Effect of increasing methanol concentrations on physiology and cytology ofCandida boidinii

Concentration of methanol in the medium strongly affected not only the physiology but also the cytology ofCandida boidinii strain 2 cells in a methanol-limited chemostat at a constant dilution rateD 0.1/h and at low pH 3.0. The formation of large cubic peroxisomes with high alcohol oxidase (AO) activity observed at low methanol concentration (S ₀ 3 g/L) disappeared on increasing the methanol concentration in the inflow medium. The AO activity in the cells sharply decreased, followed by accumulation of riboflavin phosphate and residual methanol in the medium. The activity of catalase was relatively stable. At methanol concentrationS ₀>K _I (K ₁ equal to 12 g methanol per L), which included a substantial increase in methanol dissimilation, documented by higher formaldehyde and formate dehydrogenase activities and by lower yield coefficient on methanol, the yeast cells contained large lobe-shaped peroxisomes and a smaller number of larger mitochondria. The cells formed pseudomycelium with a thick septum between the mother and daughter cells.     

Effect of temperature on the physiology and cytology of the methylotrophic yeastCandida boidinii growing in methanol-limited chemostat

All deviations from optimum cultivation temperature affect strongly the physiology and morphology of cells ofCandida boidinii strain 2 during growth in methanol-limited chemostat. The optimum cultivation temperature was 28–30 °C at which maximum cell concentration and maximum cell yield (Y _S 0.4 g/g) were achieved. At suboptimal growth temperatures the cells were rich in cell protein, RNA, alcohol oxidase (AO) and in peroxisomes. Formation of cubic peroxisomes and a 20 % decrease of budding cells in the population was observed. At supraoptimal growth temperatures (>30 °C) a sharp decrease in AO activity was accompanied by degradation of peroxisomes in the cells. The culture forms pseudomycelium: at 34 °C the cells stop growing and they are washed out of the bioreactor.     

Direct observation of multiple protonation states in recombinant human purple acid phosphatase

To date, most spectroscopic studies on mammalian purple acid phosphatases (PAPs) have been performed at a single pH, typically pH 5. The catalytic activity of these enzymes is, however, pH dependent, with optimal pH values of 5.5–6.2 (depending on the form). For example, the pH optimum of PAPs isolated as single polypeptides is around pH 5.5, which is substantially lower that of proteolytically cleaved PAPs (ca. pH 6.2). In addition, the catalytic activity of single polypeptide PAPs at their optimal pH values is four to fivefold lower than that of the proteolytically cleaved enzymes. In order to elucidate the chemical basis for the pH dependence of these enzymes, the spectroscopic properties of both the single polypeptide and proteolytically cleaved forms of recombinant human PAP (recHPAP) and their complexes with inhibitory anions have been examined over the pH range 4 to 8. The EPR spectra of both forms of recHPAP are pH dependent and show the presence of three species: an inactive low pH form (pH<pK _a,1), an active form (pK _a,1<pH<pK _a,2), and an inactive high pH form (pH>pK _a,2). The pK _a,1 values observed by EPR for the single polypeptide and proteolytically cleaved forms are similar to those previously observed in kinetics studies. The spectroscopic properties of the enzyme–phosphate complex (which should mimic the enzyme–substrate complex), the enzyme–fluoride complex, and the enzyme–fluoride–phosphate complex (which should mimic the ternary enzyme–substrate–hydroxide complex) were also examined. EPR spectra show that phosphate binds to the diiron center of the proteolytically cleaved form of the enzyme, but not to that of the single polypeptide form. EPR spectra also show that fluoride binds only to the low pH form of the enzymes, in which it presumably replaces a coordinated water molecule. The binding of fluoride and phosphate to form a ternary complex appears to be cooperative.Electronic Supplementary Material Supplementary material is available for this article at     

CHARACTERISTICS OF PHOSPHORUS LIMITATION IN CHLAMYDOMONAS REINHARDTH (CHLOROPHYCEAE) AND ITS PALMELLOIDS1

Chlamydomonas reinhardtii Dang, was grown in a chemostat culture under phosphate limitation. The steady state concentration of phosphate was below the detection limit (< 1 μg P/L) in all runs. The cellular content of phosphorus (Q_p), polyphosphate (Q_pp) and chlorophyll a increased with increasing dilution rate, and the growth rate of the alga was described by Q_p as well as Q_pp in the Droop model. The ratio Q_pp/Q_p and the activity of alkaline phosphatase were maximal at high and low growth rates, respectively. Palmelloids of Chlamydomonas were found at high dilution rates (D > 0.12 h^?1) and became attached to the wall of the culture vessel. They differed from the vegetative stage in both chemical composition and growth rate. Their contents of phosphorus and chlorophyll a were low, as in the vegetative cells, which grew at a low growth rate, whereas the ration Q_pp/Q_p and the activity of alkaline phosphatase were comparable with those of fast growing vegetative cells. The growth rate of the palmelloids was 0.03 h^?1 whereas maximum growth rate (μ_m) for the vegetative cells was 0.21 h^?1.     

Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH

 Exogenous H₂/CO₂ and glucose were consumed simultaneously by Butyribacterium methylotrophicum when grown under glucose-limited conditions. CO₂ reduction to acetate was coupled to H₂ consumption. The addition of either H₂ or CO₂ to glucose batch fermentation resulted in an increase in cell density, hydrogenase (H₂-consuming and -producing) activities and fatty acid production by B. methylotrophicum as compared to when N₂ was the feed gas. Hydrogenase activities appeared to be tightly regulated and were produced at higher rates during the exponential phase when CO₂ was the feed gas as compared to H₂ or N₂. The increase in H₂-consuming activity and decrease in H₂-producing activity was correlated with an increase in butyrate synthesis. H₂-consuming and ferredoxin (Fd)–NAD reductase activities increased while H₂-producing and NADH–Fd reductase activities decreased in cells grown at pH 5.5 compared to those at pH 7.0. The molar ratio of butyrate/acetate was shifted from 0.35 at pH 7.0 to 1.22 at pH 5.5. The addition of exogenous H₂ did not decrease the butyrate/acetate ratio at pH 7.0 nor at pH 5.5. The results indicated that growth pH values regulated both hydrogenase and Fd–NAD oxidoreductase activities such that, at acid pH, more intermediary electron flow was directed towards butyrate synthesis than H₂ production. Received: 22 August 1995/Received revision: 18 December 1995/Accepted: 22 January 1996     

Aluminium toxicity and nodulation ofTrifolium repens

Summary Effects of aluminium on theTrifolium repens var Huia-Rhizobium trifolii strain HP3 symbiosis were studied using an axenic solution-culture system. With, 10 μM phosphate, 50 μM aluminium reduced or inhibited root elongation at pH<5.0, root hair formation at pH< 5.0–5.5, and Rhizobium multiplication in the rhizosphere and nodule formation at pH<6.0. In the absence of aluminium, root elongation and root hair formation were reduced at pH<4.3, and Rhizobium multiplication and nodule formation were inhibited at pH<5.0. Root hair formation was more sensitive to aluminium at pH<5 than was root elongation. No effect of aluminium on Rhizobium multiplication and nodule formation at pH<5 was detected because both were sensitive to pH alone. At pH 5.5 most of the aluminium changed immediately to a form which was susceptible to low-speed centrifugation, but which was detected by the aluminon method of analysis, and after 24 h a precipitate formed. the concentration of phosphate was reduced also, to approximately 1μM. Toxicity was overcome by either increasing the phosphate concentration from 10 to 50 μM, or by increasing the pH to 6.0 and the calcium, concentration to 1000μM.     

Distribution of cells in different stages of the cell cycle and some age-related physiological characteristics in dependence on the dilution rate in chemostat cultures ofCandida utilis

Position of cells in their cell cycle was determined microscopically in chemostat cultures ofCandida utilis. Proportion of cells in phase G₁ decreased in a linear manner from 86% to 58% with dilution rate. Proportion of cells in phase S increased in the same range ofD from 5.6 to 13.5% and in the (G₂+M) phase from 8.4 to 28.5%, again linearly. Differential centrifugation was used to separate chemostat cultures to mother and daughter cells. Analyses showed that, relative to mother cells, daughter cells contain 2.1–11.9% more protein and 25.5–34.6% more RNA in dry matter. Their mass is 34.4–5.6% lower and volume is 154–19% smaller.     

Chemostat studies on the assimilation of hexadecane by the yeastCandida tropicalis

Summary The yeastCandida tropicalis was grown in a chemostat with hexadecane as the sole carbon source. The influence of the dilution rate, oxygen and ammonium limitation on s, x, Y_s, Q_{O 2}, Q_{CO 2}, and Q_s was investigated. When the extracellular hexadecane concentration exceeded 120 mg l^–1 (at dilution rates close to D_c, at pO₂ below 2.54 kPa and at ammonium limited growth) Y_s decreased and Q_{O 2}, Q_{CO 2}, and Q_s increased. It was concluded that uncoupling of the mitochondrial respiratory chain occurred under these conditions. The Q_{O 2} was determined by two different methods: first in situ, with a gasanalyzer directly connected to the bioreactor to analyze the outcoming gas, and second, with a sample from the bioreactor transferred to a Clark-type oxygen electrode. When cell growth was not oxygen limited in the chemostat (pO₂ above 2.54 kPa), no apparent difference between the in situ and the dynamically determined Q_{O 2} was observed. In contrast, when cell growth was oxygen limited in the chemostat, the Q_{O 2} measured in the Clark-type oxygen electrode was remarkably higher than the in situ Q_{O 2}. This indicates that the electron transport chains are limited bythe oxidases, when the cells lack oxygen.     

A novel plant-associated thermotolerant alkaliphilic methylotroph of the genusParacoccus

Strain GB isolated from the maize rhizosphere is a gram-negative, aerobic, non-spore-forming, nonpigmented, nonmotile, chemolithotrophic, facultatively methylotrophic bacterium. Cells are cocci or short rods. The strain does not require vitamins. Optimum growth in a medium with methanol occurs at 38–42°C at pH 8.0–9.2. The doubling time is 12 h. In addition to methanol, the bacterium can grow on methylamine, dimethylformamide, acetone, thiosulfate + NaHCO₃, and in an atmosphere of H₂ + CO₂ + O₂. Methanol and methylamine are oxidized by the respective dehydrogenases to CO₂ via formaldehyde and formate, respectively. The CO₂ produced is assimilated via the ribulose bisphosphate pathway. Fatty acids are dominated by cyclopropanoic (58–61%), palmitic (24–26%), and octadecanoic (8–9%) acids. The main phospholipids are phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. The major ubiquinone is Q₁₀. The bacterial genome contains genes controlling the synthesis and secretion of cytokinins. The culture liquid exhibits cytokinin activity. The G+C content of DNA is 62.5 mol %, as determined from the DNA thermal denaturation temperature T_m). Strain GB shows a moderate degree of DNA-DNA homology (<40%) with the type representatives of the genusParacoccus. Based on the data obtained, the bacterium was classified as a new species of this genus, namedP. kondratievae.     

Rapid evolution of arginine deiminase for improved anti-tumor activity

Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas. Based on our preliminary results, it was noticed that the optimum pH of ADI from Pseudomonas plecoglossicida (PpADI) was 6.0, and less than 10% of the activity was retained at pH 7.4 (pH of human plasma). Additionally, the K _m value for wild-type ADI (WT-ADI) was 2.88 mM (pH 6.0), which is over 20 times of the serum arginine level (100–120 μM). These are two major limitations for PpADI as a potential anti-cancer drug. A highly sensitive and efficient high-throughput screening strategy based on a modified diacetylmonoxime–thiosemicarbazide method was established to isolate ADI mutants with higher activity and lower K _m under physiological pH. Three improved mutants was selected from 650 variants after one round of ep-PCR, among which mutant 314 (M314: A128T, H404R, I410L) exhibiting the highest activity. Interestingly, sequence alignment shows that three amino acid substitutes in M314 are coincident with corresponding residues in ADI from Mycoplasma arginini. The specific activity of M314 (9.02 U/mg) is over 20-fold higher than that of WT-ADI (0.44 U/mg) at pH 7.4, and the K _m value was reduced to 0.65 mM (pH 7.4). Noticeably, the pH optimum was shifted from 6.0 to 6.5 in M314. Homology model of M314 was constructed to understand the molecular basis of the improved enzymatic properties. This work could provide promising drug candidate for curing arginine-auxotrophic cancers.     

Effects of NH4 +, NO3 − and HCO3 − on apoplast pH in the outer cortex of root zones of maize, as measured by the fluorescence ratio of fluorescein boronic acid

A fluorimetric ratio technique was elaborated to measure apoplastic pH in the outer root cortex of maize (Zea mays L.) grown hydroponically. A newly synthesized fluorescent probe, fluorescein boronic acid (pK_a = 5.48), which covalently binds to the cell wall of the outer cell layers, was used. Under conditions of saturating ion concentrations the apoplastic pH was determined along the root axis ranging from 1 to 30 mm behind the root tip. Apoplastic pH was recorded for root segment areas (1 mm²), and pH values of high statistical significance were obtained. With an external solution of pH 5, the apoplastic pH was about pH 5.1 in the division zone, between pH 4.8 and 4.9 in the elongation region and about pH 4.9 in the root hair zone. At an external pH of 8.6, the difference between the external pH and the apoplastic pH was considerably more, with a pH of 5.2–5.3 in all root zones. Addition of 1 mM NH₄ ⁺ caused a small apoplastic pH decrease (0.05 of a pH unit) in all root zones. Apoplastic alkalization upon application of 6 mM NO₃ ⁻ was highest (0.3 of a pH unit) in the zone where root hairs emerge; in the division and early elongation zones, apoplastic pH increased only transiently. In the presence of 10 mM HCO₃ ⁻, NO₃ ⁻ elicited a higher and persistent alkalization (0.06–0.25 of a pH unit) in all root zones. Application of fusicoccin reduced apoplastic pH from 4.85 to 4.75 in the elongation zone, while inhibition of the H⁺-ATPase with vanadate alkalized the apoplast in the root hair zone from pH 5.4 to 5.6. The observed pH differences along the root axis upon differential N supply and application of HCO₃ ⁻ provide evidence that this new pH technique is a useful tool with which to measure apoplastic pH, and in future may permit measurements at microsites at the cell level by use of microscope imaging. Received: 26 August 1998 / Accepted: 4 May 1999     

Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha: a physiological study

We have studied methanol-utilization in a peroxisome-deficient (PER) mutant of Hansenula polymorphoa. In spite of the fact that in carbon-limited chemostat cultures under induced conditions the enzymes involved in methanol metabolism were present at wild-type (WT) levels, this mutant is unable to grow on methanol as a sole carbon and energy source. Addition of methanol to glucose-limited (S_R=12.5mM) chemostat cultures of the PER mutant only resulted in an increase in yield when small amounts were used (up to 22.5 mM). At increasing amounts however, a gradual decrease in cell density was observed which, at 80 mM methanol in the feed, had dropped below the original value of the glucose-limited culture. This reduction in yield was not observed when increasing amounts of formate instead of methanol were used as supplements for the glucose-limited mutant culture and also not in WT cells, used as control in these experiments. The effect of addition of methanol to a glucose-limited PER culture was also studied in the transient state during adaptation of the cells to methanol. The enzyme patterns obtained suggested that the ultimate decrease in yield observed at enhanced methanol concentrations was due to an inefficient methanolmetabolism as a consequence of the absence of peroxisomes. The absence of intact peroxisomes results in two major problems namely i) in H₂O₂-metabolism, which most probably is no longer mediated by catalase and ii) the inability of the cell to control the fluxes of formaldehyde, generated from methanol. The energetic consequences of this metabolism, compared to the WT situation with intact peroxisomes, are discussed.Abbreviations AO alcohol oxidase - DHAS dihydroxyacetone synthase - WT wild-type - PER peroxisome-deficient - GSH reduced glutathione - GSSG glutathione disulphide     

A novel low-temperature-active β-glucosidase from symbiotic <Emphasis Type="Italic">Serratia</Emphasis> sp. TN49 reveals four essential positions for substrate accommodation

To maximize the production of flag-tagged cartilage oligomeric matrix protein angiopoietin-1 (FCA1) from Chinese hamster ovary (CHO) cells, the effects of culture pH and temperature on cell growth and FCA1 production were investigated. Cells were cultivated in a bioreactor at different culture pH (6.7, 6.9, 7.2, and 7.5) and temperatures (33 and 37 °C). Lowering the culture temperature suppressed cell growth while allowing maintenance of high cell viability for a longer culture period. The specific FCA1 productivity (q _FCA1) was increased at low culture temperature. Accordingly, the highest FCA1 concentration was obtained at pH 7.2 and 33 °C, and was approximately 4.0-fold higher than that at pH 7.2 and 37 °C. However, aggregates and a monomeric form of FCA1, which are undesirable due to reduced biological activity or immunogenicity, were significant at pH 7.2 and 33 °C. It was also found that the expression pattern of FCA1 was affected more significantly by culture pH than by the culture temperature. FCA1 aggregation dramatically decreased at culture pH 7.5 regardless of the culture temperature. Furthermore, the monomeric form of FCA1 was not observed. Taken together, optimization of culture temperature and culture pH (33 °C and pH 7.5) significantly improves the production of biologically active FCA1 with tetrameric or pentameric forms from CHO cells.     

Growth kinetics and Pho84 phosphate transporter activity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under phosphate-limited conditions

The effect of phosphate (P _i ) concentration on the growth behavior of Saccharomyces cerevisiae strain CEN.PK113-5D in phosphate-limited batch and chemostat cultures was studied. The range of dilution rates used in the present study was 0.08–0.45 h⁻¹. The batch growth of yeast cells followed Monod relationship, but growth of the cells in phosphate-limited chemostat showed change in growth kinetics with increasing dilution rates. The difference in growth kinetics of the yeast cells in phosphate-limited chemostat for dilution rates below and above approximately 0.2 h⁻¹ has been discussed in terms of the batch growth kinetic data and the change in the metabolic activity of the yeast cells. Immunological detection of a C-terminally myc epitope-tagged Pho84 fusion protein indicated derepressive expression of the Pho84 high-affinity P _i transporter in the entire range of dilution rates employed in this study. Phosphate transport activity mediated by Pho84 transporter was highest at very low dilution rates, i.e. 0.08–0.1 h⁻¹, corresponding to conditions in which the amount of synthesized Pho84 was at its maximum.     

An obligate methylotrophic, methane-oxidizing Methylomicrobium species from a highly alkaline environment

A new, obligately methylotrophic, methane-oxidizing bacterium, strain AMO 1, was isolated from a mixed sample of sediments from five highly alkaline soda lakes (Kenya). Based on its cell ultrastructure and high activity of the hexulose-6-phosphate synthase, the new isolate belongs to the type I methanotrophs. It differed, however, from the known neutrophilic methanotrophs by the ability to grow and oxidize methane at high pH values. The bacterium grew optimally with methane at pH 9–10. The oxidation of methane, methanol, and formaldehyde was optimal at pH 10, and cells were still active up to pH 11. AMO 1 was able to oxidize ammonia to nitrite at high pH. A maximal production of nitrite from ammonia in batch cultures at pH 10 was observed with 10% of CH₄ in the gas phase when nitrate was present as nitrogen source. Washed cells of AMO 1 oxidized ammonia most actively at pH 10–10.5 in the presence of limiting amounts of methanol or CH₄. The bacterium was also capable of oxidizing organic sulfur compounds at high pH. Washed cells grown with methane exhibited high activity of CS₂ oxidation and low, but detectable, levels of DMS and DMDS oxidation. The GC content of AMO 1 was 50.9 mol%. It showed only weak DNA homology with the previously described alkaliphilic methanotroph "Methylobacter alcaliphilus" strain 20 Z and with the neutrophilic species of the genera Methylobacter and Methylomonas. According to the 16S rRNA gene sequence analysis, strain AMO 1 was most closely related to a neutrophilic methanotroph, Methylomicrobium pelagicum (98.2% sequence similarity), within the gamma-Proteobacteria. Received: July 26, 1999 / Accepted: January 4, 2000     

Influence of increasing methanol concentrations on alcohol oxidase activity inCandida boidinii

In methanol-limited chemostat cultures methanol concentration >K _I for growth decreased sharply the alcohol oxidase activity in yeast cells. This effect was accompanied by accumulation of riboflavin phosphate in the medium. Purified alcohol oxidase showed higherK _m for methanol, change in absorbance maxima in the riboflavin area and chemical modification of enzyme structure discovered by means of partial proteolysis.     

Studies on the accumulation of heavy metal elements in biological systems

Summary Cells of a Daucus carota suspension culture were entrapped in a matrix of calcium alginate. The immobilised cells, incubated in a buffer mixture of sucrose, nitrate, KCl, CaCl₂, 2-(N-morpholino)-ethane sulphonic acid at pH 5.5, hydroxylated digitoxigenin. When compared under the same incubation conditions, freely suspended cells biotransformed digitoxigenin at a faster rate. Periplogenin formation was maximal at pH 5.3 and temperatures of 26°–34°C. The hydroxylase activity of the entrapped cells adapted to the presence of 20 mM CaCl₂ over a 12 day incubation. The diffusion barrier established on entrapment of the cells could not be overcome by addition of detergents or methanol. Controlled addition of chloroform (at 1/4 and 1/2 saturation) did stimulate hydroxylation of digitoxigenin without adversely affecting cell viability. The rate of hydroxylation of digitoxigenin was linear over an immobilised cell concentration of 0–7 mg dry weight and a digitoxigenin concentration of 0–20 mg/L. Five consecutive batch bioconversions at a rate greater than 60% could be achieved before the biocatalyst was inactivated. The results are discussed in relation to improving the hydroxylation reaction by immobilised D. carota and other reactions performed by immobilised plant cells.     

Inducing autoflocculation in the diatom <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> through CO<Subscript>2</Subscript> regulation

The effect of pH on flocculation was studied using the diatom Phaeodactylum tricornutum and the green algae Scenedesmus cf. obliquus as surrogate species. There was a distinct, species-specific threshold of pH where flocculation started. P. tricornutum started to flocculate at pH 10.5 and S. cf. obliquus at pH 11.3. Above this threshold, settling rates up to 360 cm h⁻¹ were observed for P. tricornutum and the concentrating factor was up to 60-fold. The combined effect of pH, turbulence, and cell density on flocculation of P. tricornutum was additionally studied in a factorial 5³-design experiment. pH was the most important factor affecting flocculation, but at the pH threshold (pH 10.5), the concentrating factor was increased by increasing cell density and turbulence. Algae increases the pH during photosynthesis, and the P. tricornutum and S. cf. obliquus cultures increased the pH to a maximum of 10.8 and 9.5, respectively, after discontinuing the CO₂ supply. For P. tricornutum, this was above the flocculation threshold, and rapid settling of this species due to increased pH was observed in a matter of hours after the CO₂ supply was turned off. This could be used as a simple, low-cost, initial dewatering step for this species.     

High rates of microbial sulphate reduction in a mesophilic ethanol-fed expanded-granular-sludge-blanket reactor

In a mesophilic (30–35 °C), sulphidogenic, ethanol-fed expanded-granular-sludge-blanket reactor, sulphate, at loading rates of up to 10.0–12.0 g Sl⁻¹␣day⁻¹, was removed with an average efficiency of more than 80%. The pH was between 7.7 and 8.3 and the maximal total dissolved sulphide concentration was up to 20 mM S (650 mg S/l). The alkaline pH was maintained by either a pH-control unit with sodium hydroxide or by stripping part of the sulphide and CO₂ from the recycle with nitrogen gas. The superficial upstream liquid velocity (v _up) was 3.0–4.5 m/h. The ratio of ethanol to sulphur was near stoichiometry. At alkaline pH, the activity of the acetotrophic sulphate-reducing bacteria, growing on acetate, was strongly enhanced, whereas at pH below 7.7 the acetotrophic sulphate-reducing bacteria were inhibited by aqueous H₂S. With regard to the removal efficiency and operational stability, external stripping with N₂ and pH control were equally successful. Received: 2 December 1996 / Received revision: 13 March 1997 / Accepted: 15 March 1997     

Pyranose 2-oxidase from Phanerochaete chrysosporium– further biochemical characterisation

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (k _cat) of 111.0 s⁻¹ and an affinity constant (K _m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k _cat/K _m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k _cat decreased to 60.9 s⁻¹ and the K _m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl₂, AgNO₃ and ZnCl₂, and 50% by lead acetate, CuCl₂ and MnCl₂. Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 1996