Reduction of Hg2+ with Reduced Mammalian Cytochrome c by Cytochrome c Oxidase Purified from a Mercury-Resistant Acidithiobacillus ferrooxidans Strain,MON-1

Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 μM and 5 μM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 μM, and 70% by 10 μM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.     

Nickel Inhibition of the Growth of a Sulfur-oxidizing Bacterium Isolated from Corroded Concrete

A sulfur-oxidizing bacterium strain NB1-3 isolated from corroded concrete was a Gram negative, non-spore-forming, and rod-shaped bacterium (0.5–1.0x 1.5–2.0μm) with a polar flagellum. Strain NB1-3 had its optimum temperature and pH for growth at 30°C and 3.0–4.0, respectively. Strain NB1-3 had enzyme activities that oxidized elemental sulfur, thiosulfate, tetrathionate, and sulfide and the activity to incorporate ¹⁴CO₂ into the cells. The mean G+C content of the DNA was 52.9 mol%. These results indicate that strain NB1-3 is Thiobacillus thiooxidans. Since nickel has been known to protect concrete from corrosion, the effect of Ni on the growth of strain NB1-3 was studied. The cell growth on tiosulfate-, elemental sulfur-, or tetrathionate-medium was completely inhibited by 0.1% metal nickel or 5mM NiSO₄. Both cellular activities of elemental sulfur oxidation and CO₂ incorporation were strongly inhibited by 5mM NiSO₄. The amounts of Ni in cells with or without nickel treatment were 1.7 and 160.0 nmol/mg protein, respectively. These results indicate that nickel binds to strain NB1-3 cells and inhibits enzymes involved in sulfur oxidation of this bacterium, and as a result, inhibits cell growth.     

Dihydrosterigmatocystin and Dihydrodemethylsterigmatocystin,New Metabolites from Aspergillus versicolor

L-Arabinose isomerase (L-arabinose ketol-isomerase, EC 5.3.1.4) was demonstrated from the L-arabinose-grown cells of Streptomyces sp. which was isolated from sea water. The enzyme was purified by MnCl₂ treatment, fractionation by polyethylene glycol and by column chromatographies on Sephadex G-150 and DEAE-cellulose. The purified enzyme was specific only for L-arabinose and the Michaelis constant for L-arabinose was 40 mM at pH 7.5. Manganese or cobalt ions were effective for the enzyme activity after dialysis against EDTA. The enzyme activity was inhibited competitively by L-arabitoI, ribitol and xylitol, of which inhibition constants were 1.1, 1.0, and 15 mM, respectively.     

Enzymatic activity of L-amino acid oxidase from snake venom Crotalus adamanteus in supercritical CO2

L-amino acid oxidase (L-AAO) from snake venom Crotalus adamanteus was successfully tested as a catalyst in supercritical CO₂ (SC-CO₂). The enzyme activity was measured before and after exposure to supercritical conditions (40°C, 110 bar). It was found that L-AAO activity slightly increased after SC-CO₂ exposure by up to 15%. L-AAO was more stable in supercritical CO₂ than in phosphate buffer under atmospheric pressure, as well as in the enzyme membrane reactor (EMR) experiment. 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) oxidation was performed in a batch reactor made of stainless steel that could withstand the pressures of SC-CO₂, in which L-amino acid oxidase from C. adamanteus was able to catalyze the reaction of oxidative deamination of L-DOPA in SC-CO₂. For the comparison L-DOPA oxidation was performed in the EMR at 40°C and pressure of 2.5 bar. Productivity expressed as mmol-s of converted L-DOPA after 3?h per change of enzyme activity after 3?h was the highest in SC-CO₂ (1.474?mmol?U^?1), where catalase was present, and the lowest in the EMR (0.457?mmol?U^?1).     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

A DNA Region That Complements on Escherichia coli cysG Mutation in Thiobacillus Ferrooxidans

Thiobacillus ferrooxidans AP19-3 has a novel NADH-dependent sulfite reductase in the periplasmic space. The gene responsible for the appearance of NADH-dependent sulfite reductase activity was cloned into a vector plasmid pBR322 to give a 5.7-kb hybrid plasmid, pTHS1, which contains a 1.3-kb DNA fragment of T. ferrooxidans AP19-3. When pTHS1 was used to transform sulfite reductase deficient E. coli mutants, strain AT2455 (cysG), JM246 (cysl), and AT2427 (cysJ), it complemented only the E. coli cysG mutation. Since cysG codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase, the enzyme involved in siroheme synthesis, the results indicate that the DNA region that codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase is present in a T. ferrooxidans 1.3 kb DNA fragment on pTHS1.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

Studies on the Pentose Isomerases of Lactic Acid Bacteria

Production of d-xylose and l-arabinose isomerases by lactic acid bacteria was greatly promoted by the addition of manganese ions in cultural medium. Effective concentration of the ions was 5 × 1O^-3 m. Ferrous ions were also effective for the production of d-xylose isomerase and cobaltous ions were somewhat effective for the production of l-arabinose isomerase. Zinc and cadmium ions inhibited bacterial growth. It was possible to increase the production of isomerase by changing MnSO₄ concentration to 5× 10^-3 m (0.l1 %) in place of 0.001 per cent in the normal medium.

Column chromatographic procedures for the purification of pentose isomerases were carried out. Cation and anion exchange resins were not suitable because of their low exchange capacities and instability of the enzyme at acidic pH range. But the isomerases were successfully purified by DEAE-cellulose column chromatography with high recovery (85~90%). Using a Tris buffer, KCl concentration was increased in gradient. d-Xylose isomerase was eluted at pH 7.0 at 0~0.2 m KCl, and l-arabinose isomerase at pH 8.0 at 0~0.4 m KCl. The purified isomerases, d-xylose isomerase and l-arabinose isomerase, both required manganese ions specifically for their activities.

D-Xylose isomerase and l-arabinose isomerase are different enzymes which can be separated from each other with acetone fractionation at pH 4.8~5.0, heat treatment or chromatography on a colnmn of DEAE-cellulose. In DEAE-cellulose chromatography with a linear gradient elution method, d-xylose isomerase is recovered in the first peak at pH 7.0 (Tris bnffer) with 0~0.2 m KCl, and l-arabinose isomerase is eluted in the second peak at pH 8.0 (Tris buffer) with a larger ionic strength.     

Properties of Tyrosinase from Pseudomonas melanogenum

The properties of the tyrosinase from Pseudomonas melanogenum was investigated with the crude enzyme preparation. Optimum temperature and pH of the enzyme were 23°C and 6.8, respectively. l-Tyrosine, d-tyrosine, m-tyrosine, N-acetyl-l-tyrosine and l-DOPA were utilized as a substrate by the enzyme. The value for Km obtained were as follows: l-tyrosine 6.90 × 10^?4 m, d-tyrosine 1.43 ×10^?3 m and l-DOPA 9.90 × 10^?4 m. The enzyme was inhibited by chelating agents of Cu²⁺ l-cysteine, l-homocysteine, thiourea and diethyl-dithiocarbamate and the inhibition was completely reversed by the addition of excess Cu²⁺ From these results it is concluded that the enzyme is a copper-containing oxidase.     

Isolation and Characterization of Acidithiobacillus ferrooxidans Strain D3-2 Active in Copper Bioleaching from a Copper Mine in Chile

Acidithiobacillus ferrooxidans strain D3-2, which has a high copper bioleaching activity, was isolated from a low-grade sulfide ore dump in Chile. The amounts of Cu²⁺ solubilized from 1% chalcopyrite (CuFeS₂) concentrate medium (pH 2.5) by A. ferrooxidans strains D3-2, D3-6, and ATCC 23270 and 33020 were 1360, 1080, 650, and 600 mg·l ^?1·30 d^?1. The iron oxidase activities of D3-2, D3-6, and ATCC 23270 were 11.7, 13.2, and 27.9 μl O₂ uptake·mg protein^?1·min^?1. In contrast, the sulfite oxidase activities of strains D3-2, D3-6, and ATCC 23270 were 5.8, 2.9, and 1.0 μl O₂ uptake·mg protein^?1·min^?1. Both of cell growth and Cu-bioleaching activity of strains D3-6 and ATCC 23270, but not, of D3-2, in the chalcopyrite concentrate medium were completely inhibited in the presence of 5 mM sodium bisulfite. The sulfite oxidase of strain D3-2 was much more resistant to sulfite ion than that of strain ATCC 23270. Since sulfite ion is a highly toxic intermediate produced during sulfur oxidation that strongly inhibits iron oxidase activity, these results confirm that strain D3-2, with a unique sulfite resistant-sulfite oxidase, was able to solubilize more copper from chalcopyrite than strain ATCC 23270, with a sulfite-sensitive sulfite oxidase.     

A New Enzyme Species of p-Hydroxybenzoate Hydroxylase during Oxygenating Process Observed by the Stopped-flow Method

Sulfite ion (HSO₃ ^-) is one of the products when elemental sulfur is oxidized by the hydrogen sulfide:ferric ion oxidoreductase of Thiobacillus ferrooxidans AP19-3. Under the conditions in which HSO₃ ^- is accumulated in the cells, the iron oxidase of this bacterium was strongly inhibited by HSO₃ ^-. Since cytochrome c oxidase is one of the most important components of the iron oxidase enzyme system in T. ferrooxidans, effects of HSO₃ ^- on cytochrome c oxidase activity were studied with the plasma membranes of HSO₃ ^--resistant and -sensitive strains of T. ferrooxidans, OK1-50 and AP19-3. The enzyme activity of AP19-3 compared with OK1-50 was strongly inhibited by HSO₃ ^-. To investigate the inhibition mechanism of HSO₃ ^- in T. ferrooxidans, cytochrome c oxidases were purified from both strains to an electrophoretically homogeneous state. Cytochrome c oxidase activity of a purified OK1-50 enzyme was not inhibited by 5 mM HSO₃ ^-. In contrast, the same concentration of HSO₃ ^- inhibited the enzyme activity of AP19-3 50%, indicating that the cytochrome c oxidase of OK1-50 was more resistant to HSO₃ ^- than that of AP19-3. Cytochrome c oxidases purified from both strains were composed of three subunits. However, the molecular weight of the largest subunit differed between OK1-50 and AP19-3. Apparent molecular weights of the three subunits of cytochrome c oxidases were 53,000, 24,000, and 19,000 for strain AP19-3 and 55,000, 24,000, and 19,000 for strain OK1-50, respectively.     

Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (K_m=12.5 mM), N-acetyl (K_m=2.52 mM), N-propionyl (K_m=0.194 mM), N-butyryl (K_m=0.033 mM), and N-glycyl (K_m =1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg²⁺, Ni²⁺, Cu²⁺) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.     

Biosynthetic Threonine Deaminase from Proteus morganii

Biosynthetic threonine deaminase was purified to an apparent homogeneous state from the cell extract of Proteus morganii, with an overall yield of 7.5%. The enzyme had a s⁰_20,w of 10.0 S, and the molecular weight was calculated to be approximately, 228,000. The molecular weight of a subunit of the enzyme was estimated to be 58,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seemed to have a tetrameric structure consisting of identical subunits. The enzyme had a marked yellow color with an absorption maximum at 415 nm and contained 2 mol of pyridoxal 5′-phosphate per mol. The threonine deaminase catalyzed the deamination of l-threonine, l-serine, l-cysteine and β-chloro-l-alanine. Km values for l-threonine and l-serine were 3.2 and 7.1 mm, respectively. The enzyme was not activated by AMP, ADP and ATP, but was inhibited by l-isoleucine. The Ki for l-isoleucine was 1.17 mm, and the inhibition was not recovered by l-valine. Treatment with mercuric chloride effectively protected the enzyme from inhibition by l-isoleucine.     

Purification and Crystallization of d-Arabinose (l-Fucose) Isomerase from Aerobacter aerogenes by Polyethylene Glycol

d-Arabinose(l-fucose) isomerase (d-arabinose ketol-isomerase, EC 5.3.1.3) was purified from the extracts of d-arabinose-grown cells of Aerobacter aerogenes, strain M-7 by the procedure of repeated fractional precipitation with polyethylene glycol 6000 and isolating the crystalline state. The crystalline enzyme was homogeneous in ultracentrifugal analysis and polyacrylamide gel electrophoresis. Sedimentation constant obtained was 15.4s and the molecular weight was estimated as being approximately 2.5 × 10⁵ by gel filtration on Sephadex G-200.

Optimum pH for isomerization of d-arabinose and of l-fucose was identical at pH 9.3, and the Michaelis constants were 51 mm for l-fucose and 160 mm for d-arabinose. Both of these activities decreased at the same rate with thermal inactivation at 45 and 50°C. All four pentitols inhibited two pentose isomerase activities competitively with same Ki values: 1.3–1.5 mm for d-arabitol, 2.2–2.7 mm for ribitol, 2.9–3.2 mm for l-arabitol, and 10–10.5 mm for xylitol. It is confirmed that the single enzyme is responsible for the isomerization of d-arabinose and l-fucose.     

Partial Purification and Characterization of Polyphenol Oxidase Isozymes in Banana Bud

Polyphenol oxidase was extracted from banana buds in the presences of Triton X-100, isoascorbate, and Polyclar AT, and two isozymes I and II have been separated and partially purified by chromatographies on Butyl Toyopearl 650 and DEAE-cellulose. I and II had different mobility in polyacrylamide gel electrophoresis with optimum pHs of 6.8 and 5.5, respectively. Both enzymes showed the apparent K_m values of 0.5 mM for dopamine with substrate inhibitions at its higher concentrations. I and II were inhibited competitively by NaCI with the K_i values of 140 mM and 40 mM, respectively. I and II have a high heat stability, and 88 and 95% of the initial activities were retained after 1-hr incubation at 70°C, respectively.     

Studies on the Metabolism of d-Amino Acid in Microorganisms

It is confirmed by a new method for the determination of d-glutamic acid, that Aerobacter strain A rapidly metabolizes d-glutamic acid, while it only shows feeble metabolic activity towards l-glutamic acid when it is grown on a dl-glutamate-K₂HPO₄ medium. A specific d-glutamic oxidase is demonstrated in the cell-free extracts of Aerobacter strain A. This enzyme seems to be different from d-glutamic-aspartic oxidase obtained from Aspergillus ustus by the authors, since the former has no activity towards d-aspartic acid.     

Distinct Physiological Roles of Two Membrane-Bound Dehydrogenases Responsible for D-Sorbitol Oxidation in Gluconobacter frateurii

Two different membrane-bound enzymes oxidizing D-sorbitol are found in Gluconobacter frateurii THD32: pyroloquinoline quinone-dependent glycerol dehydrogenase (PQQ-GLDH) and FAD-dependent D-sorbitol dehydrogenase (FAD-SLDH). In this study, FAD-SLDH appeared to be induced by L-sorbose. A mutant defective in both enzymes grew as well as the wild-type strain did, indicating that both enzymes are dispensable for growth on D-sorbitol. The strain defective in PQQ-GLDH exhibited delayed L-sorbose production, and lower accumulation of it, corresponding to decreased oxidase activity for D-sorbitol in spite of high D-sorbitol dehydrogenase activity, was observed. In the mutant strain defective in PQQ-GLDH, oxidase activity with D-sorbitol was much more resistant to cyanide, and the H⁺/O ratio was lower than in either the wild-type strain or the mutant strain defective in FAD-SLDH. These results suggest that PQQ-GLDH connects efficiently to cytochrome bo ₃ terminal oxidase and that it plays a major role in L-sorbose production. On the other hand, FAD-SLDH linked preferably to the cyanide-insensitive terminal oxidase, CIO.     

Microbial Production of Glucose Oxidase

Productivity of extracellular glucose oxidase was examined for various microorganisms and it was found in strains belonging to genus Penicillium except one species of Tallalomyces.

As the best glucose oxidase producer, Penicillium purpurogenum No. 778 was isolated from natural source. This microorganism produced 32,000 units per ml broth of glucose oxidase in a simple medium containing beet molasses, NaNO³ and KH²PO⁴ by submerged culture for 3 days. That value was about 10-times of that of Penicillium amagasakiense which has been known as an excellent glucose oxidase producer.

Culture conditions for glucose oxidase production were examined, which were extremely different among microbial species. In the case of Penicillium chrysogenum AJ 7007 and Penicillium purpurogenum No. 778, the effects of aeration and carbon sources were remarkably different from each other.

Penicillium purpurogenum No. 778 produces catalase sufficiently in a culture broth for glucose oxidase application in food industry.

Glucose oxidase was purified about 25-fold from culture supernatants of Penicillium purpurogenum No. 778, and some properties of the enzyme were examined. The optimum temperature and pH for the activity were 35°C and 5.0, respectively. The enzyme was stable at pH 5.0 to 7.0 when it was incubated at 40°C for 2 hr, while it was stable at temperature lower than 50°C when incubated at pH 5.6 for 15 min. The enzyme was specific for d-glucose and apparent Michaelis constant for d-glucose was 12.5 mm. The enzyme was inhibited by 1 mm of HgCl², CuSO⁴, NaHSO⁴ and phenylhydrazine, but not inhibited by 1 mm of p-hydroxy-mercuribenzoate, EDTA, hydroxylamine and dimedone. Four percents NaCl inhibited the activity about 50%, while the addition of ethanol (from 0 to 16%) increased oxygen uptake more than that expected from the peroxidase activity of catalase.     

Utilization of Amino Acids as a Sole Source of Nitrogen by Obligate Chemolithoautotroph Thiobacillus ferrooxidans

An obligate chemolithoautotroph, Thiobacillus ferrooxidans API 9–3, could utilize amino acids, other than glycine, methionine and phenylalanine, as a sole source of nitrogen. However, both the growth rate and growth yield were lower than those in Fe²⁺-NH4 -salts medium, suggesting that the ammonium ion was a superior nitrogen source for the strain compared to amino acids. Methionine and phenylalanine strongly inhibited the cell growth on Fe²⁺-NH4-salts medium at 10 mm. [¹⁴C]Glycine could not be taken up into the cells, and this meant the strain could not use glycine as a sole source of nitrogen. The uptake of [¹⁴C]leucine into the cells was dependent on the presence of Fe^{2 +}. When the strain was cultured on Fe^{2 +} - leucine (lOmm)-salts medium lacking an inorganic nitrogen source for 5 days at 30°C, 83.5% and 16.5% of the cellular carbon were derived from carbon dioxide and leucine, respectively, indicating that carbon dioxide was a superior carbon source for the bacterium compared to leucine. The ammonium ion did not inhibit the utilization of leucine for cellular carbon. Leucine uptake was markedly inhibited by inhibitors of protein synthesis, such as chloramphenicol (94.3% at 1 mm), streptomycin (57.2% at 5mm) and rifampin (77.2% at 0.1 mm), respectively. Carbon dioxide uptake was also completely inhibited by chloramphenicol at 4mm. These results suggest that the transport of both amino acids and carbon dioxide into the cells was dependent on protein synthesis.     

Food Composition and Emulsifying Activity of Lipoxygenase Deficient Mutant Soybeans

A newly found methanol-using bacterium, Mycobacterium gastri MB19, is a facultative methylotroph which assimilates methanol via the ribulose monophosphate pathway. 3-Hexulose phosphate synthase was purified from the organism and characterized. This enzyme was found to use glycolaldehyde (Km = 4.3 mm) and methylglyoxal (Km = 5.7 mm) as well as formaldehyde (Km = 1.4 mm) in the presence of d-ribulose 5-phosphate as an acceptor. The product of the condensation of glycolaldehyde with d-ribulose 5-phosphate was isolated by ion-exchange chromatography. The dephosphorylated product was tentatively identified as a heptulose with the molecular formula C₇H₁₄O₇ from its spectrophotometric properties and GC-MS results.