Electrophoretic study on peroxidase,indoleacetic acid oxidase,and o-diphenol oxidase fractions in extracts from different growth zones ofvicia faba L. roots

Using electrophoresis in acrylamide gel, fractions of peroxidase, indoleacetic acid oxidase, and o-diphenol oxidase were investigated in extracts from three growth zones ofVicia faba L. roots. Three peroxidase fractions (zones) moving towards the anode were revealed as well as four peroxidase fractions (zones) migrating towards the cathode. Three peroxidase fractions showed detectable indoleacetic acid oxidase activity. The o-diphenol oxidase activity was revealed in all peroxidase fractions moving towards the anode, in those moving towards the cathode the o-diphenol oxidase activity differred according to the substrate used. One fraction with both peroxidase and o-diphenol oxidase activity occurred only in electrophoreograms of extracts from the maturation zone; in this fraction no indoleacetic acid oxidase activity was demonstrable.     

Acid phosphatase fractions from various growth zones of broad bean root revealed by disc electrophoresis

Fractions of acid phosphate (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) were studied in extracts of segments from three growth zones of broad bean roots by means of electrophoresis in acrylamide gel. The azocoupling reaction with α-naphtyl phosphate was used for detection. The phosphatase activity was investigated in the range of pH 3·6–7·2. Altogether nine fractions moving towards the anode were revealed. Some fractions differed slightly in their pH optimum. The presence of Mg⁺⁺ in the incubation medium resulted in the activation of two fractions, Mn⁺⁺ showed activation of three fractions and inhibition of the rest of the fractions; the presence of Zn⁺⁺ resulted in a slight inhibition of all fractions. Between electrophoreograms of extracts of segments from the division zone and electrophoreograms of extracts of segments from the enlargement zone and from the maturation zone considerable quantitative differences were found with one fraction; proportions of the other fractions were approximately identical in electrophoreograms of all three growth zones. The response to the presence of Mg⁺⁺, Mn⁺⁺ and Zn⁺⁺ in the incubation medium as well as the pH optima of the individual fractions were identical for all three growth zones.     

Electrophoretic investigation of ribonuclease in roots and leaves ofVicia faba L.

Isozyme patterns and specific activity of ribonuclease (ribonucleate pyridinenucleotido-2′-transferase, E. C. 2.7.7.16) were followed in the extracts of segments from three growth zones of the root and in extracts of young and senescent leaves ofVicia faba L. Electrophoreograms of extracts from all three investigated root zones were identical, in the electrophoreograms of extracts from senescent leaves however one new ribonuclease occurred which could not be detected in the electrophoreograms of extracts from young leaves. Extracts from senescent leaves had higher specific activity of ribonuclease than extracts from young leaves. Extracts from the enlargement zone of the root and those from the maturation zone had a three times higher specific activity of RNase than extracts from the division zone.     

α-1,4-Glucan phosphorylase forms in the green alga Eremosphaera viridis

In extracts of the unicellular green alga Eremosphaera viridis DeBary (Chlorococcales, Chlorophyceae) the average specific activity of α-1,4-glucan phosphorylase (E.C. 2.4.1.1) was 200 nmol glucose 1-phosphate formed per min and mg protein. Using continuous and discontinuous electrophoresis on polyacrylamide gels, three phosphorylase forms were found. When the log of the relative mobility of the three enzyme forms was plotted versus the acrylamide gel concentration (Ferguson plot) parallel lines were obtained, indicating that the three enzymes were indiscernible with respect to molecular weight. Electrophoresis on density gradient gels resulted in three activity zones lying close to each other. The relative molecular mass ( M _r) of the three enzymes was estimated to be around 180,000 with a difference of less than 7,000 between the small and the large forms.     

Effects of Ionic and Osmotic Strength on the Glucosyltransferase of Rhizobium meliloti Responsible for Cyclic beta-(1,2)-Glucan Biosynthesis

The cyclic beta-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic beta-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic beta-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic beta-(1,2)-glucan biosynthesis.     

Glucans in the Cell Walls Regenerated from Vinca rosea Protoplasts

Protoplasts prepared from suspension-cultured Vinca rosea cellswere cultured for 5 days. The cell walls regenerated from theprotoplasts were mainly composed of glucans having 1,3- and1,4-linkages. To investigate the molecular species, these glucanswere separated into four fractions: EDTA (50 mM, pH 4.5)-soluble(fraction E), KOH (24%)- soluble but not precipitatable by neutralizationwith acetic acid (fraction K-S), KOH (24%)-soluble and precipitatableby neutralization with acetic acid (fraction K-P), and KOH (24%)-insoluble(fraction C). By means of sugar composition analysis, methylationanalysis, periodate oxidation and enzymatic digestion, the molecularspecies of the glucans contained in the regenerated cell wallswere deduced to be ß-1,4-glucan (cellulose) and ß-1,3-glucan.Fraction C was mainly composed of ß-1,4-glucan; ß-1,3-glucanwas mainly recovered in fraction K-P. The ß-l,3-glucanwas soluble in dilute alkali solution, but was only slightlysoluble in water. The ß-1,3-glucan had an essentiallyunbranched structure, and its weight average molecular weightestimated by gel permeation chromatography was 4.5–5.0x 10⁴. ¹ Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Tsukuba, Ibaraki305, Japan (Received May 21, 1981; Accepted October 13, 1981)     

外源酚对凤眼莲体内酚含量和与酚代谢有关酶的影响

凤眼莲能够吸收和在体内聚集外源苯酚,体内的酸含量随着环境中酚浓度的上升而上升。从生长于合酚培养液中的凤眼莲体内能够检测到酚糖苷,说明凤眼莲体内有酚精苷转移酶的存在。浓度小于５０ｍｇ／Ｌ的外源酚能提高凤眼莲体内的多酚氧化酶和过氧化物酶的活性。多酚氯化酶与过氧化物酶在线粒体和微粒体中均有不同程度的分布,而酚糖苷转移酶则不存在于这些细胞器中。     

Isomaltulose production using free cells: optimisation of a culture medium containing agricultural wastes and conversion in repeated-batch processes

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-alpha-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by intramolecular transglucosylation. The experimental designs and response surface methodology (RSM) were applied for the optimisation of the nutrient concentrations in the culture medium for the production of glucosyltransferase by Erwinia sp. D12 in shaken flasks at 200 rpm and 30 degrees C. A statistical analysis of the results showed that, in the range studied, the factors had a significant effect (P < 0.05) on glucosyltransferase production and the highest enzyme activity (10.84 U/ml) was observed in culture medium containing sugar cane molasses (150 g l(-1)), corn steep liquor (20 g l(-1)), yeast extract Prodex Lac SD (15 g l(-1)) and K2HPO4 (0.5 g l(-1)) after 8 h at 30 degrees C. The production of cell biomass by the strain of Erwinia sp. D12 was carried out in a 6.6-l fermenter with a mixing rate of 200 rpm and an aeration rate of 1 vvm. Fermentation time, cellular growth, medium pH and glucosyltransferase production were observed. The greatest glucosyltransferase activity was 22.49 U/ml, obtained after 8 h of fermentation. The isomaltulose production from sucrose was performed using free Erwinia sp. D12 cells in a batch process using an orbital shaker. The influence of the parameters sucrose concentration, temperature, pH, and cell concentration on the conversion of sucrose into isomaltulose was studied. The free cells showed a high conversion rate of sucrose into isomaltulose using batch fermentation, obtaining an isomaltulose yield of 72.11% from sucrose solution 35% at 35 degrees C.     

Antimicrobial Activity of Propolis on Oral Microorganisms

Formation of dental caries is caused by the colonization and accumulation of oral microorganisms and extracellular polysaccharides that are synthesized from sucrose by glucosyltransferase of Streptococcus mutans. The production of glucosyltransferase from oral microorganisms was attempted, and it was found that Streptococcus mutans produced highest activity of the enzyme. Ethanolic extracts of propolis (EEP) were examined whether EEP inhibit the enzyme activity and growth of the bacteria or not. All EEP from various regions in Brazil inhibited both glucosyltransferase activity and growth of S. mutans, but one of the propolis from Rio Grande do Sul (RS2) demonstrated the highest inhibition of the enzyme activity and growth of the bacteria. It was also found that propolis (RS2) contained the highest concentrations of pinocembrin and galangin. Received: 8 June 1997 / Accepted: 7 July 1997     

A protein-glucan intermediate during paramylon synthesis.

A sodium deoxycholate extract containing glucosyltransferase activity was obtained from a particulate preparation from Euglena gracilis. It transferred glucose from UDP-[14C]glucose into material that was precipitated by trichloroacetic acid. This material released beta-(1 leads to 3)-glucan oligosaccharides into solution on incubation with weak acid, weak alkali and beta-(1 leads to 3)-glucosidase. The products of the incubation of the deoxycholate extract with UDP-[14C]glucose were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioactive bands were obtained that had the properties of beta-(1 leads to 3)-glucan covalently linked to protein by a bond labile to weak acid. High-molecular-weight material containing a beta-(1 leads to 3)-glucan was also shown to be present by gel filtration. The bond linking glucan to aglycone is possibly a pyrophosphate linkage. It is proposed that in Euglena gracilis beta-(1 leads to 3)-glucan (paramylon) is synthesized on a protein primer.     

Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose:sterol glucosyltransferase (EC 2. 4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-beta-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.     

Effects of Ionic and Osmotic Strength on the Glucosyltransferase of Rhizobium meliloti Responsible for Cyclic β-(1,2)-Glucan Biosynthesis

The cyclic β-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic β-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic β-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic β-(1,2)-glucan biosynthesis.     

Antimicrobial activities of eucalyptus leaf extracts and flavonoids from Eucalyptus maculata

AIMS: We investigated the antimicrobial activities of eucalyptus leaf extracts to find effective antibacterial agents. METHODS AND RESULTS: The antimicrobial activities of leaf extracts from 26 species of eucalyptus were measured. Extracts of Eucalyptus globulus, E. maculata and E. viminalis significantly inhibited the growth of six Gram-positive bacteria (Staphylococcus aureus, MRSA, Bacillus cereus, Enterococcus faecalis, Alicyclobacillus acidoterrestris, Propionibacterium acnes), and of a fungus (Trichophyton mentagrophytes), but they did not show strong antibacterial activity against Gram-negative bacteria (Escherichia coli, Pseudomonas putida). 2',6'-dihydroxy-3'-methyl-4'-methoxy-dihydrochalcone, eucalyptin and 8-desmethyl-eucalyptin, isolated from E. maculata extracts, exhibited potent antimicrobial activities against seven micro-organisms with minimum inhibitory concentrations (MIC) ranging from 1.0 to 31 mg l(-1). CONCLUSIONS: The eucalyptus extracts and three compounds from E. maculata were found to be effective against micro-organisms that cause food poisoning, acne and athlete's foot. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows potential uses of extracts from E. globulus, E. maculata and E. viminalis, and antimicrobial compounds isolated from E. maculata.     

Purification and Some Properties of a Novel α-Amylase Produced by a Strain of Thermoactinomyces vulgaris

An α-amylase[α-l,4-glucan 4-glucanohydrolase, EC 3.2.1.1.], found in the culture filtrate of a strain of Thermoactinomyces vulgaris, was purified by ammonium sulfate fractionation, and DEAE-cellulose and CM-cellulose chromatographies. The purified enzyme showed a single band on disc gel electrophoresis. The optimum reaction pH and temperature were determined to be around pH 5.0 and 70°C. The isoelectric point was determined to be pH 5.2. The α-amylase was stabilized by Ca²⁺.

The α-amylase was found to hydrolyze pullulan to panose. Therefore, the hydrolytic pattern of this enzyme is different from those of pullulanase and isopullulanase.     

Properties of an α-1,3-Glucanase from Streptomyces sp. KI-8

Srome properties were examined of purified α-l,3-glucanase isolated from the culture supernatant of the soil microorganism Streptomyces KI-8.

The optimum pH and temperature were pH 5.4 and 60°C, respectively. The α-1,3-glucanase was stable up to 50°C on heating for 10 min. This enzyme hydrolyzed the substrate α-l,3-glucan into glucose and nigerose by an endo-type of action. Nigerotriose, nigerotetraose and nigeropentaose were hydrolyzed into glucose and nigerose, whereas nigerose was not attacked. The degree of hydrolysis of pseudonigeran, Lentinus α-1,3-glucan, mutan IG-1 (less soluble fraction) and IG-2 (more soluble fraction) by the α-1,3-glucanase were 28.5%, 14.3%, 8.8% and 10.0%, respectively. Km values (mg/ml) for pseudonigeran, Lentinus α-l,3-glucan, mutan IG-1 and IG-2 were 1.12, 1.98, 8.00 and 5.00. The enzyme solubilized 50 to 80% of mutan by concerted action with dextranase.     

Purification and characterization of basic glucosyltransferase from Streptococcus mutans serotype c

Streptococcus mutans Ingbritt (serotype c) was found to secrete basic glucosyltranserase (sucrose: 1,6-α-^D-glucan 3-α- and 6-α- glucosyltransferase). The enzyme preparation obtained by ethanol fractionation, DEAE Bio-Gel A chromatography, chromatofocusing and preparative isoelectric focusing was composed of three isozymes with slightly different isoelectric points (pI 8.1–8.4). The molecular weight was estimated to be 151 000 by SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 9.8 IU per mg of protein and the optimum pH was 6.5. The enzyme was activated 2.4-fold by commercial dextran T10, and had K_m values of 7.1 μM for the dextran and 4.3 mM for sucrose. Glucan was de novo synthesized from sucrose by the enzyme and found to be 1,6- α-D-glucan with 17.7% of 1,3,6-branching structure by a gas-liquid chromatography-mass spectroscopy.     

Enzymatic hydrolysis of cellulosic materials: A kinetic study

A kinetic study of the enzymatic hydrolysis of two celluloses with different structural features was performed at various temperatures (26-50 degrees C). The enzymatic system consisted of three types of enzymes: E(1)-beta-1,4-glucan glucanohydrolase; E(2)-beta-1,4-glucan cellobiohydrolase; and E(3)-beta-glucosidase. A mathematical model for the mechanism of the hydrolysis of cellulosic materials catalyzed by a multienzymatic system was checked and a good rationalization of the experimental results was achieved. Uncompetitive and competitive glucose inhibition on E(1) and E(2), respectively, appeared to occur for both substrates. Inhibition by cellobiose was checked at 34 degrees C on one substrate. The V(max), K(m), and glucose inhibition constants were optimized and their dependence on temperature determined.     

Effect of an inhibitor of glucosylceramide synthesis on cultured rabbit skin fibroblasts

1-Phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an effective inhibitor of UDP-glucose:ceramide glucosyltransferase, caused growth inhibition of cultured rabbit skin fibroblasts in a dose-dependent manner. At 50 microM both threo and erythro isomers of PDMP completely suppressed the cell growth. Major gangliosides of the fibroblasts, GM3 and GD3, were greatly reduced in amounts in the presence of threo-PDMP and accumulation of ceramides was observed. Surface labeling with galactose oxidase and [3H]NaBH4 demonstrated that neural glycosphingolipids with four or more sugars present on the surface of control cells were not detectable when the fibroblasts were grown in medium containing threo-PDMP. Metabolic labeling of cellular glycosphingolipids with [14C]-galactose showed reduced incorporation of radioactivity into gangliosides and neutral glycosphingolipids when threo-PDMP was present in the medium. In contrast, the erythro isomer of PDMP did not affect the biosynthesis of glycosphingolipids, a result suggesting that the inhibitory effect of erythro-PDMP on cell growth was due to a mechanism other than the inhibition of glucosyltransferase.     

KRE5 gene null mutant strains of Candida albicans are avirulent and have altered cell wall composition and hypha formation properties

The UDP-glucose:glycoprotein glucosyltransferase (UGGT) is an endoplasmic reticulum sensor for quality control of glycoprotein folding. Saccharomyces cerevisiae is the only eukaryotic organism so far described lacking UGGT-mediated transient reglucosylation of N-linked oligosaccharides. The only gene in S. cerevisiae with similarity to those encoding UGGTs is KRE5. S. cerevisiae KRE5 deletion strains show severely reduced levels of cell wall beta-1,6-glucan polymer, aberrant morphology, and extremely compromised growth or lethality, depending on the strain background. Deletion of both alleles of the Candida albicans KRE5 gene gives rise to viable cells that are larger than those of the wild type (WT), tend to aggregate, have enlarged vacuoles, and show major cell wall defects. C. albicans kre5/kre5 mutants have significantly reduced levels of beta-1,6-glucan and more chitin and beta-1,3-glucan and less mannoprotein than the WT. The remaining beta-1,6-glucan, about 20% of WT levels, exhibits a beta-1,6-endoglucanase digestion pattern, including a branch point-to-linear stretch ratio identical to that of WT strains, suggesting that Kre5p is not a beta-1,6-glucan synthase. C. albicans KRE5 is a functional homologue of S. cerevisiae KRE5; it partially complements both the growth defect and reduced cell wall beta-1,6-glucan content of S. cerevisiae kre5 viable mutants. C. albicans kre5/kre5 homozygous mutant strains are unable to form hyphae in several solid and liquid media, even in the presence of serum, a potent inducer of the dimorphic transition. Surprisingly the mutants do form hyphae in the presence of N-acetylglucosamine. Finally, C. albicans KRE5 homozygous mutant strains exhibit a 50% reduction in adhesion to human epithelial cells and are completely avirulent in a mouse model of systemic infection.     

Light dependency of the cytokinin-induced bud initiation in protonemata of the moss Funaria hygrometrica

The activities of starch synthesizing enzymes were investigated in wheat grains ( Triticum aestivum L. cv. Kolibri) throughout the grain development period. Starch phosphorylase (E.C. 2.4.1.1.) activity was especially high during the early period of grain development, while starch synthase I (ADP glucose α-glucan 4-α-glucosyl-transferase, E.C. 2.4.1.21) had a maximum activity during the later stage of grain filling. The synthetic potential of starch phosphorylase measured in vitro was about 16 times higher than the quantity of starch actually produced. It is therefore suggested that starch phosphorylase is of substantial importance in grain starch synthesis, particularly in the early period of grain growth. The synthetic potential of starch synthase I measured in vitro made up 25 to 50% of the starch production and the synthetic potential of starch synthase II (UDP glucose α-glucan 4α-glucosyl-transferase. E.C. 2.4.1.11) only about 5%.
Reducing light intensity (shading) during the grain filling period depressed grain growth and starch production by about 20%. Starch phosphorylase was not significantly affected by the reduced light intensity if enzyme activity is calculated on unit grain weight and not as activity per grain. Starch synthase I activity, however, was depressed by shading during the later stage of grain development. The depressed starch production found under low light conditions, however, cannot only be explained by an affected starch synthase I activity, but probably was also related to other still unknown factors limiting grain growth under low light conditions. The poor starch production in the shaded plants was not due to an insufficient supply of assimilates.