共查询到20条相似文献,搜索用时 8 毫秒
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The whole cell tyrosine phenol-lyase (TPL, E.C. 4.1.99.2) activity of Erwinia herbicola (ATCC 21434) was microen-capsulated. We studied the use of this for the conversion of ammonia and pyruvate along with phenol or catechol, respectively, into L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa). The reactions are relevant to the development of new methods for the production of L-tyrosine and L-dopa. The growth of E. herbicola at temperatures from 22 degrees C to 32 degrees C is stable, since at these temperatures the cells grow up to the stationary phase and remain there for at least 10 h. At 37 degrees C the cells grow rapidly, but they also enter the death phase rapidly. There is only limited growth of E. herbicola at 42 degrees C. Whole cells of E. herbicola were encapsulated within alginate-polylysine-alginate microcapsules (916 +/- 100 mum, mean +/- std. dev.). The TPL activity of the cells catalyzed the production of L-tyrosine or dihydroxyphenyl-L-alanine (L-dopa) from ammonia, pyruvate, and phenol or catechol, respectively. In the production of tyrosine, an integrated equation based on an ordered ter-uni rapid equilibrium mechanism can be used to find the kinetic parameters of TPL. In an adequately stirred system, the apparent values of-the kinetic parameters of whole cell TPL are equal whether the cells are free or encapsulated. The apparent K(M) of tyrosine varies with the amount of whole cells in the system, ranging from 0.2 to 0.3 mM. The apparent K(M) for phenol is 0.5 mM. The apparent K(M) values for pyruvate and ammonia are an order of magnitude greater for whole cells than they are for the cell free enzyme. (c) 1995 John Wiley & Sons, Inc. 相似文献
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K I Vo?vodov I V Tysiachnaia L S Gubnitski? V I Iakovleva I V Berezin 《Prikladnaia biokhimiia i mikrobiologiia》1985,21(2):161-166
The Citrobacter freundii 62 cells immobilized in PAAG and possessing the tyrosine-phenol-lyase (TPL) activity catalyse the synthesis of 3,4-dihydroxyphenyl-L-alanine (DOPA) from pyrocatechol and ammonium pyruvate. The synthesis of DOPA was studied using both free and immobilized bacterial cells. When the concentration of pyrocatechol is over 0.1 M the TPL activity of the cells is inhibited. The concentration of pyrocatechol can be increased up to 0.3 M by using an equimolar mixture of pyrocatechol and boric acid. The addition of ascorbic acid as an antioxidant results in a lower TPL activity of both free and immobilized bacterial cells. 相似文献
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Treatment of partially hepatectomized male rats with urethan 6 hr after operation resulted in 50–55% inhibition of the incorporation of orotic acid-5-3H into nuclear ribosomal RNA and heterogeneous RNA 18 hr later. Neither partially hepatectomized female rats similarly treated with urethan nor operated male animals treated with an equitoxic dose of butyl carbamate presented evidence of an impairment of nuclear RNA synthesis. 相似文献
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As an alternative approach to the production of L-DOPA from a cheap raw material, we constructed a hybrid pathway consisting of toluene dioxygenase, toluene cis-glycol dehydrogenase, and tyrosine phenol-lyase. In this pathway, catechol is formed from benzene through the sequential action of toluene dioxygenase and toluene cis-glycol dehydrogenase, and L-DOPA is synthesized from the resulting catechol in the presence of pyruvate and ammonia by tyrosine phenol-lyase cloned from Citrobacter freundii. When the hybrid pathway was expressed in E. coli, production of L-DOPA was as low as 3 mM in 4 h due to the toxic effect of benzene on the cells. In order to reduce lysis of cells, Pseudomonas aeruginosa was employed as an alternative, which resulted in accumulation of about 14 mM L-DOPA in 9 h, showing a stronger resistance to benzene. 相似文献
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Enzymatic oxidative dehydrogenation of biomass-derived l-lactate in the presence of O2 is regarded as a green alternative for pyruvate production, but the process also results in H2O2 accumulation. To remove the effect of H2O2 on pyruvate production, various potential catalases from different species were screened for their efficiency in H2O2 degradation. Then, an in vitro cascade biocatalysis was designed using lactate oxidase from Aerococcus viridans (AvLOX) and catalase from Ureibacillus thermosphaericus (UtCAT). The in situ removal of H2O2 by UtCAT significantly improved the yield and productivity of pyruvate. To simplify the technological processes and reduce production costs, a whole cell biocatalyst without the addition of catalase for the production of pyruvate was established by coexpressing AvLOX and UtCAT in Escherichia coli. By applying suitable coexpression strategies and changing the ribosome binding site (RBS) strengths, the optimal ratio of AvLOX:UtCAT(12.6:4127.3) in E. coli-AvLOX-(rbs3)UtCAT was finally achieved. Under the optimized transformation conditions, pyruvate was produced at a titer of 59.9 g/L and a yield of 90.8% in a substrate fed-batch process, promising an alternative route for the green production of pyruvate. 相似文献
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David A. Fell Peter F. Liddle Arthur R. Peacocke Raymond A. Dwek 《The Biochemical journal》1974,139(3):665-675
A new method is described for the preparation of pyruvate kinase from yeast. This eliminates proteolysis during the preparation. The molecular weight of yeast pyruvate kinase is 215000, and it is composed of four subunits. Such properties of the enzyme as its extinction coefficient, cold-lability, thiol-group reactivity and binding of Mn(2+) ions are compared with those previously reported for yeast pyruvate kinase prepared by different methods. The specific activity is significantly higher than previously observed, but otherwise the enzyme is similar, apart from its molecular weight and Mn(2+)-binding characteristics, to preparations from Saccharomyces cerevisiae obtained in this laboratory (e.g. Fell et al., 1972, and references therein) and that of C. H. Suelter (e.g. Kuczenski & Suelter, 1971, and references therein), and is different from the enzyme isolated from Saccharomyces carlsbergensis by B. Hess and his co-workers (e.g. Wieker & Hess, 1972, and references therein). 相似文献
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Jeongeun Kim Yejin Song Soo Jeong Lee Jung Eun Lee Min-Yu Chung In-Hwan Kim Byung Hee Kim 《Biotechnology progress》2020,36(1):e2910
l -α-Glycerylphosphorylcholine (l -α-GPC) is a biosynthetic precursor for the neurotransmitter acetylcholine in humans, making it a useful as a cognitive enhancer for treating patients with stroke and dementia, including Alzheimer's disease. The aim of this study was to prepare l -α-GPC via Novozym 435 (an immobilized Candida antarctica lipase B)-catalyzed hydrolysis of soy phosphatidylcholine or a fractionated soy lecithin, from which triacylglycerols were completely removed, followed by food-grade solvent extraction of l -α-GPC from the reaction products. The reaction was performed in n-hexane–water biphasic media in a stirred-batch reactor. Phosphatidylcholine was completely hydrolyzed to l -α-GPC under optimal conditions: temperature, 55°C; water content, 100 wt% of the substrate weight; enzyme loading, 10 wt% of the substrate weight; and reaction time of 6 hr (for soy phosphatidylcholine) or 8 hr (for fractionated soy lecithin). Water-soluble fractions of the reaction products containing 98.6 area% l -α-GPC (from soy phosphatidylcholine) or 52.4 area% glycerophosphodiesters, including l -α-GPC (from fractionated soy lecithin), were obtained after phase separation of the media. The resulting products would be suitable for use as food-grade cognitive enhancers because of the use of enzymatic reaction and food-grade solvent extraction. 相似文献
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1. Pyruvate kinase from the rumen ciliates genus Entodinium was partially purified and the enzymatic properties were investigated. 2. Three types of pyruvate kinase (type I, II and III) on DEAE-cellulose column were eluted with a linear gradient of KCl. The enzymatic properties differed among the types of enzyme, especially type I and type III displayed different kinetic properties to each other. The enzymatic property of type II enzyme was an intermediate between type I and III. 3. The principal enzyme, type I, required a divalent cation, Mg2+ and was activated with AMP and FDP. ATP was a potent inhibitor. The saturation curves for the substrates, PEP and ADP, were hyperbolic and Km values were 0.15 and 0.27 mM, respectively. 相似文献
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Soy protein hydrolysates with lower molecular weight were enzymatically prepared by several commercially available proteases (Alcalase 2.4L, Flavourzyme, Trypsin, Papain, Protease A and Peptidase R) with protein recovery varied from 42.59% to 79.87%. Relative content of positively charged peptides was determined on SP Sephadex C-25 using gradient sodium chloride solution as eluents. Immunomodulating properties were evaluated by measuring their effect on in vitro proliferation of murine spleen lymphocytes and phagocytic activity of peritoneal macrophages. The results showed that soy protein hydrolysates (SPHs) prepared with Alcalase and insoluble soy protein (InSP), preferable to other enzymes and soy proteins, have the highest immunomodulating activity and the optimum conditions were determined as follows: E/S=2% (Alcalase), 60 degrees C, pH 8.0, InSP concentration 6% and 225min. Positive correlations were obtained between the immunomodulating activity and content of positively charged peptides. The results suggested that lower molecular weight and positively charged peptides released from soy protein were effective in stimulating immunomodulating activity, thus provided insights into the preparation of potent immunomodulating products. 相似文献
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In phosphate buffer (pH = 7.0) containing sodium dodecyl sulfate (SDS), an environmentally friendly system, enzymatic polymerization of phenol catalyzed by horseradish peroxidase (HRP) was efficiently performed. The obtained phenol polymer is partly soluble in common solvents, such as acetone, THF and DMF. IR analysis shows that the polymer is composed of phenylene and oxyphenylene units. The functionalization of the phenol polymer was performed by reacting with epoxy chloropropane and triethylene-tetramine following, and then insoluble aminated phenol polymer was obtained. The aminated phenol polymer was adopted as carrier to prepare a novel supported palladium catalyst (PP-N-Pd) for Heck reaction. PP-N-Pd shows high catalytic performance for Heck reactions of aryl iodides with acrylic acid or styrene and the yields of trans-products were higher than 90%. Under the optimized conditions, aryl bromides and activated aryl chloride also reacted with alkenes to give the yields of above 80%. XPS analysis indicates that the main coordination atom in PP-N-Pd is N and the chemical valence of palladium in PP-N-Pd is Pd2+. The novel supported catalyst also shows good recyclability for Heck reaction. 相似文献
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D. J. Swift 《Journal of fish biology》1981,19(1):45-61
Rainbow trout were exposed to sublethal phenol or non-ionized ammonia concentrations or to hypoxia. Blood samples were taken after various exposure periods and the packed cell volume (PCV) value, the whole blood glucose concentration and the plasma cortisol and chloride ion concentrations measured. At low pollutant concentrations there were no significant changes in the blood components compared to control fish values. At higher concentrations the general response to the stressors was significant increases in the PCV value and the glucose and cortisol concentrations during the first few hours of exposure, followed by a gradual return to normal values in the subsequent exposure time. The increases in glucose and cortisol concentrations were approximately proportional to the pollutant concentrations no such correlation was found for the PCV values. No clear pattern of plasma chloride ion changes was found in any experiment. Levels of no acute effect, in terms of toxic units (TU) based on the pollutants' 48 h LC50 values, were estimated for phenol as 0.3 TU and for un-ionized ammonia as 0.1 TU, using the plasma cortisol concentration measurements. The use of fish blood component measurements as general indicators of a stress response is discussed. 相似文献
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The product of the incorporation of [14C]tyrosine as single unit into a protein of the soluble fraction of rat brain homogenate was purified by following a procedure used to purify tubulin. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified material showed a single protein band containing all the radioactivity. Purification data indicate that this protein accounts for 10.2% of the total protein of the supernatant fraction. This is in good agreement with the amount found for tubulin by the [3H]colchicine-binding method (10.5% of the total protein). The incorporated [14C]-tyrosine was found in the alpha-subunit of tubulin. Protein labelled with [3H]colchicine and [14C]tyrosine was precipatated with vinblastine sulphate and the radioactivity of 3H and that of 14C were quantitatively recovered in the precipitate (98%). Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the vinblastine precipitate showed that the 14C radioactivity moved with the tubulin band. Results obtained in experiments with phenylalanine and 3,4-dihydroxyphenylalanine were identical to those obtained for tyrosine. Bineing of colchicine did not interfere with the incorporation of tyrosine. About 30% of tubulin from rat brain supernatant fraction can incorporate tyrosine as single unit. 相似文献
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Hitoshi Enei Hidetsugu Nakazawa Shinji Okumura Hideaki Yamada 《Bioscience, biotechnology, and biochemistry》2013,77(4):725-735
The synthesis of l-tyrosine or 3,4-dihydroxyphenyl-l-alanine (l-dopa) from pyruvate, ammonia and phenol or pyrocatechol was studied with intact cells of Erwinia herbicola ATCC 21434 containing high tyrosine phenol lyase activity. By elemental analyses and determination of optical activity, the tyrosine or dopa synthesized was confirmed to be entirely of l-form. Maximum amount of l-tyrosine (60.5 g/liter) or l-dopa (58.5 g/liter) was formed using this enzymatic method by feeding sodium pyruvate and phenol or pyrocatechol. However, large amounts of by-products were formed in the l-dopa synthetic reaction mixture. By-products were proved to be formed from l-dopa and pyruvate by a nonenzymic reaction. pH and the temperature of reaction had intensive effects on the formation of by-products. A simple method using a boric acid-pyrocatechol complex was devised, as the feeding procedure of substrates was complicated. 相似文献