholoma matsutake, to reveal its ability to utilize carbohydrates such as starch as a growth substrate and to survey the saprotrophic aspects. We found β-glucosidase activity in the static culture filtrate of this fungus. The β-glucosidase was purified and characterized. The purified enzyme was obtained from about 2.1 l static culture filtrate, with 9.0% recovery, and showed a single protein band on SDS-PAGE. Molecular mass was about 160 kDa. The enzyme was most active around 60°C and pH 5.0, and stable over a pH of 4.0–8.0 for 30 min at 37°C. The purified enzyme was activated by the presence of Ca²⁺ and Mn²⁺ ions (about 2–3 times that of the control). The enzyme readily hydrolyzed oligosaccharides having a β-1,4-glucosidic linkage such as cellobiose and cellotriose. However, it did not hydrolyze polysaccharides such as avicel and CM-cellulose or oligosaccharides having an α-glucosidic linkage. Moreover, cellotriose was hydrolyzed by the enzyme for various durations, and the resultant products were analyzed by TLC. We concluded that the enzyme from T. matsutake seems to be a β-glucosidase because cellotriose with a β-1,4-glucosidic linkage decomposed to glucose during the enzyme reaction.