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1.
Chemical analysis of leaf base tissue of Dactylis glomerate failed to detect any low MW oligosaccharide intermediates during fructosan synthesis. Extracts of tissue harvested at various times after the incorporation of 14CO2 showed a decline in radioactivity in sucrose and an equivalent rise in high MW fructosan with no significant accumulation of radioactivity in oligosaccharides. No evidence was obtained for the existence of nucleotide fructose in the tissue, indicating that fructosan synthesis occurs by direct transfer of fructosyl residues from sucrose to the polymer.  相似文献   

2.
A 10-fold purification of sucrose sucrose fructosyl transferase from Cichorium intybus roots was achieved by ammonium sulphate fractionation and DEAE-cellulose column chromatography. The energy of activation for this enzyme was ca 48 kJ/mol sucrose. Sucrose sucrose fructosyl transferase and invertase were prominent during early months of growth. Evidence obtained from: (1) the changes in carbohydrate composition at monthly intervals; (2) comparative studies on fructosyl transferase and invertase at different stages of root growth; and (3) incubation studies with [14C]glucose, [14C]fructose and [14C]sucrose revealed that, during the later stages of root growth, fructosan hydrolase is responsible for fructosan hydrolysis. No evidence for the direct transfer of fructose from sucrose to high Mr glucofructosans was obtained.  相似文献   

3.
A sucrose:sucrose 1F-β-d-fructosyltransferase (EC 2.4.1.99) has been purified from onion seeds by fractionation with ammonium sulphate and then by chromatography on DEAE-cellulose, CM-cellulose, octyl-Sepharose and Sephadex G-200. The purified enzyme which showed a single protein band on polyacrylamide gel electrophoresis was free from the other fructosyltransferases, catalysed fructosyltransfer from sucrose to another sucrose to form 1-kestose and glucose, and also in some degree transferred a fructosyl residue from sucrose to raffinose and stachyose but did not to 1-kestose and nystose. The enzyme had an Mr of ca 68 000, an optimum pH of 5.4, and Km of 0.083 M, was stable at 20–37° for 10 min, and was inhibited by Hg2+, Ag+, Mn2+ and p-chloromercuribenzoate.  相似文献   

4.
An acid-tolerant α-galactosidase (CVGI) was isolated from the fruiting bodies of Coriolus versicolor with a 229-fold of purification and a specific activity of 398.6 units mg?1. It was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration chromatography. The purified enzyme gave a single band corresponding to a molecular mass of 40 kDa in SDS-PAGE and gel filtration. The α-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The optimum temperature and pH of the enzyme were determined as 60 °C and 3.0, respectively. The enzyme was very stable at a temperature range of 4–50 °C and at a pH range of 2–5. Among the metal ions tested, Cu2+, Cd2+ and Hg2+ ions have been shown to partially inhibit the activity of α-galactosidase, while the activity of CVGI was completely inactivated by Ag+ ions. N-bromosuccinamide inhibited enzyme activity by 100 %, indicating the importance of tryptophan residue(s) at or near the active site. CVGI had wide substrate specificity (p-nitrophenyl galactoside, melidiose, raffinose and stachyose). After treatment with CVGI, raffinose family oligosaccharide was hydrolyzed effectively to yield galactose and sucrose. The results showed that the general properties of the enzyme offer potential for use of this α-galactosidase in several production processes.  相似文献   

5.
A novel dioscin-α-l-rhamnosidase was isolated and purified from fresh bovine liver. The activity of the enzyme was tested using diosgenyl-2,4-di-O-α-l-rhamnopyranosyl-β-d-glucopyranoside as a substrate. It was cleaved by the enzyme to two compounds, rhamnoses and diosgenyl-O-β-d-glucopyranoside. The optimal conditions for enzyme activity were that temperature was at 42 °C, pH was at 7, reaction time was at 4 h, and the substrate concentration was at 2%. Furthermore, metal ions such as Fe3+, Cu2+, Zn2+, Ca2+ and Mg2+ showed different effects on the enzyme activity. Mg2+ acted as an activator whereas Cu2+, Fe3+, and Zn2+ acted as strong inhibitors in a wide range of concentrations from 0 to 200 mM. It was interesting that Ca2+ played a role as an inhibitor when its concentration was at 10 mM and acted as an activator at the other concentrations for the enzyme. Moreover, the molecular weight of enzyme was determined as 75 kDa.  相似文献   

6.
Bush DR 《Plant physiology》1989,89(4):1318-1323
Sucrose is the predominant form of photosynthetically reduced carbon transported in most plant species. In the experiments reported here, an active, proton-coupled sucrose transport system has been identified and partially characterized in plasmalemma vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves. The isolated vesicles concentrated sucrose fivefold in the presence of an imposed pH gradient (basic interior). The presence of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, prevented sucrose accumulation within the vesicles. ΔpH-dependent sucrose transport exhibited saturation kinetics with an apparent Km of 1.20 ± 0.40 millimolar, suggesting translocation was carrier-mediated. In support of that conclusion, two protein modifiers, diethyl pyrocarbonate and p-chloromercuribenzenesulfonic acid, were found to be potent inhibitors with 50% inactivation achieved at 750 and 30 micromolar, respectively. ΔpH-Dependent sucrose transport was not inhibited by glucose, fructose, raffinose, or maltose suggesting the transport system was specific for sucrose. Transport activity was associated with the plasmalemma because ΔpH-dependent sucrose transport equilibrated on a linear sucrose gradient at 1.17 grams per cubic centimeter and comigrated with a plasmalemma enzyme marker, vanadate-sensitive K+, Mg2+-ATPase. Taken together, these results provide the first In vitro evidence in support of a sucrose-proton symport in the plasmalemma of mature leaf tissue.  相似文献   

7.
A soluble enzyme preparation from the leaves of fennel (Foeniculum vulgare M.) has been shown to catalyze the cation-dependent cyclization of both geranyl pyrophosphate and neryl pyrophosphate to the bicyclic rearranged monoterpene l-endo-fenchol (R. Croteau, M. Felton, and R. Ronald, 1980 Arch. Biochem. Biophys.200, 524–533). To examine the possible presence of free intermediates between the acyclic precursors and fenchol, and to remove competing cyclase and pyrophosphatase activities, the soluble preparation was partially purified by ammonium sulfate fractionation followed by gel filtration on Sephadex G-150 and ion exchange chromatography on O-diethylaminoethyl-cellulose. Activities for the cyclization of geranyl pyrophosphate and neryl pyrophosphate to fenchol were coincident on Chromatographic fractionation suggesting that the same enzyme was capable of cyclizing both acyclic substrates. No interconversion of the acyclic precursors was detected. Although bornyl pyrophosphate is a free intermediate in the biosynthesis of the related bicyclic monoterpenol borneol, both protein fractionation and isotopic dilution experiments ruled out endo-fenchyl pyrophosphate as a free intermediate in fenchol biosynthesis. Similarly, while construction of the fenchane skeleton was demonstrated to involve the rearrangement of an intermediate pinane skeleton, isotopic dilution experiments ruled out both optical antipodes of α-pinene, β-pinene, cis-2-pinanol, trans-2-pinanol, and the corresponding 2-pinyl pyrophosphates as free intermediates of the enzyme-catalyzed reaction. Furthermore, exhaustive search of the enzymatic reaction products provided no evidence to suggest the involvement of any free intermediate between the acyclic precursor and fenchol. The endo-fenchol synthetase has an apparent molecular weight of 60,000, shows a pH optimum near 7.0, and requires Mn2+ (1 mm) for catalytic activity. Co2+ can partially substitute for Mn2+, but other divalent cations are ineffective. The partially purified synthetase is inhibited by p-hydroxymercuribenzoate and by phenylglyoxal, and it exhibits a preference for geranyl pyrophosphate over neryl pyrophosphate as substrate. An integrated scheme is proposed for the cyclization and rearrangement catalyzed by fenchol synthetase.  相似文献   

8.
The production of β-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of β-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular β-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 °C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 °C. β-Fructofuranosidase activity was slightly activated by Cu2+, Mn2+, Mg2+, and Na+ at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with Kd values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively.  相似文献   

9.
Bifidobacterium lactis is a moderately oxygen-tolerant, saccharolytic bacterium often used in combination with fructooligosaccharides (FOS) as a probiotic supplement in diverse dairy products. This is the first report describing the gene structure and enzymatic properties of a β-fructofuranosidase [EC 3.2.1.26] from Bifidobacteria. BfrA was identified in Bifidobacterium lactis DSM 10140T and heterologously expressed in Escherichia coli. The G+C content was identical with the G+C content as determined for the total genomic DNA (61.9 mol %). The gene codes for a 532-aa residue polypeptide of 59.4 kDa. Surprisingly, the deduced aa sequence revealed only minor similarity to other fructofuranosidases (18% to E. coli cscA). The enzyme was purified to homogeneity after incorporation of a C-terminal 6xHIS affinity tag. It hydrolased sucrose, 1-kestose, Raftilose®, Actilight®, inulin, and raffinose (100%, 91%, 84%, 80%, 37%, 4%). Fructose moieties were released in an exo-type fashion. Substrates with α-glycosidic linkages or residues other than fructose were not attacked. The kinetic parameters K m and V max for sucrose hydrolysis were 10.3 mM and 0.031 μM/min (pH 7.6; 37°C). The activity was abolished by Zn2+ (1 mM) and significantly inhibited by Fe2+ and Ni2+ (10 mM). The enzyme showed its maximal activity at 40°C.  相似文献   

10.
Fuhrer J 《Plant physiology》1982,70(1):162-167
Stress ethylene production in bean (Phaseolus vulgaris L., cv. Taylor's Horticultural) leaf tissue was stimulated by Cd2+ at concentrations above 1 micromolar. Cd2+-induced ethylene biosynthesis was dependent upon synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Activity of ACC synthase and ethylene production rate peaked at 8 h of treatment. The subsequent decline in enzyme activity was most likely due to inactivation of the enzyme by Cd2+, which inhibited ACC synthase activity in vitro at concentrations as low as 0.1 micromolar. Decrease in ethylene production rate was accompanied by leakage of solutes and increasing inhibition of ACC-dependent ethylene production. Ca2+, present during a 2-hour preincubation, reduced the effect of Cd2+ on leakage and ACC conversion. This suggests that Cd2+ exerts its toxicity through membrane damage and inactivation of enzymes. The possibility of an indirect stimulation of ethylene biosynthesis through a wound signal from injured cells is discussed.  相似文献   

11.
The filamentous fungus Aspergillus terreus secretes both invertase and β-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a β-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0–6.0 and 55–65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0–10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn2+ (161 %), Co2+ (68 %) and Mg2+ (61 %) and was inhibited by Al3+, Ag+, Fe2+ and Fe3+. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (KM = 22 mM). However, in the presence of Mn2+, the apparent affinity and Vmax for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in Vmax was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and β-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.  相似文献   

12.
Dimethylallylpyrophosphate:l-tryptophan dimethylallyltransferase (DMAT synthetase), the first pathway-specific enzyme of ergot alkaloid biosynthesis, has been isolated from mycelia of Claviceps sp., strain SD 58, and purified to apparent homogeneity. The enzyme reaction products were identified as l-4-(γ,γ-dimethylallyl)tryptophan and inorganic pyrophosphate. DMAT synthetase is a single subunit protein of molecular weight 70,000–73,000 and has an isoelectric point at pH 5.8. The enzyme is activated by Fe2+, Mg2+, and particularly Ca2+; Km values for l-tryptophan and dimethylallylpyrophosphate were determined to be 0.067 and 0.2 mm, respectively. Kinetic analysis indicated that the DMAT synthetase reaction proceeds by a sequential rather than a ping-pong mechanism.  相似文献   

13.
An enzyme synthesizing stachyose, galactinol-raffinose galactosyltransferase (EC2.4.1.67), has been purified ca 40-fold from mature leaves of Cucurbita pepo using ammonium sulphate precipitation, Sephadex gel filtration and DEAE-Sephadex gel chromatography. The purified enzyme fraction was separated from all but 2 % of the total,α-galactosidase activity extracted from the tissue. The enzyme was optimally active at pH 6.9 and was stable for at least a month at 4° in the presence of 20 mM 2-mercaptoethanol. The enzyme displayed high specificity for the donor galactinol (Km 7.7 mM) and the acceptor raffinose (Km 4.6 mM) and was unable to effect synthesis of any other member of the raffinose series of galactosyl-sucrose oligosaccharides. Co2+, Hg2+, Mn2+ and Ni2+ ions were particularly inhibitory; no metal ion promotion was observed and 5 mM EDTA was ineffective. Myo-inositol was strongly inhibitory (Ki 2 mM), melibiose weakly so. Tris buffer (0. 1 M) was also inhibitory. Galactinol hydrolysis occurred in the absence of the acceptor raffinose but there was no hydrolysis of either raffinose or stachyose in the absence of the donor galactinol. The reaction was readily reversible and exchange reactions were detected between substrates and products. It is proposed that the synthesis of stachyose in mature leaves ofC. pepo proceeds via this galactosyltransferase and not via α-galactosidase.  相似文献   

14.
Some of the in vitro properties of PAL from gherkin hypocotyls were investigated. No metal ion requirement for this enzyme could be demonstrated but there were indications that PAL was a sulphydryl enzyme. Kinetic analysis suggested that PAL exhibited negative homotropic cooperativity. Two Km values were determined, these were KmH, 2·9 × 10?4 M and KmL, 4·3 × 10?5 M. Strong inhibition of the enzyme was exerted by d-phenylalanine, trans-cinnamic acid, o-coumaric acid, gallic acid, quercetin and kaempferol. Kinetic studies on the inhibition patterns of these compounds established that d-phenylalanine linearized the curvilinear kinetics, trans-cinnamic acid and o-coumaric acid acted as competitive inhibitors whilst gallic acid, quercetin and kaempferol acted as mixed inhibitors. Using a number of desensitization techniques PAL was partially desensitized to inhibition by the mixed inhibitors. These results led to the conclusion that PAL may have a regulatory role in phenol, coumarin and flavonoid biosynthesis.  相似文献   

15.
The synthesis and accumulation of lychnose and isolychnose in leaves of Cerastium arvense during late fall and winter were shown to be controlled mainly by the temperature regime, while the photoperiodic regime had only a quantitative effect. Labelled lychnose and isolychnose were formed from [14C]raffinose in a cell-free enzyme preparation of leaves of Cerastium arvense in accordance with the equation: raffinose+raffinose (iso-)lychnose+sucrose.Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday  相似文献   

16.
Chloromethane (CH3Cl), a gaseous natural product released as a secondary metabolite by many woodrotting fungi of the family Hymenochaetaceae, has been shown to act as a methyl donor for biosynthesis of methyl esters of benzoic and furoic acid in the primary metabolism of Phellinus pomaceus. The broad-specificity methylating system could esterify a wide range of aromatic and aliphatic acids. In addition to CH3Cl, both bromo- and iodomethanes acted as methyl donors. Methylation did not appear to proceed via methanol or a coenzyme A intermediate. The initial growth-related accumulation of methyl benzoate during culture of P. pomaceus was paralleled by an increase in activity of the methylating system in the mycelium. Changes in percent incorporation of C2H3 from exogenous C2H3Cl during growth indicated that although utilization of CH3Cl was initially closely coupled to biosynthesis of the compound, the system became less tightly channeled later in growth. This phase coincided with release of gaseous CH3Cl by the fungus. A biochemically distinct CH3Cl-utilizing system capable of methylating phenols and thiophenol was also identified in the fungus, but in contrast with the carboxylic acid-methylating system, it attained maximum activity in the idiophase. Preliminary investigations of a non-CH3Cl-releasing fungus, Fomitopsis pinicola, have shown the presence of a CH3Cl-utilizing system capable of methylating benzoic acid, suggesting that CH3Cl biosynthesis may occur in non-hymenochaetaceous fungi. Halogenated compounds hitherto found in nature are mainly stable end products of metabolism. The participation of CH3Cl in primary fungal metabolism demonstrates that some halometabolites may have a previously unrecognized role as intermediates in the biosynthesis of nonhalogenated natural products.  相似文献   

17.
Sulphate activation in the unicellular red alga Rhodella   总被引:1,自引:0,他引:1  
ATP-sulphurylase (ATP: sulphate adenylyl-transferase, E.C. 2.7.74) from the unicellular red alga Rhodella has been purified 14-fold by (NH4)2SO4 fractionation. It exhibits a temperature optimum of 31°, an activation energy of 10.8 kcal, has a pH optimum between 7.5 and 9.0 and forms unstable intermediates when incubated with ATP and group VI anions (CrO42?, MoO42?), WO42?), resulting in the accumulation of pyrophosphate. Of the nucleotides tested, only ATP is acted upon by the enzyme. A divalent ion is required for activity and stimulation of the enzyme is 5 times higher with Mg2+ than any other ion tested. The actual substrate for the reaction is a Mg-ATP2? complex. Free ATP inhibits the reaction. APS-[35S] and traces of PAPS-[35S] are formed when cell-free extract from Rhodella is incubated with ATP and sulphate-[35S]. This indicates the existence of APS-kinase (ATP:adenylyl-sulphate 31-phosphotransferase, E.C. 2.7.1.25) as well as ATP-sulphurylase.  相似文献   

18.
A family-4 α-galactosidase Mel4A of Bacillus halodurans was expressed in Escherichia coli and characterized. Recombinant enzyme rMel4A depended on NAD+, some divalent cations such as Mn2+, and reducing reagents such as dithiothreitol. rMel4A was active on small saccharides such as raffinose but not on highly polymerized galactomannan. Immunological analysis indicated that raffinose induced the production of Mel4A in B. halodurans.  相似文献   

19.
A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni2+, Ba2+ and Fe2+. The apparent K m and V max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min?1 mg?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min?1 mg?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.  相似文献   

20.
《Phytochemistry》1986,25(12):2765-2768
Incorporation of [14C]sucrose into difructosyl glucose (F2G), trifructosyl glucose (F3G) and tetrafructosyl glucose (F4G) in the presence of various nucleoside triphosphates revealed that formation of F4G and F3G is retarded in the presence of ATP, and formation of F3G and F2G is significantly enhanced in the presence of CTP, whereas UTP has no effect on the synthesis of these oligosaccharides. Different fructosyl transferases seem to be responsible for the different fructosylation steps and self transfer seems to be the major pathway for fructosan synthesis. Utilization of added glucose, which is formed by sucrose sucrose fructosyl transferase action in vivo, is completely inhibited in acetate buffer whereas in phosphate, citrate and citrate-phosphate buffers glucose is actively utilized. In the presence of fluoride ions both glucose utilization and its conversion to CO2 is inhibited by ca 50%. CO2 production from [14C]glucose is completely inhibited in acetate ions. No evidence for the incorporation of 14C from [14C]glucose into [14C]sucrose is observed. The ratio of bound fructose to bound glucose is the same in the entire length of the root indicating that there is no preferential zone for fructosan synthesis.  相似文献   

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