Stability and refractoriness of the high catalase activity in the oxidative-stress-resistant fission yeastSchizosaccharomyces pombe

Effect of oxygen and metabolic substrates (glucose, ethanol) on the catalase activity of anaerobically grownSchizosaccharomyces pombe cells was assessed, and compared with that ofSaccharomyces cerevisiae in order to determine the catalase activity regulation inS. pombe. In contrast toS. cerevisiae, the total catalase activity of permeabilizedS. pombe anaerobically grown cells is higher than that found in aerobically grown cells, is stable and constant under all circumstances (i.e. it is not induced by oxygen and/or substrates), and only a negligible part (3–5%) of it is contributed byde novo protein synthesis during aeration with or without substrates. The patent catalase activity of intact cells rises 2-fold during 6-h aeration without substrate and 7–8-fold in the presence of glucose or ethanol. The increase is not inhibited by cycloheximide and is thus not due tode novo catalase synthesis, but may reflect enhanced transport of catalase to the cell surface or a permeabilization of the plasma membrane during the aeration.     

Haemoprotein formation in yeast

Summary A procedure was described for the isolation of mutants affected in the regulation of catalase activity. Two such mutants, cgr 1 and cgr 2 were obtained. Both of them show catalase activity that is resistant to repression by glucose, but is sensitive to anoxia to the same extent as the wild type.     

Efficient Expression of Peroxidatic Activity of Catalase in the Coupled System with Glucose Oxidase—a model of Yeast Peroxisomes

Catalase functioned exclusively to degrade hydrogen peroxide in a reaction mixture containing methanol and hydrogen peroxide, while, when the enzyme was coupled with glucose oxidase, successful conversion of methanol to formaldehyde occurred at the optimized ratio of glucose oxidase to catalase: activity, 1.0 × 10 ^?3; number of molecules, 1.3; protein content, 1. These values in the coupled system were very similar to the ratio of alcohol oxidase to catalase in peroxisomes, one of the subcellular organelles from a methanol-assimilating yeast, Kloeckera sp. 2201, in which these enzymes were coupled to metabolize methanol efficiently. The presence of the optimum ratio in the coupled system in vitro was confirmed by the kinetic analysis of the expression of the peroxidatic activity of catalase coupled with glucose oxidase. Construction of the immobilized system of the coupled enzymes at the optimum ratio demonstrated that the oxidation of methanol through the peroxidatic function of catalase could be continuously and stably operated, the results indicating the usefulness of the system as a model of yeast peroxisomes. Thus, the coupled reaction with glucose oxidase brought out the latent function of catalase, which could not be expected in the system including only catalase.     

Simultaneous production of glucose oxidase and catalase by Alternaria alternata

Summary A number of factors affecting simultaneous production of cell-bound glucose oxidase and catalase by the fungus Alternaria alternata have been investigated. Consecutive optimization of the type and concentration of nitrogen and carbon source, the initial pH and growth temperature resulted in a simultaneous increase in glucose oxidase and catalase by 780% and 68% respectively. Two second-order equations, describing the combined effect of pH and temperature on the activity of each enzyme, revealed that glucose oxidase had its optima at pH 7.9 and 32.3°C and catalase at pH 8.5 and 18.1°C. Under certain growth conditions, yields as high as 23.5 and 18,100 units/g carbon source for glucose oxidase and catalase, respectively, were simultaneously obtained.Offprint requests to: B. J. Macris     

Sugar repression in the methylotrophic yeast Hansenula polymorpha studied by using hexokinase-negative, glucokinase-negative and double kinase-negative mutants

Two glucose-phosphorylating enzymes, a hexokinase phosphorylating both glucose and fructose, and a glucose-specific glucokinase were electrophoretically separated in the methylotrophic yeastHansenula polymorpha. Hexokinase-negative mutants were isolated inH. polymorpha by using mutagenesis, selection and genetic crosses. Regulation of synthesis of the sugar-repressed alcohol oxidase, catalase and maltase was studied in different hexose kinase mutants. In the wild type and in mutants possessing either hexokinase or glucokinase, glucose repressed the synthesis of maltase, alcohol oxidase and catalase. Glucose repression of alcohol oxidase and catalase was abolished in mutants lacking both glucose-phosphorylating enzymes (i.e. in double kinase-negative mutants). Thus, glucose repression inH. polymorpha cells requires a glucose-phosphorylating enzyme, either hexokinase or glucokinase. The presence of fructose-phosphorylating hexokinase in the cell was specifically needed for fructose repression of alcohol oxidase, catalase and maltase. Hence, glucose or fructose has to be phosphorylated in order to cause repression of the synthesis of these enzymes inH. polymorpha suggesting that sugar repression in this yeast therefore relies on the catalytic activity of hexose kinases.     

The effect of growth medium on the antioxidant defense of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We compared the oxidation of dihydrorhodamine 123, glutathione contents and activities of superoxide dismutase (SOD) and catalase for three wild-type strains of Saccharomyces cerevisiae grown on media with different carbon sources. The rate of oxidation of dihydrorhodamine 123 was much higher in respiring cells grown on ethanol or glycerol media than in fermenting cells grown on glucose medium. The total SOD activity was highest on glycerol medium and lowest on ethanol medium, while the catalase activity was highest on glycerol medium. The sequence of glutathione content values was: glucose > ethanol > glycerol.     

Glucose oxidase and catalase activities ofPenicillium variabile P16 immobilized in polyurethane sponge

Conidia ofPenicillium variabile P16 were immobilized in polyurethane sponge and used in repeated-batch processes in a fluidized-bed reactor. Optimal conditions for production of glucose oxidase and catalase were: inoculum size, 10%; glucose concentration, 80 g L^–1; Ca-carbonate concentration, 15 g L^–1; temperature, 28°C and aeration rate, 4 VV^–1 min^–1. In an extended repeated-batch process, glucose oxidase activity was highest after the fourth batch and catalase activity was highest after the fifth batch. Scanning electron microscopy showed that the fungus grew only in the interior of carrier particles.     

Regulation of catalase synthesis in Saccharomyces cerevisiae by carbon catabolite repression.

Summary A number of strains of Saccharomyces cerevisiae, wild type or respiratory deficient, were grown on glucose, galactose or raffinose. Specific activities of catalase T were about tenfold higher in late stationary wild type cells grown on glucose than in wild type cells harvested when glucose had just disappeared completely from the medium, or in respiratory deficient strains (rho^–, mit^–, pet) grown to stationary phase.Catalase A activity is completely absent in wild type cells grown to zero percent glucose or in respiratory deficient cells grown on glucose to stationary phase. High catalase A activity was detected in derepressed wild type cells and in a strain carrying the op 1 (pet 9) mutation, although this strain is unable to grow on nonfermentable carbon sources. All respiratory deficient strains tested have low, but significant catalase A activities after growth on galactose or raffinose.Wild type cells harvested during growth on glucose and rho^–-cells grown on low glucose to stationary phase contain enzymatically inactive catalase A protein. The apoprotein of the enzyme is apparently accumulated in rho^–-cells whereas glucose-repressed wild type cells seem to contain a mixture of apoprotein and heme-containing catalase A monomer.These results show that a source of chemical energy, probably ATP, is required for derepression of yeast catalase from catabolite repression. At least in the case of catalase A, energy produced by respiration is necessary if catabolite repression is caused by glucose. If less repressing sugars are utilized, ATP derived from fermentation appears sufficient for partial derepression. Formation of the active enzyme can apparently be influenced by carbon catabolite repression at different points: (1) at the level of protein synthesis, (2) at the stage of heme incorporation, (3) at the level of formation of the enzymatically active tetramer.     

n-Alkane oxidation byCandida tropicalis

Summary The specific activity of the enzyme catalase was investigated in batch cultures ofCandida tropicalis on the following substrates: Gelsenberg 14/18, n-hexandecane, 1-octadecene, oleyl alcohol, oleic acid and glucose. The catalase activity does not change with the different oxidation levels of the hydrocarbon substrates. A correlation between specific activity and growth rate was established.Inhibition experiments with 2-amino-1,3,4-triazol (AT), a specific catalase inhibitor, gave no evidence for two kinds of hydrogen peroxide-degrading enzymes in yeast cells.Growth on hydrocarbons instead of on glucose enhanced the specific activities of both isocitratelyase (ICL) and catalase to about the same extent.On the other hand the succinate-cytochrome C-oxidoreductase, a mitochondrial enzyme, showed more or less the same activity on both substrates.All these facts suggest that the catalase enzyme is related to the degradation (- or -oxidation) or to the gluconeogenesis (glyoxylate cycle) of fatty acids, but not to the initial oxidation of the alkanes.     

Induction of Catalase Activity in a Methanol-utilizing Yeast,Kloeckera sp. No. 2201

The methanol-grown cells of Kloeckera sp. No. 2201 exhibit a markedly high catalase activity as compared with the glucose-grown and ethanol-grown cells. In this connection, specific organelles (“microbodies”) appear only in the methanol-grown cells. When the yeast cells harvested from a methanol medium (cells whose catalase activity had been enhanced to an appreciable extent: “partially induced cells”) were transferred into media containing glucose, ethanol or methanol as the sole carbon and energy source, further increase of catalase activity was mediated only by methanol. This induction of catalase activity was partially inhibited by cycloheximide at its high concentration, but chloramphenicol did not show any effect. Glucose inhibited strongly the induction by methanol, while galactose gave no effect. Electron microscopical observation revealed that the development of microbodies in the cells growing on methanol was hardly affected by cycloheximide. Disappearance of microbodies was observed electron microscopically after the methanol-grown cells (partially induced cells) were transferred to a methanol-glucose medium and cultivated for 8 hr. 3′,5′-Cyclic AMP or dibutyryl-3′,5′-cyclic AMP could not eliminate the inhibitory effect of glucose on the catalase induction. Addition of caffeine or theophylline did not promote the action of the cyclic nucleotides. 3-Amino-1,2,4-triazole inhibited only 40% of the hydrogen peroxide-decomposing activity in the cell homogenate of methanol-grown cells even at its concentration of as high as 10 mm, while sodium azide inhibited the enzyme activity completely at the concentration of 1 mm.     

Cytochemical localization of glucose oxidase in peroxisomes of Aspergillus niger

Summary The subcellular localization of glucose oxidase (E.C. 1.1.3.4) in mycelia of Aspergillus niger has been investigated using cytochemical staining techniques. Mycelia from fermenter cultures, which produced gluconic acid from glucose, contained elevated levels of glucose oxidase and catalase. Both enzymes were located in microbodies. In addition, when the organism was grown on glucose with methylamine as a nitrogen source, amine oxidase activity was detected in the microbodies. These organelles can therefore be designated as peroxisomes.     

Induction of Catalase Activity by Hydrocarbons in Candida tropicalis рK 233

1. The catalase activity of Candida tropicalis pK 233 was induced by hydrocarbons but not by glucose, galactose, ethanol, acetate or lauryl alcohol.

2. The induction of the catalase activity depending upon hydrocarbons was sensitive to cycloheximide but not to chloramphenicol.

3. Glucose repressed strongly the induction of the catalase activity by hydrocarbons but galactose did not affect seriously.

4. When C. tropicalis was incubated with hydrocarbons, the appearance of microbodies was observed electronmicroscopicaliy.

     

Survival of Fungi in Hyperbaric Oxygen in Relation to Changes in Catalase Activity

Survival of replacement cultures of Mucor species in oxygenat 10 atm was markedly affected by the carbon source; culturesreplaced on glucose media survived for a much shorter periodthan cultures on water while 1 per cent ethanol increased thesurvival time. This effect of carbon source on survival wascorrelated with changes in catalase activity; in the presenceof glucose and other carbohydrates, catalase activity decreasedwhereas on 1 per cent ethanol a large increase in catalase activityoccurred which was maintained for several days. An increasein catalase activity also occurred on methanol, pyruvate, glycerol,and formate. Induced changes in peroxidase activity were similarto those shown by catalase but the activity of three other enzymesdecreased in hyperbaric oxygen on all media.     

Serratia marcescens SYBC08 catalase isolated from sludge containing hydrogen peroxide shows increased catalase production by regulation of carbon metabolism

A high‐catalase‐producing strain, which was isolated from sludge containing hydrogen peroxide, was identified as Serratia marcescens SYBC08 by 16S rDNA sequence analysis. Serratia spp. was reported as non‐spore‐forming bacterium (except S. marcescens spp. sakuensis), but in our study electron microscopic observation revealed that the strain did produce spores. The content of the main fatty acid C_16:0 (14.8%) was significantly different from that of S. marcescens spp. sakuensis (33.2%) and S. marcescens spp. marcescens DSM 30121^T (34.8%), and the biochemical characteristics were not identical to those of S. marcescens spp. sakuensis. We speculate that the relatively high catalase activity and the spore structures may enable the strain to survive in a hydrogen peroxide environment. The most suitable carbon and nitrogen sources for the catalase production by S. marcescens SYBC08 were citric acid and corn steep liquor powder. A strategy of carbon metabolism regulation to enhance the catalase production was exploited. In the 7‐L fermenter, catalase production (20 353 U/mL) obtained in the presence of glucose and citric acid was 1.68‐ and 1.31‐fold higher than that obtained in the presence of glucose or citric acid, at equimolar carbon concentration. This production yield was much higher than that of many catalase‐producing strains, but only slightly lower than the production by Micrococcus luteus (34 601 U/mL). The results suggest that the new spore‐forming S. marcescens SYBC08 is a potential candidate for the production of catalase.     

Catalase Activities of Hydrocarbon-utilizing Candida Yeasts

The catalase activities of the Candida cells grown on hydrocarbons were generally much higher than those of the cells grown on Iauryl alcohol, glucose or ethanol. Km values for hydrogen peroxide of the enzymes from the glucose- and the hydrocarbon-grown cells of Candida tropicalis were the same level. The enzyme activities of the yeasts were higher at the exponential growth phase, especially of the hydrocarbon-grown cells, than at the stationary phase. Profuse appearance of microbodies having homogeneous matrix surrounded by a single-layer membrane has also been observed electronmicroscopically in the hydrocarbon- grown cells of several Candida yeasts. Cytochemical studies using 3,3′-diaminobenzidine (DAB) revealed that the catalase activity was located in microbodies. These facts suggest that the catalase activities would be related to the hydrocarbon metabolism in the yeasts.     

Hypoxic acclimation to anoxia in Avena roots

Low molecular weight phenolic compounds, identified in two differentsoils with vegetative cover, Fagus sylvatica L. andPinus laricio Poiret, spp. calabrica, were tested atdifferent concentrations on seed of Pinus laricio. Wedetermined the activity of catalase, responsible for degradation of fatty acidto provide substrates for gluconeogenesis and malate synthase,phosphoenolpyruvate carboxykinase and isocitrate lyase, enzymes of theglyoxylicpathway. The data obtained show that all the phenolic compounds bioassayedaffected seed germination inhibiting catalase and glyoxylic enzymes. Theresultsshow that the inhibition of Pinus laricio seed germinationby phenolic compounds is clearly linked to low utilisation of storage lipidswith a consequent deficit in glucose, the main respiratory substrate.     

Haemoprotein formation in yeast

Summary Catalase A and T activities were investigated in two standard strains and three catalase regulatory cgr mutants of yeast in respiratory competent and incompetent states, which were under various degrees of glucose repression.The formation of catalase A was very sensitive to glucose repression and was characterized by a long delay in derepression. Deprivation of the energy source in respiratory incompetent cells prevented the derepression of catalase A. The lack of catalase A in respiratory incompetent cells can be overcome by growing the cells in raffinose or by the prolongation of the fermentative phase of derepression.Catalase T is under control of different regulatory systems probably common with some other haemoproteins.     

Catalase anabolism in yeast: Loss of regulation by oxygen of catalase apoprotein synthesis after mutation

Summary A mutant of Saccharomyces cerevisiae which displays catalase activity when grown under strictly anaerobic conditions has been selected on solid media.Although some preformed holoenzyme has accumulated in anaerobic cells, a sharp increase of activity is still measured during adaptation to oxygen in glucose-buffer; however, a striking difference with the wild-type strain is that in the mutant, catalase formation is observed in the presence of cycloheximide that totally inhibits cytoplasmic translation. It is concluded that kat 80 mutant has lost the regulatory control by oxygen of apocatalase synthesis; the latter precursor, characterized as apocatalase T, is thought to be activated in vivo, under aerobic conditions, by inclusion of prosthetic group.Regulation of enzyme synthesis by catabolite repression (glucose effect) persists, unmodified by reference to the wild-type parental strain.Mutation kat 80 specifically hits catalase anabolism, as no significant variations were observed for the edification of the respiratory system and (apo)cytochrome c peroxidase production.Genetic analysis shows that kat 80 phenotype, recessive in heterozygotes, results from a single nuclear mutation.Abbreviations Enzymes. Catalase or hydrogen-peroxide hydrogen-peroxide oxidoreductase (EC 1.11.1.6) - Cytochrome c peroxidase or ferrocytochrome c hydrogen-peroxide oxidoreductase (EC 1.11.1.5)     

Development of catalase activity in yeast in relation to growth and respiration

1. 1) When yeast cells grown anaerobically were adapted to aerobicculture in a normal medium, catalase formation was markedlyenhanced after the earlier stage of exponential growth of thecells. The same thing occurred with cells transferred from ananaerobic culture into a nitrogen deficient medium.
2. 2) Thecatalase activity of aerobically grown cells declinedprogressivelyuntil glucose, which had been added to the mediumwas profoundlyexhausted. This decline was followed by a progressiverecoveryof activity to a normal level with the growth of thecells.Similar behavior of catalase was also seen at low concentrationsof glucose, except that an abrupt rise in activity was observedat the beginning of incubation. Even when cells which had declinedto a minimum of catalase activity were aerated in phosphatebuffer, they continued to synthesize catalase.
3. 3) The patternof alteration of catalase activity during cellgrowth was accompaniedby a comparable pattern of alterationin respiratory capacity.On the basis of this finding, togetherwith the fact that antimycinA causes intensive depression incatalase formation, it maybe inferred that the formation ofthe respiratory chain conductsthe formation of catalase.
4. 4) In the presence of ethyl alcoholas the carbon source inplace of glucose, a rise in both catalaseactivity and respiratorycapacity occurred from initiation ofincubation. This fact canbe interpreted to mean that the repressiveeffect of glucoseon catalase formation depends on the aerobiccharacter of thecells.

(Received February 26, 1968; )