Transient responses of <Emphasis Type="Italic">Wickerhamia</Emphasis> sp. yeast continuous cultures to qualitative changes in carbon source supply: induction and catabolite repression of α-amylase synthesis

The purpose of this study was to evaluate the inductive effect of starch and maltose, and the repressive/inhibitory effect of glucose, on amy-1 gene expression and α-amylase production by Wickerhamia sp., using continuous culture under transient-state conditions at a dilution rate (D) of 0.083 h^?1. Induction and repression kinetics of α-amylase were studied by changing the medium feed from glucose to maltose or starch in the induction experiments and vice versa in the repression experiments. Expression levels of amy-1 gene were measured by RT-qPCR. Results showed that starch was a more efficient inducer of α-amylase synthesis compared to maltose, with maximum accumulation rate constants of 0.424 and 0.191 h^?1, respectively. In contrast, α-amylase synthesis in starch and maltose cultures was partially repressed by glucose as indicated by a specific activity close to basal levels and a decay constant rate (??0.065 and ??0.069 h^?1, respectively) higher than ??D. A linear dependence of the specific rate of α-amylase production on mRNA relative abundance of amy-1 gene was observed. An inhibitory effect of glucose was not observed even at a concentration of 30 g L^?1. In conclusion, the transient continuous culture is a useful tool to determine the qualitative and quantitative effects of maltose and starch on α-amylase induction and of glucose on enzyme repression, as well as to obtain a detailed understanding of the dynamic behavior of the yeast culture. Furthermore, results showed that amylaceous substrates can be very effective carbon sources for the production of α-amylase without being inhibited by glucose.     

Starch degradation by the mould Trichoderma viride I. The mechanism of starch degradation

The mechanism of starch degradation by the fungus Trichoderma viride was studied in strain CBS 354.44, which utilizes glucose, starch and dextrins but is unable to assimilate maltose. It was shown that the amylolytic enzyme system is completely extracellular, equally well induced by starch, amylose or amylopectin and that it consists mainly of enzymes of the glucoamylase type which yield glucose as the main product of starch hydrolysis. Small amounts of -amylase are produced also. The enzymes produced in starch cultures degrade starch, amylose and amylopectin equally well.Enzyme synthesis in starch media takes place to a considerable extent after exhaustion of the carbon source when maximum growth has been attained.Low-molecular dextrins are degraded by extracellular enzymes of the glucoamylase type. These enzymes are produced in media containing starch or dextrins. Maltotriose is consumed for only one third leaving maltose in the culture filtrate. Maltose is hardly attacked and hardly induces any amylolytic enzyme activity. No stable -glucosidase appears to be produced.     

Specificity of the dehydrogenases of maize endosperm

By means of starch gel electrophoresis and spectrophotometric assays, several different, specific dehydrogenases have been detected in liquid endosperm of maize 16 days after pollination. The typical alcohol dehydrogenase (ADH) bands on the starch gel appear when ethanol is used as substrate in the reaction mixture. However, some activity does appear with no substrate and with galactose or lactic acid as substrates, though not to the extent previously found and probably not due to the presence of a general type dehydrogenase as previously suggested (Scandalios, 1967). No specific activity appears with glucose, glucose 6-phosphate, galactose, malic acid, and isocitric acid when these are substituted for ethanol in the spectrophotometric assay for ADH. However, more specific spectrophotometric assays do show activity for malic acid and for isocitric acid. Both these enzymes, malate dehydrogenase (MDH) and isocitrate dehydrogenase, show definite patterns on the zymogram, with only a slight overlap with the ADH bands. MDH shows zymogram overlap with lactic acid as substrate.This work was supported by the U.S. Atomic Energy Commission, under contract No. AT(11-1)-1338.     

Kinetic study on starch hydrolysis using a glucose/maltose sensing system

Summary A dual-enzyme electrode flow injection system that can simultaneously determine glucose and maltose is used for an on-line study of starch hydrolyses catalysed by amylases. With the working system, determinations can be made every 2 minutes. A 10 L sample size with recycled back-flow minimises any loss of the reaction medium. The production, growth and decay of glucose and maltose concentrations during starch hydrolysis under various enzymatic conditions can thus be closely monitored, making it useful for the study of the catalytic kinetics of amylases and in screening and analysing enzyme systems.     

Regulation of the amylase secretion by the yeast Filobasidium capsuligenum and a 2-deoxy-d-glucose resistant mutant

Summary The regulation of extracellular amylase production by the basidiomycetous yeast Filobasidium capsuligenum CCY 64-5-1 was characterized using growing and resting cells. A basal level of amylolytic activity was produced with various carbon sources including glucose. Amylase secretion was repressed by glucose and, more severely, by 2-deoxy-d-glucose, whereas compounds with -1,4-linked glucose, such as methyl glucoside, maltose, -cyclodextrin and soluble starch, served as inducers. Repression was not relieved by exogenously added cAMP. The effects of several metabolic inhibitors on amylase secretion were studied. Following UV-mutagenesis a mutant strain (FC-5) capable of growing in a 2-deoxy-d-glucose supplemented corn starch medium was selected for further characterization. This strain produced more amylase, had acquired an increased resistance against repression by glucose, and retained a growth rate comparable to the wild type. FC-5 was also characterized by a reduced glucokinase activity and an increased hexokinase activity.     

Efficient production of l-lactic acid by newly isolated thermophilic Bacillus coagulans WCP10-4 with high glucose tolerance

A thermophilic Bacillus coagulans WCP10-4 with tolerance to high concentration of glucose was isolated from soil and used to produce optically pure l-lactic acid from glucose and starch. In batch fermentation at pH?6.0, 240 g/L of glucose was completely consumed giving 210 g/L of l-lactic acid with a yield of 95 % and a productivity of 3.5 g/L/h. In simultaneous saccharification and fermentation at 50 °C without sterilizing the medium, 200 g/L of corn starch was completely consumed producing 202.0 g/L of l-lactic acid. To the best of our knowledge, this strain shows the highest osmotic tolerance to glucose among the strains ever reported for lactic acid production. This is the first report of simultaneous saccharification and fermentation of starch for lactic acid production under a non-sterilized condition.     

Influence of different carbon sources on growth,lipid content and fatty acid composition in four strains belonging to mucorales

Summary Mucor mucedo, Mucor plumbeus, Mortierella ramanniana and Rhizopus arrhizus were tested for growth, lipid production and fatty acid composition on maltose, lactose, glucose, soluble starch and sodium acetate as a sole carbon source. A comparison of lipid production and fatty acid composition of tested strains showed, that glucose and maltose provided fat with a large proportion of saturated fatty acids and lactose, glycerol or starch gave unsaturated lipid with a high content of -linolenic acid.     

Production of native-starch-degrading enzymes by a Bacillus firmus/lentus strain

Summary A bacterium belonging to the Bacillus firmus/lentus-complex and capable of growth on native potato starch was isolated from sludge of a pilot plant unit for potato-starch production. Utilization of a crude enzyme preparation obtained from the culture fluid after growth of the microorganism on native starch, resulted in complete degradation of native starch granules from potato, maize and wheat at a temperature of 37°C. Glucose was found as a major product. Production of maltose, maltotriose and maltotetraose was also observed. Native-starch-degrading activity (NSDA) could be selectively adsorbed on potato-starch granules, whereas soluble-starch-degrading activity (SSDA) remained mainly in solution. The use of such a starch-adsorbed enzyme preparation on native starch resulted in a completely changed product pattern. An increase in oligosaccharides concomitant with less glucose formation was observed. An increased conversion of soluble starch to maltopentaose was possible with this starch-adsorbed enzyme preparation. It is concluded that NSDA comes from -amylase(s) and SSDA from glucoamylase(s) and/or -glucosidase(s). Cultivation of B. firmus/lentus on glucose, maltose, or soluble starch resulted in substantially smaller quantities of (native) starch-degrading activity.Offprint requests to: D. J. Wijbenga     

Xyloglucan (amyloid) formation in the cotyledons of Tropaeolum majus L. seeds

Mature seeds of Tropaeolum majus L. contain the cell wall polysaccharide xyloglucan (amyloid), protein and lipid as storage substances. The transitory occurrence of starch during the process of seed development could be substantiated.[U-¹⁴C]-labelled xylose, glucose and glucuronic acid were fed to ripening seeds and the incorporation of radioactivity into xyloglucan, starch and the sugar nucleotide fraction of the cotyledons was determined. The results indicate that exogenous supplied xylose is not incorporated directly into xyloglucan, but is transformed to glucose before incorporation into xyloglucan and starch. Radioactivity from glucuronic acid was predominantly found in the xylose moiety of xyloglucan. Incubation of seeds with [6-¹⁴C]-labelled glucose resulted in an incorporation of labelled hexoses into amyloid and starch, whereas xylose residues of amyloid remained unlabelled.Abbreviations p.a. post anthesis - UDP uridine 5-diphosphate - GDP guanosine 5-diphosphate - TLC thin layer chromatography - HPLC high pressure liquid chromatography     

Formation of a raw starch-hydrolyzing alpha-amylase by Clostridium 2021: Effect of carbon sources

Summary Clostridium 2021 was found to produce -amylase effective at hydrolyzing raw starch. Of the carbohydrates examined, starch at 3 % concentration was found to be the best carbon source for enzyme production. The products of -amylase action on starch were: maltose. glucose and higher dextrins.     

A highly branched storage polyglucan in the thermoacidophilic red microalga <Emphasis Type="Italic">Galdieria maxima</Emphasis> cells

The thermoacidophilic red alga Galdieria maxima is capable of heterotrophic growth. The content of carbohydrates in G. maxima grown heterotrophically increases by a factor of 4, reaching as much as 60% of cell dry weight. The increase in the level of carbohydrates in cells is due to accumulation of a storage α-glycan. According to a specific cleavage to glucose catalyzed by amyloglucosidase and the high positive specific optical rotation characteristic of polyglucans, this polysaccharide can be classified as a floridean starch. The data of ¹H NMR spectroscopy and the results of methylation showed that the average length of the unbranched regions of the polysaccharide molecule is six to seven glucose residues. The degree of branching of the starch molecule of G. maxima is greater than that of storage polysaccharides of other red algae, glycogens of yeast, and phytoglycogens of cyanobacteria.     

The raw starch-degrading alkaline amylase ofBacillus sp IMD 370

The amylase ofBacillus sp IMD 370 is the first report of an alkaline amylase with the ability to digest raw starch. The amylase could degrade raw corn and rice starches more effectively than raw potato starch. It showed no adsorb-ability to any type of raw starch at any pH value tested. The enzyme digested raw corn starch to glucose, maltose, maltotriose and maltotetraose. The maximum pH for raw starch hydrolysis was pH 8.0 compared to pH 10.0 for soluble starch hydrolysis. The metal chelator, ethylenediaminetetraacetic acid, strongly inhibited raw starch-digestion and its effect was reversed by the addition of divalent cations. Degradation of raw starch was stimulated six-fold in the presence of -cyclodextrin (17.5 mM).     

Pigment production in chemostat cultures of Streptococcus bovis

Summary Streptococcus bovis 2B altered its morphology and metabolism in response to changes in the rate of glucose addition. Specific pigment (measured by a simple spectrophotometric method) and lactic acid production increased with dilution rate (D) in a glucose-limited chemically defined medium. Lactic acid production increased immediately after a D stepup or a glucose pulse; increase of pigment production was slower and occurred with a lag of one generation. No increase in specific pigment production was seen after a starch pulse. Electron micrographs of thin sections showed that slow growing cells had thick cell walls. Faster growing cells had thinner cell walls but more bound pigment, suggesting a tradeoff between cell wall thickening and pigment production. Ammonia-limited cells continued to use all the glucose available and lactate and pigment production increased only slightly with D. Thin sections again showed thick cell walls for slow growing cells. Division irregularities characteristic of unbalanced growth were seen, which may explain the early washout of the ammonia-limited culture. Cystine in the medium was not used as a nitrogen source.     

Growth and carotenoid production by pH-stat cultures of Phaffia rhodozyma

To optimize biomass and carotenoid production by Phaffia rhodozyma in pH-stat cultures, two methods of feeding glucose were studied. In the first method, which is comparatively simple to operate, the glucose feeding set point (pH 5.02) was higher than the culture pH (5.00) and P. rhodozyma grew at a low specific growth rate (=0.055 h^–1). In the second method, the glucose feeding set point (pH 4.98) was lower than the culture pH (5.00) and the yeast grew at a specific growth rate of =0.095 h^–1. With the second method of glucose feeding, which is more complex and in order to prevent overfeeding of glucose, a time interval was added to the control strategy of the glucose pump and allowed to expire before the next dose of glucose was added. The length of the time interval affected biomass and carotenoid production. A critical time interval (T_c) was defined. In pH-stat cultures of P. rhodozyma, it was found that if the time interval was set longer than the critical time interval, the yeast did not grow.     

Starch synthesis by isolated amyloplasts from wheat endosperm

The aim of this work was to discover which compound(s) cross the amyloplast envelope to supply the carbon for starch synthesis in grains of Triticum aestivum L. Amyloplasts were isolated, on a continuous gradient of Nycodenz, from lysates of protoplasts of endosperm of developing grains, and then incubated in solutions of ¹⁴C-labelled: glucose, glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate and glycerol 3-phosphate. Only glucose 1-phosphate gave appreciable labelling of starch that was dependent upon the integrity of the amyloplasts. Incorporation into starch was linear with respect to time for 2 h. At the end of the incubations, 98% of the ¹⁴C in the soluble fraction of the incubation mixture was recovered as [¹⁴C]glucose 1-phosphate. Thus it is unlikely that the added [¹⁴C glucose 1-phosphate was extensively metabolized prior to uptake by the amyloplasts. It is argued that the behaviour of the isolated amyloplasts, and previously published data on the labelling of starch by [¹³C]glucose, are consistent with the view that in wheat grains it is a C-6, not a C-3, compound that enters the amyloplast to provide the carbon for starch synthesis.Abbreviations PPase alkaline inorganic pyrophosphatase - UDPglucose uridine 5-diphosphoglucose     

Sodium phosphate enhancement of starch hydrolysis by a diastatic strain of Saccharomyces cerevisiae

Summary Low concentrations of inorganic phosphate stimulate by up to 2-fold aerobic growth ofS. cerevisiae N.C.Y.C. 625 in starch medium. No such stimulation of growth is seen in a glucose medium. Enhanced growth on starch appears to result from increased secretion of glucoamylase (1,4--D-glucan glucohydrolase, EC 3.2.1.3) into the culture medium.     

A Cluster of Thermotoga neapolitana Genes Involved in the Degradation of Starch and Maltodextrins: the Expression of the aglB and aglA Genes in E. coli and the Properties of the Recombinant Enzymes

The aglB and aglA genes from the starch/maltodextrin utilization gene cluster of Thermotoga neapolitana were subcloned into pQE vectors for expression in Escherichia coli. The recombinant proteins AglB and AglA were purified to homogeneity and characterized. Both enzymes are hyperthermostable, the highest activity was observed at 85°. AglB is an oligomer of identical 55-kDa subunits capable of aggregation. This protein hydrolyses cyclodextrins and linear maltodextrins to glucose and maltose by liberating glucose from the reducing end of the molecules, and it is a cyclodextrinase with -glucosidase activity. The pseudo-tetrasaccharide acarbose, a potent -amylase and -glucosidase inhibitor, does not inhibit AglB but, on the contrary, acarbose is degraded quantitatively by AglB. Recombinant AglB is activated in the presence of CaCl₂, KCl, and EDTA, as well as after heating of the enzyme. AglA is a dimer of two identical 54-kDa subunits, and it hydrolyses the -glycoside bonds of disaccharides and short maltooligosaccharides, acting on the substrate from the non-reducing end of the chain. It is a cofactor-dependent -glucosidase with a wide action range, hydrolysing both oligoglucosides and galactosides with -links. Thereby, the enzyme is not specific with respect to the configuration at the C4 position of its substrare. For the enzyme to be active, the presence of NAD⁺, DTT, and Mn²⁺ is required. Enzymes AglB and AglA supplement one another in substrate specificity and ensure complete hydrolysis to glucose for the intermediate products of starch degradation.     

Selection of microorganisms which produce raw-starch degrading enzymes

Summary Microorganisms which produce strong raw-starch degrading enzymes were isolated from soil using a medium containing a unique carbon source, -amylase resistant starch (-RS), which is insoluble in water and hardly digested with Bacillus amyloliquefaciens -amylase. Among the isolates, three strains showing high activities were characterized. Two of them, K-27 (fungus) and K-28 (yeast), produced -amylase and glucoamylase, and the final product from starch was only glucose. The third strain, K-2, was a bacterium and produced -amylase, which produced glucose and malto-oligosaccharides from starch. The enzyme preparation of these strains degraded raw corn starch rapidly.     

Symplasmic and apoplasmic radial ion transport in plant roots

Enzymes of starch synthesis and degradation were identified in crude extracts of the unicellular green alga Dunaliella marina (Volvocales). By polyacrylamide gel electrophoresis and specific staining for enzyme activities, 4 multiple forms of starch synthase, 2 amylases, and at least 2 forms of -glucan phosphorylase were visible. Using specific -glucans incorporated into the gel before electrophoresis we have tentatively correlated -amylase and -amylase with both hydrolytic activities. The activities of -glucan phosphorylase and amylase(s) were measured quantitatively in crude extracts, and the concomitant action of -glucan phosphorylase and amylase(s) was found to account for the fastest rate of starch mobilization observed in vivo. Isolated chloroplasts retained both typical plastid marker enzymes and ADPglucose pyrophosphorylase, starch synthase, amylase(s), and -glucan phosphorylase to a similar percentage. Gel electrophoretic analysis followed by staining for enzyme activity of a stromal fraction resulted in a pattern of multiple forms of starch-metabolizing enzymes analogous to that found in a crude extract. We interpret the combined data as indicating the exclusive location in vivo of starch-metabolizing enzymes in chloroplasts of D. marina.Abbreviations Chl chlorophyll - DEAE-dextran diethylaminoethyl-dextran - DDT dithiothreitol - EDTA ethylenediamine tetraacetic acid - FBPase fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11 - G1P glucose 1-phosphate - G6P-DH glucose 6-phosphate dehydrogenase, EC 1.1.1.49 - HEPES N-2-hydroxyethylpiperazine-N-ethanesulphonic acid - MES 2-(N-morpholino)ethanesulphonic acid - P_i inorganic orthophosphate - RuBP carboxylase ribulose-1,5-bisphosphate carboxylase, EC 4.1.1.39     

Über die chemische Natur der Reserve-Polysaccharide in Acetabularia-Chloroplasten

Summary The reserve polysaccharides in chloroplasts of several species of Acetabularia have been identified as starch.The starch granules in situ as well as the isolated and purified particles stain with Lugol-reagent. They show the characteristic birefringence.Acid hydrolysis and degradation by -amylase followed by acid hydrolysis showed that the starch is composed only of glucose units.The statements of Vanden Driessche and Bonotto (1967) concerning the inulin character of the reserve polysaccharides in Acetabularia chloroplasts need correction.