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1.
An alternative microbiological method for the production of malate from fumarate is presented. The yeast Dipodascus magnusii was used for this bioconversion. The optimum cell growth temperature was 28°C and the working volume 120 ml. The highest level of fumarase activity during bioconversion was achieved at a pH of 7.5 and a temperature of 37°C. These conditions were determined as optimal. Using sodium fumarate (1M), the maximum specific productivity of malic acid obtained was 1.72 g/(gDCW × h) for intact cells. In the case of ammonium fumarate, it was 2.25 g/(gDCW × h). 相似文献
2.
Isao Takata Kozo Yamamoto Tetsuya Tosa Ichiro Chibata 《Enzyme and microbial technology》1980,2(1):30-36
Extensive experiments were carried out to improve the productivity ofl-malic acid from fumaric acid using Brevibacterium flavum immobilized with carrageenan. The most favourable preparation for the continuous production ofl-malic acid was obtained when 16 g of B. flavum cells was entrapped in 100 ml 3.4% carrageenan gel. However, the immobilized cells produced an unwanted by-product, succinic acid. Treatment of the immobilized cells with 0.6% bile extract suppressed the side reaction and gave the highest operational stability of fumarase activity. By the immobilization of intact cells, the optimal temperature of the enzyme reaction shifted to 10°C higher, the optimal pH became broader, and the operational stability of fumarase activity increased. The effect of temperature on the stability of fumarase activity in the immobilized cell column was investigated under conditions of continuous enzyme reaction. The decay of fumarase activity during continuous enzyme reaction was expressed by an exponential relationship. The productivity of the immobilized B. flavum using carrageenan was as high as 5.2 times that of the conventional immobilized B. ammoniagenes using polyacrylamide. 相似文献
3.
Of various yeasts tested in the conversion of fumaric to L-malic acid, Saccharomyces bayanus had the highest activity of fumarase. Cells permeabilized with 0.2% (w/v) CTAB for 5 min gave maximum enzyme activity. Under non-growth conditions, fumarase activity in the permeabilized cells was four times higher (271 U/g) than that of the intact cells (67 U/g). The proposed mathematical model for the batch production of L-malic acid was validated at different initial fumaric acid concentrations. The average conversion of fumaric acid was up to 82% and gave 21, 40, 83 and 175 mM L-malic acid from respectively, 25, 50, 100 and 210 mM: fumaric acid. 相似文献
4.
Effects of Fe2+ ions on the levels of two enzymes (fumarase and mesaconase) with fumarase activity in two Pseudomonads grown under various nutritional conditions were investigated. Fe2+ ions decreased fumarase but increased mesaconase. A high level of mesaconase was found in Ps. arvilla which was unable to metabolize itaconate. The level of mesaconase in the itaconate-grown cells of Ps. fluorescens was almost the same as that in the glucose-grown cells. This suggests that mesaconase is not an enzyme involved in the metabolism of C5-branched-chain dicarboxylates but presumably, taking the place of fumarase, plays a role in the operation of the tricarboxylic acid cycle in the cells grown in the medium containing Fe2+ ions more than 10 nmol/ml. 相似文献
5.
Two different types of fumarase were found in sonic extracts of Escherichia coli; one required Fe-S for the enzyme activity, and the other did not. When the cells were grown without aeration, the Fe-S-independent enzyme occupied over 80% of the overall fumarase activity. Highly purified Fe-S-independent enzyme was suggested to be composed of four subunits (Mr = 48 kDa) by SDS-polyacrylamide gel electrophoresis and gel filtration. Amino acid and N-terminal sequence analyses supported the possibility that the enzyme is a product of fumC gene (FUMC). In aerobically grown cells, however, the content of FUMC was low and the Fe-S-dependent fumarase occupied over 80% of the overall activity. The Fe-S-dependent enzyme appeared to be labile and the activity was rapidly lost during purification. Although the spontaneous inactivation was previously ascribed to thermal lability (S.A. Woods & J.R. Guest (1987) FEMS Microbiol. Lett. 48, 219), the activity could be restored by anaerobic incubation with ferrous ions and SH-compounds. 相似文献
6.
The Bradyrhizobium japonicum fumarase gene (fumC-like) was cloned and sequenced, and a fumC deletion mutant was constructed. This mutant had a Nod+ Fix+ phenotype in symbiosis with the host plant, soybean, and growth in minimal medium with fumarate as sole carbon source was also not affected. The cloned B. japonicum fumC gene fully complemented an Escherichia coli Fum- mutant, strain JH400, for growth in minimal medium with fumarate. The predicted amino acid sequence of the FumC protein showed strong similarity to the E. coli FumC protein, Bacillus subtilis CitG protein, Saccharomyces cerevisiae Fum1 protein, and the mammalian fumarases. The B. japonicum FumC protein accounted for about 40% of the total fumarase activity in aerobically grown cells. The remaining 60% was ascribed to a temperature-labile fumarase. These data suggest that B. japonicum possesses two different fumarase isoenzymes, one of which is encoded by fumC. Besides E. coli, which has three fumarases, B. japonicum is thus the second bacterium for which there is genetic evidence for the existence of more than one fumarase. 相似文献
7.
Molecular characterization of potato fumarate hydratase and functional expression in Escherichia coli. 总被引:1,自引:0,他引:1
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The tricarboxylic acid cycle enzyme fumarase (fumarate hydratase; EC 4.2.1.2) catalyzes the reversible hydration of fumarate to L-malate. We report the molecular cloning of a cDNA (StFum-1) that encodes fumarase from potato (Solanum tuberosum L.). RNA blot analysis demonstrated that StFum-1 is most strongly expressed in flowers, immature leaves, and tubers. The deduced protein contains a typical mitochondrial targeting peptide and has a calculated molecular mass of 50.1 kD (processed form). Potato fumarase complemented a fumarase-deficient Escherichia coli mutation for growth on minimal medium that contains acetate or fumarate as the sole carbon source, indicating that functional plant protein was produced in the bacterium. Antiserum raised against the recombinant plant enzyme recognized a 50-kD protein in wild-type but not in StFum-1 antisense plants, indicating specificity of the immunoreaction. A protein of identical size was also detected in isolated potato tuber mitochondria. Although elevated activity of fumarase was previously reported for guard cells (as compared with mesophyll cells), additional screening and genomic hybridization data reported here do not support the hypothesis that a second fumarase gene is expressed in potato guard cells. 相似文献
8.
Intracellular distribution of fumarase in various animals 总被引:2,自引:0,他引:2
The subcellular distribution of fumarase was investigated in the liver of various animals and in several tissues of the rat. In the rat liver, fumarase was predominantly located in the cytosolic and mitochondrial fractions, but not in the peroxisomal fraction. The amount of fumarase associated with the microsomes was less than 5% of the total enzyme activity. The investigation of the intracellular distribution of hepatic fumarase of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp revealed that the amount of the enzyme located in the cytosol was comparable to that in the mitochondria of all these animals. The subcellular distribution of the enzyme in the kidney, brain, heart, and skeletal muscle of rat, and in hepatoma cells (AH-109A) was also investigated. Among these tissues, the brain was the only exception, having no fumarase activity in the cytosolic fraction, and the other tissues showed a bimodal distribution of fumarase in the cytosol and the mitochondria. The mitochondrial fumarase was predominantly located in the matrix. About 10% of the total fumarase was found in the outer and inner membrane, although it was unclear whether this fumarase was originally located in these fractions. No fumarase activity was detected in the intermembranous space. 相似文献
9.
A. C. M. Weijman 《Antonie van Leeuwenhoek》1977,43(3-4):323-331
Carbohydrates released during acid hydrolysis of intact cells ofDipodascus were studied by gas-liquid chromatographic analysis as their trimethylsilyl derivatives. In addition, cells were characterized
by pyrolysis gas-liquid chromatography and pyrolysis mass spectrometry.
The data obtained support the classification ofDipodascus uninucleatus in a separate genusDipodascopsis. Glucuronic acid is present inD. uninucleatus and, therefore, a possible affinity to fungi classified in the Zygomycetes is considered.Dipodascus aggregatus andDipodascus australiensis were found to be rather different, but very close toGeotrichum candidum and related species. 相似文献
10.
11.
Exposure of dark-grown restingEuglena gracilis Klebs var.bacillaris Cori to light, ethanol, or malate produced an increase in the specific activity of fumarase (EC. 4.2.1.2) and succinate dehydrogenase (EC. 1.3.99.1) during the first 8–12 h of exposure to inducer, followed by a decrease in the specific activity of both mitochondrial enzymes between 12 and 72 h. The increased specific activity represented a net increase in the level of active enzyme, and it was dependent upon cytoplasmic protein synthesis. The photoinduction of fumarase required continuous illumination while the subsequent decrease in fumarase specific activity was independent of light. Light had little effect on the ethanol and malate induction of fumarase and succinate dehydrogenase. In the mutant W3BUL, which has no detectable protochlorophyll(ide) and chloroplast DNA, light induced both mitochondrial enzymes and the kinetics of enzyme induction were similar to the induction kinetics in wild-type cells. The induction of mitochondrial enzymes appears to be controlled by a non-chloroplast photoreceptor. Dark-grown resting cells of the plastidless mutant W10SmL have lost the ability to regulate fumarase levels. In this mutant, the specific activity of fumarase fluctuated and light had little effect on these fluctuations, indicating that fumarase synthesis was uncoupled from the nonchloroplast photoreceptor. Ethanol addition produced transient changes in fumarase specific activity in W10SmL indicating that in this mutant, mitochondrial enzymes are still inductible by metabolites. Fumarase synthesis in wild-type cells was not induced in the dark by levulinic acid, a chemical inducer of the breakdown ofEuglena storage carbohydrates. Taken together, our results indicate that the photoinduction of mitochondrial enzyme synthesis is not a result of the photoinduction of carbohydrate breakdown. The mechanisms by which light and organic carbon induce the synthesis ofEuglena mitochondria may differ. 相似文献
12.
Kozo Yamamoto Tetsuya Tosa Kiyokazu Yamashita Ichiro Chibata 《Applied microbiology and biotechnology》1976,3(3):169-183
Summary Continuous production ofL-malic acid from fumaric acid using immobilized microbial cells was investigated. Several microorganisms having fumarase activity were immobilized into a polyacrylamide gel lattice. Among the microorganisms tested, immobilizedBrevibacterium ammoniagenes IAM 1645 showed the highest enzyme activity, but produced an unwanted by-product, succinic acid. Conditions for suppression of this side reaction were investigated, and bile extract treatment of immobilized cells was found to be effective.The bile extract treatment of immobilized cells also resulted in a marked increase of reaction rate forL-malic acid formation.No difference was observed between the native enzyme and immobilized cells in optimal pH and temperature of the enzyme reaction.The effect of temperature on the reaction rate and the stability of fumarase activity of an immobilized cell column were investigated under conditions of continuous enzyme reaction. The decay of enzyme activity during continuous enzyme reaction was expressed by an exponential relationship. Half-life of the fumarase activity of the immobilized cell column at 37°C was calculated to be 52.5 days.Presented at the Annual Meeting of the Society of Fermentation Technology, Japan, Osaka, Japan, October 30, 1975. 相似文献
13.
Isao Umemura Satoru Takamatsu Tadashi Sato Tetsuya Tosa Ichiro Chibata 《Applied microbiology and biotechnology》1984,20(5):291-295
Summary In our laboratory, EAPc-7 a strain having higher aspartase activity was derived from Escherichia coli ATCC 11303. For the improvement of l-aspartic acid productivity using EAPc-7 cells immobilized in -carrageenan, it was necessary to eliminate the fumarase activity which converts fumaric acid to l-malic acid. Several treatments for specifically eliminating fumarase activity from EAPc-7 cells were tested and it was found that when EAPc-7 cells were treated in a culture broth (pH 4.9) containing 50 mM l-aspartic acid at 45° C for 1 h, fumarase activity was almost completely eliminated without inactivation of the aspartase.The treated cells, immobilized in -carrageenan, were used for continuous production of l-aspartic acid from ammonium fumarate. The formation of l-malic acid was negligible and the half-life of the immobilized preparation was 126 days.Productivity of immobilized preparation of treated EAPc-7 cells in l-aspartic acid production was six times of that of the parent cell preparation. 相似文献
14.
Continuous enzymatic synthesis of L-malic acid from potassium fumarate in packed-bed flow reactors was investigated. Carrageenan-immobilized Escherichia coli cells were used as a biocatalyst. The operational stability of the biocatalyst fumarase activity was studied, and conditions for preserving high activity of the biocatalyst were determined. 相似文献
15.
Carrot suspension cells were found to be unable to transport malate directly into the cell but utilized it as a single carbon source in a unique manner -they converted malate extracellularly to fumarate and subsequently used it instead. The uptake of fumarate proved to be inducible and sensitive to pH and protonophore. Immuno-blot experiments using an antibody raised against Arabidopsis fumarase showed that fumarase polypeptide appeared in the medium. Fumarase was not detected in medium when fumarate or glucose was used as a carbon source. The activity of fumarase, which catalyzes the reversible hydration reactions, was induced both in the medium (malate into fumarate, releasing protons) and in the cells (fumarate into malate, requiring protons) and resulted in an increase in the pH gradient across the plasma membrane. The reason for the participation of fumarase in the utilization of malate is discussed. 相似文献
16.
Studies on yeast fumarase provide the main evidence for dual localization of a protein in mitochondria and cytosol by means of retrograde translocation. We have examined the subcellular targeting of yeast and human fumarase in live cells to identify factors responsible for this. The cDNAs for mature yeast or human fumarase were fused to the gene for enhanced green fluorescent protein (eGFP) and they contained, at their N-terminus, a mitochondrial targeting sequence (MTS) derived from either yeast fumarase, human fumarase, or cytochrome c oxidase subunit VIII (COX) protein. Two nuclear localization sequences (2x NLS) were also added to these constructs to facilitate detection of any cytosolic protein by its targeting to nucleus. In Cos-1 cells transfected with these constructs, human fumarase with either the native or COX MTSs was detected exclusively in mitochondria in >98% of the cells, while the remainder 1-2% of the cells showed varying amounts of nuclear labeling. In contrast, when human fumarase was fused to the yeast MTS, >50% of the cells showed nuclear labeling. Similar studies with yeast fumarase showed that with its native MTS, nuclear labeling was seen in 80-85% of the cells, but upon fusion to either human or COX MTS, nuclear labeling was observed in only 10-15% of the cells. These results provide evidence that extramitochondrial presence of yeast fumarase is mainly caused by the poor mitochondrial targeting characteristics of its MTS (but also affected by its primary sequence), and that the retrograde translocation mechanism does not play a significant role in the extramitochondrial presence of mammalian fumarase. 相似文献
17.
Isao Takata Kozo Yamamoto Tetsuya Tosa Ichiro Chibata 《Applied microbiology and biotechnology》1979,7(2):161-172
Summary To develop an efficient method for continuous production of L-malic acid from fumaric acid using immobilized microbial cells, screening of microorganisms having high fumarase activity was carried out and cultural conditions of selected microorganisms were investigated. As a result of screening microorganisms belonging to the genera Brevibacterium, Proteus, Pseudomonas, and Sarcina were found to produce fumarase in high levels. Among these microorganisms Brevibacterium ammoniagenes, B. flavum, Proteus vulgaris, and Pseudomonas fluorescens were further selected for their high fumarase levels in the cultivation on several media. These 4 microorganisms were entrapped into a k-carrageenan gel lattice, and the resultant immobilized B. flavum showed the highest fumarase activity and operational stability.Cultural conditions for the fumarase formation and the operational stability of fumarase activity of immobilized B. flavum are detailed. Productivity for L-malic acid using immobilized B. flavum with k-carrageenan was 2.3 fold of that using immobilized B. ammoniagenes with polyacrylamide.Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Nagoya, April 3, 1978 相似文献
18.
W T Johnson A C Thomas 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1999,221(2):147-152
Cytochrome-c oxidase is the copper-dependent terminal respiratory complex (complex IV) of the mitochondrial electron transport chain whose activity in a variety of tissues is lowered by copper deficiency. Because inhibition of respiratory complexes increases the production of reactive oxygen species by mitochondria, it is possible that copper deficiency increases oxidative stress in mitochondria as a consequence of suppressed cytochrome-c oxidase activity. In this study, the activities of respiratory complex I + III, assayed as NADH:cytochrome-c reductase, complex II + III, assayed as succinate:cytochrome-c reductase, complex IV, assayed as cytochrome-c oxidase, and fumarase were measured in mitochondria from HL-60 cells that were grown for seven passages in serum-free medium that was either unsupplemented or supplemented with 50 n M CuSO4. Fumarase activity was not affected by copper supplementation, but the complex I + III:fumarase and complex IV:fumarase ratios were reduced 30% and 50%, respectively, in mitochondria from cells grown in the absence of supplemental copper. This indicates that copper deprivation suppressed the electron transfer activity of copper-independent complex I + III as well as copper-dependent complex IV. Manganese superoxide dismutase (MnSOD) content was also increased 49% overall in the cells grown in the absence of supplemental copper. Furthermore, protein carbonyl groups, indicative of oxidative modification, were present in 100-kDa and 90-kDa proteins of mitochondria from copper-deprived cells. These findings indicate that in cells grown under conditions of copper deprivation that suppress cytochrome-c oxidase activity, oxidative stress in mitochondria is increased sufficiently to induce MnSOD, potentiate protein oxidation, and possibly cause the oxidative inactivation of complex I. 相似文献
19.
Escherichia coli contains three biochemically distinct fumarases which catalyze the interconversion of fumarate to L-malate in the tricarboxylic acid cycle. Batch culture studies indicated that fumarase activities varied according to carbon substrate and cell doubling time. Growth rate control of fumarase activities in the wild type and mutants was demonstrated in continuous culture; FumA and FumC activities were induced four- to fivefold when the cell growth rate (k) was lowered from 1.2/h to 0.24/h at 1 and 21% O(2), respectively. There was a twofold induction of FumA and FumC activities when acetate was utilized instead of glucose as the sole carbon source. However, these fumarase activities were still shown to be under growth rate control. Thus, the activity of the fumarases is regulated by the cell growth rate and carbon source utilization independently. Further examination of FumA and FumC activities in a cya mutant suggested that growth rate control of FumA and FumC activities is cyclic AMP dependent. Although the total fumarase activity increased under aerobic conditions, the individual fumarase activities varied under different oxygen levels. While FumB activity was maximal during anaerobic growth (k = 0.6/h), FumA was the major enzyme under anaerobic cell growth, and the maximum activity was achieved when oxygen was elevated to 1 to 2%. Further increase in the oxygen level caused inactivation of FumA and FumB activities by the high oxidized state, but FumC activity increased simultaneously when the oxygen level was higher than 4%. The same regulation of the activities of fumarases in response to different oxygen levels was also found in mutants. Therefore, synthesis of the three fumarase enzymes is controlled in a hierarchical fashion depending on the environmental oxygen that the cell encounters. 相似文献
20.
Yoav Peleg Emil Battat Michael C. Scrutton Israel Goldberg 《Applied microbiology and biotechnology》1989,32(3):334-339
Summary Electrophoretic studies of fumarase and nicotine adenine dinucleotide (NAD)-malate dehydrogenase were carried out in the fumaric acid-accumulating fungus Rhizopus oryzae. The analyses revealed two fumarase isoenzymes, one localised solely in the cytosol and the other found both in the cytosol and in the mitochondrial fraction. The activity of the cytosolic isoenzyme of fumarase was higher during the acid production stage than during growth. Addition of cycloheximide inhibited fumaric acid production and decreased the activity of the cytosolic isoenzyme of fumarase. These results suggested that de novo protein synthesis is required for increase in the activity of the cytosolic isoenzyme and that such an increase in activity is essential for fumaric acid accumulation. Three distinct isoenzymes of NAD-malate dehydrogenase could be detected in R. oryzae. No changes were observed in the isoenzyme pattern of malate dehydrogenase during fumaric acid production. 相似文献