Regulation of the synthesis of methanol oxidizing enzymes inKloeckera sp. 2201 andHansenula polymorpha,a comparison

A comparative study was made of the regulation of the synthesis of methanol dissimilating enzymes inkloeckera sp. 2201 andHansenula polymorpha using chemostat and batch growth conditions and methanol or glucose as carbon sources. During growth in methanol-limited chemostat cultures similar enzyme patterns for alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two yeasts were found. When growing in batch culture with glucoseH. polymorpha, but notKloeckera sp. 2201, was found to produce ethanol which might affect the synthesis of these enzymes.     

Effects of cellobiose concentration on the anaerobic growth ofCellulomonas uda

Summary Anaerobic growth ofCellulomonas uda on cellobiose was investigated. With uncontrolled pH, a total metabolic inhibition due to the combined effect of low pH and acid accumulation occurred after 2 g/l cellobiose had been used. This inhibition was partly reduced when the culture pH was maintained at 7.0, but then the growth rate and growth yield were found to be both reduced. Endoglucanase synthesis was related to the cellobiose concentration under these batch growth conditions and markedly influenced by the dilution rate in chemostat.     

The influence of the culture pH value on the direct glucose oxidative pathway in Klebsiella pneumoniae NCTC 418

Klebsiella pneumoniae NCTC 418 was cultured aerobically in chemostat cultures (D=0.3 h^-1; 35°C) under respectively carbon-, phosphate-, potassium-, sulphate-, and ammonia-limited conditions with glucose as the sole carbon and energy source. The effect of the external pH value on glucose metabolism and on the enzymes of the direct glucose oxidative pathway was examined. The pH value of the medium had a profound influence on both the activity and the synthesis of the glucose dehydrogenase and the gluconate dehydrogenase. At pH values ranging from pH 5.5 to pH 6.0 maximal activity and synthesis of these enzymes resulted in a more than 80% conversion of the glucose consumed into gluconate and 2-ketogluconate under potassium-or phosphate-limited conditions. On the other hand, no gluconate and/or 2-ketogluconate production could be detected when K. pneumoniae was cultured at pH 8.0. Whereas the synthesis of gluconate dehydrogenase seemingly was completely repressed, still some glucose dehydrogenase was present. The lack of glucose dehydrogenase activity at pH 8.0 was shown not to be due to the dissociation of the cofactor PQQ from the enzyme.Abbreviations DCIP dichlorophenol indophenol - PQQ pyrroloquinoline quinone [2,7,9-tricarboxy-1H-pyrrolo (2,3-f) quinoline-4,5-dione] - WB Wurster's Blue [1,4-bis-(dimethylamino)-benzene perchlorate]     

Involvement of pyruvate dehydrogenase in product formation in pyruvate-limited anaerobic chemostat cultures of Enterococcus faecalis NCTC 775

Enterococcus faecalis NCTC 775 was grown anaerobically in chemostat culture with pyruvate as the energy source. At low culture pH values, high in vivo and in vitro activities were found for both pyruvate dehydrogenase and lactate dehydrogenase. At high culture pH values the carbon flux was shifted towards pyruvate formate lyase. Some mechanisms possibly involved in this metabolic switch are discussed. In particular attention is paid to the NADH/NAD ratio (redox potential) and the fructose-1,6-bisphosphate-dependent lactate dehydrogenase activity as possible regulatory factors.Abbreviations PDH pyruvate dehydrogenase complex (EC 1.2.2.2) - PFL pyruvate formate lyase (EC 2.3.1.54) - LDH lactate dehydrogenase (EC 1.1.1.27) - FBP fructose-1,6-bisphosphate - MTT 3-(4,5-dimethyl-thiazoyl-2)-2,5-diphenyltetrazolium bromide - TPP thiamine pyrophosphate     

Characterization of the cellulolytic enzyme system from the brown-rot fungus Coniophora puteana

Summary The cellulolytic enzymes of various strains of the brown-rot fungus Coniophora puteana were studied. The organism was grown in an air-lift fermentor in mineral medium containing glucose, cellobiose or amorphous cellulose. The specific growth rate varied between 0.082 and 0.062 h^–1. On amorphous cellulose as sole carbon source, the organism secreted various proteins, some of which were characterized. The mixture contained inter alia four endocellulases, two exo-cellobiohydrolases and a cellobiose dehydrogenase. Three endocellulases (named type I) were active on soluble cellulose derivatives but inactive on p-nitrophenyllactoside (p-NPL), whereas a fourth endocellulase (named type II) was active on both. The two exo-cellobiohydrolases released cellobiose from amorphous cellulose; they were inactive on soluble cellulose derivatives but hydrolyzed p-NPL with strong cellobiose inhibition. A cellobiose dehydrogenase having spectral characteristics compatible with a flavo b-cytochrome was also identified. Neither the exo-cellobiohydrolase nor the type II endocellulase were secreted during growth on cellobiose whereas type I endocellulases and cellobiose dehydrogenase were formed at a reduced rate. No formation of cellulolytic enzymes was observed during growth on glucose alone. Correspondence to: G. Canevascini     

Growth yields and glucose metabolism of N<Subscript>2</Subscript>-fixing <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> at different culture pH values

Gluconacetobacter diazotrophicus was grown in chemostat under N₂-fixing conditions at different culture pH values (from 2.5 to 7.5) with glucose as the C-source. Maximum glucose and oxygen utilization yields were observed at pH values between 5.0 and 6.5. Yields, although lower, were not severely affected at acidic (2.5–4.5) and moderate alkaline (7.5) pH values. But, at pH values just over 7.5, cultures became unstable and washed out. Maximum biomass yields coincided with optimal activity (and minimal synthesis) of pyrroloquinoline quinone (PQQ)-linked glucose dehydrogenase (PQQ-GDH). At external pH values of 7.0 and above, whereas PQQ-GDH was actively synthesized, a very low in situ activity could be detected. The lack of PQQ-GDH activity at moderate alkaline pH values seems to be the cause of lack of growth of this organism under these conditions.     

Cellobiose-oxidizing enzymes from the lignocellulose-degrading basidiomycete Phanerochaete chrysosporium: interaction with microcrystalline cellulose

Summary Cellulose-degrading cultures of the white-rot fungus Phanerochaete chrysosporium produce two extracellular cellobiose-oxidizing enzymes, cellobiose oxidase and cellobiose: quinone oxidoreductase. These two enzymes bind strongly to microcrystalline cellulose (MCC) in the pH range 4–7; above neutral pH their affinity for MCC decreases. Cellulose-bound enzymes could not be eluted with phosphate buffer (20 mM, pH 6) containing polyols (10%), KCl (1 M), urea (1 M) or 1% ionic or non-ionic detergent. TRIS or borate buffer at pH 9 eluted 30%–35% of the cellobiose-oxidizing enzyme activity. The cellulose-immobilized enzymes oxidized cellobiose actively, suggesting that the catalytic sites are not involved in cellulose binding. These results suggest that the cellobiose-oxidizing enzymes of P. chrysosporium may be organized into two domains: a cellulose-binding domain and a catalytic domain.Offprint requests to: V. Renganathan     

Degradation of a Lignin–Carbohydrate Substrate by Soil Fungi Producing Laccase and Cellobiose Dehydrogenase

The growth of nonsporulating mycelial fungi INBI 2-26(+), a producer of laccase; INBI 2-26(–), a producer of cellobiose dehydrogenase; and their mixed culture on lignin–carbohydrate substrates under conditions of submerged fermentation was studied. The degrees of degradation of lignin, cellulose, and hemicellulose of cut straw over 23 days amounted to 29.8, 51.4, and 72% for the laccase producer; 15.8, 33.9, and 59.1% for the cellobiose dehydrogenase producer; and 15.8, 39.4, and 64.5% for the mixed culture, respectively. The laccase activity in the medium when strain 2-26(+) was cultivated individually reached its maximum on day 28; the activity of cellobiose dehydrogenase of strain 2-26(–), on days 14–28. A method for determining cellobiose dehydrogenase activity in the presence of laccase was developed. In the mixed culture, both enzymes were formed; however, the level of laccase synthesis was 1.5-fold lower compared to that of strain 2-26(+), while synthesis of cellobiose dehydrogenase was similar to that of the corresponding producer. Cellobiose dehydrogenase failed to boost the action of laccase while degrading the lignin of straw.     

Degradation of lignin-carbohydrate substrate by soil fungi--producers of laccase and cellobiose dehydrogenase

The growth of nonsporulating mycelial fungi INBI 2-26(+), producer of laccase; INBI 2-26(-), producer of cellobiose dehydrogenase; and their mixed culture on lignin-carbohydrate substrates under conditions of submerged fermentation were studied. The degrees of degradation of lignin, cellulose, and hemicellulose of cut straw over 23 days amounted to 29.8, 51.4, and 72% for the laccase producer; 15.8, 33.9, and 59.1% for the cellobiose dehydrogenase producer; and 15.8, 39.4, and 64.5% for the mixed culture, respectively. The laccase activity in the medium when strain 2-26(+) was cultivated individually reached its maximum on day 28; the activity of cellobiose dehydrogenase of strain 2-26(-), on days 14 to 28. A method for determining cellobiose dehydrogenase activity in the presence of laccase was developed. In the mixed culture, both enzymes were formed; however, the level of laccase synthesis was 1.5-fold lower compared to that of strain 2-26(+), while synthesis of cellobiose dehydrogenase was similar to that of the corresponding producer. Cellobiose dehydrogenase failed to boost the action of laccase while degrading the lignin of straw.     

Hyperbolic growth of Thermoanaerobacter thermohydrosulfuricus (Clostridium thermohydrosulfuricum) increases ethanol production in pH-controlled batch culture

Thermoanaerobacter thermohydrosulfuricus Rt8.B1 exhibited hyperbolic growth (i.e. a continuous rate of growth, without diauxie, during growth and utilization of two carbon sources) on mixed carbohydrate substrates when grown in pH-controlled batch culture. Hyperbolic growth was observed with xylose in combination with either glucose or cellobiose. Diauxic growth ways observed when T. thermohydrosulfuricus Rt8.B1 was grown on a glucose plus cellobiose substrate mix. The major fermentation end-products under all substrate conditions were ethanol and acetate. Ethanol production varied depending on the substrate supplied and was always greatest on mixtures that included xylose (i.e. hyperbolic growth). High ethanol-to-acetate ratios could not be explained on the basis of a greater substrate uptake and thus more ethanol production under these conditions, or by variations in the levels of acetate kinase and NADP-linked alcohol dehydrogenase synthesis. The high ethanol-to-acetate ratio could not be increased by growing T.thermohydrosulfuricus Rt8.B1 under a partial pressure of hydrogen (1 atm) or by growth at different pH. Growth under these conditions decreased the ethanol-to-acetate ratio.Correspondence to: G. M. Cook     

Enzymatic regulation of glucose catabolism by Lactobacillus plantarum in response to pH shifts in a chemostat

Summary The influence of medium pH on the regulation of glucose catabolism by Lactobacillus plantarum 8014 was examined in anaerobic chemostat cultures. When L. plantarum was grown in a chemostat at pH 5.5, and the pH shifted to pH 7.5, acetate was produced in addition to lactate and acetoin. After the shift, acetate kinase and NAD-dependent lactate dehydrogenase activities increased while the acetoin dehydrogenase and alpha-acetolactate synthase activities decreased. The high acetate kinase activity together with low acetoin dehydrogenase and alpha-acetolactate synthase activities may explain why L. plantarum made more acetate at the expense of acetoin in response to alkaline conditions.Offprint requests to: T.J. Montville     

Synthesis and localization of α-amylase and α-glucosidase in Bacillus licheniformis grown in batch and continuous culture

In batch and continuous cultures of Bacillus licheniformis NC1B 6346 α-amylase was invariably extracellular and could not be detected in the cytoplasm or cell surface. α-Glucosidase however, was largely intracellular but at the end of exponential growth and during slow growth under Mg²⁺ limitation it was detected in the culture fluid. Both enzymes were susceptible to catabolite repression and glucose totally inhibited their synthesis in batch culture. In maltose-limited chemostat culture, synthesis of both enzymes was maximal at D = 0.2/h and declined at higher growth rates. α-Amylase synthesis was constitutive but α-glucosidase synthesis was induced by maltose and maltotriose but not by methyl-α-D-glucoside or phenyl-α-D-glucoside. α-Amylase was synthesized at pH 6.5 and above in maltose-limited chemostat culture but not below this pH. Intracellular α-glucosidase synthesis varied little with pH. Increasing temperature decreased the synthesis of both enzymes in chemostat culture to the extent that α-glucosidase was undetectable at 50° C. Polar lipid composition varied with pH and temperature but there was no correlation between this and enzyme secretion. Moreover cerulenin, an antibiotic that inhibits protein secretion in some bacteria by interacting with the membrane had no effect on α-amylase secretion but decreased the release of α-glucosidase upon protoplast formation.     

Cyclic AMP and cyclic GMP control of synthesis of constitutive enzymes in Escherichia coli

Escherichia coli was grown in chemostat culture under glycerol-limited and ammonium-limited conditions at growth rates between 0.1 and 0.5 h-1. At steady state, the concentrations of cyclic AMP and cyclic GMP and the activities of four constitutive enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, NADH oxidase and cyclic phosphodiesterase) were determined in the organism. Addition of exogenous cyclic AMP, cyclic GMP or phencyclidine perturbed the steady state and caused inhibition or stimulation of synthesis of phosphodiesterase and isocitrate dehydrogenase. A novel hypothesis is proposed to account for the ability of bacteria to regulate the synthesis of constitutive enzymes with cyclic nucleotides and possibly other small molecules.     

Purification and properties of an endo-cellulase of avicelase type from Irpex lacteus (Polyporus tulipiferae).

A culture filtrate of Irpex lacteus (Polyporus tulipiferae) was fractionated initially by salting out with ammonium sulfate, and a cellulase [EC 3.2.1.4.] fraction with high Avicel-hydrolyzing activity (formerly called Avicelase) was extensively purified by a series of column chromatography procedures. This purified endo-cellulase showed a less random hydrolytic mechanism, and was obtained in a yield of 0.04% with respect to the starting material. Its specific activity was enhanced approximately 30 times over that of the starting material. The cellulase component showed a single peak on both ultracentrifugal and acrylamide disc electrophoretic analyses. Its molecular weight was estimated to be 56,000. It contained 12.2% carbohydrate; the major sugar constituents were glucose and mannose. Regarding the amino acid composition, the contents of aspartic acid and glycine were highest, followed by those of glutamic acid, serine, and theonine. The cellulase component was not markedly inhibited by most metal ions tested excepted for Hg2+. This purified endo-cellulase attacked a series of cellooligosaccharides, beta-cellobioside, CM-cellulose, and insoluble, cellulosic substrates. In the digests from insoluble substrates, glucose, cellobiose, cellotriose, and cellotetraose were detectable, but the amount of cellobiose was the largest by far. In constrast, cellobiose and glucose were produced in almost equal amounts from beta-cellobioside.     

Formation and release of cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol

Summary Production and release of cellulolytic enzymes by Trichoderma reesei QM 9414 were studied under induced and non-induced conditions. For that purpose, a method was developmed to produce cellulases by Trichoderma reesei QM 9414 using the soluble inducer, cellobiose, as the only carbon source. The production was based on continuous feeding of cellobiose to a batch culture. For optimum production, the cellobiose supply had to be adjusted according to the consumption so that cellobiose was not accumulated in the culture. With a proper feeding program the repression and/or inactivation by cellobiose could be avoided and the cellulase production by Trichoderma reesei QM 9414 was at least equally as high as with cellulose as the carbon source.During the cultivation, specific activities against filter paper, carboxymethyl cellulose (CMC) and p-nitrophenyl glucoside were analyzed from the culture medium as well as from the cytosol and the cell debris fractions. There was a base level of cell debris bound hydrolytic activity against filter paper and p-nitrophenyl glucoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper and CMC hydrolyzing enzymes, which were actively released into the medium even in the early stages of cultivation. -Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing.     

Regulation by ammonium and glucose of Arthrobacter fluorescens alanine dehydrogenase

Alanine dehydrogenase in Arthrobacter fluorescens exhibited an allosteric behaviour and two K _m values for ammonium were estimated. In batch cultures at different ammonium concentrations and in continuous culture following an NH₄ ⁺ pulse, the level of ADH activity seems to be regulated by the ammonium concentration, high activities being observed when extracellular ammonium was in excess. The response to the growth rate of an ammonium-limited chemostat culture of A. fluorescens seems to indicate that alanine dehydrogenase and glutamine synthetase activities were inversely related. High activities of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase have been found in crude extract of ammonium-limited cultures. From the results obtained in batch cultures grown at different glucose concentrations and in carbon-limited chemostat culture it appeared that the limitation by glucose influenced alanine dehydrogenase activity negatively. No glutamate dehydrogenase activity and no glutamate synthase activity could be detected with either NADH or NADPH as coenzymes.Abbreviations ADH alanine dehydrogenase - GS glutamine synthetase - GDH glutamate dehydrogenase - GOGAT glutamine oxoglutarate aminotransferase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase     

Production of quinoprotein d-glucose dehydrogenase in the culture medium of Acinetobacter calcoaceticus

On addition of low concentrations (0.005%) of Triton X-100 to a mineral medium supplemented with 0.5% heptadecane, a marked stimulation of growth rate was observed for Acinetobacter calcoaceticus strains able to grow on alkanes while appreciable amounts of soluble quinoprotein d-glucose dehydrogenase [d-glucose: (pyrroloquinoline-quinone) 1-oxidoreductase, EC 1.1.99.17] were found in the culture medium. At higher Triton X-100 concentrations (0.04%), still larger amounts of d-glucose dehydrogenase and also cytoplasmic enzyme activities appeared in the culture medium. Although combinations of other carbon sources plus non-ionic detergents also produced these enzymes in the medium, the combination of heptadecane and Triton X-100 gave higher levels and had a stabilizing effect on d-glucose dehydrogenase. Therefore, by using this combination and culturing within certain pH limits, a stable enzyme solution, having already a high specific activity, is produced while the cell harvesting and disruption steps can be circumvented. The results indicate that d-glucose dehydrogenase in this organism is a periplasmic enzyme, coupled to a cytochrome b.     

pH oscillations and constant low pH delay the appearance of highly branched (colonial) mutants in chemostat cultures of the quorn(R) myco-protein fungus, Fusarium graminearum A3/5

At pH 5.8, highly branched (colonial) mutants appear in glucose-limited chemostat cultures of Fusarium graminearum A3/5 after ca. 400 h (ca. 107 generations) of growth. The appearance of these mutants was delayed by up to 144 h (45 generations) when the culture was switched at intervals of 120 h between pH 4.8 and 6.6. The concentration of cycloheximide-resistant macroconidia in the culture was used as an indicator of the periodic selection of advantageous mutants and it was found that, in chemostat populations subjected to pH oscillations, the interval (210 +/- 20 h) between peaks was nearly double that observed in chemostat populations cultured at constant pH (124 +/- 12 h at constant pH 5.8 and 120 h +/- 17 h at constant pH 4.5), indicating that the population evolved more slowly under oscillating pH than under constant pH. When grown in mixed culture with the parental strain (A3/5), the selective advantage of two colonial mutants isolated from chemostat cultures grown under conditions of oscillating pH was found to be pH dependent. Compared to cultures grown at constant pH 5.8, a delay of ca. 312 h (87 generations) in the appearance of colonial mutants was observed when F. graminearum A3/5 was grown in glucose-limited chemostat culture at constant pH 4.5. (c) 1996 John Wiley & Sons, Inc.     

Heterologous expression of an Agaricus meleagris pyranose dehydrogenase-encoding gene in Aspergillus spp. and characterization of the recombinant enzyme

Pyranose dehydrogenase (PDH) is a flavin-dependant sugar oxidoreductase found in the family Agaricaceae, basidiomycetes that degrade lignocellulose-rich forest litter, and is catalytically related to the fungal enzymes pyranose 2-oxidase and cellobiose dehydrogenase. It has broad substrate specificity and displays similar activities with most sugar constituents of lignocellulose including disaccharides and oligosaccharides, a number of (substituted) quinones, and metal ions are suitable electron acceptors rather than molecular oxygen. In contrast to pyranose 2-oxidase and cellobiose dehydrogenase, which oxidize regioselectively at C-2 and C-1, respectively, PDH is capable of oxidation on C-1 to C-4 as well as double oxidations, depending on the nature of the substrate. This makes it a very interesting enzyme for biocatalytic applications, as many of the reaction products are otherwise unaccessible by chemical or enzymatic means. PDH was characterized in detail in a limited number of fungi, and the first encoding genes were isolated only recently. We report here, for the first time, the heterologous expression of one of these genes, encoding the major PDH protein in Agaricus meleagris, in the filamentous fungi Aspergillus nidulans, and Aspergillus niger.     

Regulation of the tricarboxylic acid cycle and poly-beta-hydroxybutyrate metabolism in Azotobacter beijerinckii grown under nitrogen or oxygen limitation.

Azotobacter beijerinckii was grown in ammonia-free glucose/mineral salts media in chemostat culture under oxygen or nitrogen limitation. Selected enzymes of the tricarboxylic acid cycle and poly-beta-hydroxybutyrate metabolism were monitored in relation to oxygen supply for both steady and transition states. Two dissolved oxygen concentrations were used for the nitrogen-limited steady state to investigate the possible effects of respiratory protection of nitrogenase on these enzymes. The levels of NADH oxidase, isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase increased markedly on relaxation of oxygen limitation while pyruvate dehydrogenase and citrate synthase were relatively unaffected. beta-Ketothiolase and acetoacetyl-CoA reductase levels decreased as oxygen limitation was relaxed. Respiratory activity, as measured by the QO2 value, increased with oxygen supply rate. Imposition of oxygen limitation on a nitrogen-limited culture caused an immediate increase in the NADH/NAD ratio but this rapidly readjusted to its previous steady-state value. These changes are discussed in relation to respiratory protection of nitrogenase and poly-beta-hydroxybutyrate metabolism in A. beijerinckii.