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1.
Resting cells of Pseudomonas oleovorans PO-1R that had been grown on octane oxidized 1-alkenes containing 6 to 12 carbon atoms and 1,7-octadiene to their corresponding 1,2-epoxides. The microorganism was capable of growing on 1-octene but not on 1,7-octadiene as a sole carbon source. The optimal temperature, pH, and 1-octene concentration for 1,2-epoxyoctane production by the resting cells were 34 to 40 C, pH 7 to 8, and 1.5 mg of 1-octene per ml, respectively. Epoxide concentration reached a maximum after 150 min of incubation and subsequently declined. In the absence of 1-octene, the epoxide was metabolized readily by the resting cells. The amount of 1,2-epoxyoctane produced was dependent on the initial cell concentration. With larger cell populations, the amount of epoxide present after 60 min of incubation was less than the amount observed at lower population densities after the same time period. This relationship was attributed to the rapid depletion of 1-octene at high biomass concentrations and the resultant early initiation of epoxide degradation by the resting cells. 相似文献
2.
Marie-Jose de Smet Jaap Kingma Hans Wynberg Bernard Witholt 《Enzyme and microbial technology》1983,5(5):352-360
The bioconversion of hydrocarbons by Pseudomonas oleovorans has been studied in two-phase systems. In these systems, the hydrocarbon substrate is present in sufficient amounts to form the bulk apolar phase. High cell densities (up to 20 mg dry mass per ml water phase) are reached when the apolar phase consists of n-octane, 1-octene or 1-decene. There is considerable cell damage after incubation for 50–70 h. Loss of cell viability and membrane damage as observed by freeze-fracture electron microscopy correlate with a loss of hydrocarbon oxidation, measured as the conversion of 1-octene to 1,2-epoxyoctane. The final yield of oxidized hydrocarbon in the apolar substrate phase can be increased substantially by replacing the damaged cells with freshly grown cells. Yields up to 150 mg 1,2-epoxyoctane per ml 1-octene and up to 20–25 mg 1,2-epoxyoctane per ml culture were obtained with four cycles of the cell renewal procedure. Several other substrates in addition to octene were tested in the optimized two-phase system. Of these, 1-decene was converted into (R)-1,2-epoxydecane with an optical purity of 60%, while allylbenzene was converted into chiral 1,2-epoxy-3-phenylpropane. Some of the future applications of the conversion products are discussed. 相似文献
3.
Enzymatic epoxidation: synthesis of 7,8-epoxy-1-octene, 1,2-7,8-diepoxyoctane, and 1,2-Epoxyoctane by Pseudomonas oleovorans. 总被引:2,自引:2,他引:0
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The kinetics of the enzymatic formation of 7,8-epoxy-1-octene, 1,2-7,8-diepoxyoctane, and 1,2-epoxyoctane by growing and resting cell suspensions of Pseudomonas oleovorans are described. Formation of 1,2-epoxyoctane occurs concurrently with exponential growth on 1-octene, providing that 1-octene is in excess. Conversion of 1,7-octadiene to 7,8-epoxy-1-octene by cells growing on octane lags behind exponential growth and continues into the stationary phase, terminating upon cell death. Formation of 1,2-7,8-diepoxyoctane does not begin until the cells are well into the stationary phase and also continues until cell death. Results with growing and resting cell suspensions suggest that the various substrates compete for the same enzyme system; that viable cells are essential for substrate transport and epoxidation by whole cells; and that whole cells may concentrate and sequester the epoxides, rendering them unrecoverable by our current methods. 相似文献
4.
Immobilization of Nocardia corallina B-276 was examined for production of 1,2-epoxyoctane from 1-octene in the presence of n-hexadecane as the organic solvent. Hydrophobic silicone polymer was the most suitable material for entrapment of the cells. Coentrapment of aqueous reaction medium into the silicone polymer matrix improved the epoxide productivity. It was also effective to immobilize the cells in a mixed matrix composed of silicone polymer and calcium alginate gel involving the reaction medium. In the organic monophase, the amount of epoxide accumulated with the cells immobilized in an almost equivolumetric composite of both materials was 2 and 7 times the amounts in the silicone and alginate single matrices, respectively, and it became larger than with the free cells in the aqueous-organic two-liquid phase after a longer period of batch operation. The use of such an optimized composite matrix enabled us to perform a relatively simple operation of the continuous three-phase bioreactor. 相似文献
5.
Yoshikazu Izumi Yoshiaki Matsumura Yoshiki Tani Hideaki Yamada 《Bioscience, biotechnology, and biochemistry》2013,77(11):2673-2679
For the purpose of producing pyruvic acid from 1,2-propanediol (PD), three PD-utilizing and thiamin-requiring microorganisms were isolated from soil. All the isolated strains were found to be pyruvic acid producers. Among them, strain 80-M was the best producer and was identified as an Acinetobacter sp. With this bacterium, the conditions were optimized for pyruvic acid production from PD by two methods: growing cell and resting cell methods. The amount of thiamin added to the medium remarkably affected the pyruvic acid production using either method. Under optimal conditions, 14.6 and 10.0 mg/ml of pyruvate as a sodium salt were produced from 20 mg/ml of PD by the growing and resting cell methods, respectively. In the resting cell system, only PD-grown cells showed a significant pyruvate productivity from PD. 相似文献
6.
《Bioscience, biotechnology, and biochemistry》2013,77(9):1957-1964
Ralstonia sp. Ba-0323, a wild strain isolated from soil, produced catechol from benzoate and accumulated it outside the cells. The bacterium produced a maximal amount of catechol (1.6 mg/ml) from 3 mg/ml of sodium benzoate in a 20-h growing culture. The conversion rate of benzoate to catechol was 70% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 3 mg/ml of sodium benzoate reached 1.9 mg/ml at the conversion rate of 83% after 8 h of incubation. Catechol 1,2-dioxygenase, which catalyzed the ring cleavage of catechol, was purified to homogeneity from a cell extract of Ralstonia sp. Ba-0323 growing on benzoate and characterized. The specific activity of the purified enzyme was much lower than those of the dioxygenases from other microorganisms reported. The Km for catechol of the purified enzyme was much higher than those of other dioxygenases. In addition, the NH2-terminal amino acid sequence of the enzyme was less similar to the other catechol 1,2-dioxygenases than they are to each other. 相似文献
7.
A hydrophobic silicone polymer could be effectively applied to immobilization of two kinds of biocatalysts operating in organic media. Horse liver alcohol dehydrogenase, which was solubilized in a small amount of water, or deposited on water-filled hydrophilic particles, was immobilized in this material. This configuration of the preparation involving finely dispersed aqueous phase permitted a simple packed-bed operation for the enzymatic oxidation of alcohol and reduction of aldehyde with a coupled-substrate NAD(H) recycling in n-hexane. Another example was the immobilization of Nocardia corallina which catalysed epoxidation of liquid alkenes such as 1-tetradecene, 1-octene, and styrene in the presence of n-hexadecane. In order to adjust the hydrophobicity-hydrophilicity balance of the support, it was effective to immobilize the cells in a mixed matrix composed of silicone polymer and Ca-alginate gel. The optimum composition of the mixed matrix, which yielded the highest productivity of epoxide, was 80-90% silicone + 20-10% alginate for the production of 1,2-epoxytetradecane, 40-50% silicone + 60-50% alginate for 1,2-epoxyoctane, and almost 0% silicone + 100% alginate for styrene oxide. This significant change of the optimum composition was primarily associated with the degree of substrate inhibition. 相似文献
8.
A.L. Botes 《Biotechnology letters》1999,21(6):511-517
Purification of the membrane-associated epoxide hydrolase from the yeast Rhodosporidium toruloides CBS 0349 to electrophoretic homogeneity was achieved in a single chromatographic step employing the affinity ligand adsorbent Mimetic Green. More than 68% of the total epoxide hydrolase activity present in the whole cells was recovered from the membrane fraction. The enzyme was purified 26-fold with respect to the solubilized membrane proteins and was obtained in a 90% yield. The purified epoxide hydrolase has an apparent monomeric molecular weight of 54 kDa, and a pI of 7.3. The enzyme was optimally active at 30–40 °C, and pH 7.3–8.5. The enzyme is highly glycosylated with a carbohydrate content >42%. The specific activity of the purified enzyme for (±)-1,2-epoxyoctane is 172 mol min–1 mg protein–1. The amino acid composition of the protein was determined. This is the first report of a yeast epoxide hydrolase purified to homogeneity in milligram amounts. 相似文献
9.
10.
Asymmetric hydrolysis of a homologous range of straight chain 1,2-epoxyalkanes was achieved using whole cells of Chryseomonas luteola. Depending on the chain length, hydrolyses of the racemic epoxides afforded optically active epoxides and diols with varying degrees of optical purity. In the case of 1,2-epoxyoctane, the enantiomeric excess of the remaining (S)-epoxide and formed (R)-diol was excellent (ees > 98% and eep = 86%). This is the first report of a bacterial epoxide hydrolase with such unusual enantioselectivity for terminal mono-substituted epoxides bearing no directing group on the chiral C-2 carbon. Benzyl glycidyl ether and the 2,2-disubstituted epoxide, 2-methyl-1,2-epoxyheptane, were hydrolysed, but no enantioselectivity was observed. © Rapid Science Ltd. 1998 相似文献
11.
In resting cells of Cephalosporium acremonium CW19, protein synthesis was inhibited completely by 100 μg cycloheximide per ml. Furthermore, ongoing protein synthesis halted abruptly when cycloheximide was added after 20 min of incubation. Although cycloheximide did not affect the specific rate of penicillin N production, it markedly inhibited the specific rate of cephalosporin C production. The effect of cycloheximide was not influenced by the carbon source used to prepare the cells for the resting cell system. 相似文献
12.
Production of catechol from benzoate by the wild strain Ralstonia species Ba-0323 and characterization of its catechol 1,2-dioxygenase. 总被引:2,自引:0,他引:2
C L Wang S Takenaka S Murakami K Aoki 《Bioscience, biotechnology, and biochemistry》2001,65(9):1957-1964
Ralstonia sp. Ba-0323, a wild strain isolated from soil, produced catechol from benzoate and accumulated it outside the cells. The bacterium produced a maximal amount of catechol (1.6 mg/ml) from 3 mg/ml of sodium benzoate in a 20-h growing culture. The conversion rate of benzoate to catechol was 70% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 3 mg/ml of sodium benzoate reached 1.9 mg/ml at the conversion rate of 83% after 8 h of incubation. Catechol 1,2-dioxygenase, which catalyzed the ring cleavage of catechol, was purified to homogeneity from a cell extract of Ralstonia sp. Ba-0323 growing on benzoate and characterized. The specific activity of the purified enzyme was much lower than those of the dioxygenases from other microorganisms reported. The Km for catechol of the purified enzyme was much higher than those of other dioxygenases. In addition, the NH2-terminal amino acid sequence of the enzyme was less similar to the other catechol 1,2-dioxygenases than they are to each other. 相似文献
13.
Nitrilase-Catalyzed Production of Nicotinic Acid from 3-Cyanopyridine in Rhodococcus rhodochrous J1 总被引:2,自引:1,他引:2
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Caluwadewa Deepal Mathew Toru Nagasawa Michihiko Kobayashi Hideaki Yamada 《Applied microbiology》1988,54(4):1030-1032
The nitrilase which occurs abundantly in cells of Rhodococcus rhodochrous J1 catalyzes the direct hydrolysis of 3-cyanopyridine to nicotinic acid without forming nicotinamide. By using resting cells, the reaction conditions for nicotinic acid production were optimized. Under the optimum conditions, 100% of the added 3-cyanopyridine could be converted to nicotinic acid, the highest yield achieved being 172 mg of nicotinic acid per 1.0 ml of reaction mixture containing 2.89 mg (dry weight) of cells in 26 h. 相似文献
14.
The interactions of rat liver epoxide hydrolase (EC 3.3.2.3) with itself and with cytochromes P-450 and NADPH-cytochrome P-450 reductase were investigated in microsomal preparations and in reconstituted systems in which all of the enzymes are functionally active. Hydrodynamic measurements indicated that purified epoxide hydrolase behaves as a single aggregate of approximately 16 monomeric units and that further aggregation of the protein only occurs in the presence of high concentrations of phospholipid. Neither guanidine-HCl nor the nonionic detergent Lubrol PX was able to completely dissociate the aggregate into monomers. The interactions of epoxide hydrolase with NADPH-cytochrome P-450 reductase and the major forms of cytochrome P-450 isolated from phenobarbital- and 5,6-benzoflavone-treated rats were studied by Soret difference spectroscopy, by perturbation of the fluorescence of NADPH-cytochrome P-450 reductase and fluorescein-labeled epoxide hydrolase, and by CD spectroscopy. The spectra provided evidence that binding of the proteins to each other occurs and some of the results suggest that affinity constants are on the order of 107, m?1. The spectral perturbations were not observed with other intrinsic membrane proteins. When microsomes were treated with the crosslinking reagent dimethylsuberimidate and solubilized with detergents, epoxide hydrolase could be precipitated with antibodies raised to cytochromes P-450 or NADPH-cytochrome P-450 reductase. Transient times were determined for the conversion of 1-octene to octene-1,2-dihydrodiol in a reconstituted enzyme system and for the conversion of naphthalene to naphthalene-1,2-dihydrodiol in rat liver microscomes and compared to the transient times predicted from the enzymatic rates of hydrolysis of the intermediate epoxides. In all cases the observed transient times were shorter than expected, in support of the view that coupling of epoxide hydrolase with cytochromes P-450 occurs. These results support the view that epoxide hydrolase couples with cytochrome P-450-containing mixed-function oxidase systems and may have relevance to the metabolism of potentially harmful xenobiotics by these enzymes. 相似文献
15.
Biological Process for Converting Naphthalene to cis-1,2-Dihydroxy-1,2-Dihydronaphthalene 总被引:4,自引:3,他引:1
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A biological process for converting naphthalene to cis-1,2-dihydroxy-1,2-dihydronaphthalene (DHD) catalyzed by Pseudomonas putida strain 119 was optimized in flask experiments. These studies revealed the following: (i) P. putida 119 can propagate efficiently and produce DHD when supplied one of several carbon sources and naphthalene; (ii) maximum DHD production by P. putida 119 occurs in logarithmic-growth-phase cells and decreases at various rates in the stationary growth phase, depending upon the carbon source used; (iii) several analogs of salicylic acid can be used as effective inducers of naphthalene metabolism in P. putida cells growing on glucose; and (iv) the addition of chemical surfactants to naphthalene-cell (P. putida 119) mixtures stimulates DHD production. 相似文献
16.
Toru Nagasawa Gurdev Singh Dhillon Takafumi Ishii Hideaki Yamada 《Journal of biotechnology》1985,2(6):365-377
The regulatory properties of serine-O-transacetylase and O-acetylserine sulfhydrylase have been investigated with 3-chloro-l-alanine resistant Bacillus sphaericus L-118. The enhancement of O-acetylserine sulfhydrylase formation by 3-chloro-l-alanine was observed and this effect was counteracted by corepressor l-cysteine. O-Acetylserine sulfhydrylase occurring in B. sphaericus L-118 can catalyse β-replacement reaction of 3-chloro-l-alanine in the presence of a high concentration of sodium hydrosulfide to form l-cysteine. The optimal reaction conditions for l-cysteine production were studied using resting cells. Under optimal conditions, about 80% of the added 3-chloro-l-alanine could be converted to l-cysteine. The highest yield achieved was 70 mg of l-cysteine per 1.0 ml of the reaction mixture. 相似文献
17.
Isolation and Characterization of the Epoxide Hydrolase-Encoding Gene from Xanthophyllomyces dendrorhous 总被引:1,自引:0,他引:1
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The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the α/β hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system. 相似文献
18.
Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis 总被引:1,自引:0,他引:1
Visser H Vreugdenhil S de Bont JA Verdoes JC 《Applied microbiology and biotechnology》2000,53(4):415-419
We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated
molecular mass of 46.3 kDa. The amino acid sequence was similar to that of microsomal epoxide hydrolase, which suggests that
the epoxide hydrolase of R. glutinis also belongs to the α/β hydrolase fold family. EPH1 cDNA was expressed in Escherichia coli and resting cells showed a specific activity of 200 nmol min−1 (mg protein)−1 towards 1,2-epoxyhexane.
Received: 2 August 1999 / Received revision: 4 October 1999 / Accepted: 10 October 1999 相似文献
19.
Summary Immobilized growing yeast cells were prepared in kappa-carra-geenan gel. Gel beads containing a small number of cells were incubated in a complete medium. The cells grew very well in the gel and the number of living cells per ml of gel increased to over 10 times that of free cells per ml of culture medium. After growing in the gel, the cells formed a dense layer of cells near the gel surface and produced large amounts of ethanol. The conditions for continuous production of ethanol using immobilized growing yeast cells were investigated. The supply of appropriate nutrients for growth was essential for the continuous production. The living cells in the gel were maintained at the high level of 109 per ml of gel and continuous production of ethanol using the complete medium containing 10% glucose was carried out with a retention time of 1 h. In this operation, a stable steady state was maintained for longer than 3 months. The ethanol concentration was 50 mg/ml and the conversion of glucose utilized to ethanol produced was almost 100% of the theoretical yield. 相似文献
20.
Cytochrome Content of Two Pseudomonads Containing Mixed-Function Oxidase Systems 总被引:5,自引:1,他引:4
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Julian A. Peterson 《Journal of bacteriology》1970,103(3):714-721
The cytochrome and nonheme iron protein content of two pseudomonads, Pseudomonas oleovorans and P. putida, containing mixed function oxidase systems was examined. The mixed function oxidase system of P. oleovorans and P. putida had previously been shown to be present in cells which had been grown on hexane and camphor, respectively, as energy source. The content of protoheme was found to increase significantly when the organisms were grown on the substrates for mixed function oxidation. The nonheme iron protein content increased significantly in the case of P. putida and was constant in P. oleovorans. The cytochrome c content was essentially constant in both pseudomonads. The content of cytochrome P-450 in P. putida increased from an immeasurably low amount to 0.15 nmoles per mg (dry weight). The content of cytochrome o in P. oleovorans increased by a factor of 4.5. P. oleovorans was not found to contain detectable quantities of cytochrome P-450 either in the presence or absence of the mixed function oxidase system. 相似文献