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1.
D. F. E. Richter  G. O. Kirst 《Planta》1987,170(4):528-534
d-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and d-mannitol dehydrogenase (EC 1.1.1.67) were estimated in a cell-free extract of the unicellular alga Platymonas subcordiformis Hazen (Prasinophyceae), d-Mannitol dehydrogenase had two activity maxima at pH 7.0 and 9.5, and a substrate specifity for d-fructose and NADH or for d-mannitol and NAD+. The K m values were 43 mM for d-fructose and 10 mM for d-mannitol. d-Mannitol-1-phosphate dehydrogenase had a maximum activity at pH 7.5 and was specific for d-fructose 6-phosphate and NADH. The K m value for d-fructose 6-phosphate was 5.5 mM. The reverse reaction with d-mannitol 1-phosphate as substrate could not be detected in the extract. After the addition of NaCl (up to 800 mM) to the enzyme assay, the activity of d-mannitol dehydrogenase was strongly inhibited while the activity of d-mannitol-1-phosphate dehydrogenase was enhanced. Under salt stress the K m values of the d-mannitol dehydrogenase were shifted to higher values. The K m value for d-fructose 6-phosphate as substrate for d-mannitol-1-phosphate dehydrogenase remained constant. Hence, it is concluded that in Platymonas the d-mannitol pool is derectly regulated via alternative pathways with different activities dependent on the osmotic pressure.Abbreviations Fru6P d-fructose 6-phosphate - Mes 2-(N-morpholino)ethanesulfonic acid - MT-DH d-mannitol-dehydrogenase - MT1P-DH d-mannitol-1-phosphate dehydrogenase - Pipes 1,4-piperazinediethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol  相似文献   

2.
The catabolism of d-galactose in yeast depends on the enzymes of the Leloir pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD+-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD+-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.  相似文献   

3.
In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l−1) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.  相似文献   

4.
Cell-free extracts of d-fructose grown cells of Pseudomonas putida, P. fluorescens, P. aeruginosa, P. stutzeri, P. mendocina, P. acidovorans and P. maltophila catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose and contained 1-P-fructokinase activity suggesting that in these species fructuse-1-P and fructose-1,6-P2 were intermediates of d-fructose catabolism. Neither the 1-P-fructokinase nor the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose was present in significant amounts in succinate-grown cells indicating that both activities were inducible. Cell-free extracts also contained activities of fructose-1,6-P2 aldolase, fructose-1,6-P2 phosphatase, and P-hexose isomerase which could convert fructose-1,6-P2 to intermediates of either the Embden-Meyerhof pathway or Entner-Doudoroff pathway. Radiolabeling experiments with 1-14C-d-fructose suggested that in P. putida, P. aeruginosa, P. stutzeri, and P. acidovorans most of the alanine was made via the Entner-Doudoroff pathway with a minor portion being made via the Embden-meyerhof pathway. An edd - mutant of P. putida which lacked a functional Entner-Doudoroff pathway but was able to grow on d-fructose appeared to make alanine solely via the Embden-Meyerhof pathway.Non-Standard Abbreviations cpm counts per min - edd - mutant lacking Entner-Doudoroff dehydrase (6-PGA dehydrase) - EDP Entner-Doudoroff pathway - EMP Embden-Meyerhof pathway - FDP fructose-1,6-P2 - FDPase FDP phosphatase - F-1-P fructose-1-P - F-6-P fructose-6-P - FPTs PEP: d-fructose phosphotransferase system - G-6-P glucose-6-P - KDPG 2-keto-3-deoxy-6-P-gluconate - PEP P-enolpyruvate - 1-PFK 1-P-fructokinase - 6-PFK 6-P-fructokinase - 6-PGA 6-P-gluconate  相似文献   

5.
A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn2+. At pH 9 and 40°C, 118 g d-psicose l−1 was produced from 700 g d-fructose l−1 after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

6.
Clostridium sphenoides was grown on glucose in a phosphate-limited medium. Below 80 M phosphate two new products were formed in addition to ethanol, acetate, H2 and CO2: d(-)-1,2-propanediol and d(-)-lactate. These compounds were apparently synthesized via the methylglyoxal by-pass. The activity of the enzymes involvedmethylglyoxal synthase, methylglyoxal reductase, 1,2-propanediol dehydrogenase and glyoxalase-could be demonstrated in cell extracts of C. sphenoides. The formation of 1,2-propanediol from methylglyoxal proceeded via lactaldehyde. The enzyme methylgloxal synthase was inhibited by phosphate. Clostridium glycolicum, C. nexile, C. cellobioparum, C. oroticum and C. indolis did not produce propanediol under the condition of phosphate limitation. The latter two species, however, formed d(-)-lactate.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday  相似文献   

7.
Utilization of d-amino acids being substrates of d-amino acid dehydrogenase of Salmonella typhimurium was examined. The experiments were done with wild type strains and the mutants dadA missing the enzyme activity and dadR in which its synthesis is released from catabolite repression. Growth on d-tryptophan, d-histidine and d-methionine used as precursors of the l-amino acids was faster when the respective auxotrophs carried dadR mutations. The dadR mutants grew faster when d-or l-alanine was present as a sole source of nitrogen. Experiments with d-amino acid dehydrogenase in vitro provided evidence that d-tryptophan is its substrate with a very low affinity to the dehydrogenase.  相似文献   

8.
Summary Inducible resistance to the glycopeptide antibiotics vancomycin and teicoplanin is mediated by plasmid pIP816 in Enterococcus faecium strain BM4147. Vancomycin induced the synthesis of a ca. 40 kDa membrane-associated protein designated VANA. The resistance protein was partially purified and its N-terminal sequence was determined. A 1761 by DNA restriction fragment of pIP816 was cloned into Escherichia coli and sequenced. When expressed in E. coli, this fragment encoded a ca. 40 kDa protein that comigrated with VANA from enterococcal membrane fractions. The ATG translation initiation codon for VANA specified the methionine present at the N-terminus of the protein indicating the absence of signal peptide processing. The amino acid sequence deduced from the sequence of the vanA gene consisted of 343 amino acids giving a protein with a calculated Mr of 37400. VANA was structurally related to the d-alanyl-d-alanine (d-ala-d-ala) ligases of Salmonella typhimurium (36% amino acid identity) and of E. coli (28%). The vanA gene was able to transcomplement an E. coli mutant with thermosensitive d-ala-d-ala ligase activity. Thus, the inducible resistance protein VANA was structurally and functionally related to cytoplasmic enzymes that synthesize the target of glycopeptide antibiotics. Based on these observations we discuss the possibility that resistance is due to modification of the glycopeptide target.  相似文献   

9.
Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 1018 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC50 values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC50 values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

10.
d-Cysteine desulfhydrase of Escherichia coli W3110 trpED102/F trpED102 was physiologically characterized. It was found to be located in the cytosolic fraction, as 3-chloro-d-alanine dehydrochlorinase is. d-Cysteine desulfhydrase catalyzed not only the ,-elimination reaction of O-acetyl-d-serine to form pyruvate, acetic acid and ammonia, but also the -replacement reaction of O-acetyl-d-serine with sulfide to form d-cysteine. However, these reactions appeared not to proceed in vivo. No other activity of d-cysteine synthesis from O-acetyl-d-serine and sulfide was detected in a crude cell extract of E. coli which was immunotitrated with antibodies raised against the purified d-cysteine desulfhydrase. Although d-cysteine desulfhydrase catalyzes the degradation (,-elimination reaction) of 3-chloro-d-alanine, which is an effective antibacterial agent, E. coli W3110 trpED102/F trpED102 did not show resistance against 3-chloro-d-alanine. Therefore, d-cysteine desulfhydrase does not contribute to 3-chloro-d-alanine detoxification in vivo.  相似文献   

11.
Park CS  Yeom SJ  Kim HJ  Lee SH  Lee JK  Kim SW  Oh DK 《Biotechnology letters》2007,29(9):1387-1391
The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l1 was produced without by-products from 500 g d-psicose l−1 after 6 h.  相似文献   

12.
Summary A polysaccharide producing strain ofBacillus licheniformis was isolated from exudate of raffia palm,Raffia vinifera. The optimum conditions for growth and polysaccharide production have been investigated and established. No appreciable polysaccharide was formed on glucose. It grew best in Czapek-Dox media with sucrose as the carbon source. The polysaccharide has been characterized as a heteropolymer containingd-glucose,d-mannose andd-xylose.  相似文献   

13.
Corynebacterium glutamicum R was metabolically engineered to broaden its sugar utilization range to d-xylose and d-cellobiose contained in lignocellulose hydrolysates. The resultant recombinants expressed Escherichia coli xylA and xylB genes, encoding d-xylose isomerase and xylulokinase, respectively, for d-xylose utilization and expressed C. glutamicum R bglF 317A and bglA genes, encoding phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) β-glucoside-specific enzyme IIBCA component and phospho-β-glucosidase, respectively, for d-cellobiose utilization. The genes were fused to the non-essential genomic regions distributed around the C. glutamicum R chromosome and were under the control of their respective constitutive promoter trc and tac that permitted their expression even in the presence of d-glucose. The enzyme activities of resulting recombinants increased with the increase in the number of respective integrated genes. Maximal sugar utilization was realized with strain X5C1 harboring five xylA–xylB clusters and one bglF 317A bglA cluster. In both d-cellobiose and d-xylose utilization, the sugar consumption rates by genomic DNA-integrated strain were faster than those by plasmid-bearing strain, respectively. In mineral medium containing 40 g l−1 d-glucose, 20 g l−1 d-xylose, and 10 g l−1 d-cellobiose, strain X5C1 simultaneously and completely consumed these sugars within 12 h and produced predominantly lactic and succinic acids under growth-arrested conditions.  相似文献   

14.
The fermentation of d-glucose and d-xylose mixtures by the yeast Candida tropicalis NBRC 0618 has been studied under the most favourable operation conditions for the culture, determining the most adequate initial proportion in these sugars for xylitol production. In all the experiments a synthetic culture medium was used, with an initial total substrate concentration of 25 g L−1, a constant pH of 5.0 and a temperature of 30 °C. From the experimental results, it was deduced that the highest values of specific rates of production and of overall yield in xylitol were achieved for the mixtures with the highest percentage of d-xylose, specifically in the culture with the initial d-glucose and d-xylose concentrations of 1 and 24 g L−1, respectively, with an overall xylitol yield of 0.28 g g−1. In addition, the specific rates of xylitol production declined over the time course of the culture and the formation of this bioproduct was favoured by the presence of small quantities of d-glucose. The sum of the overall yield values in xylitol and ethanol for all the experiments ranged from 0.26 to 0.56 g bioproduct/g total substrate.  相似文献   

15.
Cell-free extracts of d-fructose grown cells of marine species of Alcaligenes as well as Pseudomonas marina contained an activity which catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose in the 1-position as well as activities of the following enzymes: 1-P-fructokinase, fructose-1,6-P2 aldolase, PPi-dependent 6-P-fructokinase, fructokinase, glucokinase, P-hexose isomerase, glucose-6-P dehydrogenase, 6-P-gluconate dehydrase, and 2-keto-3-deoxy-6-P-gluconate aldolase. The presence of these enzyme activities would allow d-fructose to be degraded by the Embden-Meyerhof pathway and/or the Entner-Doudoroff pathway. In cell-free extracts of d-glucose grown cells, the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose as well as 1-P-fructokinase activity were reduced or absent while the remaining enzymes were present at levels similar to those found in d-fructose grown cells. Radiolabeling experiments suggested that both d-fructose and d-glucose were utilized primarily via the Entner-Doudoroff pathway. Alteromonas communis, a marine species lacking 1-P-fructokinase and the PPi-dependent 6-P-fructokinase, contained all the enzyme activities necessary for the catabolism of d-fructose and d-glucose by the Entner-Doudoroff pathway; the involvement of this pathway was also consistent with the results of the radiolabeling experiments.Non-Standard Abbreviations EDP Entner-Doudoroff pathway - EMP Embden-Meyerhof pathway - FDP fructose-1,6-P2 - FDPase FDP phosphatase - F-1-P fructose-1-P - F-6-P fructose-6-P - FPTS PEP: d-fructose phosphotransferase system - PPi-6-PFK PPi dependent 6-PFK - G-6-P glucose-6-P - KDPG 2-keto-3-deoxy-6-P-gluconate - PEP P-enolpyruvate - 1-PFK 1-P-fructokinase - 6-PFK 6-P-fructokinase - 6-PGA 6-P-gluconate  相似文献   

16.
Sinorhizobium sp., which can convert d-fructose into d-psicose, was isolated from soil. The optimal pH, temperature, and cell concentration for d-psicose production with the isolated strain were 8.5, 40°C, and 60 mg/ml, respectively. The toluene-treated cells showed 2.5- and 4.8-fold increases in the d-psicose concentration and productivity compared with untreated washed cells. Under the optimal conditions, the toluene-treated cells produced 37 g d-psicose/l from 70% (w/v) (3.9 M) d-fructose after 15 h.  相似文献   

17.
l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn2+, Fe3+, Fe2+, Ca2+ and inhibited by Cu2+, Ag+, Hg2+, Pb2+. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.  相似文献   

18.
Summary Urine of mutant ddY/DAO mice lackingd-amino-acid oxidase activity contained more serine and proline than that of normal ddY/DAO+ mice.d-Amino-acid oxidase treatment of urinary amino acids decreased the serine and proline, suggesting that they containedd-isomers. An HPLC analysis confirmed the presence ofd-serine. Urinary serine and proline contents were not decreased when the ddY/DAO mice were fed a diet which did not contain supplementaryd-methionine or when they were given water containing antibiotics. These results suggest that thed-serine andd-proline do not derive from thed-methionine supplemented in the diet or from intestinal bacteria. In urine of the ddY/DAO mice, a substance which seemed to bed-methionine sulfoxide and/ord-methionine sulfone was present. It is probably a metabolite of thed-methionine supplemented in the diet. Thed-aminoaciduria in the mutant mice lackingd-amino-acid oxidase activity indicates that this enzyme is involved in the metabolism of thed-amino acids in normal mice.  相似文献   

19.
Summary 1. MC has been shown to inhibit the uptake ofl-glutamate and increased-aspartate release from preloaded astrocytes in a dose-dependent fashion.2. Two sulfhydryl (SH-)-protecting agents; reduced glutathione (GSH), a cell membrane-nonpenetrating compound, and the membrane permeable dithiothreitol (DTT), have been shown consistently to reverse the above effects. MC-inducedd-aspartate release is completely inhibited by the addition of 1 mM DTT or GSH during the actual 5-min perfusion period with MC (5µM); when added after MC treatment, DTT fully inhibits the MC-inducedd-aspartate release, while GSH does not.3. Neither DTT nor GSH, in the absence of MC, have any effect on the rate of astrocyticd-aspartate release. Other studies demonstrate that although MC treatment (5µM) does not induce astrocytic swelling, its addition to astrocytes swollen by exposure to hypotonic medium leads to their failure to volume regulate.4. Omission of calcium from the medium greatly potentiates the effect of MC on astrocyticd-aspartate release, an effect which can be reversed by cotreatment of astrocytes with the dihydropyridine Ca2+-channel antagonist nimodipine (10µM), indicating that one possible route of MC entry into the cells is through voltage-gated L-type channels.  相似文献   

20.
S. Asakura  Dr. R. Konno 《Amino acids》1997,12(3-4):213-223
Summary Urine of ddY/DAO mice lackingd-amino-acid oxidase contained 5.7 times more serine than that of normal ddY/DAO+ mice. Most of the serine wasd-isomer. The origin of thisd-serine was examined. Oral administration of 0.02% amoxicillin and 0.004% minocycline to the ddY/ DAO- mice for 7 days did not reduce the urinaryd-serine, indicating that thed-serine was not of intestinal bacterial origin. When the mouse diet was changed to one with different compositions, the urinaryd-serine was considerably reduced. Furthermore, starvation of the ddY/DAO- mice for 24 hours reduced the urinaryd-serine to 33% of the original level. These results indicate that most of the urinaryd-serine comes from the diet. However, the urine of the starved ddY/DAO- mice still contained 4.6 times mored-serine than that of the ddY/DAO+ mice, suggesting a part of the D-serine have an endogenous origin.  相似文献   

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