Utilization of Carbohydrates by Rhynchosporium secalis. I. Growth and Sporulation on Glucose, Galactose, and Galacturonic acid

The fungus Rhynchosporium secalis (Oud.) Davis grew and speculated in liquid nutrient media that contained glucose, galactose or galacturonic acid, or a pair of those substances, as the sole carbon source. Sporulation was inhibited by high concentrations of glucose and galacturonic acid. Growth and sporulation were greatest on glucose, and least on galactose. Growth was increased when glucose and galacturonic acid were mixed. Nitrogen concentration affected sporulation but not growth.     

Development and analysis of pectic screening media for use in the detection of pectinase mutants

Summary Two solid pectic media were devised for mutually exclusive detection of extracellular polygalacturonase and pectin lyase produced by fungi including the vascular parasite of tomato Verticillium albo-atrum. These media allowed detection of pectinase-defective mutants. Polygalacturonase detection medium contains non-methylated polygalacturonan (sodium polypectate) is buffered at pH 5.0 (Na citrate, 0.05 M) and is calcium-free. In contrast pectin lyase medium contains polymethylgalacturonan (pectin), is buffered at pH 8.0 (HEPES, 0.05 M) and contains calciumrich agar. When glucose was added to the media for selection of catabolite-resistant mutants, enzyme synthesis was still evident, whereas in comparable conditions in liquid culture production was almost completely repressed. This apparent discrepancy is discussed in terms of the influence of basal synthesis, colony biomass and accumulation of oligouronides which repress induced synthesis and activity.Abbreviations CR catabolite repression - CTAB cetyltrimethyl ammoniumbromide - GALA galacturonic acid - NAPP sodium polypectate - PG polygalacturonase - PL pectin lyase - TBA thiobarbituric acid - UGALA unsaturated galacturonic acid     

Effects of different carbon sources on the synthesis of pectinase by Aspergillus niger in submerged and solid state fermentations

A study was made to compare the production of pectinase by Aspergillus niger CH4 in solid-state (SSF) and submerged (SmF) fermentations. Production of endo- (endo-p) and exo-pectinase (exo-p) by SSF was not reduced when glucose, sucrose or galacturonic acid (up to 10%) were added to a culture medium containing pectin. Moreover, both activities increased when concentrations of the carbon sources were also increased. In SmF, these activities were strongly decreased when glucose or sucrose (3%) was added to culture medium containing pectin. The addition of galacturonic acid affected endo-p activity production to a lesser extend than exo-p. Final endo-p and exo-p activities in SSF were three and 11 times higher, respectively, than those obtained in SmF. The overall productivities of SSF were 18.8 and 4.9 times higher for endo-p and exo-p, respectively, than those in SmF. These results indicate that regulatory phenomena, such as induction-repression or activation-inhibition, related to pectinase synthesis by A. niger CH4 are different in the two types of fermentation. Correspondence to: E. Favela-Torres     

Exopolysaccharide production by a unicellular cyanobacterium isolated from a hypersaline habitat

The unicellular cyanobacterial strain 16Som2, isolated from a Somaliland saltpan and identified asCyanothece sp., is characterized by cells surrounded by a thick polysaccharidic capsule, the external part of which dissolves into the medium during growth, causing a progressive increase in culture viscosity. In spite of this, the thickness of the capsule remained almost constant under all the culture conditions tested, demonstrating that the processes of its synthesis and solubilization occurred at a similar rate. The synthesis of carbohydrates was neither enhanced by increasing salinity (sea-water enriched with NaCl in the range 0 to 2.0 M) nor by Mg²⁺, K⁺ or Ca²⁺ deficiencies. In contrast, N-limitation and, to a lesser extent, P-limitation induced a significant enhancement of carbohydrate synthesis; in particular, N-deficiency stimulated the synthesis of all the carbohydrate fractions (intracellular, capsular and soluble). The soluble polysaccharide, separated from the culture medium and hydrolyzed with 2N trifluoroacetic acid, showed a sugar composition consisting of glucuronic acid: galacturonic acid: galactose: glucose: mannose: xylose: fucose in a molar ratio of 1: 2: 2.4: 6.8: 4.8: 2.9: 1.6.Cyanothece sp. culture subjected to nitrogen starvation synthesized polysaccharide with a mean productivity of 115 mg (EPS) l^–1d^–1, for the polymer solubilized into the medium, and of 15 mg (CPS) l^–1d^–1 for the capsular polysaccharide.     

Differential induction of enzyme in soybean cell cultures by elicitor or osmotic stress

Soybean cell cultures were challenged either by glucan elicitor from Phytophthora megasperma f.sp. glycinea or by osmotic stress (0.4 M glucose). Osmotic stress induced production of a microsomal NADPH-dependent flavone synthase (flavone synthase II) which catalyses conversion of (2S)-naringenin to apigenin. In one of our cell-lines this enzyme activity was not detected either in unchallenged cells or in cells treated with glucan elicitor. Inducibility of flavone synthase II by 0.4 M glucose was highest at the end of the linear growth phase. Changes in the activities of a number of other enzymes were determined after treatment of the cells with elicitor or 0.4 M glucose. The activities of phenylalanine ammonialyase, cinnamate 4-hydroxylase, chalcone synthase and dihydroxypterocarpan 6a-hydroxylase all increased with elicitor and with osmoticum, albeit to a different degree. The rise in enzyme activity occurred later with osmoticum than with elicitor. The prenyltransferase involved in glyceollin synthesis was induced strongly by elicitor but only very weakly by osmoticum, whereas isoflavone synthase and NADPH: cytochrome-c reductase were only induced by elicitor. The activity of glucose-6-phosphate dehydrogenase did not change with elicitor or with osmoticum. Different product patterns were also obtained: whereas with elicitor, glyceollin I was the major product, intermediates of the glyceollin pathway (7,4-dihydroxyflavanone, trihydroxypterocarpan) accumulated with osmoticum.     

Stimulation of the production of extracellular pectinolytic activities of Aspergillus sp. by galacturonic acid and glucose addition

We studied the effect of the addition of galacturonic acid and of glucose on the production of the pectinolytic activities by Aspergillus sp. The results showed that a differential response in the production of the activities was produced. The production of the pectinolytic activity as measured by reducing groups was not negatively affected when low concentrations of eithr glucose or galacturonic acid were added to growing pectin containing cultures of Aspergillus sp. regardless of the time of addition. The production of the activity, measured by viscosimetry, was stimulated by addition of galacturonic acid at 24 h, suggesting that galacturonic acid participates in the induction of pectinolytic activities of Aspergillus sp. When the addition was made at t = 0, the increase in pH seems to affect the production of this activity. The addition of glucose at 24 h had no effect on the production of the viscosimetric activity; however, catabolic repression was observed upon the addition at t = 0. The production of both pectinolytic activities was strongly repressed when glucose was added at high concentration and was affected less when galacturonic acid was added at the same high concentration.     

Degradation of a washed carrot preparation by cellulases and pectinases

A washed carrot substrate, prepared with high yields and easy handling properties, was found to be a suitable substrate for studying cellulolytic and pectinolytic degradation processes. A cellulase from Trichoderma reesei, and Rohament P, a macerating enzyme from Aspergillus alleaceus in endopolygalacturonase, degraded the washed carrot substrate to an extent of 60%. With the combined action of both enzymes, degradation was more than 80%. Simultaneous action of both enzymes was more efficient than their sequential use. The effect of temperature, pH, incubation time, enzyme concentration, and substrate concentration on the degradation by the single enzymes and their mixture were studied. Gas chromatographic sugar analysis revealed that Rohament P liberated glucose, arabinose, and galactose in the low-molecular-weight fraction obtained by ultrafiltration, in addition to high amounts of galacturonic acid. These carbohydrates were also found in the high-molecular-weight fraction (retentate) together with rhamnose and mannose. Cellulase BC released mainly glucose, although galacturonic acid, arabinose, xylose, and mannose were also detected both in the ultrafiltrate and retentate. Morphologically, during Rohament P degradation of the washed carrot substrate, damaged tissues and disintegrated cells were seen, whereas on cellulase BC action mainly disintegrated cell walls were observed.     

Implication of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in the production of pectinolytic enzymes during cocoa fermentation

The role of bacilli in cocoa fermentation is not well known. Their potential of production of pectinolytic enzymes during this process was evaluated. Bacillus growth was monitored and pectinolytic strains were screened for their use of pectin as sole carbon source. Effects of cocoa fermentation parameters susceptible to influence on enzyme production were analysed. Among 98 strains isolated, 90 were positive for pectin degradation and 80% of them presented detectable pectinolytic activities in submerged fermentation. Forty-eight strains produced polygalacturonase (PG), 47 yielded pectin lyase (PL) and 23 strains produced both enzymes. Bacilli growth was not significantly affected during fermentation. PL production was favoured by galactose, lactose, glucose as sugars, and arginine, glutamine, cysteine and ammonium sulphate as nitrogen compounds. Pectin at low concentration (0.05%) and iron stimulated PL production. It was strongly repressed by galacturonic acid (1%), and negatively affected by nitrogen starvation, zinc and temperatures above 45°C. PL yield was very weak below pH 4.0 and in anaerobic conditions. PG production was weakened by sucrose and cation depletion. It was increased slightly by cysteine, ammonium nitrate and nitrogen starvation and significantly above 40°C. PG synthesis was not affected by acidic pH (3.0–6.0) or oxygen availability. As fermentation products, lactate and acetate lowered the production of both enzymes while ethanol had no effect. The high proportion of pectinolytic producers among the strains studied and analysis of factors influencing pectinolytic enzymes production, suggest that Bacillus sp. is liable to produce at least one enzyme during cocoa fermentation.     

Starch degradation by the mould Trichoderma viride II. Regulation of enzyme synthesis

The synthesis of amylolytic enzymes by the maltose not-utilizing Trichoderma viride strain CBS 354.44 requires the presence of starch or dextrins. Several readily utilizable carbon sources such as glucose and glutamic acid were shown to exert a strong catabolite repression which completely inhibited enzyme induction by starch or dextrins.Enzyme synthesis occurs in the exponential and in the stationary growth phase. In the latter, the ratio between saccharifying and dextrinizing enzyme activity is invariably high. In the exponential growth phase this ratio depends on the nature of the inducing substrate. Growth on starch results in an initially high production of dextrinizing activity, the saccharifying one becoming predominant in the course of exponential growth. The latter activity in dextrin DE 30 cultures is predominant from the very beginning. Thus, the amylolytic enzyme system of T. viride consists of at least two different enzymes, the synthesis of each being controlled specifically. The careful regulation of the synthesis of the dextrinizing enzyme is discussed with special reference to the production of non-utilizable maltose by the latter.     

Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

Comparison of Sucrose-Hydrolyzing Enzymes Produced by Rhizopus oryzae and Amylomyces rouxii

Rhizopus oryzae produces lactic acid from glucose but not efficiently from sucrose, while Amylomyces rouxii, a species closely related to R. oryzae, ferments these sugars equally. The properties of two sucrose-hydrolyzing enzymes purified from culture filtrates of R. oryzae NBRC 4785 and A. rouxii CBS 438.76 were compared to assess lactic acid fermentation by the two fungi. The substrate specificity of the enzymes showed that the enzymes from strains NBRC 4785 and CBS 438.76 are to be classified as glucoamylase and invertase respectively. The entity of the enzyme from strain NBRC 4785 might be a glucoamylase, because eight residues of the N-terminal amino acid sequence coincided with those of the deduced protein from the amyB gene of R. oryzae. The enzyme from NBRC 4785 was more unstable than that from strain CBS 438.76 under conditions of lower pH and higher temperature. These observations mean that the culture conditions of R. oryzae for lactic acid production from sucrose should be strictly controlled to prevent inactivation of the glucoamylase hydrolyzing sucrose.     

Utilization of Carbohydrates by Rhynchosporium secalis II. Absorption and Metabolism of Glucose, Galactose and Galacturonic acid

Glucose, galactose and galacturonic acid were taken up at different rates by the fungus Rhynchosporium secalis and were intracellularly converted to other forms of carbohydrate at different rates. These differences explain why, when there is only a single source of nutrient carbon in the growth medium, development of the fungus is greatest when glucose is present and least when galactose is present. Glucose and galactose were taken up by the same mechanism for their uptake showed a reciprocal competitive inhibition. Uptake mechanisms had a high affinity for glucose (apparent K_m 2.76 mM) and galacturonic acid (apparent K_m 3.10 mM) and a low affinity for galactose (apparent K_m 29.67 mM). After uptake, galactose accumulated in the mycelium, whereas glucose and galacturonic acid were rapidly converted to other soluble carbohydrates, principally trehalose and mannitol. The insoluble carbohydrates within the mycelium were little affected by the type of carbohydrate that was supplied to the fungus.     

Activity of glycolytic enzymes of Saccharomyces cerevisiae in the presence of acetic acid

Summary The effect of acetic acid on transport of glucose and on the activity of glycolytic enzymes of Saccharomyces cerevisiae was investigated. Acetic acid did not affect glucose transport. The inhibitory effect of the acid on the enzymes was considered from the point of view of acidification of the cytoplasm (pH dependence of the activity) and of the direct effect of the presence of acetic acid. Enolase was the enzyme most severely affected according to these two criteria. Fermentation was monitored in vivo by ³¹P-NMR. When ATP was available, a rise in cytoplasmic pH was observed and fermentation proceeded with a lower level of sugar phosphate. This may indicate that control was exerted at one of the early phosphorylation steps. Offprint requests to: M. C. Loureiro-Dias     

Exopectic Acid Transeliminase of an Erwinia

A species of Erwinia was found to produce no other pectolytic enzyme than the two transeliminases of exo-types, namely, an oligogalacturonide transeliminase and an exopectic acid transeliminase. Of the two enzymes, the exopectic acid transeliminase was isolated and its properties were investigated. The results are as follows: (1) Pectic acids having an unsaturated galacturonic acid residue at the non-reducing end of the molecule are susceptible but oxidized or reduced pectic acids resistant to the enzyme action. (2) The enzyme has no activity toward pectinic acid and polymethylpolygalacturonate methyl glycoside. The limit of the enzymatic degradation for citrus pectic acid is 43.8%. (3) The rate of the enzyme activity was maximal with tetragalacturonic acid and followed by acid-soluble pectic acid, acid-insoluble pectic acid, pectic acid and trigalacturonic acid. Unlike the oligogalacturonide transeliminases of Pseudomonas sp. (strain S2) and Erwinia aroideae, the present enzyme shows a considerably high activity toward pectic acids of high molecular weight. (4) The pH-activity curves vary with the buffer employed. (5) The enzyme is activated by Co²⁺ and Mn²⁺ but powerfully inhibited by Cu²⁺ and Hg²⁺. Ca²⁺ has no significant effect on the enzyme activity.     

Thetrans-eliminative breakdown of Na-polygalacturonate byPseudomonas fluorescens

Three strains ofPseudomonas fluorescens were isolated, which produced an inducible enzyme capable of converting Na-polygalacturonate into several reaction products which strongly absorbed ultraviolet light with a maximum at 232 mµ. Paper and column chromatography revealed that these products constituted a homologous series of oligo-uronides. The fastest moving compound appeared to be an unsaturated mono-uronic acid, 4-deoxy-l-threo-5-hexoseulose uronic acid, whereas indirect evidence showed the others to consist of the unsaturated mono-uronic acid linked by glycosidic bonding to 1, 2, 3, or 4 galacturonic acid moieties. Therefore, the enzyme concerned appears to be an endo-polygalacturonic acidtrans-eliminase. Enzyme extracts prepared from the isolates did not show a hydrolytic activity.The effects of some factors (pH, substrate and enzyme concentrations, inhibitory substances) on enzyme kinetics were investigated.Study of the literature on bacterial pectolytic enzymes suggests that many of these have been wrongly described as depolymerases, polygalacturonases, macerating enzymes, etc., and are better classified astrans-eliminases.     

Production and characterization of polygalacturonase fromGeotrichum candidum

An extracellular polygalacturonase (EC 3.2.1.15) fromGeotrichum candidum ATCC 34614 grown onsauerkraut brine was produced and characterized. Polygalacturonic acid markedly increased the enzyme yield in the brine. The fungus produced the highest activity (290 U/l) in brine with 0.3% (w/v) polygalacturonic acid. The pH and temperature optima of the enzymes were 4.5 to 5.0 and 30°C, respectively. It was stable from pH 4.0 to 5.8 and at 30°C but lost its activity at higher temperatures. The K_m and V_max values for polygalacturonic acid were 4.2 mg/ml and 0.19mm galacturonic acid/min, respectively. The enzyme was not substrate inhibited.     

Regulation of Enzyme Synthesis of the Glyoxylate,the Citric Acid,and the Methylcitric Acid Cycles in Candida lipolytica

Syntheses of the key enzymes of the glyoxylate cycle, in Candida lipolytica, were highly repressed by glucose. Syntheses of the key enzymes of the methylcitric acid cycle were also slightly repressed by glucose but the degrees of repression in the syntheses of these enzymes were nearly equal to those of repression in the syntheses of several enzymes of the citric acid cycle. All enzyme syntheses repressed by glucose were derepressed during incubation with succinate as well as with n-alkanes: enzyme syntheses of the methylcitric acid cycle did not necessitate the addition of propionate or odd-carbon n-alkanes. The enzymes of the methylcitric acid cycle seem to be constitutive, similarly as those of the citric acid cycle.

In the parent strain, the respective enzyme levels of the cells grown on an odd-numbered n-alkane were similar to those of the cells grown on an even-numbered n-alkane. But in the mutant strain lacking 2-methylisocitrate lyase, the cells grown on the odd-numbered alkane contained aconitate hydratase, NADP-Iinked isocitrate dehydrogenase, isocitrate lyase, 2- methylcitrate synthase and 2-methylaconitate hydratase all at higher levels than the cells grown on the even-numbered alkane. Both the parent cells and the mutant cells grown on the same carbon source contained at individually similar levels of the following six enzymes; citrate synthase, NAD-linked isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase, malate dehydrogenase, and malate synthase. The pleiotropic changes of enzyme activities in the mutant cells grown on the odd-numbered alkane seem to be ascribable to direct or indirect stimulation caused by threo-d_s-2-methylisocitric acid accumulation.     

A yeast mutant with glucose-resistant formation of mitochondrial enzymes

Summary Yeast mutants with glucose-insensitive formation of mitochondrial enzymes were isolated starting with a strain completely lacking alcohol dehydrogenase activity. The mutations could uniquely be attributed to a single nuclear gene, designated CCR80. They were largely dominant. Glucose-resistant enzyme formation was most prominent with regard to mitochondrial enzymes succinate dehydrogenase and NADH: cytochrome c oxidoreductase. The effect of CCR80 ^r mutations was rather small but significant on the gluconeogenetic enzymes isocitrate lyase, malate synthase and fructose-1,6-bisphosphatase and on invertase synthesis. The repressive effect of maltose in CCR80 ^r mutants was also reduced showing that glucose-resistance is not caused by a mere hexose uptake defect. This regulatory disorders were not accompanied by reduced levels of glycolytic enzymes or drastically altered levels of glycolytic intermediates.Aerobic fermentation of glucose was almost completely inhibited in the mutants; anaerobic glucose degradation was reduced but not completely abolished. Therefore, the mutants appear to be altered in the regulation of glycolysis. A largely glucose-resistant synthesis of respiratory enzymes is obviously a corollary of this alteration.     

Influence of fructose and glucose on the production of exopolysaccharides and the activities of enzymes involved in the sugar metabolism and the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772

 The effect of fructose and glucose on the growth, production of exopolysaccharides and the activities of enzymes involved in the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus grown in continuous culture was investigated. When grown on fructose, the strain produced 25 mg l^-1 exopolysaccharide composed of glucose and galactose in the ratio 1:2.4. When the carbohydrate source was switched to a mixture of fructose and glucose, the exopolysaccharide production increased to 80 mg l^-1, while the sugar composition of the exopolysaccharide changed to glucose, galactose and rhamnose in a ratio of 1:7.0:0.8. A switch to glucose as the sole carbohydrate source had no further effect. Analysis of the enzymes involved in the synthesis of sugar nucleotides indicates that in cell-free extracts of glucose-grown cells the activity of UDP-glucose pyrophosphorylase was higher than that in cell-free extracts of fructose-grown cells. The activities of dTDP-glucose pyrophosphorylase and the rhamnose synthetic enzyme system were very low in glucose-grown cultures but could not be detected in fructose-grown cultures. Cells grown on a mixture of fructose and glucose showed similar enzyme activities as cells grown on glucose. Analysis of the intracellular level of sugar nucleotides in glucose-grown cultures of L. delbrueckii subsp. bulgaricus showed the presence of UDP-glucose and UDP-galactose in a ratio of 3.3:1, respectively, a similar ratio and slightly lower concentrations were found in fructose-grown cultures. The lower production of exopolysaccharides in cultures grown on fructose may be caused by the more complex pathway involved in the synthesis of sugar nucleotides. The absence of activities of enzymes leading to the synthesis of rhamnose nucleotides in fructose-grown cultures appeared to result in the absence of rhamnose monomer in the exopolysaccharides produced on fructose. Received: 1 February 1996/Received revision: 31 May 1996/Accepted: 2 June 1996     

Enzymes of arginine utilization and their formation in Aeromonas formicans NCIB 9232

In Aeromonas formicans two inducible catabolic pathways of L-arginine have been characterized. The arginine decarboxylase is induced by arginine which also induces the three enzymes of the arginine deiminase pathway but only in stress conditions such as a shift from aerobic growth conditions to very low oxygen tension. Addition of glucose to medium containing arginine leads to repression of the enzymes involved in the arginine deiminase pathway while exogenous cAMP prevents that repression of enzyme synthesis by glucose. This suggests that the induction of arginine deiminase pathway is regulated by carbon catabolite repression and the energetic state of the cell.