Production of <Emphasis Type="SmallCaps">d</Emphasis>-arabitol by a newly isolated <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced d-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30°C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 ± 1.1 g d-arabitol from 175 ± 1.1 g glucose per liter at pH 5.0, 30°C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced d-arabitol from fructose, galactose, and mannose. The yeast produced d-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 ± 1.9 g d-arabitol from 175 ± 1.8 g glucose per liter in 210 h at pH 5.0, 30°C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via d-arabitol route. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. department of Agriculture.     

Regulation of calcium fluxes in rat pancreatic islets: Calcium extrusion by sodium-calcium countertransport

Summary The mechanisms by which glucose regulates calcium fluxes in pancreatic endocrine cells were investigated by monitoring the efflux of⁴⁵Ca from prelabeled and perifused rat pancreatic islets. In the absence of both extracellular calcium and glucose, partial or total removal of extracellular sodium decreases the efflux of⁴⁵Ca from prelabeled islets. Glucose also reduces the efflux of⁴⁵Ca from islets perifused in the absence of extracellular calcium. This inhibitory effect of glucose on⁴⁵Ca efflux is decreased by half when the extracellular concentration of sodium is lowered to 24mm. In the absence of extracellular calcium but presence of glucose, partial or even total removal of extracellular sodium fails to decrease the efflux of⁴⁵Ca. At normal extracellular calcium concentration (1mm) partial removal of extracellular sodium dramatically increases⁴⁵Ca efflux from pancreatic islets. This increase in⁴⁵Ca efflux is partially but not totally suppressed by either 16.7mm glucose or cobalt. It is totally suppressed by 4.4mm glucose or by the combination of 16.7mm glucose and cobalt. At normal extracellular calcium concentration, glucose initially reduces and subsequently increases⁴⁵Ca efflux. The initial fall is unaffected by tetrodotoxin but decreased by 50% at low extracellular sodium concentration (24mm). The present results suggest the existence in pancreatic endocrine cells of a glucose-sensitive process of sodium-calcium counter-transport. By inhibiting such a process, glucose may decrease the efflux of calcium from islet cells. The effect of glucose is not mediated by an increase in intracellular sodium concentration. It could contribute to the intracellular accumulation of calcium which is thought to trigger insulin release.This paper is the IVth in a series.     

Certain aspects of the pH dependence of triggering inBacillus megaterium spores

Incubation of unactivatedBacillus megaterium 14581 spores in glucose, or in glucose plusl-alanine, at or below pH 3.6 resulted in germination arrested somewhere before onset of stainability. However, triggering continued at this reduced pH, and spores thus triggered were fully capable of completing the germination sequence in the absence of the germinants once the pH was neutralized. The same spores could be triggered either by a mixture of glucose andl-alanine or by a larger concentration of glucose alone. From this it was concluded that triggering results from an adequate stimulus which can be generated in different ways.l-alanine action in triggering has a pH profile distinct from that of glucose, suggesting that these two germinants have different receptor sites as well. At a level of acidity at which a weak glucose concentration triggered relatively few spores, a much larger fraction was found apparently distributed over a range of sub-triggering levels. Some of these could be made to trigger on transfer to a secondary reagent, or mixture of reagents, which by themselves are not very efficient germinants of the strain studied. The degree of additional triggering was found to depend on the nature of the complementary germinants, as well as on the pH at which glucose stimulated them. Evidence that spores may occupy stimulated states for finite lifetimes is presented and discussed.     

Formation and partial characterization of glucose-2-oxidase,a H2O2 producing enzyme in Phanerochaete chrysosporium

Summary A sugar oxidizing enzyme which produces H₂O₂ during glucose starvation in the white-rot fungus Phanerochaete chrysosporium has been purified from mycelial extracts and somewhat characterized. Enzyme purity was confirmed by analytical isoelectric focusing and by dodecylsulfate/polyacrylamide gel electrophoresis, both techniques revealing a homogeneous protein. The enzyme is active over a broad pH range with maximum activity at pH 7.5. Of several sugars tested, glucose was the preferred substrate although -d-gluconolactone and d-xylose were also oxidized at significant rates (at 60% and 37%, respectively, of the rate observed with glucose). K _m-values for glucose and xylose are 1.03 and 20 mM respectively and the glucose oxidation product was idenitified as d-arabino-2-hexosulose. The possible importance of glucose-2-oxidase in lignin degradation is discussed.     

Extrusion of metabolites from baker’s yeast during glucose-induced acidification

Extrusion of metabolites (glycerol, lactic, malic, and succinic acid) during the medium acidification caused by resting baker’s yeast supplied with 200mm glucose was studied under aerobic and anaerobic conditions and in the absence and presence of 14mm KCl. The maximum levels of glycerol and of the sum of acids (about 13 and 8mm, respectively) were attained anaerobically; aerobiosis reduced the levels by 40–50 % and the presence of K⁺ ions by another 10–20 %. The time courses of glucose consumption and medium acidification were similar aerobically and anaerobically. The glucose consumption ourves exhibited a short plateau about 2 min after glucose addition, caused probably by a rapid osmotic equilibration of glucose across the cell mambrane. Metabolite extrusion indicates that at high glucose concentrations the alcohol dehydrogenase reaction is supplemented by other reactions aiding in the maintenance of a balanced NAD⁺/NADH ratio in the cells.     

A study of the dependence of the human erythrocyte glucose transport system on membrane sulfhydryl groups

Summary A brief review of the data relating the glucose transport system and other membrane functions of red cells to surface sulfhydryl groups is presented. The effect of a variety of sulfhydryl reagents on glucose efflux rates from loaded red cells was studied. Neither iodoacetate nor iodoacetamide at 5mm inhibited efflux. Several maleimide derivatives and disulfides inhibited efflux in 0.7 to 2.0mm concentrations. Organomercury compounds, on the other hand, were active in the 0.07 to 0.1mm range. These data suggest that, if sulfhydryl groups are important in the glucose efflux process, they are not equally accessible to the above reagents; and that the primary effect of these reagents may be on structural elements near membrane sulfhydryl groups.     

An investigation about “anticoagulase”, a ferment said to be produced by staphylococci

Summary 2.75% K-oxalate human plasma was not coagulated below a pH 5.8–5.9, either by a CaCl₂ solution, or by staphylo-coagulase. The prevention of plasma coagulation by the filtrate of a glucose broth culture of staphylococci must be ascribed to the decrease of pH.The experiments made byTillett (4) andTunnicliff andHammond (6) in studying the anticoagulation action of glucose broth cultures of streptococci are herewith confirmed. The neutralizing of the anticoagulating action of a filtrate of a glucose broth culture of staphylococci after treatment with kaolin must also be ascribed to an increase of pH caused by adsorption of acid metabolic products of the staphylococci.Staphylo-coagulase is not destroyed at pH 4.5.     

Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition

Summary The anaerobic glucose uptake (at 20°, pH 3.5) by resting cells of Saccharomyces cerevisiae followed unidirectional Michaelis-Menten kinetics and was competitively inhibited by l-sorbose; K _m and K _i were respectively 5.6×10^-4 m and 1.8×10^-1 m; V _max was 6.5×10^-8 moles mg^-1 min^-1. The aerobic uptake of glucose by resting yeast was also inhibited by l-sorbose but did not follow unidirectional Michaelis-Menten kinetics. Glucose-limited growth in the chemostat of a respiration-deficient mutant of S. cerevisiae was competitively inhibited by l-sorbose. As predicted by theory for transport-limited growth in the chemostat (van Uden, 1967) the steady state glucose concentrations were linear functions of the l-sorbose concentrations with different slopes at different dilution rates; K _m and K _i were respectively 7.2×10^-4 m and 1.8×10^-1 m. It is concluded that glucose transport was the rate-limiting step of anaerobic fermentation of S. cerevisiae and of growth of the mutant and that l-sorbose is a competitive inhibitor of active glucose transport in this yeast. The latter conclusion is accommodated in the transport model of van Steveninck and Rothstein (1965).     

Evaluation ofl-lactic acid production in batch, fed-batch, and continuous cultures ofRhizopus sp. MK-96-1196 using an airlift bioreactor

Various processes which producel-lactic acid using ammonia-tolerant mutant strain,Rhizopus sp. MK-96-1196, in a 3 L airlift bioreactor were evaluated. When the fed-batch culture was carried out by keeping the glucose concentration at 30 g/l, more than 140 g/l ofl-lactic acid was produced with a product yield of 83%. In the case of the batch culture with 200 g/l of initial glucose concentration, 121 g/L ofl-lactic acid was obtained but the low product yield based on the amount of glucose consumed. In the case of a continuous culture, 1.5 g/l/h of the volumetric productivity with a product yield of 71% was achieved at dilution rate of 0.024 h⁻¹. Basis on these results three processes were evaluated by simple variable cost estimation including carbon source, steam, and waste treatment costs. The total variable costs of the fed-batch and continuous cultures were 88% and 140%, respectively, compared to that of batch culture. The fed-batch culture with highl-lactic acid concentration and high product yield decreased variable costs, and was the best-suited for the industrial production ofl-lactic acid.     

A general amino-acid permease is inducible in Chlorella vulgaris

Glucose or non-metabolizable glucose analogues induce two systems of amino-acid transport in Chlorella vulgaris: an arginine-lysine system and a proline system. An additional third system of amino-acid transport is induced when glucose and an inorganic nitrogen source are present during glucose induction. The transport rates in glucose-NH ₄ ⁺ -treated cells are 10 to 80 times higher than in untreated cells. The transport system shows a rather broad specificity and catalyses the transport of at least ten neutral and acidic amino acids. Three of these amino acids (l-alanine, l-serine and glycine) are transported by the proline system as well. The system is specific for l-amino acids and has a pH optimum between 5 and 6. Transport by this system seems to be active, since amino acids are accumulated inside the cells.     

Enhanced l-lysine production in threonine-limited continuous culture of Corynebacterium glutamicum by using gluconate as a secondary carbon source with glucose

In order to improve the production rate of l-lysine, a mutant of Corynebacterium glutamicum ATCC 21513 was cultivated in complex medium with gluconate and glucose as mixed carbon sources. In a batch culture, this strain was found to consume gluconate and glucose simultaneously. In continuous culture at dilution rates ranging from 0.2 h⁻¹ to 0.25 h⁻¹, the specific l-lysine production rate increased to 0.12 g g⁻¹ h⁻¹ from 0.1 g g⁻¹ h⁻¹, the rate obtained with glucose as the sole carbon source [Lee et al. (1995) Appl Microbiol Biotechnol 43:1019–1027]. It is notable that l-lysine production was observed at higher dilution rates than 0.4 h⁻¹, which was not observed when glucose was the sole carbon source. The positive effect of gluconate was confirmed in the shift of the carbon source from glucose to gluconate. The metabolic transition, which has been characterized by decreased l-lysine production at the higher glucose uptake rates, was not observed when gluconate was added. These results demonstrate that the utilization of gluconate as a secondary carbon source improves the maximum l-lysine production rate in the threonine-limited continuous culture, probably by relieving the limiting factors in the lysine synthesis rate such as NADPH supply and/or phosphoenolpyruvate availability. Received: 16 May 1997 / Received revision: 28 August 1997 / Accepted: 29 August 1997     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

Lactate oxidation byTreponema pallidum

Whole cells ofTreponema pallidum consumed O₂ with lactate in a glucose-depleted medium.d(–) Lactate caused marked stimulation of O₂ uptake at a rate similar to that with glucose, whereasl(+) lactate resulted in no increase over the reduced rate observed upon glucose depletion. Lactate oxidation was specific for -hydroxy straight-chain acids of 3,4, and 5 carbons. O₂ uptake during lactate oxidation proceeded independently of pyruvate oxidation and required NAD. The product of lactate oxidation was pyruvate.d(–) Lactate-stimulate O₂ uptake was sensitive to chlorpromazine and resistant to amytal and cyanide. Glucose did not inhibit the oxidation of lactate as shown by the additive effect of both substrates on O₂ uptake. Oxidation of glucose, but not lactate, provided energy necesary for motilibty or maintenance of virulence. A mixture of lactate isomers was formed from glucose with thel(+) isomer concentration remaining constant and thed(–) isomer concentration varying inversely with dissolved O₂ concentration. The function of lactate as an oxidizable substrate is apparently quite distinct from that of glucose.     

Effect of 2-deoxy-d-glucose on maintenance in culture of neonatal B cell of rat

Summary The effect of 2-deoxy-d-glucose on maintenance in culture of B cells of the neonatal rat was examined by supplementation of Medium 199 containing 5.5 mM glucose with 1 mM 2-deoxy-d-glucose. Islets maintained in medium with 5.5 mM glucose (basal medium) for 7 d underwent remarkable decreases in glucose sensitivity, and the levels of insulin in the medium dropped. By contrast, addition of 2-deoxy-d-glucose promoted a higher insulin content in medium and an increase in the glucose-induced insulin release and biosynthesis. Moreover, the addition of the deoxysugar caused a selective deletion of fibroblasts and prevented the deterioration of islet cells in basal medium, yielding clusters mostly consisting of islet cells at the end of culture.     

Pretreatment with 3-O-methyl-d-glucose or 2-deoxy-d-glucose attenuates the post-mortem rise in rat brain lactate

The present investigation examined the effects of pretreatment with 3-O-methyl-d-glucose (3OMG) or 2-deoxy-d-glucose (2DOG) on post-mortem rise in rat brain lactate to evaluate their potential use for minimizing ischemia-induced rise in brain lactate. The results showed that iv administration of either glucose analogue (2 g/kg) at 2.5 min prior to sacrifice significantly attenuated (to 0.61 of control levels) post-mortem brain lactate rise. Pretreating rats with 2-deoxy-d-glucose (2 g/kg) 15 min prior to sacrifice resulted in a greater inhibition (to 0.52 of control) of the post-mortem lactate rise. The effects of these two analogues (3OMG and 2DOG) can be accounted for by their inhibition of brain glucose transport and inhibition of brain glucose metabolism by 2DOG. The present results suggest that intervention with either of these glucose analogues under the proper experimental procedures may minimize the cytopathological consequences of ischemia related to the rise in brain lactate.     

Rat L6 myotubes as an in vitro model system to study GLUT4-dependent glucose uptake stimulated by inositol derivatives

Some of inositol derivatives have been reported to help the action of insulin stimulating glucose uptake in skeletal muscle cells. Rat L6 myotubes were employed in an attempt to develop an in vitro model system for investigation of the possible insulin-like effect of eight inositol derivatives, namely allo-inositol, d-chiro-inositol l-chiro-inositol, epi-inositol, muco-inositol, myo-inositol, scyllo-inositol and d-pinitol. At a higher concentration of 1 mM seven inositol derivatives other than myo-inositol were able to stimulate glucose uptake, while at 0.1 mM only d-chiro-inositol, l-chiro-inositol, epi-inositol and muco-inositol could induce glucose uptake, indicating their significant insulin-mimetic activity. Immunoblot analyses revealed that at least d-chiro-inositol, l-chiro-inositol, epi-inositol, muco-inositol and d-pinitol were able to induce translocation of glucose transporter 4 (GLUT4) to plasma membrane not only in L6 myotubes but also in skeletal muscles of rats ex vivo. These results demonstrated that L6 myotubes appeared efficient as an in vitro system to identify inositol derivatives exerting an insulin-like effect on muscle cells depending on the induced translocation of GLUT4.     

Roles of glucose and acetate as carbon sources in<Emphasis Type="SmallCaps">l</Emphasis>-histidine production with<Emphasis Type="Italic">Brevibacterium flavum</Emphasis> FERM1564 revealed by metabolic flux analysis

The metabolic flux pattern forl-histidine production was analyzed when glucose and/or acetate were used as carbon sources. Totall-histidine production was enhanced when mixed substrate (glucose and acetate) was used, compared with that when either glucose or acetate was used as the sole carbon source. Theoretical maximum carbon fluxes through the main pathways forl-histidine production, cell growth, and ATP consumption for cell maintenance were obtained by the linear programming (LP) method. By comparison of the theoretical maximum carbon fluxes with actual ones, it was found that a large amount of glucose was actually used for maintenance of cell viability. On the other hand, acetate was used for cell growth. After depletion of acetate in the mixed substrate culture, the flux for glucose tol-histidine synthesis was markedly enhanced. A strategy for effectivel-histidine production using both carbon sources was proposed.     

A malic acid dependent mutant of Schizosaccharomyces malidevorans

The yeast Schizosaccharomyces malidevorans utilizes l-malate when grown on glucose as the carbon source. A mutant of this yeast has been isolated which is dependent on the presence of both l-malate and glucose for growth. The mutant utilizes l-malate as rapidly as the wildtype and the utilization of glucose is greatly reduced. Other TCA cycle intermediates do not relieve the malate dependence.To John Ingraham whose pioneering work with malolactic bucteria made me curious enough about the field of nine microbiology to enter it and whose intense instruction in scientific method has made my continued pursuit of physiological and genetic questions a joy     

Carbon sources,growth, sclerotium formation and carbohydrate composition of Sclerotinia sclerotiorum

Summary Sclerotinia sclerotiorum (Lib.) D By. was grown in stationary liquid mineral-salts medium, pH 4.3, containing various carbon sources and the weight of mycelia and sclerotia was determined at regular intervals. When grown on various glucose concentrations (0–24 g of C/l), more sclerotia were produced at 8–12 g of C/l. Sclerotia were not usually formed in shake cultures. The ability of the fungus to use other carbon sources for growth and sclerotium formation was tested at 12 g of C/l in the stationary mineral-salts medium. The highest weights of mycelia and sclerotia occurred with raffinose, sucrose, maltose, lactose, d-mannose, d-glucose, d-fructose or l-arabinose. Good growth but decreased sclerotium production were found on cellobiose and d-xylose. Reduced or poor growth, a long lag period and few or no sclerotia occurred on trehalose, melibiose, l-sorbose, l-rhamnose, d-ribose, d-arabinose, l-xylose or 8 polyols. No growth was observed with erythritol or i-inositol. A combination of glucose plus trehalose or polyols resulted in increased growth and the formation of sclerotia. Organic acids supported little or no growth and no sclerotia were produced. Generally culture filtrates which supported growth and formation of sclerotia became acid (about pH 3.5). The pH of the culture filtrate usually increased slowly during the growth period when the fungus grew poorly and no sclerotia were formed. The alcoholsoluble sugars and polyols present in culture filtrates, mycelia and sclerotia were determined by paper and thin-layer chromatography. Regardless of the carbon source, mannitol was usually present in culture filtrates. The occurrence of other compounds in the filtrates depended on the carbon source. Trehalose, mannitol and usually small quantities of glucose or fructose were present in mycelia and sclerotia from all carbon sources. Galactitol or pentitols occurred in mycelia and sclerotia when the fungus grew on galactose and oligosaccharides containing galactose or the corresponding pentose, sugars. Acid hydrolyzates of the alcohol-insoluble fraction of mycelia or sclerotia contained glucose, smaller amounts of galactose and mannose and traces of ribose and rhamnose.     

Effects of complete cell recycling on product formation byLactobacillus casei ssp.rhamnosus in continuous cultures

The lactic acid bacteriumLactobacillus casei ssprhamnosus was cultivated in a system with complete cell recycling in order to obtain information on how this cultivation technique affected the microorganisms. Cultivations at two different glucose concentrations (25 g/L and 50 g/L) were performed. Hollow fiber filters were used for separating the cells from the spent broth. The cell recycling was carried out for 128–135 h. Samples were taken three to four times daily for analysis ofd- andl-lactate, glucose concentration, and cell mass. Protein patterns were studied with two-dimensional electrophoresis. A change in the protein pattern was observed. Dry weight of cell mass of 86 g/L was obtained when cultivated on 50 g glucose/L, which was approximately 15 times more than in the batch culture. The percentage ofd-lactate of the total lactate increased with time; when cultivated on 25 g glucose/L, it increased from 3% to 13%. No racemase was detected by the methods used. The data collected from these two recycling experiments show that this is an efficient way to obtain high cell densities, but that the method can affect the product formation pattern of the microorganism.