Maximizing production of glutathione by amino acid modulation and high-cell-density fed-batch culture of Saccharomyces cerevisiae

In this paper the high-cell-density fed-batch culture and optimal amino acid modulation were combined together to enhance glutathione production in Saccharomyces cerevisiae T65. Ethanol concentration in the broth was an important parameter for feedback control in fed-batch culture. Low ethanol concentration was propitious to both the cell growth and glutathione synthesis. The feedback control of a low ethanol concentration was an efficient way to realize high-cell-density culture and the biomass reached 140 g/L after 57 h fermentation. With optimal amino acid addition to elevate the glutathione content continually, the maximum glutathione yield achieved 2190 mg/L.     

Improved glutathione production by gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic acid, 15 mmol/L cysteine, and 15 mmol/L glycine).     

Effect of coordinated addition of specific amino acids on the synthesis of recombinant glucose isomerase

The amplified expression of a recombinant protein is known to lead to an intracellular depletion of specific amino acid pools which in turn may affect the production of the desired protein. In order to counteract and overcome such a situation during the fermentation of the recombinant Escherichia coli (PMSG27) containing the glucose isomerase (GI) gene from Streptomyces sp. NCIM 2730, the effect of addition of different amino acids on the specific activity of GI was studied. The amino acid composition of GI from Streptomyces sp. NCIM 2730 reveals predominantly aspartic acid, glutamic acid, and glycine; therefore, in the present paper, the effect of coordinated addition of the assorted combinations of these three amino acids on the synthesis of recombinant GI was studied. The results were analyzed using a 2³ factorial design. The following conclusions were derived from the analysis of two-factor interactions of the three amino acids: (i) The interaction between the aspartic and glutamic acid is independent of aspartic acid concentration but is affected by the increasing concentrations of glutamic acid, (ii) The effect of aspartic acid concentration is more than that of glycine, and (iii) During the interaction of glutamic acid and glycine, the effect of glutamic acid is more prominent than that of glycine. The three-factor interaction analyses reveal that the effect of the three amino acids is in the order aspartic acid > glutamic acid > glycine.     

The amino acid requirements of rabbit fibroblasts,strain RM3-56

Strain RM3-56 of rabbit fibroblasts was found to require arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine for growth in a medium containing 2 per cent dialyzed serum as the only undefined component. The requirement for serine is less specific than that of the other 13 amino acids and it is partially replaced by glycine, or alanine, or by several combinations of so called accessory amino acids. The concentrations of essential amino acids which permit maximal proliferation range from 0.005 to 0.3 mM. Cystine, glutamine, lysine, tryptophan, tyrosine, valine are toxic at concentrations of 5 mM. The rate of proliferation of RM3-56 in a medium containing all 14 essential amino acids is increased significantly by the addition of alanine and to a lesser extent by the addition of aspartic and glutamic acids and glycine. A deficiency of cystine or glutamine results in cellular degeneration within 3 to 5 days, whereas the cells remain in good condition for 2 to 3 weeks in the absence of each of the remaining 12 essential amino acids. The results obtained with RM3-56 are compared with strains HeLa, L, and U12, whose amino acid requirements have been investigated under similar conditions.     

Asparaginase production by a recombinant Pichia pastoris strain harbouring Saccharomyces cerevisiae ASP3 gene

The therapeutic enzyme asparaginase, which is used for the treatment of acute lymphoblastic leukaemia, is industrially produced by the bacteria Escherichia coli or Erwinia crysanthemi. In spite of its effectiveness as a therapeutic agent, the drug causes severe immunological reactions. As asparaginase is also produced by the yeast Saccharomyces cerevisiae, this microorganism could be considered for the production of the enzyme, providing an alternative antitumoral agent. In this study the ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOX1 gene promoter. Similarly to S. cerevisiae the heterologous enzyme was addressed to the P. pastoris cell periplasmic space. Enzyme yield per dry cell mass reached 800 U g⁻¹, which was seven fold higher than that obtained using a nitrogen de-repressed ure2 dal80 S. cerevisiae strain. High cell density cultures performed with P. pastoris harbouring the ASP3 gene using a 2 l instrumented bioreactor, where biomass concentration reached 107 g l⁻¹, resulted in a dramatic increase in volumetric yield (85,600 U l⁻¹) and global volumetric productivity (1083 U l⁻¹ h⁻¹).     

Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro

Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1-cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids—glutamine, taurine, and glycine—were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium-5. Moreover, addition of another eight amino acids—asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM-6)—had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM-5 and ?6 (chemically defined, protein-free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley-Liss, Inc.     

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Abbreviations Ala alanine - Asn asparagine - 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic callus - nEC non-embryogenic callus - Gaba gamma-aminobutyric acid - Glu glutamic acid - Gly glycine - Pro proline - Ser serine     

Analysis of glutathione-enhanced differentiation by microfilariae of Onchocerca lienalis (Filarioidea: Onchocercidae) in vitro

Reduced glutathione (GSH), but not its oxidized form (GSSG), stimulated development of Onchocerca lienalis microfilariae to the late first-larval stage in vitro. The degree and frequency of development was dose-related with a peak of activity at 15 mM, a concentration that is similar to known intracellular levels of GSH. To determine the mode(s) of action of this multifunctional compound, other reducing agents (L-cysteine, dithiothreitol), cysteine delivery agents (N-acetyl-L-cysteine, L-thiazolidine-4-carboxylic acid, L-2-oxothiazolidine-4-carboxylic acid), cysteine analogues (S-methyl-L-cysteine, D-glucose-L-cysteine, cysteine ethyl ester), free-component amino acids of GSH (glutamic acid, cysteine, and glycine), a specific metabolic inhibitor of gamma-glutamyl synthetase (buthionine sulfoximine), and an inhibitor of gamma-glutamyl transpeptidase (gamma-glutamyl glutamic acid) were also tested at concentrations of 0.01-50 mM in this system. N-acetyl-L-cysteine at 1-5 mM and D-glucose-L-cysteine at 2.5-10 mM significantly enhanced development. In contrast to those worms maintained in GSH-supplemented medium, microfilariae exposed to GSH for only the first 24 hr showed no enhancement by day 7 in culture. Neither buthionine sulfoximine nor gamma-glutamyl glutamic acid at 0.01-35 mM inhibited the effects of 15 mM GSH or 1 mM N-acetyl-L-cysteine. Results indicate that GSH or other cysteine analogues possessing a free sulfhydryl group must be present in the extranematodal environment to support microfilarial differentiation in vitro.     

Protein and free amino acids in wheat grain as affected by soil types and salinity levels in irrigation water

Summary The effects of four lysimeter soil series under three salinity levels were evaluated for grain yield, wt/1000 seeds, protein, and amino acids in Mexican dwarf wheat (Triticum aestivum L. var. Cajeme 71). The soil series consisted of: Holtville clay loam, Greenfield sandy loam, San Emigdio sandy loam, and Altamont clay loam. The irrigation water salinity levels were designated: low –2.2 mmho, medium –4.2 mmho, and high –7.1 mmho.No significant differences were found in the amount of grain harvested or wt/1000 seeds in the 1976 crop produced on the differential soil series. The yield of the 1977 crop was significantly affected by the soil types.Effects of soil type on the protein amino acids in the grain in both years were similar. Significantly higher protein amino acid levels of histidine, arginine, aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine were found in the grain grown on Altamont clay loam soil than the other types.The free amino acids in grain from the 1976 and 1977 crops were similarly affected by the soil types, except that the quantitative values of the free amino acids were substantially lower in 1977 than in 1976. The free amino acids significantly influenced by soil types were tryptophane, lysine, arginine, aspartic acid, threonine, serine, glycine, alanine, valine, isoleucine, tyrosine, and phenylalanine. In both years' crops, the sum of the free amino acid fractions was significantly higher in the grain produced on the Altamont soil than on the other soils.Salinity level in the irrigation water did not affect the 1976 crop yield or wt/1000 seeds. Although yields of the 1977 crop were significantly reduced by salinity, the wt/1000 seeds was not. The sum of protein amino acids was significantly higher in the 1976 and 1977 grain crops irrigated with high salinity water than in low salinity irrigated crops.An increased salinity irrigation water significantly reduced the sum of free amino acid fractions in the 1976 grain crop. Since some of the free amino acids in the 1977 grain crop increased while the others decreased due to the salinity level in the irrigation water, the sum of the free amino acid fractions was not significantly influenced.Significant interactions were found between soil types and salinity levels on free arginine, threonine, serine, glutamic acid, and alanine, and also on the sum of the free amino acids in the 1976 wheat grain. In the 1977 wheat grain, there were significant interactions between soil types and salinity levels on the free glutamic acid, valine, leucine, tyrosine, and phenylalanine, and on protein serine, glutamic acid, glycine, alanine, and the sum of the protein amino acids.The amounts of essential amino acids expressed as mg of amino acid/g of protein were not affected by the soil types or salinity levels. With the exception of lysine, and possibly threonine and methionine plus cystine, the essential amino acids were present in the grain at concentrations equal to or greater than recommended by WHO and FAO.     

Glucose and amino acid metabolism in neonatal rat skeletal muscle in tissue culture

The characteristics of glucose and amino acid metabolism over a 98-hour incubation period were studied in a primary culture of neonatal rat skeletal muscle cells. The cells formed large myotubes in culture, were spontaneously highly contractile, and had cell phosphocreatine levels exceeding ATP concentrations. Medium glucose fell from 7.2±0.2 to 1.5±0.1 mM between 0 and 98 hours; intracellular glucose was readily detectable, indicating glycolysis was limited by phosphorylation, not glucose transport. Alanine levels in the medium increased from 0.06±0.01 to 0.82±0.04 mM between 0 and 48 hours and decreased to 0.72±0.04 mM by 98 hours. The period of net alanine production correlated with the rise in the cell mass action ratio of the alanine aminotransferase reaction. Cell aspartate, glutamate, and calculated oxalacetate levels were inversely related to the cell NADH/NAD⁺ ratio, as represented by the intracellular lactate/pyruvate ratio (r=0.78–0.88). The branched chain amino acids (leucine, isoleucine, valine) were actively utilized, e.g., medium leucine fell from 0.70±0.01 to 0.30±0.06 mM between 0 and 98 hours. In addition, arginine and serine consumption was observed in conjunction with ornithine, proline, and glycine production. Conclusions: (1) A major driving force for the high rates of alanine production by skeletal muscle cells in tissue culture is the active utilization of branched chain amino acids. (2) Intracellular aspartate and glutamate pools are linked, probably via the malate-aspartate shuttle, to the cell NADH/NAD⁺ redox state. (3) Muscle cells in tissue culture metabolize significant amounts of arginine and serine in association with the production of ornithine and proline, and these pathways may possibly be related to creatine production.     

Effect of nicotine on the level of extracellular amino acids in the hippocampus of rat

Using microdialysis, we compared intracerebral and subcutaneous administration of nicotine for the effect on the levels of extracellular amino acids in the hippocampus of anesthetized rats. Administration by microdialysis of 10 mM nicotine, resulting in a nicotine concentration of 0.134 μmol/g in the hippocampus, increased the extracellular levels of aspartic acid, glutamic acid, and serine by 26–60%. At 50 mM nicotine the increases in the levels of aspartic acid, glutamic acid, serine, glycine, and glutamine were between 76% and 141%. Subcutaneous administration of nicotine at a dose of 6 μmol/kg caused a 57% increase in the extracellular level of glutamic acid. After a dose of 12 μmol/kg that resulted in a nicotine level of 0.015 μmol/kg in the hippocampus, the extracellular level of glutamic acid was increased by 100%, and that of aspartic acid by 24%. Thus, higher cerebral nicotine levels were needed with intracerebral than with subcutaneous administration to obtain similar amino acid changes. Prior administration of mecamylamine or L-kynurenine prevented the subcutaneous nicotine-induced elevation of the extracellular levels of aspartic acid and glutamic acid. Our results indicate that receptor interactions modulate nicotine effects and that both nicotinic cholinergic and NMDA/glycine glutamatergic receptors participate in the action of nicotine in increasing extracellular amino acid levels.     

Amino acid concentrations in fluids from the bovine oviduct and uterus and in KSOM-based culture media.

Amino acids in bovine oviductal and uterine fluids were measured and compared with those in modified simplex optimized medium (KSOM) supplemented with either fetal calf serum or Minimum Essential Medium amino acids in addition to bovine serum albumin, fetal calf serum or polyvinyl alcohol. Concentrations of cysteine, threonine, tryptophan, alanine, aspartate, glycine, glutamate, proline, beta-alanine, and citrulline were higher in oviductal fluids than in KSOM-based culture media. Nonessential and essential amino acids were present in ratios of 5:1 and 2:1 in oviductal and uterine fluids, respectively. Concentrations of alanine (3.7 mM), glycine (14.1 mM) and glutamate (5.5 mM) were high in oviductal fluids, comprising 73% of the free amino acid pool. Of the amino acids measured in uterine fluids, alanine (3.1 mM), glycine (12.0 mM), glutamate (4.2 mM), and serine (2.7 mM) were highest in concentration, and the first three comprised 43% of the free amino acid pool. In conclusion, amino acid concentrations in the bovine reproductive tract were substantially higher than those in embryo culture media. Certain amino acids, particularly alanine, glutamate, glycine and taurine, are present in strikingly high concentrations in both oviductal and uterine fluids, suggesting that they might play important roles in early embryo development. The particular pattern of amino acid concentrations may be an important factor to be considered for the improvement of embryo culture media.     

Quantitative analysis of free amino acids and carbohydrates in spring barley anthers at different stages of maturity

The content of the carbohydrates glucose, fructose and sucrose was determined in spring barley anthers at different stages of maturity. During maturation the sucrose content of the anthers increased markedly. The following 17 free amino acids were detected in anthers of different stages of maturity: aspartic acid, glutamic acid, serine, alanine, arginine, leucine, isoleucine, lysine, α-aminobutyric acid, glutamine, proline, tyrosine, phenylalanine, valine, threonine, cystine and glycine. Quantitative analysis was only carried out in amino acids present in higher concentrations in the analysed samples. These were: aspartic acid, glutamic acid, α-aminobutyric acid, proline, serine, valine and glutamine, and a mixture of amino acids (leucine, isoleucine, valine and phenylalanine). The total content of free amino acids increased with increasing maturity of the anthers. However, not all amino acids followed contributed to this increase, but only proline, glutamic acid, aspartic acid and glutamine. A small difference was found in the variety Gopal in which the aspartic acid content did not increase significantly, but the content of the mixture of amino acids and serine did. With the exception of green anthers of the variety Firlbecks Union, proline was present in the highest concentration in all samples analysed.     

Intrabiliary glutathione hydrolysis. A source of glutamate in bile

High concentrations of glutathione (GSH) and two of its constituent amino acids, glutamate and glycine, are normally found in rat bile. To examine the role of intrabiliary GSH hydrolysis as a source of these amino acids, as well as of cystine in bile, the biliary excretion of GSH and free amino acids was measured in normal male Sprague-Dawley rats; in animals given either phenol 3,6-dibromphthalein disulfonate or diethyl maleate, inhibitors of GSH secretion into bile; and after a retrograde intrabiliary infusion of (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), an irreversible inhibitor of gamma-glutamyl transferase activity. Total concentration of amino acids in normal rat bile ranged from 4 to 7 mM and was more than double the concentration in plasma (2-3 mM). Although most amino acids were detected in bile, glutamate and glycine were the most prevalent (1.2 and 1.0 mM, respectively), followed by the branched chain amino acids valine and leucine. The administration of phenol 3,6-dibromphthalein disulfonate (180 mumol/kg, intravenous), or of diethyl maleate (1 mmol/kg, intraperitoneal), resulted in a marked decrease in the biliary excretion of GSH, as well as a decrease in the excretion of glutamate, cystine, and glycine; however, the effects of these agents were not specific for the amino acid constituents of GSH. Following retrograde intrabiliary infusion of AT-125 (10 mumol/kg), there was an immediate and sustained doubling in the rate of biliary excretion of both GSH and glutathione disulfide and a marked decrease in the rate of excretion of glutamate. Varying the dose of AT-125 (0-20 mumol/kg) resulted in an inverse linear relation between hepatic gamma-glutamyl transferase activity and the biliary excretion of intact GSH. These findings suggest that most, if not all, of the free glutamate in excreted bile is formed from the intrabiliary hydrolysis of GSH. Prior to hydrolysis within the biliary tree, substantial concentrations of GSH must be transported from liver cells into bile; minimal canalicular concentrations of this tripeptide are estimated at 5 mM.     

Discovery of alcohol dehydrogenase from mushrooms and application to alcoholic beverages

Saccharomyces cerevisiae is the main microorganism used in alcoholic beverage brewing, because this microbe has alcohol dehydrogenase (ADH) activity. We have recently discovered that some genera of mushrooms produce alcohol dehydrogenase, and made wine, beer and sake using mushrooms in place of S. cerevisiae. The highest alcohol concentrations in the wine, beer and sake were achieved with Pleurotus ostreatus (2648 mM, 12.2%), Tricholoma matsutake (1069 mM, 4.6%) and Agaricus blazei (1736 mM, 8.0%). In the case of wine made using A. blazei, the same alcohol concentration (1736 mM, 8.0%) was produced under both aerobic and anaerobic conditions. This wine produced by A. blazei contained about 0.68% β- -glucan, which is known to have preventive effects against cancer. The wine made using Flammulina velutipes showed thrombosis-preventing activity, giving a prolonged thrombin clotting time 2.2-fold that of the control. Thus, alcoholic beverages made using mushrooms seem to be a functional food source which can be expected to have preventive effects against cancer and thrombosis.     

The Role of Low-Molecular-Weight Nitrogen Compounds in the Osmotolerance of Rhodococcus erythropolis and Arthrobacter globiformis

Investigations showed that Rhodococcus erythropolis E-15 and Arthrobacter globiformis 2F cells respond to osmotic shock by increasing the synthesis of free amino acids, primarily glutamic acid (80% of the intracellular free amino acid pool). The osmoprotective role of glutamic acid follows from its beneficial effect on the growth of bacteria in high-salinity media. It was found that the addition of this amino acid to the growth medium at a concentration of 2 mM shortened the lag phase and increased the growth rate and biomass yield of either of the two bacteria. The addition of another osmoprotectant, trehalose, to the high-salinity growth medium of R. erythropolis E-15 at the same concentration (2 mM), restored the growth parameters of this bacterium to the control values.     

A daily study of the diatom spring bloom at Roscoff (France) in 1985. III. Free amino acids composition studied by HPLC analysis

The growth of a natural phytoplankton population was studiedduring a monospecific spring bloom of the diatom Rhizosoleniadelicatula at Roscoff (western English Channel). Direct examinationof the intracellular pool of 19 free amino acids (FAA) was usedas an index of the physiological status of the cells. TotalFAA in the particulate matter shows a general decrease duringthe bloom, and FAA cell content varies from 200 mM 1 cell volume^–1to 10 mM at the time of maximum biomass. FAA-N/particulate Nis <3% during the study, slightly decreasing at the timeof maximum biomass. Individual free amino acids appear reliablefor the development of the diatom biomass and good indicatorsof the growing population. At maximum biomass, major compoundsare glutamic acid (30.8 mol%), glutamine (11.7%), alanine (9.9%),isoleucine (6.6%) and lysine (6.4%). Serine, glycine, arginineand aspartic acid appear also as major components during otherperiods of the study. We suggest that the observed changes arean indication of the physiological state of the cells duringthe bloom. In particular glutamine (GLN), glutamic acid (GLU)and the GLN/GLU ratio allow the metabolic evolution of the naturalpopulation to be characterized. GLN is strictly linked to theliving biomass (Chla) while GLU is present at high percentagethroughout the study. The ecological significance of ß-alanine(BALA) is also revealed during this study, being strongly correlatedto degraded matter represented by phaeopigments. These resultsdemonstrate the validity of using individual free amino acidsas an aid in understanding the physiological status of algaein natural bloom conditions.     

The role of low-molecular-weight nitrogen compounds in the osmotolerance of Rhodococcus erythropolis and Arthrobacter globiformis

Investigations showed that Rhodococcus erythropolis E-15 and Arthrobacter globiformis 2F cells respond to osmotic shock by increasing the synthesis of free amino acids, primarily glutamic acid (80% of the intracellular free amino acid pool). The osmoprotective role of glutamic acid follows from its beneficial effect on the growth of bacteria in high-salinity media. It was found that the addition of this amino acid to the growth medium at a concentration of 2 mM shortened the lag phase and increased the growth rate and biomass yield of either of the two bacteria. The addition of another osmoprotectant, trehalose, to the high-salinity growth medium of R. erythropolis E-15 at the same concentration (2 mM), restored the growth parameters of this bacterium to the control values.     

Biosynthesis of amino acids from sucrose and Krebs cycle metabolites by Rhizobium lupini bacteroids

Summary The possibility of amino acids biosynthesis from sucrose, metabolites of Krebs cycle or glyoxylate and ammonium by intact bacteroids has been studied. The suspension of intact Rhizobium lupini bacteroids in phosphate buffer solution pH 7.8 was shown to catalyse the biosynthesis from sucrose and ammonium of some amino acids, such as alanine, aspartic and glutamic acids, glycine and serine. The yield of alanine and aspartic acid was 2.5–3 times higher than that of other amino acids, which were formed in almost equal quantities. Intact bacteroids were also found to catalyse the biosynthesis of aspartic and glutamic acids, alanine and glycine from ammonium and Krebs cycle metabolites such as fumaric acid (FA), oxaloacetic acid (OAA), pyruvic acid (PA), a-ketoglutaric acid (a-KGA), malic acid (MA), as well as from glyoxylic acid (GOA). The biosynthesis of aspartic acid from fumaric acid was dominant. Besides that, the suspension of intact bacteroids catalysed transamination of aspartic and glutamic acids, the transamination of aspartic acid being especially intense with -KGA and GOA. Aspartic acid was synthesized most efficiently through the amination of fumaric acid, while glutamic acid was better synthesized through the transamination of aspartic acid with -KGA than through reductive amination of -KGA.The experimental data proved that intact bacteroids posess Krebs cycle enzymes and primary ammonia assimilation enzymes. This enzyme complex permits bacteroids to detoxify ammonia, which they produce using sucrose and metabolites of Krebs cycle as the sources of carbon.The data obtained are of great interest as they prove the importance of bacteroids in the synthesis of amino acids from ammonium which is formed in the course of N₂-fixation, and sucrose available from leaves.     

重组大肠杆菌生产谷胱甘肽发酵条件的研究

研究了重组Ｅ．Ｃｏｌｉ产ＧＳＨ的发酵条件,重点考察了添加酵母膏、前体氨基酸和ＡＴＰ的影响。结果发现,前体氨基酸和ＡＴＰ均能促进胞内ＧＳＨ的积累,若在发酵０ｈ和１２ｈ分别加入２０ｇ／ＬＡＴＰ和９ｍｍｏｌ／Ｌ前体氨基酸,则细胞干重和胞内ＧＳＨ含量可分别比对照提高２４％和１４倍。应用正交试验得出的针对细胞干重和ＧＳＨ总量的最佳组合,最大细胞干重和ＧＳＨ总量比原试验中的最好结果分别提高了１０％和２６％。在分析了该菌对葡萄糖利用情况的基础上,对该菌进行了指数流加培养,２５ｈ细胞干重与发酵液内ＧＳＨ总量分别达到８０ｇ／Ｌ和８８０ｍｇ／Ｌ,比摇瓶最好结果分别提高了８３和４６倍。