beta-Glucose-1-Phosphate, a Possible Mediator for Polysaccharide Formation in Maltose-Assimilating Lactococcus lactis

Homolactic fermentation of glucose and heterolactic fermentation of maltose with Lactococcus lactis 65.1 were confirmed. When moles of glucose were compared, the uptake rates of the two carbon sources were similar. The intracellular concentration of fructose-1,6-diphosphate (FDP) in maltose-assimilating cells was half of that in glucose-assimilating cells. Similarly, formation of FDP and lactate from maltose by extracts of maltose-grown cells was half of that formed from glucose by extracts of glucose-grown cells, indicating a difference in the utilization of the two carbon sources for energy metabolism. Concentrations of adenine nucleotides were similar in both types of cells. Glucose-1-phosphate was found in extracts of maltose-grown cells given maltose and, in addition, an inducible and low beta-specific phosphoglucomutase activity was observed. beta-Glucose-1-phosphate was not metabolized by cell extracts to either FDP or lactate, suggesting an alternative metabolic route. The amount of [C]maltose incorporated into the cell material of maltose-grown cells was four times greater than that of [C]glucose incorporated into the cell material of glucose-grown cells. The intracellular concentration of UTP was lower in maltose-assimilating cells than in glucose-assimilating cells. Cells grown on maltose were more spherical and less fragile than cells grown on glucose.     

Physiological Role of β-Phosphoglucomutase in Lactococcus lactis

A β-phosphoglucomutase (β-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h⁻¹, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h⁻¹ on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

Some aspects of catabolite repression of mitochondrial enzymes inSaccharomyces cerevisiae

The synthesis of mitochondrial enzymes inSaccharomyces cerevisiae is partly derepressed during growth with maltose as compared with glucose. The present investigations were aimed at finding any more differences between maltose- and glucose-grown cultures that might indicate the nature of the effector(s) of catabolite repression.The capacity of the pentose-phosphate pathway in resting cells is the same whether they have been grown on maltose or on glucose; about 9% of the sugar is metabolized via this pathway. The activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are the same in both cultures. There is, however, a correlation between the repression of aconitase synthesis by glucose and the intracellular level of glucose-6-phosphate.There are no differences between maltose- and glucose-grown cultures in the concentration of structural and reserve carbohydrates, RNA and proteins, except for glycogen. In young maltose-grown cultures the level of this polysaccharide is about 25% higher.The growth rate and the rate of sugar consumption per mg of cells in maltose-grown cultures are about 30% lower than in glucose-grown cultures. Probably the restriction of sugar consumption in maltose-grown cultures results in less accumulation of catabolites and in partial derepression of the synthesis of mitochondrial enzymes.The author is much indebted to Mrs. M. Vermeulen-Verdam for her skillful and enthusiastic assistance. He also thanks Mr. J. H. M. Bex, Mr. L. van Dijk, Mr. E. A. H. Huisman and Mr. A. de Wit for their cooperation in some parts of the present work.     

Glycogen Formation by the Ruminal Bacterium Prevotella ruminicola

Prevotella ruminicola is an important ruminal bacteria. In maltose-grown cells, nearly 60% of cell dry weight consisted of high-molecular-weight (>2 x 10(sup6)) glycogen. The ratio of glycogen to protein (grams per gram) was relatively low (1.3) during exponential growth, but when cell growth slowed during the transition to the stationary phase, the ratio increased to 1.8. As much as 40% of the maltose was converted to glycogen during cell growth. Glycogen accumulation in glucose-grown cells was threefold lower than that in maltose-grown cells. In continuous cultures provided with maltose, much less glycogen was synthesized at high (>0.2 per h) than at low dilution rates, where maltose was limiting (28 versus 60% of dry weight, respectively). These results indicated that glycogen synthesis was stimulated at low growth rates and was also influenced by the growth substrate. In permeabilized cells, glycogen was synthesized from [(sup14)C]glucose-1-phosphate but not radiolabelled glucose, indicating that glucose-1-phosphate is the initial precursor of glycogen formation. Glycogen accumulation may provide a survival mechanism for P. ruminicola during periods of carbon starvation and may have a role in controlling starch fermentation in the rumen.     

The influence of limiting and non-limiting growth conditions on glucose and maltose metabolism in Lactococcus lactis ssp. lactis strains

Three strains of Lactococcus lactis ssp. lactis, a dairy strain 65.1, a type strain ATCC 19435, and a mutant AS 211, were grown on glucose and on maltose under chemostat conditions. When the culture was shifted from glucose-limiting to non-limiting conditions, the product shifted from mixed acids to lactate. Mixed acids were obtained in all maltose cultures; however, an enhanced lactate formation was observed in 19435 and AS 211. An inorganic-phosphate (P_i)-dependent maltose phosphorylase activity was found to be responsible for the initial conversion of maltose. The activation of maltose phosphorylase by Pi was strain-specific. When growth was on maltose under non-limiting conditions, a correlation was found between high initial maltose phosphorylase and -phosphoglucomutase activities and lactate production. No such correlation was observed in maltose-limited cells. In glucose-grown cells under non-limiting conditions, homo-fermentative lactate formation coincided with high concentrations of fructose 1,6-bisphosphate (Fru1,6P ₂) and pyruvate (Pyr) and low concentrations of phosphoenolpyruvate (PPyr). Under limiting conditions, mixed acid formation coincided with low concentrations of Fru1,6P ₂ and Pyr and high concentrations of PPyr. In maltose-grown cells there was no correlation between intracellular intermediary metabolite concentrations and product formation. Therefore, in addition to intracellular intermediary metabolite concentrations, the product formation on maltose is suggested to be regulated by the transport and initial phosphorylating steps.     

Purification and characterization of two phosphoglucomutases from Lactococcus lactis subsp. lactis and their regulation in maltose- and glucose-utilizing cells.

Two distinct forms of phosphoglucomutase were found in Lactococcus lactis subsp. lactis, strains 19435 and 65.1, growing on maltose: beta-phosphoglucomutase (beta-PGM), which catalyzes the reversible conversion of beta-glucose 1-phosphate to glucose 6-phosphate in the maltose catabolism, and alpha-phosphoglucomutase (alpha-PGM). beta-PGM was purified to more than 90% homogeneity in crude cell extract from maltose-grown lactococci, and polyclonal antisera to the enzyme were prepared. The molecular mass of beta-PGM was estimated by gel filtration to be 28 kDa; its isoelectric point was 4.8. The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively. The expression of both PGM enzymes was investigated under different growth conditions. The specific activity and amount of beta-PGM per milliliter of cell extract increased with time in lactococci grown on maltose, but the enzyme was absent in lactococci grown on glucose, indicating enzyme synthesis to be induced by maltose in the growth medium. When glucose was added to maltose-grown lactococci, both the specific activity and amount of beta-PGM per milliliter of cell extract decreased rapidly. This suggests that synthesis of beta-PGM is repressed by glucose in the medium. Although the specific activity of alpha-PGM did not change during growth on maltose or glucose, lactococcal strain 19435 showed a much higher specific activity of both alpha- and beta-PGM than strain 65.1 when grown on maltose.     

High-Affinity Maltose Binding and Transport by the Thermophilic Anaerobe Thermoanaerobacter ethanolicus 39E

Thermoanaerobacter ethanolicus is a gram-positive thermophile that produces considerable amounts of ethanol from soluble sugars and polymeric substrates, including starch. Growth on maltose, a product of starch hydrolysis, was associated with the production of a prominent membrane-associated protein that had an apparent molecular weight of 43,800 and was not detected in cells grown on xylose or glucose. Filter-binding assays revealed that cell membranes bound maltose with high affinity. Metabolic labeling of T. ethanolicus maltose-grown cells with [¹⁴C]palmitic acid showed that this protein was posttranslationally acylated. A maltose-binding protein was purified by using an amylose resin affinity column, and the binding constant was 270 nM. Since maltase activity was found only in the cytosol of fractionated cells and unlabeled glucose did not compete with radiolabeled maltose for uptake in whole cells, it appeared that maltose was transported intact. In whole-cell transport assays, the affinity for maltose was approximately 40 nM. Maltotriose and α-trehalose competitively inhibited maltose uptake in transport assays, whereas glucose, cellobiose, and a range of disaccharides had little effect. Based on these results, it appears that T. ethanolicus possesses a high-affinity, ABC type transport system that is specific for maltose, maltotriose, and α-trehalose.     

Glycogen biosynthesis via UDP-glucose in the ruminal bacterium Prevotella bryantii B1(4).

Prevotella bryantii is an important amylolytic bacterium in the rumen that produces considerable amounts of glycogen when it is grown on maltose. Radiolabel studies indicated that glucose-1-phosphate was converted to UDP-glucose, and this latter intermediate served as the immediate precursor for glycogen synthesis. High levels of UDP-glucose pyrophosphorylase activities (> 1,492 nmol/min/mg of protein) were detected in cells grown on maltose, cellobiose, glucose, or sucrose, and activity was greatly stimulated (by approximately 60-fold) by the addition of fructose-1,6-bis phosphate (half-maximal activation concentration was 240 microM). However, ADP-glucose pyrophosphorylase activity was not detected in any of the cultures. Glycogen synthase activity in maltose-grown cultures (48 nmol/min/mg of protein) was higher than that in cellobiose-, sucrose-, and glucose-grown cultures (< 26 nmol/min/mg of protein). This is the first report of a bacterium that exclusively uses UDP-glucose to synthesize glycogen. The elucidation of this unique glycogen biosynthesis pathway provides information necessary to further investigate the role of bacterial glycogen accumulation in rumen metabolism.     

Physiological role of beta-phosphoglucomutase in Lactococcus lactis

A beta-phosphoglucomutase (beta-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of beta-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h(-1), while the deletion of beta-PGM resulted in a maximum specific growth rate of 0.05 h(-1) on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as beta-glucose 1-phosphate in the medium. Furthermore, the beta-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of alpha-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the beta-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded beta-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

Mechanism of maltose uptake and glucose excretion in Lactobacillus sanfrancisco.

Lactobacillus sanfrancisco LTH 2581 can use only glucose and maltose as sources of metabolic energy. In maltose-metabolizing cells of L. sanfrancisco, approximately half of the internally generated glucose appears in the medium. The mechanisms of maltose (and glucose) uptake and glucose excretion have been investigated in cells and in membrane vesicles of L. sanfrancisco in which beef heart cytochrome c oxidase had been incorporated as a proton-motive-force-generating system. In the presence of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), and cytochrome c, the hybrid membranes facilitated maltose uptake against a concentration gradient, but accumulation of glucose could not be detected. Similarly, in intact cells of L. sanfrancisco, the nonmetabolizable glucose analog alpha-methylglucoside was taken up only to the equilibration level. Selective dissipation of the components of the proton and sodium motive force in the hybrid membranes indicated that maltose is transported by a proton symport mechanism. Internal [14C]maltose could be chased with external unlabeled maltose (homologous exchange), but heterologous maltose/glucose exchange could not be detected. Membrane vesicles of L. sanfrancisco also catalyzed glucose efflux and homologous glucose exchange. These activities could not be detected in membrane vesicles of glucose-grown cells. The results indicate that maltose-grown cells of L. sanfrancisco express a maltose-H+ symport and glucose uniport system. When maltose is the substrate, the formation of intracellular glucose can be more rapid than the subsequent metabolism, which leads to excretion of glucose via the uniport system.     

Presence of lactate dehydrogenase and lactate racemase in Megasphaera elsdenii grown on glucose or lactate.

Activity of D-lactate dehydrogenase (D-LDH) was shown not only in cell extracts from Megasphaera elsdenii grown on DL-lactate, but also in cell extracts from glucose-grown cells, although glucose-grown cells contained approximately half as much D-LDH as DL-lactate-grown cells. This indicates that the D-LDH of M. elsdenii is a constitutive enzyme. However, lactate racemase (LR) activity was present in DL-lactate-grown cells, but was not detected in glucose-grown cells, suggesting that LR is induced by lactate. Acetate, propionate, and butyrate were produced similarly from both D- and L-lactate, indicating that LR can be induced by both D- and L-lactate. These results suggest that the primary reason for the inability of M. elsdenii to produce propionate from glucose is that cells fermenting glucose do not synthesize LR, which is induced by lactate.     

Phosphorus-31 NMR studies of maltose and glucose metabolism in Streptococcus lactis

Summary Streptococcus lactis ferments glucose in a homolactic fashion but a heterolactic fermentation pattern is observed when it is grown on maltose. Using in vivo phosphorus-31 and carbon-13 NMR studies of glucose-metabolizing cells we confirmed that fructosediphosphate (FDP) is the major glycolytic intermediate and that the production of lactate causes major changes both in the intra- and extracellular pH values. Starved cells contain mainly 3-phosphoglycerate (3-PGA) and some phosphoenolpyruvate (PEP). Metabolism of maltose also brings about major changes in pH, but it was unclear from the poorly resolved in vivo spectra if FDP was the main glycolytic intermediate present. This question was further studied by analyzing perchloric acid extracts by phosphorus-31 NMR. These studies showed that glucose-metabolizing cells have higher levels of FDP and lower levels of inorganic phosphate (P _i ) than maltose-metabolizing cells. 3-PGA always remained present in the latter cells suggesting that these exist in a semi-starved state which is probably the reason for their heterolactic fermentation pattern. In the course of these studies we also examined the effects of the inhibitors 2-deoxyglucose, fluoride and iodoacetate. We could demonstrate that by judicious choice of carbon sources and inhibitors one could completely reduce the intracellular P _i pool. This suggests that one should be able to regulate the shift from heterolactic to homolactic fermentation, as P _i is considered to be the most potent inhibitor of pyruvate kinase in these cells.Sponsored by grants from the Swedish Natural Sciences Research Council (NFR) (to BHH and HJV), the Biotechnology Research Foundation (SBF) (to BHH) and the Canadian Natural Sciences Research Council (NSERC) (to HJV)     

Molecular subtyping within a single Salmonella typhimurium phage type, DT204c, with a PCR-generated probe for IS200

Abstract Pullulan is an industrial biopolymer produced by the yeast-like fungus Aureobasidium , usually by direct fermentation of starch. Despite evidence that autogenous amylases produced during these fermentations are detrimental to the final molecular mass of the product, fundamental studies of these enzymes have not been reported. Total extracellular amylases were studied from the promising production strain NRRL Y-12,974. Growth rates and yields were equivalent in cultures grown on glucose, maltose, soluble starch, or cornstarch. Total amylase levels were low and varied only three-fold, from 0.01 IU ml⁻¹ in glucose-grown cultures to 0.03 IU ml⁻¹ in soluble-starch-grown cultures. All cultures showed both α-amylase activity and activity against pullulan. Synthetic oligosaccharide substrates were apparently attacked by an α-glucosidase, produced in highest levels by maltose-grown cultures.     

Intermediary Metabolite Concentrations in Xylulose- and Glucose-Fermenting Saccharomyces cerevisiae Cells

Glucose and xylulose fermentation and product formation by Saccharomyces cerevisiae were compared in batch culture under anaerobic conditions. In both cases the main product was ethanol, with glycerol, xylitol, and arabitol produced as by-products. During glucose and xylulose fermentation, 0.74 and 0.37 g of cell mass liter⁻¹, respectively, were formed. In glucose-fermenting cells, the carbon balance could be closed, whereas in xylulose-fermenting cells, about 25% of the consumed sugar carbon could not be accounted for. The rate of sugar consumption was 3.94 mmol g of initial biomass⁻¹ h⁻¹ for glucose and 0.39 mmol g of initial biomass⁻¹ h⁻¹ for xylulose. Concentrations of the intermediary metabolites fructose-1,6-diphosphate (FDP), pyruvate (PYR), sedoheptulose 7-phosphate (S7P), erytrose 4-phosphate, citrate (CIT), fumarate, and malate were compared for both types of cells. Levels of FDP, PYR, and CIT were lower, and levels of S7P were higher in xylulose-fermenting cells. After normalization to the carbon consumption rate, the levels of FDP were approximately the same, whereas there was a significant accumulation of S7P, PYR, CIT, and malate, especially of S7P, in xylulose-fermenting cells compared with in glucose-fermenting cells. In the presence of 15 μM iodoacetate, an inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), FDP levels increased and S7P levels decreased in xylulose-assimilating cells compared with in the absence of the inhibitor, whereas fermentation was slightly slowed down. The specific activity of transaldolase (EC 2.2.1.2), the pentose phosphate pathway enzyme reacting with S7P and glyceraldehyde-3-phosphate, was essentially the same for both glucose- and xylulose-fermenting cells. It was, however, several orders of magnitude lower than that reported for a Torula yeast and Candida utilis. The presence of iodoacetate did not influence the activity of transaldolase in xylulose-fermenting cells. The results are discussed in terms of a competition between the pentose phosphate pathway and glycolysis for the common metabolite, glyceraldehyde-3-phosphate, which would explain the low rates of xylulose assimilation and ethanol production from xylulose by S. cerevisiae.     

Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures.

Lactic streptococci, classically regarded as homolactic fermenters of glucose and lactose, became heterolactic when grown with limiting carbohydrate concentrations in a chemostat. At high dilution rates (D) with excess glucose present, about 95% of the fermented sugar was converted to l-lactate. However, as D was lowered and glucose became limiting, five of the six strains tested changed to a heterolactic fermentation such that at D = 0.1 h(-1) as little as 1% of the glucose was converted to l-lactate. The products formed after this phenotypic change in fermentation pattern were formate, acetate, and ethanol. The level of lactate dehydrogenase, which is dependent upon ketohexose diphosphate for activity, decreased as fermentation became heterolactic with Streptococcus lactis ML(3). Transfer of heterolactic cells from the chemostat to buffer containing glucose resulted in the nongrowing cells converting nearly 80% of the glucose to l-lactate, indicating that fine control of enzyme activity is an important factor in the fermentation change. These nongrowing cells metabolizing glucose had elevated (ca. twofold) intracellular fructose 1,6-diphosphate concentrations ([FDP](in)) compared with those in the glucose-limited heterolactic cells in the chemostat. [FDP](in) was monitored during the change in fermentation pattern observed in the chemostat when glucose became limiting. Cells converting 95 and 1% of the glucose to l-lactate contained 25 and 10 mM [FDP](in), respectively. It is suggested that factors involved in the change to heterolactic fermentation include both [FDP](in) and the level of lactate dehydrogenase.     

Phosphorylating enzymes involved in glucose fermentation of Actinomyces naeslundii.

Enzymatic activities involved in glucose fermentation of Actinomyces naeslundii were studied with glucose-grown cells from batch cultures. Glucose could be phosphorylated to glucose 6-phosphate by a glucokinase that utilized polyphosphate and GTP instead of ATP as a phosphoryl donor. Glucose 6-phosphate was further metabolized to the end products lactate, formate, acetate, and succinate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase was only PPi. Phosphoglycerate kinase, pyruvate kinase, and acetate kinase coupled GDP as well as ADP, but P(i) compounds were not their phosphoryl acceptor. Cell extracts showed GDP-dependent activity of phosphoenolpyruvate carboxykinase, which assimilates bicarbonate and phosphoenolpyruvate into oxaloacetate, a precursor of succinate. Considerable amounts of GTP, polyphosphate, and PPi were found in glucose-fermenting cells, indicating that these compounds may serve as phosphoryl donors or acceptors in Actinomyces cells. PPi could be generated from UTP and glucose 1-phosphate through catalysis of UDP-glucose synthase, which provides UDP-glucose, a precursor of glycogen.     

Involvement of oxygen-sensitive pyruvate formate-lyase in mixed-acid fermentation by Streptococcus mutans under strictly anaerobic conditions

Streptococcus mutans JC2 produced formate, acetate, ethanol, and lactate when suspensions were incubated with an excess of galactose or mannitol under strictly anaerobic conditions. The galactose- or mannitol-grown cell suspensions produced more formate, acetate, and ethanol than the glucose-grown cells even when incubated with glucose. The levels of lactate dehydrogenase and fructose 1,6-bisphosphate were not significantly different in these cells, but the level of pyruvate formate-lyase was higher in the galactose- or mannitol-grown cells, and that of triose phosphate was lower in the galactose-grown cells. This suggests that the regulation of pyruvate formate-lyase may play a major role in the change of the fermentation patterns. The cells of S. mutans grown on glucose produced a significant amount of volatile products even in the presence of excess glucose under strictly anaerobic conditions. However, when the anaerobically grown cells were exposed to air, only lactate was produced from glucose. When cells were anaerobically grown on mannitol and then exposed to air for 2 min, only trace amounts of fermentation products were formed from mannitol under anaerobic conditions. It was found that the pyruvate formate-lyase in the cells was inactivated by exposure of the cells to air.     

Metabolism of the reserve polysaccharide of Streptococcus mitis: Properties of a transglucosylase

1. A transglucosylase has been separated from cell extracts of Streptococcus mitis, and has been partially purified by chromatography on DEAE-cellulose. 2. The transglucosylase was present in the six strains of Streptococcus mitis that were examined, and the activity of the enzyme was the same whether the cells had grown on glucose or on maltose. Four of the strains could store intracellular iodophilic polysaccharide when grown on high concentrations of glucose or maltose (1%), but none of the strains stored polysaccharide during growth on 0·1% glucose. The activity of transglucosylase in cell extracts was the same whether or not the cells had stored polysaccharide. 3. The transglucosylase degrades amylose in the presence of a suitable acceptor, transferring one or more glucosyl residues from the non-reducing end of the donor to the non-reducing end of the acceptor. With [¹⁴C]glucose as acceptor the maltodextrins produced were labelled in the reducing glucose unit only. 4. The enzyme can synthesize higher maltodextrins from maltose and maltotriose. Maltotetraose is disproportionated to give products of sufficient chain length to give a stain with iodine. 5. The action pattern of S. mitis during the degradation of synthetic amylose was shown to be intermediate between the single-chain and multi-chain mechanism.     

Improvement in lactic acid production from starch using α-amylase-secreting <Emphasis Type="Italic">Lactococcus lactis</Emphasis> cells adapted to maltose or starch

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l⁻¹ h⁻¹ lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l⁻¹ h⁻¹ lactate). Maximum volumetric lactate productivity was further increased (1.57 g l⁻¹ h⁻¹ lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.     

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