d-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637

Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. d-Amino acid dehydrogenase is a flavoenzyme that digests free neutral d-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 d-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial d-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to d-proline. The enzyme mediated electron transport from d-proline to coenzyme Q₁, thus distinguishing it from d-amino acid oxidase. The apparent K _m and V _max values were 40.2 mM and 25.0 μmol min⁻¹ mg⁻¹, respectively, for dehydrogenation of d-proline, and were 8.2 μM and 12.3 μmol min⁻¹ mg⁻¹, respectively, for reduction of Q₁. The respective pH and temperature optima were 8.0 and 37°C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian d-amino acid oxidase than other bacterial d-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from d-proline to a c-type cytochrome was suggested spectrophotometrically.     

Studies on the inactivation of the flavoproteind-amino acid oxidase fromTrigonopsis variabilis

Inactivation ofd-amino acid oxidase occured by different mechanisms. The enzyme showed a rapid loss of activity in the presence of micromolar amounts of Cu²⁺ and Hg²⁺. It was also sensitive to oxidative inactivation by Fe²⁺ and H₂O₂ when both reagents were added in millimolar amounts. When oxidatively inactivatedd-amino acid oxidase and a corresponding non-treated control were modified with the sulfhydryl-modifying, fluorescent reagent monobromobimane and subsequently digested with endoproteinase Glu-C, Cys-298 was identified to be a target for oxidative modification according to differences in the known peptide profile of fluorescence intensity. Another reason for the observed loss of enzyme activity in crude extracts was the specific proteolytic digestion ofd-amino acid oxidase, which was dependent on the growth phase of the cells used. This cleavage was catalyzed by a serine-type proteinase and was the introductory step for the further complete degradation of the enzyme. In addition, a coenriched 50-kDa protein, identified as NADPH-specific glutamate dehydrogenase, significantly decreased the stability of thed-amino acid oxidase activity. Treatment of apo-d-amino acid oxidase fromT. variabilis with monobromobimane resulted in a significantly increased fluorescence of two peptides, neither of which contained any cysteine residue. Thus, an involvement of cysteine residues in binding the FAD coenzyme should be excluded.     

Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001

α-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca²⁺ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The K _m, V _max and k _cat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min^–1 and 117,000 · min^–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far. Received: 24 May 1999 / Accepted: 7 October 1999     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.     

Purification and characterization of dihydroorotase fromPseudomonas putida

Dihydroorotase was purified to homogeneity fromPseudomonas putida. The relative molecular mass of the native enzyme was 82 kDa and the enzyme consisted of two identical subunits with a relative molecular mass of 41 kDa. The enzyme only hydrolyzed dihydro-l-orotate and its methyl ester, and the reactions were reversible. The apparentK _m andV _max values for dihydro-l-orotate hydrolysis (at pH 7.4) were 0.081 mM and 18 μmol min⁻¹ mg⁻¹, respectively; and those forN-carbamoyl-dl-aspartate (at pH 6.0) were 2.2 mM and 68 μmol min⁻¹ mg⁻¹, respectively. The enzyme was inhibited by metal ion chelators and activated by Zn²⁺. However, excessive Zn²⁺ was inhibitory. The enzyme was inhibited by sulfhydryl reagents, and competitively inhibited byN-carbamoylamino acids such asN-carbamoylglycine, with aK _i value of 2.7 mM. The enzyme was also inhibited noncompetitively by pyrimidine-metabolism intermediates such as dihydrouracil and orotate, with aK _i value of 3.4 and 0.75 mM, respectively, suggesting that the enzyme activity is regulated by pyrimidine-metabolism intermediates and that dihydroorotase plays a role in the control of pyrimidine biosynthesis.     

Pyranose 2-dehydrogenase, a novel sugar oxidoreductase from the basidiomycete fungus Agaricus bisporus

A novel C-2-specific sugar oxidoreductase, tentatively designated as pyranose 2-dehydrogenase, was purified 68-fold to apparent homogeneity (16.4 U/mg protein) from the mycelia of Agaricus bisporus, which expressed maximum activity of the enzyme during idiophasic growth in liquid media. Using 1,4-benzoquinone as an electron acceptor, pyranose 2-dehydrogenase oxidized d-glucose to d-arabino-2-hexosulose (2-dehydroglucose, 2-ketoglucose), which was identified spectroscopically through its N,N-diphenylhydrazone. The enzyme is highly nonspecific. d-,l-Arabinose, d-ribose, d-xylose, d-galactose, and several oligosaccharides and glycopyranosides were all converted to the corresponding 2-aldoketoses (aldosuloses) as indicated by TLC. d-Glucono-1,5-lactone, d-arabino-2-hexosulose, and l-sorbose were also oxidized at significant rates. UV/VIS spectrum of the native enzyme (λ_max 274, 362, and 465 nm) was consistent with a flavin prosthetic group. In contrast to oligomeric intracellular pyranose 2-oxidase (EC 1.1.3.10), pyranose 2-dehydrogenase is a monomeric glycoprotein (pI 4.2) incapable of reducing O₂ to H₂O₂ (> 5 × 10⁴-fold lower rate using a standard pyranose oxidase assay); pyranose 2-dehydrogenase is actively secreted into the extracellular fluid (up to 0.5 U/ml culture filtrate). The dehydrogenase has a native molecular mass of ∼79 kDa as determined by gel filtration; its subunit molecular mass is ∼75 kDa as estimated by SDS-PAGE. Two pH optima of the enzyme were found, one alkaline at pH 9 (phosphate buffer) and the other acidic at pH 4 (acetate buffer). Ag⁺, Hg²⁺, Cu²⁺, and CN^– (10 mM) were inhibitory, while 50 mM acetate had an activating effect. Received: 19 August 1996 / Accepted: 21 November 1996     

Purification and properties of the membrane-bound NADH oxidase of a facultatively anaerobic alkaliphile

A membrane-bound NADH oxidase of an anaerobic alkaliphile, M-12 (a strain of Amphibacillus sp.), was solubilized with decanoyl N-methylglucamide and purified by chromatography on DEAE-Sepharose and hydroxyapatite. The purified enzyme appears to consist of a single polypeptide component with an apparent molecular mass of 56 kDa. The enzyme catalyzed the oxidation of NADH with the formation of H₂O₂ and exhibited a specific activity of 46 μmol NADH min^–1 (mg protein)^–1. NADPH did not serve as a substrate for the enzyme. The K _m for NADH was estimated to be 0.05 mM. The enzyme exhibited a pH dependence for activity, with a pH optimum at approximately 9.5. The enzyme required a high concentration of salt and exhibited maximum activity in the presence of 600 mM NaCl. Received: 3 August 1998 / Accepted: 23 December 1998     

Characterization of a novel ginsenoside-hydrolyzing α-l-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T

The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054^T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K _m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V _max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min⁻¹ mg⁻¹ of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd.     

Characterization of an acid-labile, thermostable β-glycosidase from Thermoplasma acidophilum

A recombinant putative β-galactosidase from Thermoplasma acidophilum was purified as a single 57 kDa band of 82 U mg⁻¹. The molecular mass of the native enzyme was 114 kDa as a dimer. Maximum activity was observed at pH 6.0 and 90°C. The enzyme was unstable below pH 6.0: at pH 6 its half-life at 75°C was 28 days but at pH 4.5 was only 13 h. Catalytic efficiencies decreased as p-nitrophenyl(pNP)-β-d-fucopyranoside (1067) > pNP-β-d-glucopyranoside (381) > pNP-β-d-galactopyranoside (18) > pNP-β-d-mannopyranoside (11 s⁻¹ mM⁻¹), indicating that the enzyme was a β-glycosidase.     

Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases

We cloned and sequenced a xylanase gene named xylD from the acidophilic fungus Bispora sp. MEY-1 and expressed the gene in Pichia pastoris. The 1,422-bp full-length complementary DNA fragment encoded a 457-amino acid xylanase with a calculated molecular mass of 49.8 kDa. The mature protein of XYLD showed high sequence similarity to both glycosyl hydrolase (GH) families 5 and 30 but was more homologous to members of GH 30 based on phylogenetic analysis. XYLD shared the highest identity (49.9%) with a putative endo-1,6-β-d-glucanase from Talaromyces stipitatus and exhibited 21.1% identity and 34.3% similarity to the well-characterized GH family 5 xylanase from Erwinia chrysanthemi. Purified recombinant XYLD showed maximal activity at pH 3.0 and 60 °C, maintained more than 60% of maximal activity when assayed at pH 1.5–4.0, and had good thermal stability at 60 °C and remained stable at pH 1.0–6.0. The enzyme activity was enhanced in the presence of Ni²⁺ and β-mercaptoethanol and inhibited by some metal irons (Hg²⁺, Cu²⁺, Pb²⁺, Mn²⁺, Li⁺, and Fe³⁺) and sodium dodecyl sulfate. The specific activity of XYLD for beechwood xylan, birchwood xylan, 4-O-methyl-d-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020, and 1,429 U mg⁻¹, respectively. The apparent K _m and V _max values for beechwood xylan were 5.6 mg ml⁻¹ and 3,622 μmol min⁻¹ mg⁻¹, respectively. The hydrolysis products of different xylans were mainly xylose and xylobiose.     

Characterization of a recombinant cellobiose 2-epimerase from <Emphasis Type="BoldItalic">Caldicellulosiruptor saccharolyticus</Emphasis> and its application in the production of mannose from glucose

A putative N-acyl-d-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min^–1 mg^–1 for d-glucose with a 47-kDa monomer. The epimerization activity for d-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35, 18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as d-glucose, d-xylose, l-altrose, l-idose, and l-arabinose, to their C2 epimers, such as d-mannose, d-lyxose, l-allose, l-gulose, and l-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for mannose among monosaccharides. Thus, mannose at 75 g l^–1 and fructose at 47.5 g l^–1 were produced from 500 g l^–1 glucose at pH 7.5 and 75°C over 3 h by the enzyme.     

Engineering lower inhibitor affinities in β-<Emphasis Type="SmallCaps">d</Emphasis>-xylosidase of <Emphasis Type="Italic">Selenomonas ruminantium</Emphasis> by site-directed mutagenesis of Trp145

β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-β-d-xylooligosaccharides to d-xylose. One property that could use improvement is its relatively high affinities for d-glucose and d-xylose (K _i ~ 10 mM), which would impede its performance as a catalyst in the saccharification of lignocellulosic biomass for the production of biofuels and other value-added products. Previously, we discovered that the W145G variant expresses K _i ^d-glucose and K _i ^d-xylose twofold and threefold those of the wild-type enzyme. However, in comparison to the wild type, the variant expresses 11% lower k _cat ^d-xylobiose and much lower stabilities to temperature and pH. Here, we performed saturation mutagenesis of W145 and discovered that the variants express K _i values that are 1.5–2.7-fold (d-glucose) and 1.9–4.6-fold (d-xylose) those of wild-type enzyme. W145F, W145L, and W145Y express good stability and, respectively, 11, 6, and 1% higher k _cat ^d-xylobiose than that of the wild type. At 0.1 M d-xylobiose and 0.1 M d-xylose, kinetic parameters indicate that W145F, W145L, and W145Y catalytic activities are respectively 46, 71, and 48% greater than that of the wild-type enzyme.     

Overproduction and characterization of a recombinant <Emphasis Type="SmallCaps">D</Emphasis>-amino acid oxidase from <Emphasis Type="Italic">Arthrobacter protophormiae</Emphasis>

A screening of soil samples for d-amino acid oxidase (d-AAO) activity led to the isolation and identification of the gram-positive bacterium Arthrobacter protophormiae. After purification of the wild-type d-AAO, the gene sequence was determined and designated dao. An alignment of the deduced primary structure with eukaryotic d-AAOs and d-aspartate oxidases showed that the d-AAO from A. protophormiae contains five of six conserved regions; the C-terminal type 1 peroxisomal targeting signal that is typical for d-AAOs from eukaryotic origin is missing. The dao gene was cloned and expressed in Escherichia coli. The purified recombinant d-AAO had a specific activity of 180 U mg protein⁻¹ for d-methionine and was slightly inhibited in the presence of l-methionine. Mainly, basic and hydrophobic d-amino acids were oxidized by the strictly enantioselective enzyme. After a high cell density fermentation, 2.29 × 10⁶ U of d-AAO were obtained from 15 l of fermentation broth.     

A highly stable Cu/Zn superoxide dismutase from <Emphasis Type="Italic">Withania somnifera</Emphasis> plant: gene cloning,expression and characterization of the recombinant protein

A gene from Withania somnifera (winter cherry), encoding a highly stable chloroplastic Cu/Zn superoxide dismutase (SOD), was cloned and expressed in Escherichia coli. The recombinant enzyme (specific activity of ~4,200 U mg⁻¹) was purified and characterized. It retained ~90 and ~70% residual activities after 1 h at 80 and 95°C, respectively. At 95°C, thermal inactivation rate constant (K _d) of the enzyme was 2.46 × 10⁻³ min⁻¹ and half-life of heat inactivation was 4.68 h. The enzyme was stable against a broad pH range (2.5–11.0). It also showed a high degree of resistance to detergent, ethanol and protease digestion. This recombinant Cu/Zn SOD could therefore have useful applications.     

A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris

Using degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR, a 1,347-bp full-length complementary DNA fragment encompassing the gene man5A, which encodes a 429-amino acid β-mannanase with a calculated mass of 46.8 kDa, was cloned from acidophilic Bispora sp. MEY-1. The deduced amino acid sequence (catalytic domain) displayed highest identity (54.1%) with the Emericella nidulans endo-β-1,4-d-mannanase, a member of the glycoside hydrolase family 5. Recombinant MAN5A was overexpressed in Pichia pastoris, and its activity in the culture medium reached 500 U ml⁻¹. The enzyme was acidophilic, with highest activity at pH 1.0–1.5, lower than any known mannanases, and optimal temperature for activity was 65°C. MAN5A had good pH adaptability, excellent thermal and pH stability, and high resistance to both pepsin and trypsin. The specific activity, K _m, and V _max for locust bean gum substrate was 3,373 U mg⁻¹, 1.56 mg ml⁻¹, and 6,587.6 μmol min⁻¹ mg⁻¹, respectively. The enzymatic activity was not significantly affected by ions such as Ca²⁺, Cr³⁺, Co²⁺, Zn²⁺, Na⁺, K⁺, and Mg²⁺ and enhanced by Ni²⁺, Fe³⁺, Mn²⁺ and Ag⁺. These favorable properties make MAN5A a potential candidate for use in various industrial applications.     

Purification and properties of the cell-associated beta-xylosidase from Aureobasidium

β-Xylosidase was extracted from Aureobasidium sp. ATCC 20524 and purified to homogeneity. The molecular mass was estimated at 411 kDa. The enzyme contained 15.3% (w/w) carbohydrate. The optimum pH and temperature were pH 3.5 and 80°C, respectively. The enzyme was stable at pH 3.5–9 after 3 h and at 80°C after 15 min. The Michaelis constant (K _m) and maximum velocity (V _max) toward p-nitrophenyl-β-D-xyloside were 2.0 mmol l⁻¹ and 0.94 mmol min⁻¹ mg⁻¹ protein, respectively. The enzyme was inhibited strongly by mercury, lead, and copper ions. Journal of Industrial Microbiology & Biotechnology (2001) 26, 276–279. Received 02 August 2000/ Accepted in revised form 15 December 2000     

The role of an NAD-independent lactate dehydrogenase and acetate in the utilization of lactate byClostridium acetobutylicum strain P262

Clostridium acetobutylicum strain P262 utilized lactate at a rapid rate [600 nmol min^–1 (mg protein)^–1], but lactate could not serve as the sole energy source. When acetate was provided as a co-substrate, the growth rate was 0.05 h^–1. Butyrate, carbon dioxide and hydrogen were the end products of lactate and acetate utilization, and the stoichiometry was 1 lactate + 0.4 acetate → 0.7 butyrate + 0.6 H₂ + 1 CO₂. Lactate-grown cells had twofold lower hydrogenase than glucose-grown cells, and the lactate-grown cells used acetate as an alternative electron acceptor. The cells had a poor affinity for lactate (K_s = 1.1 mM), and there was no evidence for active transport. Lactate utilization was catabolyzed by an inducible NAD-independent lactate dehydrogenase (iLDH) that had a pH optimum of 7.5. The iLDH was fivefold more active with d-lactate than l-lactate, and the K _m for d-lactate was 3.2 mM. Lactate-grown cells had little butyraldehyde dehydrogenase activity, and this defect did not allow the conversion of lactate to butanol. Received: 17 October 1994 / Accepted: 30 January 1995     

Characterization of a tryptophan 6-halogenase from <Emphasis Type="Italic">Streptomyces toxytricini</Emphasis>

Tryptophan (Trp) halogenases are found in various bacteria and play an important role in natural product biosynthesis. Analysis of the genome of Streptomyces toxytricini NRRL 15443 revealed an ORF, stth, encoding a putative Trp halogenase within a non-ribosomal peptide synthetase gene cluster. This gene was cloned into pET28a and functionally overexpressed in Escherichia coli. The enzyme halogenated both l- and d-Trp to yield the corresponding 6-chlorinated derivatives. The optimum activity was at 40°C and pH 6 giving k _cat /K _M value of STTH of 72,000 min⁻¹ M⁻¹. The enzyme also used bromide to yield 6-bromo-Trp.     

Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst

The glyA gene encoding a serine hydroxymethyl transferase (SHMT) with threonine aldolase activity was isolated from Streptococcus thermophilus YKA-184 chromosomal DNA. This aldolase is a pyridoxal 5′-phosphate-dependent enzyme that stereospecifically catalyzes the interconversion of l-threonine to glycine and acetaldehyde. The enzyme was overexpressed in Escherichia coli M15 as a recombinant protein of 45 kDa with a His6-tag at its N-terminus. The recombinant enzyme was purified to homogeneity by a single chromatographic step using Ni-nitrilotriacetic acid affinity, obtaining a high activity-recovery yield (83%). Lyophilized and precipitated enzymes were stable at least for 10 weeks when stored at −20°C and 4°C. It was observed that the K _m for l-allo-threonine was 38-fold higher than that for l-threonine, suggesting this enzyme can be classified as a specific l-allo-threonine aldolase. The optimum pH range of threonine aldolase activity for the recombinant SHMT was pH 6–7. When tested for aldol addition reactions with non-natural aldehydes, such as benzyloxyacetaldehyde and (R)-N-Cbz-alaninal, two possible β-hydroxy-α-amino acid diastereoisomers were produced, but with moderate stereospecificity. The enzyme showed potential as a biocatalyst for the stereoselective synthesis of β-hydroxy-α-amino acids.     

Spectrophotometric kinetic study and analytical implications of the glucose oxidase-catalyzed reduction of [MIII(LL)2Cl2]+ complexes by d-glucose (M=Os and Ru, LL=2,2′-bipyridine and 1,10-phenanthroline type ligands)

 Glucose oxidase-catalyzed reduction of cis[M^III (LL)₂Cl₂]⁺ (M=Os and Ru) complexes to cis[M^II (LL)₂Cl₂] (LL=2,2′-bipyridine and 1,10-phenanthroline type ligands) by d-glucose is a first-order process in the complex and the enzyme in aqueous buffered solution. The reaction follows MichaelisMenten kinetics in d-glucose and the rate is independent of d-glucose concentration above 0.03 M. The reactivity decreases in the series [Ru(bpy)₂Cl₂]⁺ > [Os(phen)₂Cl₂]⁺ > [Os(4,4′-Me₂bpy)₂Cl₂]⁺ > [Os(4,7Me₂phen)₂Cl₂]⁺. The measured second-order rate constant for the oxidation of reduced glucose oxidase by [Os(phen)₂Cl₂]⁺ in air equals 1.2×10⁵ M^–1 s^–1 at pH 6.7, [d-glucose] 0.05 M, and 25  °C, which is ca. 20% less than that when the reaction solutions are purged with argon. In the case of [Ru(bpy)₂Cl₂]⁺ the rate constant equals 1.8×10⁵ M^–1 s^–1 under similar conditions in air, showing higher reactivity of Ru complexes compared with Os ones. The reduction is pH-dependent with a maximum around 7. Added for solubilization of poorly soluble metal complexes, surfactants decrease the rates of the enzymatic reaction. The retardation effect increases in the series: cetyltrimethylammonium bromide < Triton X-100 < sodium dodecyl sulfate, i.e. on going from positively charged to neutral and then to negatively charged surfactants. The behavior of the Os^III and Ru^III complexes toward reduced glucose oxidase contrasts to that of recently studied ferricenium cations. As opposed to the latter, the former do not show kinetically meaningful binding with the enzyme, and the Michaelis kinetics typical of the ferricenium case is not realized for the Os^III, and Ru^III species. The systems Os^III- or Ru^III-glucose oxidase are convenient for routine "one pot" spectrophotometric monitoring of the d-glucose content in samples, since the metal reduction to M^II is accompanied by a strong increase in absorbance in the visible spectral region. Received: 1 July 1998 / Accepted: 13 January 1999