Biochemical Studies on Buckwheat α-Glucosidase

The transglucosylation action of buckwheat α-glucosidase on soluble starch, maltose maltotriose and maltotetraose are described and discussed. The transglucosylation products of soluble starch were isolated by carbon-Celite column chromatography and by paper chromatography. Among the products were found follows: nigerose, maltose, kojibiose and isomaltose as disaccharides and 2-α-isomaltosylglucose, 2-α-nigerosylglucose, nigerotriose. 3-α-maltosylglucose and maltotriose as trisaccharides.

Furthermore, the existence of 6-α-nigerosylglucose, 4-α-kojibiosylglucose, panose, isopanose and 3-α-isomaItosylglucose was suspected. A new trisaccharide, 2-α-nigerosylglucose which was obtained in a crystalline form (monohydrate) melted at 186~188°C and gave [α]_D+ 178.3 (c = 0.6, in water).

These experimental results on the reaction products seem to indicate that the activated glucosyl group from the substrate (starch, in this case) is transferred to any position of C–2,3,4 or 6 of glucose released from the substrate and the same type of transglucosylation occurrs upon the non-reducing terminal of disaccharides just produced, which leads to the formation of various kinds of trisaccharide, tetrasaccharide etc. The synthesis of α-oligosaccharides from free glucose could not be detected by paper chromatography.     

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6′‐phosphate phosphatase (MapP)

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose‐specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6‐phospho‐α‐glucosidase, which in B. subtilis hydrolyses maltose 6′‐P into glucose and glucose 6‐P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6‐P into glucose 1‐P and glucose 6‐P. However, purified MalP phosphorolyses maltose but not maltose 6′‐P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6′‐P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1‐P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6′‐P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6′‐P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1‐P. MapP therefore connects PTS‐mediated maltose uptake to maltose phosphorylase‐catalysed metabolism. Dephosphorylation assays with a wide variety of phospho‐substrates revealed that MapP preferably dephosphorylates disaccharides containing an O‐α‐glycosyl linkage.     

Characterization of starch breakdown in the intact spinach chloroplast

Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia oleracea) chloroplast which had been preloaded with ¹⁴C-starch photosynthetically from ¹⁴CO₂. Starch breakdown was dependent upon inorganic phosphate and the ¹⁴C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of ¹⁴C from triose-phosphate to glycerate 3-phosphate. Iodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3-phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was concluded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic cleavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate.     

Uptake and metabolism of carbohydrates by epidermal tissue

Isolated epidermis of Commelina communis L. and Tulipa gesneriana L. assimilated ¹⁴CO₂ into malic acid and its metabolites but not into sugars or their phosphates; epidermis could not reduce CO₂ by photosynthesis and therefore must be heterotrophic (Raschke and Dittrich, 1977). If, however, isolated epidermis of Commelina communis was placed on prelabelled mesophyll (obtained by an exposure to ¹⁴CO₂ for 10 min), radioactive sugars appeared in the epidermis, most likely by transfer from the mesophyll. Of the radioactivity in the epidermis, 60% was in sucrose, glucose, fructose, 3-phosphoglyceric acid and sugar phosphates. During a 10-min exposure to ¹⁴CO₂, epidermis in situ incorporated 16 times more radioactivity than isolated epidermal strips. Isolated epidermis of Commelina communis and Tulipa gesneriana took up ¹⁴C-labelled glucose-1-phosphate (without dephosphorylation), glucose, sucrose and maltose. These substances were transformed into other sugars and, simultaneously, into malic acid. Carbons-1 through-3 of malic acid in guard cells can thus be derived from sugars. Radioactivity appeared also in the hydrolysate of the ethanol-insoluble residue and in compounds of the tricarboxylic-acid cycle, including their transamination products. The hydrolysate contained glucose as the only radioactive compound. Radioactivity in the hydrolysate was therefore considered an indication of starch. Starch formation in the epidermis began within 5 min of exposure to glucose-1-phosphate. Autoradiograms of epidermal sections were blackened above the guard cells. Formation of starch from radioactive sugars therefore occurred predominantly in these cells. Epidermis of tulip consistently incorporated more ¹⁴C into malic and aspartic acids than that of Commelina communis (e.g. after a 4-h exposure to [¹⁴C]glucose in the dark, epidermis, with open stomata, of tulip contained 31% of its radioactivity in malate and aspartate, that of Commelina communis only 2%). The results of our experiments allow a merger of the old observations on the involvement of starch metabolism in stomatal movement with the more recent recognition of ion transfer and acid metabolism as causes of stomatal opening and closing.Abbreviation G-1-P glucose-1-phosphate     

Differential amylosaccharide metabolism of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum.

Clostridium thermosulfurogenes displayed faster growth on either glucose, maltose, or starch than Clostridium thermohydrosulfuricum. Both species grew faster on glucose than on starch or maltose. The fermentation end product ratios were altered based on higher ethanol and lactate yields on starch than on glucose. In C. thermohydrosulfuricum, glucoamylase, pullulanase, and maltase were mainly responsible for conversion of starch and maltose into glucose, which was accumulated by a putative glucose permease. In C. thermosulfurogenes, beta-amylase was primarily responsible for degradation of starch to maltose, which was accumulated by a putative maltose permease and then hydrolyzed by glucoamylase. Regardless of the growth substrate, the rates of glucose, maltose, and starch transformation were higher in C. thermosulfurogenes than in C. thermohydrosulfuricum. Both species had a functional Embden-Meyerhof glycolytic pathway and displayed the following catabolic activities: ferredoxin-linked pyruvate dehydrogenase, acetate kinase, NAD(P)-ethanol dehydrogenase, NAD(P)-ferredoxin oxidoreductase, hydrogenase, and fructose-1,6-diphosphate-activated lactate dehydrogenase. Ferredoxin-NAD reductase activity was higher in C. thermohydrosulfuricum than NADH-ferredoxin oxidase activity, but the former activity was not detectable in C. thermosulfurogenes. Both NAD- and NADP-linked ethanol dehydrogenases were unidirectional in C. thermosulfurogenes but reversible in C. thermohydrosulfuricum. The ratio of hydrogen-producing hydrogenase to hydrogen-consuming hydrogenase was higher in C. thermosulfurogenes. Two biochemical models are proposed to explain the differential saccharide metabolism on the basis of species enzyme differences in relation to specific growth substrates.     

Red cell enzymes of primates (Anthropoidea)

The patterns of five red cell enzymes (acid phosphatase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, adenylate kinase, and phosphoglucomutase) in various Anthropoidea were compared on starch gels. Small samples of gorilla, orang, and gibbon, six genera of Old World monkeys, and seven genera of New World monkeys were examined. Among the hominoids the gibbon was the most divergent. The langur differed markedly from macaques and baboons in certain enzyme patterns. Most of the ceboids had relatively high G6PD activities. Individual variations in 6PGD, AP, AK, and PGM but not in G6PD were seen in some species. Many genera could be distinguished by the mobility of the achromatic bands.     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

Distribution of Yeast Cell Wall Lytic Activity among Microorganisms

An α-glucosidase has been isolated from the mycelia of Penicillium purpurogenum in electrophoretically homogeneous form, and its properties have been investigated. The enzyme had a molecular weight of 120,000 and an isoelectric point of pH 3.2. The enzyme had a pH optimum at 3.0 to 5.0 with maltose as substrate. The enzyme hydrolyzed not only maltose but also amylose, amylopectin, glycogen, and soluble starch, and glucose was the sole product from these substrates. The Km value for maltose was 6.94×10^?4 m. The enzyme hydrolyzed phenyl α-maltoside to glucose and phenyl α-glucoside. The enzyme had α-glucosyltransferase activity, the main transfer product from maltose being maltotriose. The enzyme also catalyzed the transfer of α-glucosyl residue from maltose to riboflavin.     

Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.)

The aim of this work was to investigate the capacity for synthesis of starch and fatty acids from exogenous metabolites by plastids from developing embryos of oilseed rape (Brassica napus L.). A method was developed for the rapid isolation from developing embryos of intact plastids with low contamination by cytosolic enzymes. The plastids contain a complete glycolytic pathway, NADP-glucose-6-phosphate dehydrogenase, NADP-6-phosphogluconate dehydrogenase, fructose-1,6-bisphosphatase, NADP-malic enzyme, the pyruvate dehydrogenase complex (PDC), and acetyl-CoA carboxylase. Organelle fractionation studies showed that 67% of the total cellular PDC activity was in the plastids. The isolated plastids were fed with ¹⁴C-labelled carbon precursors and the incorporation of ¹⁴C into starch and fatty acids was determined. ¹⁴C from glucose-6-phosphate (G-6-P), fructose, glucose, fructose-6-phosphate and dihydroxyacetone phosphate (DHAP) was incorporated into starch in an intactness- and ATP-dependent manner. The rate of starch synthesis was highest from G-6-P, although fructose gave rates which were 70% of those from G-6-P. Glucose-1-phosphate was not utilized by intact plastids for starch synthesis. The plastids utilized pyruvate, G-6-P, DHAP, malate and acetate as substrates for fatty acid synthesis. Of these substrates, pyruvate and G-6-P supported the highest rates of synthesis. These studies show that several cytosolic metabolites may contribute to starch and/or fatty acid synthesis in the developing embryos of oilseed rape.     

Exchange of Metabolites in Cyanophora paradoxa and its Cyanelles*

The flagellate Cyanophora paradoxa contains blue-greenish, organelle-like inclusions termed cyanelles which perform photosynthetic CO₂-fixation in place of chloroplasts. By the use of the HPLC-technique, Cyanophora was shown to form glucose, sucrose, maltose, mannitol, ribose, glycerol and trehalose. Extracts from the whole organism and from the eucaryotic host, but not from the cyanelles, convert ¹⁴C-labelled UDP-glucose to polyglucan. Synthesis of sucrose from UDP-glucose and fructose-6-P or fructose could not be demonstrated in any extract from Cyanophora. The transfer of metabolites into cyanelles was monitored by the silicone oil filtering technique. The solute spaces for ¹⁴C-labelled sorbitol and ³H₂O were the same indicating that sorbitol freely penetrated the plasma membrane of cyanelles in contrast to the situation found in chloroplasts. The measurements of the solute spaces for the different compounds showed that maltose and sucrose were not accumulated by isolated cyanelles. Other compounds like fructose, fucose, glutamine or glycine had intermediate sizes of their solute spaces. Cyanelles apparently possess a rapidly transporting glucose carrier and not a malate/oxaloacetate shuttle and also not an ATP/ADP translocator. The carrier composition at the plasma membrane of cyanelles and at the inner envelope membrane of chloroplasts seems to be totally different.     

Thermostable glucose-tolerant glucoamylase produced by the thermophilic fungus Scytalidium thermophilum

Glucoamylase produced byScytalidium thermophilum was purified 80-fold by DEAE-cellulose, ultrafiltration and CM-cellulose chromatography. The enzyme is a glycoprotein containing 9.8% saccharide, pI of 8.3 and molar mass of 75 kDa (SDS-PAGE) or 60 kDa (Sepharose 6B). Optima of pH and temperature with starch or maltose as substrates were 5.5/70 °C and 5.5/65 °C, respectively. The enzyme was stable for 1 h at 55 °C and for about 8 d at 4 °C, either at pH 7.0 or pH 5.5. Starch, amylopectin, glycogen, amylose and maltose were the substrates preferentially hydrolyzed. The activity was activated by 1 mmol/L Mg²⁺ (27%), Zn²⁺ (21%), Ba²⁺ (8%) and Mn²⁺ (5%).K _m and {ie11-1} values for starch and maltose were 0.21 g/L, 62 U/mg protein and 3.9 g/L, 9.0 U/mg protein, respectively. Glucoamylase activity was only slightly inhibited by glucose up to a 1 mol/L concentration.     

Protease and lipase production by a strain ofSerratia marcescens (532 S)

Summary Production of both exolipase and exoprotease activities bySerratia marcescens 532 S isolated from an aerobic fixed-biomass reactor were strongly influenced by nutritional factors which acted as inducers or repressors. In batch culture, protease and lipase activities were produced after the exponential phase. NH₄Cl, amino acids and simple carbon sources caused repression of protease activity. At a concentration of 1.5 g L^–1, the individual addition of maltose, mannitol, acetate, fructose or glucose, repressed exoprotease production, with the greatest effect by glucose. An inverse relationship existed between exoprotease synthesis and increasing glucose concentrations. Lipids activated lipase production, the most significant increase occurred when Tween 80 was added in the medium. Thus, glucidolytic, proteolytic and lipolytic activities could be efficiently expressed in batch cultures only successively.At low dilution rate of chemostat cultures with a constant glucose input concentration of 2 g L^–1, glucidolytic, proteolytic and lipolytic activities were produced, but did not have the same regulation: atD values <0.08 h^–1, the level of protease activity dropped while that of lipase showed a corresponding increase. Above these values, increasingD led to a decrease of the two hydrolase activities, at the level of the specific activities as well as in the specific rate of biosynthesis of each enzyme. Similar results were obtained in chemostat culture with a constant specific growth rate of 0.04 h^–1 with increasing glucose input concentrations, i.e. protease and lipase activities decreased when the specific glucose uptake rates were enhanced.     

Production of α-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

α-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55°C and 5.5, respectively, and the apparent K_m for starch was 0.8 mg ml⁻¹. The products of α-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of α-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial α-amylase. Activities of α-amylase up to 4.4 U ml⁻¹ (1 U represents 1 μmol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml⁻¹ in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml⁻¹), α-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed α-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of α-amylase (initial concentration, 2.5 mg ml⁻¹), they were found to be less effective than starch, but maltose was almost as effective. The fungal α-amylase was found to be stable at 60°C in the presence of low concentrations of starch (≤5%), suggesting that it may be suitable for industrial application.     

Carbohydrate and carbon metabolite accumulation responses in leaves of ozone tolerant and ozone susceptible spinach plants after acute ozone exposure

The objective of this study was to determine whether exposure of plants to ozone (O₃) increased the foliar levels of glucose, glucose sources, e.g., sucrose and starch, and glucose-6-phosphate (G6P), because in leaf cells, glucose is the precursor of the antioxidant, L-ascorbate, and glucose-6-phosphate is a source of NADPH needed to support antioxidant capacity. A further objective was to establish whether the response of increased levels of glucose, sucrose, starch and G6P in leaves could be correlated with a greater degree of plant tolerance to O₃. Four commercially available Spinacia oleracea varieties were screened for tolerance or susceptibility to detrimental effects of O₃ employing one 6.5 hour acute exposure to 25O nL O₃ L^-1 air during the light. One day after the termination of ozonation (29 d post emergence), leaves of the plants were monitored both for damage and for gas exchange characteristics. Cultivar Winter Bloomsdale (cv Winter) leaves were least damaged on a quantitative grading scale. The leaves of cv Nordic, the most susceptible, were approximately 2.5 times more damaged. Photosynthesis (Pn) rates in the ozonated mature leaves of cv Winter were 48.9% less, and in cv Nordic, 66.2% less than in comparable leaves of their non-ozonated controls. Stomatal conductance of leaves of ozonated plants was found not to be a factor in the lower Pn rates in the ozonated plants. At some time points in the light, leaves of ozonated cv Winter plants had significantly higher levels of glucose, sucrose, starch, G6P, G1P, pyruvate and malate than did leaves of ozonated cv Nordic plants. It was concluded that leaves of cv Winter displayed a higher tolerance to ozone mediated stress than those of cv Nordic, in part because they had higher levels of glucose and G6P that could be mobilized during diminished photosynthesis to generate antioxidants (e.g., ascorbate) and reductants (e.g., NADPH). Elevated levels of both pyruvate and malate in the leaves of ozonated cv Winter suggested an increased availability of respiratory substrates to support higher respiratory capacity needed for repair, growth, and maintenance.Abbreviations ADPG-PPiase ADPglucose pyrophosphorylase - ASC L-ascorbic acid - APX ascorbate peroxidase - Ce CO₂ concentration in air in the measuring cuvette during photosynthesis measurements - Ci CO₂ concentration in the leaf intercellular spaces during photosynthesis measurement - Chl chlorophyll - DHA dehydroascorbic acid - DHA reductase dehydroascorbate reductase - DHAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - Gluc glucose - GR glutathione reductase - G_sw stomatal conductance with units as mmol H₂O m^-2 s^-1 - GSSG oxidized glutathione - GSH reduced glutathione - G1P glucose-1-phosphate - G6P glucose-6-phosphate - G6P dehydrogenase glucose-6-phosphate dehydrogenase - 6PG 6-phosphogluconate - 6PG dehydrogenase 6-phosphogluconate dehydrogenase - F6P fructose-6-phosphate - FBP fructose-1,6-bisphosphate - MAL malate - MDHA reductase monodehydroascorbate reductase - PE post-emergence - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - PYR pyruvate - Pn net CO₂ photoas-similation in leaves - PPFD photosynthetic photon flux density with units of mol photons m^-2 s^-1 - PPRC pentose phosphate reductive cycle - RuBP ribulose-1,5-bisphosphate - rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SLW specific leaf weight - TCA cycle tricarboxylic acid cycle - Triose-P DHAP+GAP     

碳源对早花百子莲愈伤组织诱导及其增殖的生理特性影响

为揭示碳源对早花百子莲愈伤组织诱导与增殖的影响机理,该研究以早花百子莲的小花梗为外植体,比较分析30.0 g/L蔗糖、葡萄糖、麦芽糖在愈伤组织诱导、增殖中的效果,测定不同碳源种类处理下愈伤组织增殖相关生理特性,并根据细胞增殖效果、生理指标相关性进行优化验证。结果表明:(1)蔗糖、葡萄糖和麦芽糖碳源处理下,愈伤组织诱导率分别为86.00%、72.00%和59.67%,蔗糖碳源的愈伤组织诱导率比葡萄糖和麦芽糖分别显著提高19.44%和44.13%(P<0.05),蔗糖碳源较葡萄糖和麦芽糖碳源的愈伤组织大小分别显著增加22.44%和90.09%(P<0.05);愈伤组织增殖阶段,蔗糖碳源能够同时维持良好的细胞增殖效率及活性,而葡萄糖碳源的愈伤组织增殖快、状态差,麦芽糖处理增殖慢、状态佳;蔗糖转换葡萄糖碳源后愈伤组织细胞团大小、细胞活性明显下降;蔗糖转换蔗糖、蔗糖转换麦芽糖的效果较好。(2)培养基碳源显著调节愈伤组织增殖阶段的糖代谢、内源激素代谢和氧化胁迫平衡。(3)愈伤组织的主要糖组分为淀粉、葡萄糖;淀粉、麦芽糖含量与细胞团大小相关性高,以蔗糖为碳源的培养基中添加麦芽糖,愈伤组织增殖效果良好,细胞团颜色鲜黄,活性较强。(4)愈伤组织的结合态IAA、GA4、CTK含量与细胞团大小具有一定的相关性,培养基中添加1.0 mg/L 6-BA可显著促进愈伤组织增殖效率(P<0.05)。(5)ROS活性与POD、CAT活性以及POD活性与H2O2含量均呈显著负相关关系(P<0.05),而POD与CAT活性呈极显著正相关关系(P<0.01)。(6)验证及优化实验结果表明,增殖培养基中添加麦芽糖、6-BA可有效促进早花百子莲愈伤组织继代增殖效果,其中麦芽糖可以保持、改善细胞活性,而6-BA主要促进了细胞增殖。研究发现,蔗糖为早花百子莲愈伤组织诱导及增殖的最佳碳源,蔗糖、麦芽糖组合碳源利于愈伤组织细胞活力维持,而毒莠定(PIC)与6-BA组合利于愈伤组织细胞增殖,最佳增殖培养基为:MS+0.5 mg/L PIC+1.5 mg/L 6-BA+15.0 g/L蔗糖+15.0 g/L麦芽糖+7.0 g/L琼脂。     

A new ADP (UDP)--glucose glucosyltransferase activity in disrupted starch grains.

Disrupted potato starch granules obtained in the presence of 8 M urea were shown to increase [¹⁴C] glucose incorporation from labeled ADP-glucose or UDP-glucose into starch, as compared to intact grains. Labeled glucose or maltose units were found to be incorporated through a linkage that produced cyclic phosphate esters upon mild alkaline treatment and was sensitive to hydrolysis at pH 2.0. Both properties of this linkage strongly resembled those of the pyrophosphate bond of ADP-glucose or UDP-glucose.     

Maltose metabolism by pea chloroplasts

The aim of this work was to investigate the origin of maltose formed during starch breakdown in the dark by chloroplasts of Pisum sativum. The maximum catalytic activities of maltose phosphorylase and maltase in pea leaves were shown to be low, relative to those of enzymes known to be involved in starch breakdown. Fractionation of pea leaves indicated that the chloroplasts lack maltase but have enough maltose phosphorylase to synthesize the amounts of maltose formed when isolated chloroplasts breakdown starch. The absence of exogenous phosphate markedly reduced starch breakdown and maltose accumulation by isolated chloroplasts. When [¹⁴C]glucose was supplied to chloroplasts that were breaking down starch in the dark, maltose was labelled and most of the label was in the glucose moeity. It is suggested that maltose phosphorylase, using glucose-1-phosphate formed from starch by α-glucan phosphorylase, is responsible for, at least some of, the synthesis of maltose during starch breakdown by pea chloroplasts in vitro.     

Purification and characterization of a soluble,cytoplasmic decycling maltodextrinase from aLactobacillus species,strain 26X,isolated from kitchen waste water

A soluble, cytoplasmic decycling maltodextrinase with a relative molecular mass (M _r) of around 62 000, and an isoelectric point (pI) of about 4 was purified to 93% homogeneity from aLactobacillus species, strain 26X, that was isolated from the waste water of a kitchen and resembledLactobacillus plantarum in its physiological and biochemical characteristics, but differed from this species in producing exclusively L( + )-lactic acid. The enzyme exhibited higher activities with maltotetraose to maltoheptaose than with cyclodextrins, in which cyclomaltohexaose was hydrolysed fastest, and the amounts of the linearized cyclic substrates were in the order cyclomaltohexaose < cyclomaltoheptaose < cyclomaltooctaose. The substituents of cyclomaltoheptaose derivatives considerably blocked the enzymic activity, and the accessibility to enzymic attack depended on the degree of substitution. The main product of hydrolysis proved to be maltose. The maltodextrinase exhibited, above all with maltotetraose, some transglycosylation activity. Except for maltopentaose, maltosyl transfer predominated. Starch and pullulan were degraded at low rates, the main products from starch being glucose and maltose, and from pullulan a branched trisaccharide, presumably panose.     

Anaerobic and aerobic energy metabolism in the larvae (tetrathyridia) of Mesocestoides corti

The tetrathyridia of Mesocestoides corti produce lactate, succinate, acetate, and CO₂ as major carbon-containing end products during in vitro incubation with glucose as the substrate. Differences in the rate of glucose consumption and lactate production under anaerobic or aerobic conditions were observed, but their significance could not be determined. However, succinate production was greatly decreased in the presence of oxygen.The relative activities and intracellular distribution of various enzymes involved in energy-supplying metabolism of the larvae appear to conform to the pathways observed in other parasitic helminths known to produce lactate, succinate, and volatile fatty acids as metabolic end products. Some common features found in this respect are the relatively low pyruvate kinase activity, the presence of a highly active cytoplasmic phosphoenolpyruvate carboxylase and the capability of mitochondrial membrane bound fumarate reductase to reduce fumarate by means of NADH. Although a stimulatory effect of fructose-1,6-diphosphate on the reaction velocity of pyruvate kinase occurred, the absolute activity of this enzyme is very low.Nearly all the enzymes required for Krebs cycle activity are available in the tetrathyridia. Under the assay conditions employed by us, only NAD-dependent isocitrate dehydrogenase could not be demonstrated. The small amounts of ¹⁴CO₂ liberated from 6-¹⁴C-glucose suggest that the cycle in its classical form probably only functions at a very low rate. The incorporation of ¹⁴C from labeled glucose into glycogen indicates the presence of enzymes capable of glycogenesis. The incorporation rate was found to be higher in the presence of oxygen than under anaerobic conditions. On account of the very low NAD-linked glycerol-3-phosphate dehydrogenase activity the glycerolphosphate cycle may be of minor importance for the tetrathyridia.As a result of these studies a scheme for the main carbohydrate dissimilating pathways in the tetrathyridia is proposed and the significance of oxygen with respect to energy-supplying metabolism is discussed.     

Determination of optimal carbon source and pH value for sclerotial formation of Polyporus umbellatus under artificial conditions

Polyporus umbellatus is one of the precious medicinal fungi, with sclerotia used as a diuretic agent and antidote in China for many years. This has led to the present interest in producing sclerotia of P. umbellatus in the laboratory due to a decreased abundance in natural sources. Here, we investigated the determining factors for sclerotial formation in P. umbellatus. Five carbon sources, namely, maltose, fructose, glucose, sucrose and soluble starch with different initial pH values were evaluated for their effects on mycelial growth and sclerotial development of P. umbellatus. Maltose, fructose and glucose could induce sclerotial formation of P. umbellatus. Sucrose and soluble starch could stimulate growth of the fungus but had no effect on sclerotial formation. The most efficient sclerotial production occurred with maltose followed by fructose and a pH of 5. In addition, different macroscopically evident characteristics of sclerotial development of P. umbellatus induced by different carbon sources were also observed. Our findings could provide new insights into further research on sclerotial production in P. umbellatus under artificial cultivation.