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.     

Regulation of flavin biosynthesis in the methylotrophic yeast Hansenula polymorpha

Under various conditions of growth of the methylotrophic yeast Hansenula polymorpha, a tight correlation was observed between the levels of flavin adenine dinucleotide (FAD)-containing alcohol oxidase, and the levels of intracellularly bound FAD and flavin biosynthetic enzymes. Adaptation of the organism to changes in the physiological requirement for FAD was by adjustment of the levels of the enzymes catalyzing the last three steps in flavin biosynthesis, riboflavin synthetase, riboflavin kinase and flavin mononucleotide adenylyltransferase. The regulation of the synthesis of the latter enzymes in relation to that of alcohol oxidase synthesis was studied in experiments involving addition of glucose to cells of H. polymorpha growing on methanol in batch cultures or in carbon-limited continuous cultures. This resulted not only in selective inactivation of alcohol oxidase and release of FAD, as previously reported, but invariably also in repression/inactivation of the flavin biosynthetic enzymes. In further experiments involving addition of FAD to the same type of cultures it became clear that inactivation of the latter enzymes was not caused directly by glucose, but rather by free FAD that accumulated intracellularly. In these experiments no repression or inactivation of alcohol oxidase occurred and it is therefore concluded that the synthesis of this enzyme and the flavin biosynthetic enzymes is under separate control, the former by glucose (and possibly methanol) and the latter by intracellular levels of free FAD.Abbreviations FAD Flavin adenine dinucleotide - FMN riboflavin-5-phosphate; flavin mononucleotide - Rf riboflavin     

Oxidation of Methanol and Formaldehyde by a System Containing Alcohol Oxidase and Catalase Purified from Candida sp. N-16

The oxidation of methanol and formaldehyde was investigated by using some combination systems of alcohol oxidase, catalase, which were purified from Candida N-16, and hydrogen peroxide. The activity of alcohol oxidase was irreversibly inhibited when the enzyme was incubated with 2.5 mm hydrogen peroxide for 15 min. However, the oxidation of methanol to formaldehyde by alcohol oxidase in the presence of catalase was extremely promoted by the addition of 30 mm hydrogen peroxide. Alcohol oxidase could oxidize not only methanol but also formaldehyde as follows: HCHO + 0₂ + H₂O→HCOOH + H₂O₂. The formaldehyde oxidizing activity was inhibited by hydrogen peroxide. The system containing alcohol oxidase and catalase appears to be the entity of the oxygen-dependent oxidation system of formaldehyde previously found in the cell-free extract of the yeast.     

An Efficient Method for Separating Ascospores from Sporulating Cultures in Saccharomyces cerevisiae by Hydrostatic Pressure

A new oxidative reaction of ethylene glycol was found with two alcohol oxidases from methanol yeast, Candida sp. and Pichia pastoris. Both alcohol oxidases oxidized ethylene glycol to glyoxal via glycolaldehyde. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Candida alcohol oxidase were around 8.5 and 5.5, respectively, and their apparent K_ms were 2.96 m and 28.6 mm, respectively. The optimum temperature was 40°C at pH 7.0. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Pichia alcohol oxidase were around 8.0 and 6.0, respectively, and their optimum temperatures were 50 and 45°C, respectively, at pH 7.0. The apparent K_m for glycolaldehyde was found to be 83.3 mm. For the accumulation of glyoxal, addition of catalase was effective, and a higher amount of glyoxal was obtained at a much lower temperature than the optimum for the alcohol oxidase. When 0.1 m ethylene glycol and glycolaldehyde were incubated with 80 units of the Pichia enzyme at 10°C, both substrates were almost completely converted to glyoxal after 10 and 3h of incubation, respectively.     

Oxidation of Methanol,Formaldehyde and Formate by a Candida Species

1. The oxidation of methanol to carbon dioxide by Candida N–16 grown on methanol was investigated. The presence of enzymes which catalyze the following reaction was found in the cell-free extract of the yeast employed; CH₃OH→HCHO→HCOOH→CO₂. 2. Methanol was oxidized to formaldehyde by an alcohol oxidase. The reaction was as follows; CH₃OH+O₂→HCHO+H₂O₂. The alcohol oxidase was crystallized after purification by ammonium sulfate-precipitation and column chromatography using DEAE-Sephadex A-50. A prosthetic group of the enzyme was proved to be FAD. The enzyme possessed a broad specificity for alcohols such as methanol, ethanol, n-propanol, n-butanol and n-amylalcohol. The enzyme was inducibly formed only by the addition of methanol. 3. The oxidation of formaldehyde to formate was catalyzed by a NAD-linked dehydrogenase dependent on GSH. 4. Formate was oxidized by a NAD-linked dehydrogenase. 5. Catalase was also found in the extract, and methanol was chemically oxidized by the reaction of catalase and hydrogen peroxide which was generated by the alcohol oxidase system. 6. The oxidation pathway from methanol to carbon dioxide was also found in other methanol-utilizing yeasts such as Candida N-17, Saccharomyces H-1 and Torulopsis M-1.     

Growth of Hansenula polymorpha in a methanol-limited chemostat

Hansenula polymorpha has been grown in a methanol-limited continuous culture at a variety of dilution rates. Cell suspensions of the yeast grown at a dilution rate of 0.16 h^-1 showed a maximal capacity to oxidize excess methanol (QO ₂ ^max ) which was 1.6 times higher than the rate required to sustain the growth rate (Q _O2). When the dilution rate was decreased to 0.03 h^-1, QO ₂ ^max of the cells increased to a value of more than 20 times that of Q _O2. The enzymatic basis for this tremendous overcapacity for the oxidation of excess methanol at low growth rates was found to be the methanol oxidase content of the cells. The level of this enzyme increased from 7% to approximately 20% of the soluble protein when the growth rate was decreased from 0.16 to 0.03 h^-1. These results were explained on the basis of the poor affinity of methanol oxidase for its substrates. Methanol oxidase purified from Hansenula polymorpha showed an apparent K _mfor methanol of 1.3 mM in air saturated reaction mixtures and the apparent K _mof the enzyme for oxygen was 0.4 mM at a methanol concentration of 100 mM.The involvement of an oxygen dependent methanol oxidase in the dissimilation of methanol in Hansenula polymorpha was also reflected in the growth yield of the organism. The maximal yield of the yeast was found to be low (0.38 g cells/g methanol). This was not due to a very high maintenance energy requirement which was estimated to be 17 mg methanol/g cells x h.     

Microbial oxidation of methanol: Properties of crystallized alcohol oxidase from a yeast, Pichia sp

Alcohol oxidase (alcohol:oxygen oxidoreductase) was crystallized from a methanolgrown yeast, Pichia sp. The crystalline enzyme is homogenous as judged from polyacrylamide gel electrophoresis. Alcohol oxidase catalyzed the oxidation of short-chain primary alcohols (C₁ to C₆), substituted primary alcohols (2-chloroethanol, 3-chloro-1-propanol, 4-chlorobutanol, isobutanol), and formaldehyde. The general reaction with an oxidizable substrate is as follows: Primary alcohol + O₂ → aldehyde + H₂O₂ Formaldehyde + O₂ → formate + H₂O₂. Secondary alcohols, tertiary alcohols, cyclic alcohols, aromatic alcohols, and aldehydes (except formaldehyde) were not oxidized. The K_m values for methanol and formaldehyde are 0.5 and 3.5 mm, respectively. The stoichiometry of substrate oxidized (alcohol or formaldehyde), oxygen consumed, and product formed (aldehyde or formate) is 1:1:1. The purified enzyme has a molecular weight of 300,000 as determined by gel filtration and a subunit size of 76,000 as determined by sodium dodecyl sulfate-gel electrophoresis, indicating that alcohol oxidase consists of four identical subunits. The purified alcohol oxidase has absorption maxima at 460 and 380 nm which were bleached by the addition of methanol. The prosthetic group of the enzyme was identified as a flavin adenine dinucleotide. Alcohol oxidase activity was inhibited by sulfhydryl reagents (p-chloromercuribenzoate, mercuric chloride, 5,5′-dithiobis-2-nitrobenzoate, iodoacetate) indicating the involvement of sulfhydryl groups(s) in the oxidation of alcohols by alcohol oxidase. Hydrogen peroxide (product of the reaction), 2-aminoethanol (substrate analogue), and cupric sulfate also inhibited alcohol oxidase activity.     

Derepression of FAD Pyrophosphorylase and Flavin Changes during Growth of Kloeckera sp. No. 2201 on Methanol

A methanol-utilizing yeast Kloeckera sp. No. 2201, when grown with methanol as a sole carbon and energy source, accumulated about three times much flavin as those grown with glucose, ethanol, or glycerol. A high proportion of the total flavin was FAD in methanol-grown cells. A remarkable derepression of FAD pyrophosphorylase accompanied by an inducible formation of an FAD-dependent alcohol oxidase which catalyzes oxidation of methanol, the first step in the oxidation sequence, was observed during growth of the yeast on methanol. Significant elevations of riboflavin synthetase and flavokinase were also found. Formate, as well as methanol, effectively induced both FAD pyrophosphorylase and methanol-oxidizing enzymes (alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and catalase). Observations with other methanol-utilizing yeasts also gave essentially same results. These results led to the conclusion that cellular flavin level might be under control with level of flavoprotein physiologically required.     

Oxalic acid and oxalate oxidase enzyme in <Emphasis Type="Italic">Costus pictus</Emphasis> D. Don

The leaves of Costus pictus are sour in taste due to the presence of oxalic acid in the leaves. Different stages of leaves were collected and the samples were designated as stage one, stage two and stage three. It was found that oxalate content and oxalate oxidase activity were maximum in second leaf stage followed by first leaf stage and third leaf stage. Drying causes substantial loss of oxalate content and complete loss of oxalate oxidase activity. With various solvents water recovered more oxalate followed by methanol and ethanol while oxalate oxidase activity was maximum in ethanol followed by methanol and water. The ethanol or methanol extract of second leaf stage of C. pictus can be used for isolating active principles. The oxalate oxidase from C. pictus can be used as a cheap source of oxalate oxidase enzyme which is used in oxalate determination in biological fluids. Moreover, the sensitivity of oxalate determination employing oxalate oxidase from C. pictus will be more as oxalate oxidase in C. pictus has K _m 20 times lesser than the oxalate oxidase enzyme from barley seedling.     

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.     

Regulation of alcohol oxidase synthesis in Hansenula polymorpha: Oversynthesis during growth on mixed substrates and induction by methanol

The regulation of the synthesis of alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase was investigated in the methanol-utilizing yeast Hansenula polymorpha. The organism was found to synthesize immunologically identical alcohol oxidases during growth on glycerol and methanol. Growth on glycerol, however, was not dependent on the alcohol oxidase, as was shown with a mutant without alcohol oxidase protein. Similarly it was shown with a catalase activity negative mutant that high catalase activity during growth on glycerol was not a prerequisite for the utilization of this substrate, though absolutely required for growth on methanol.Experiments were conducted with mixed substrates to study the influence of methanol on alcohol oxidase synthesis. In batch cultures, growth on ribose plus methanol resulted in an enhanced rate of alcohol oxidase synthesis as compared to ribose alone. In continuous cultures, (D=0.1 h^-1) addition of methanol to glycerol-, glucose-, or sorbose-limited cultures gave rise to increased alcohol oxidase activity of up to 20 U/mg, which is about by 2 times higher than the specific activity used for growth on methanol alone. The increase in specific activity of the dissimilatory enzymes on the mixed substrates is partly due to methanol per se, as was shown by a mutant unable to dissimilate or assimilate methanol.     

Alcohol oxidase of methylotrophic thermo-and acidotolerant yeastHansenula sp

Electrophoretic analysis of alcohol oxidase purified from the methylotrophic thermo- and acidotolerant yeastHansenula sp. revealed the presence of two active forms of the enzyme with molar mass 440 kg/mol (major component) and 724 kg/mol (minor component). A subunitM of the enzyme was found to be 72 kg/mol. Two active forms of the enzyme found by electrophoresis seem to be caused by dissociation of the octameric form to the tetramer under alkaline conditions. Studies of alcohol oxidase showed a kinetic variability of the enzyme with respect to itsK _m. It is proposed that the variability ofK _m is caused by enzyme binding to formaldehyde.     

STUDIES ON THE PROPERTIES OF RETINAL ALCOHOL DEHYDROGENASE FROM THE RAT

An NAD-dependent alcohol dehydrogenase (alcohol:NAD oxidoreductase; EC 1.1.1.1) has been isolated and partially purified from the retinal cytosol of the rat. Its substrate specificity and sensitivity to inhibitors of hepatic alcohol dehydrogenase have been investigated. Ethanol, 1-propanol and 1-butanol served as substrates for this enzyme but the K_m values were more than 100-fold higher than those reported for hepatic alcohol dehydrogenase. Methanol and retinol were unreactive with this alcohol dehydrogenase. Inhibition by pyrazole was observed but the K_t was about 100-fold higher than the value observed for hepatic alcohol dehydrogenase. n-Butyraldoxime inhibited retinal alcohol dehydrogenase with a K_t of 2 μM, a value which approximates its K_t for hepatic alcohol dehydrogenase. 1, 10-Phenanthroline was ineffective as an inhibitor. Oxidation of retinol was observed in retinal homogenates in the presence of NADP but no inhibition was observed with ethanol, methanol or pyrazole. We conclude that oxidation of retinol is not catalysed by soluble retinal alcohol dehydrogenase.     

Formate production from methanol by formaldehyde dismutase coupled with a methanol oxidation system

Summary Formaldehyde dismutase was greatly stabilized by immobilization in a urethane prepolymer (PU-6). The immobilized enzyme exhibited stochiometrical dismutation of formaldehyde to methanol and formate in several repeated reactions. Conversion of methanol to formate occurred in a reaction with an immobilized enzyme system consisting of alcohol oxidase, catalase and formaldehyde dismutase, and with an intact cell-mixture of Hansenula polymorpha and Pseudomonas putida. Furthermore, the stability of the cell-mixture during repeated reactions was greatly improved by the immobilization, the 600 mM methanol added periodically being converted to formate in a 75% yield in 12 h. The immobilized cellsystem was also effective for the conversion of several aliphatic alcohols, C₁ to C₄, to the corresponding acids.     

Mixed substrate growth of methylotrophic yeasts in chemostat culture: Influence of the dilution rate on the utilisation of a mixture of glucose and methanol

The growth of Hansenula polymorpha and Kloeckera sp. 2201 with a mixture of glucose and methanol (38.8%/61.2%, w/w) and the regulation of the methanol dissimilating enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase were studied in chemostat culture, as a function of the dilution rate. Both organisms utilized and assimilated glucose and methanol simultaneously up to dilution rates of 0.30 h^-1 (H. polymorpha) and 0.26h^-1, respectively (Kloeckera sp. 2201) which significantly exceeded _max found for the two yeasts with methanol as the only source of carbon. At higher dilution rates methanol utilisation ceased and only glucose was assimilated. Over the whole range of mixed-substrate growth both carbon sources were assimilated with the same efficiency as during growth with glucose or methanol alone.In cultures of H. polymorpha, however, the growth yield for glucose was lowered by the unmetabolized methanol at high dilution rates. During growth on both carbon sources the repression of the synthesis of all catabolic methanol enzymes which is normally caused by glucose was overcome by the inductive effect of the simultaneously fed methanol. In both organisms the synthesis of alcohol oxidase was found to be regulated differently as compared to catalase, formaldehyde and formate dehydrogenase. Whereas increasing repression of the synthesis of alcohol oxidase was found with increasing dilution rates as indicated by gradually decreasing specific activities of this enzyme in cell-free extracts, the specific activities of this enzyme in cell-free extracts, the specific activities of catalase and the dehydrogenases increased with increasing growth rates until repression started. The results indicate similar patterns of the regulation of the synthesis of methanol dissimilating enzymes in different methylotrophic yeasts.Abbreviations and Terms C₁ Methanol - C₆ glucose; D dilution rate (h^-1) - D _c critical dilution rate (h^-1) - q _s specific, rate of substrate consumption (g substrate [g cell dry weight]^-1 h^-1) - q _CO2 and q _O2 are the specific rates of carbon dioxide release and oxygen consumption (mmol [g cell dry weight]^-1 h^-1) - RQ respiration quotient (q _CO2 q _O2 ¹ ) - s ₀(C₁) and s ₀(C₆) are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - s residual substrate concentration in the culture liquid (g l^-1) - Sp. A. enzyme specific activity - x cell dry weight concentration (gl^-1) - Y _X/C6 growth yield on glucose (g cell dry weight [g substrate]^-1     

Physiological and morphological acid resistance ofCandida boidinii

The growth ofCandida boidinii strain 2 in a methanol-limited chemostat at a dilution rate of 0.1/h and a low extracellular pH (2.8–4.0) is characterized by a maximum yield coefficient referred to the methanol consumedY _S of 0.4 g/g and a maximum cell content of nitrogenous compounds of 60%. The cell proteins are rich in essential amino acids. At pH<2.6 or >4.0 the cell concentration decreases due to lower growth rate, accompanied by increased metabolic quotientsQ _S,Q _CO2 andQ _form, and increased activities of dissimilating dehydrogenases. The activity of alcohol oxidase (AO) in intact cells (0.54 IU/mg protein) was unaffected by pH 2.8–3.8 although in a cell-free extract the AO activity decreased at these low pH values after a 10-min incubation. The lower AO activity in cells at pH<2.8 and pH>3.8 brought about increased residual methanol levels in the medium, and also an increased level of riboflavin phosphate, arising probably by the release of FAD from active AO. Catalase activity was completely pH-independent. Cell morphology also showed no changes at pH 2.8–4.2, formation of cell chains being observed only at pH<2.8. However, the ultrastructure of cells grown in the chemostat at pH 2.6, however, did not evince any changes as compared with cells grown, at higher pH apart from a lag in cytokinesis. These findings, which point to acid resistance of strain 2, make it possible to produce biomass from methanol, with a high content of valuable proteins and AO, under nonsterile conditions.     

Oxidation of formaldehyde by alcohol oxidase of Candida boidinii

A formaldehyde oxidase activity was found in cell-free extracts of methanol-grown yeast Candida boidinii. Loss of alcohol oxidase activity in a mutant, 4₈, led to loss of the formaldehyde oxidase activity, indicating that the same enzyme is probably responsible for both activities. This could be demonstrated with the purified alcohol oxidase which oxidizes, besides lower primary alcohols, formaldehyde to formate. The K _m value for formaldehyde is 5.7 mM. It seems that alcohol oxidase is not implicated in formaldehyde oxidation in vivo.     

Purification and Properties of Benzyl Alcohol Oxidase from Botrytis cinerea

A benzyl alcohol oxidase (BAO) was purified to homogeneity from Botrytis cinerea. The enzyme was found to have a molecular mass of 214 kD with a trimeric structure, and optimal pH and temperature of 5.0 and 30°C, respectively. The enzyme activity was not sensitive to metal ions or to metal ion chelators, while thiol blocking reagents strongly inhibited BAO activity. Sulfur dioxide irreversibly inhibited the enzyme activity and the inhibitory effect of ethanol was weak and reversible. Benzyl alcohol was the most effective alcohol substrate for BAO. Para or meta monosubstituted benzyl alcohol with methyl or methoxy groups were good substrates. BAO also oxidized cinnamyl alcohol, furfuryl alcohol, and some terpenic alcohols· with an alkenyl group near the reactive carbinol. Secondary alcohol, methanol and phenol were not substrates. Product inhibition studies suggested that benzaldehyde and benzyl alcohol were bound at different places to the active site. O₂ was the only electron acceptor identified and Botrytis cinerea benzyl alcohol oxidase was classified .as EC 1.1.3.7 according to stoichiometrical studies. We discuss the metabolic role of BAO in the Botrytis cinerea-grape host-parasite relationship.     

Production of hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic fungusPleurotus eryngii

Summary Production of extracellular hydrogen peroxide by fungal oxidases is been investigated as a requirement for lignin degradation. Aryl-alcohol oxidase activity is described in extracellular liquid and mycelium ofPleurotus eryngii and studied under non-limiting nitrogen conditions. This aryl-alcohol oxidase catalyses conversion of primary aromatic alcohols to the corresponding aldehydes and H₂O₂, showing no activity with aliphatic and secondary aromatic alcohols. The enzyme is stable at pH 4.0–9.0, has maximal activity at 45°–50°C and pH 6.0–6.5, is inhibited by Ag⁺, Pb²⁺ and NaN₃, and has aK _m of 1.2 mM using veratryl alcohol as substrate. A single protein band with aryl-alcohol oxidase activity was found in zymograms of extracellular and intracellular crude enzyme preparations fromP. eryngii.     

Methanol metabolism in yeasts: Regulation of the synthesis of catabolic enzymes

The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of the two organisms grown under glucose limitation at various dilution rates, suggested that the synthesis of these enzymes is controlled by derepression — represion rather than by induction — repression. Except for alcohol oxidase, the extent to which catabolite repression of the catabolic enzymes was relieved at low dilution rates was similar in both organisms. In Hansenula polymorpha the level of alcohol oxidase in the cells gradually increased with decreasing dilution rate, whilst in Kloeckera sp. 2201 derepression of alcohol oxidase synthesis was only observed at dilution rates below 0.10 h^–1 and occurred to a much smaller extent than in Hansenula polymorpha.Derepression of alcohol oxidase and catalase in cells of Hansenula polymorpha was accompanied by synthesis of peroxisomes. Moreover, peroxisomes were degraded with a concurrent loss of alcohol oxidase and catalase activities when excess glucose was introduced into the culture. This process of catabolite inactivation of peroxisomal enzymes did not affect cytoplasmic formaldehyde dehydrogenase.