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1.
Attempts were made with success to develop a chemically defined medium for 5′-purine ribonucleotide production by Brevibacterium ammoniagenes ATCC 6872 and its adenine auxotroph KY 7208.The results demonstrated that the presence of pantothenate and thiamine and a limiting level of manganese in the medium are essential for IMP production from hypoxanthine. These conditions were likewise indispensable for GMP, GDP and GTP productions and AMP, ADP and ATP productions from corresponding bases by ATCC 6872 and for direct IMP fermentation with KY 7208 strain.It was further shown that R5P accumulation by ATCG 6872 culture, in the absence of bases, was affected by the two vitamins and Mn 2+ exactly in the same way as the nucleotide synthesis. Morphogenetic alterations were induced under such conditions as two vitamins added and Mn 2+ kept deficient. 相似文献
2.
An adenine-requiring mutant (KY7208) of Brevibacterium ammoniagenes ATTC 6872 was found to accumulate an appreciable quantity of IMP and hypoxanthine in the culture liquid.Crystalline IMP was isolated from culture broth of KY7208 by the use of ion-exchange columns. The preparation obtained was definitely identified as 5′-IMP, based on the results on paperchromatography, UV and IR absorption spectra, and analyses of its hydrolysates.Growth responses of this mutant were demonstrated to adenine and adenosine, but not to 5′-AMP, 3′-AMP and 5′-AMP.Over 5 mg of IMP per ml of broth were produced by the organism in natural medium consisting of glucose, yeast extract, urea, high concentrations of phosphate and magnesim salts, and others. The chemical changes showed that hypoxanthine first accumulated in the earlier stage of fermentation, and IMP synthesis then took place with the disappearance of hypoxanthine in the later stage of fermentation. 相似文献
3.
Brevibacterium insectiphilium KY 3446 (Steinhous, Breed AHU 1401) was found to accumulate IMP from hypoxanthine and UMP from uracil, respectively. This strain is thus considered to present the fourth example in salvage-type fermentation, in addition to Micrococcus sodonensis, Arthrobacter citreus and Brevibacterium ammoniagenes reported previously.IMP from adenine and UMP from cytosine were also produced by KY 3446, respectively. Further, the addition of inosine and adenosine instead of the bases also caused IMP accumulation.This strain grew well on sucrose medium, and produced IMP and UMP in higher yields on sucrose than on glucose medium.Excessive amounts of Mn 2+ stimulated growth, but markedly inhibited IMP production. The optimal concentration of Mn 2+ for IMP accumulation induced morphogenetic alterations from normal and small to abnormal and large cells. 相似文献
4.
The effects of manganese ion (Mn 2+) and adenine on the accumulation of 5′ inosinic acid (IMP) by Brevibacterium ammoniagenes KY 13102, were examined. Adenine regulated the accumulation of IMP in the presence of limiting amounts of Mn 2+ and the accumulation of hypoxanthine (Hx) in the presence of excessive amounts of the ion. Manganese ion markedly affected IMP accumulations, cell growth and cellular morphology. These biological changes caused by Mn 2+ are related to changes in the syntheses of macromolecules. The cells cultivated under limitation of Mn 2+ showed abnormally elongated and irregular forms irrespective of adenine levels and had smaller nucleotide pools than those of the cells in the presence of excessive Mn 2+. The Mn 2+ limited cells showed ability to accumulate IMP directly in the cell suspension but the Mn 2+ excessive cells did not accumulated IMP but Hx. These results indicated that adenine and Mn 2+ affected the IMP accumulation independently each other and adenine acted as a feedback regulator on de novo synthesis of purine nucleotide and limitation of Mn 2+ caused morphological changes, resulting in changes of permeability of the cells. The fatty acid contents of the Mn 2+ limited cells were higher than those of the Mn 2+ excessive cells and the ratio of unsaturated fatty acid to saturated one was higher in the former cells. 相似文献
5.
A decoyinine resistant, KY 13501, isolated after nitrosoguanidine treatment from Brevibacterium ammoniagenes ATCC 6872 converted 5′XMP added in fermentation media to guanine derivatives and accumulated them in the media. The converted substances were identified as guanine, 5′GMP, 5′GDP, and 5′GTP. The conditions for the conversion were examined and the following points were clarified. (1) Very low concentration of manganese ion (Mn 2+) showed profound effects on the conversion and the excessive amounts of the ion severely repressed the conversion. (2) Under limitation of Mn 2+, 5′XMP was converted most efficiently when added at inoculation time. (3) The inhibition of the conversion by excessive amount of Mn 2+ was completely released by addition of a surface activating agent, polyoxyethylene stearylamine. (4) For the conversion, it was essential to maintain pH of the media at 7.5 to 8.0 and supply ammonium ion. 相似文献
6.
Enzymatic studies with Brevibacterium ammoniagenes ATCC 6872 demonstrated that 5-phosphoribose pyrophosphokinase and purinenucleotide pyrophosphorylase were involved in the nucleotide synthesis from purine base by ATCC 6872 and that its actual accumulation from base seemed to take place extracellularly through the action of the salvage enzymes leaked out of cells. Mn 2+ deficiency and the simultaneous presence of pantothenate and thiamine, essential for efficient nucleotide accumulation, caused the extracellular leakage of the two enzymes with the simultaneous excretion of R5P. In the direct IMP fermentation with the adenine auxotroph, it was verified that hypoxanthine first produced de novo was reconverted into IMP extracellularly by the salvage enzymes as speculated previously.A guanine-requiring mutant of Brevibacterium ammoniagenes ATCC 6872 accumulated a large amonnt of 5′-xanthosine-monophosphate (abbreviated as XMP).The quantity of XMP accumulated by the strain was affected significantly by guanine levels in the medium. The suppression of XMP accumulation by an excessive addition of guanine compounds was recovered by the supply of casamino acids in the medium.An enzyme in the pathway of de novo XMP synthesis, IMP dehydrogenase (IMP: NAD oxidoreductase, EC 1.2.1.14), was repressed and inhibited by guanine compounds.The facts that an exogenous xanthine was not converted to XMP by the growing cells and that the activity of XMP-pyrophosphorylase was very low or deficient suggest that XMP accumulation by the strain would be probably due to the direct excretion of the nucleotide from the cells. 相似文献
7.
The accumulation of 5′-inosinic acid (IMP) by a mutant, KY 13102, induced from Brevibacterium ammoniagenes ATCC 6872 by ultraviolet light irradiation, was examined. Although growth was stimulated by adenine or adenosine, the microorganism showed fair growth in the medium containing amino acids but no adenine. Among six kinds of natural nutrients tested, meat extract and Casamino Acids were suitable for the accumulation of IMP. Manganese ion strongly affected growth, the accumulation of IMP and hypoxanthine, and cell morphology. Among amino acids tested, L-methionine, L-proline, and L-valine stimulated IMP accumulation. In the medium containing 1.0 g of L-proline per liter, 12.8 mg of IMP per ml was accumulated. The mechanism of IMP accumulation by the mutant is discussed. 相似文献
8.
In attempts to obtain GMP producing strains, Brevibacterium ammoniagenes was treated with UV, N.T.G. or D.E.S. as a mutagen. Adenine-guanine requiring mutants were obtained from an adenine-requiring mutant of Brev. ammoniagenes, KY 3482–9 and two of them, presumably adenine-xanthine requiring mutants, were then reverted to mutants which required only adenine for their growth.Although these revertants were not able to accumulate a copious amount of GMP, most of them and of adenine-guanine requiring mutants produced larger amounts of IMP than the parent adenine-requiring strain.Effects of Mn 2+ and purine bases in the medium on IMP production by these mutants were examined and IMP productivities of these mutants were compared with the parent strain under optimal conditions.These mutagenic treatments were thus proved to be effective for the increase of de novo IMP production by Brev. ammoniagenes mutants. Brevibacterium ammoniagenes ATCC 6872 accumulates 5′-GDP and -GTP, or 5′-ADP and -ATP together with GMP or AMP in nucleotide fermentation by salvage synthesis.With cell free extract of this strain, transphosphorylating reactions of AMP or GMP were investigated.ATP-AMP transphosphorylating enzyme(s) was partially purified to 21.7 fold with acid treatment, salting-out and column chromatography.In ATP-AMP and ATP-GMP transphosphorylating reactins, optimal conditions were decided such as for concentrations of enzyme, of MgCl 2 and of phosphate donor, pH and cell age as the enzyme sources.Specificities of phosphate donors and acceptors were examined with both the partially purified enzymes or the sonicate. AMP and GMP were phosphorylated by ATP rapidly, but IMP and XMP were not, therefore supporting our previous finding that Brev. ammoniagenes could not accumulated IDP, ITP, XDP and XTP in IMP and XMP fermentation, respectively.Although ATP was the best donor for both AMP and GMP phosphorylations, other nucleoside triphosphates and PRPP were used as phosphate donors.Furthermore, phosphorylation of ADP to ATP was investigated and possible mechanisms of nucleoside di- or triphosphates synthesis in the nucleotide fermentation were discussed.From these results, it is suggested as a possible mechanism for nucleoside di- and triphosphate accumulation by Brev. Ammoniagenes, that a nucleoside monophosphate formed is phosphorylated to a nucleoside di-phosphate with ATP or other phosphate donors and then the nucleoside diphosphate is converted to a triphosphate with these phosphate donors.Both AMP and GMP were transphosphorylated rapidly to the corresponding nucleoside-diphosphates and triphosphates by ATP and by other high energy phosphate compounds with cell free extracts of Brevibacterium ammoniagenes.Some enzyme inhibitors, such as metals and PCMB were shown to inhibit the phosphorylations of AMP and GMP. Higher levels of ATP, ADP, GTP and GDP also inhibited the activity of the partially purified ATP-AMP transphosphorylating enzyme(s).In guanine nucleotides fermentation by salvage synthesis with this strain, addition of these inhibitors to the medium increased the amounts of GMP and total guanine nucleotides accumulated.On the contrary, supplement of xylene or of other organic solvents to the medium stimulated the accumulation of both GTP and total guanine compouuds in this fermentation. From enzymatic studies, these solvents are presumed to have the ability to change cell permeability.Such findings give an effective method for controlling the amounts of nucleotides accumulated in these fermentations. 相似文献
9.
The accumulation of 5′-inosinic acid (IMP) by a mutant, KY 13102, induced from Brevibacterium ammoniagenes ATCC 6872 by ultraviolet light irradiation, was examined. Although growth was stimulated by adenine or adenosine, the microorganism showed fair growth in the medium containing amino acids but no adenine. Among six kinds of natural nutrients tested, meat extract and Casamino Acids were suitable for the accumulation of IMP. Manganese ion strongly affected growth, the accumulation of IMP and hypoxanthine, and cell morphology. Among amino acids tested, L-methionine, L-proline, and L-valine stimulated IMP accumulation. In the medium containing 1.0 g of L-proline per liter, 12.8 mg of IMP per ml was accumulated. The mechanism of IMP accumulation by the mutant is discussed. 相似文献
10.
The biochemical rationale for the inhibition of citric acid fermentation by Aspergillus niger in the presence of Mn 2+ ions has been investigated using high citric acid-yielding, Mn 2+ ion-sensitive as well as Mn 2+ ion-tolerant mutant strains of A. niger. In the presence of Mn 2+ (1.5 mg/l), citric acid production by the Mn 2+ ion-sensitive strain (KCU 520) was reduced by about 75% with no apparent effect on citric acid yield by the Mn 2+ ion-tolerant mutant strain (GS-III) of A. niger. The significantly increased level of the Mn 2+ ion-requiring NADP +-isocitrate dehydrogenase activity in KCU 520 cells and the lack of effect on the activity level of the enzyme in GS-III mutant cells by Mn 2+ ions during fermentation seem to be responsible for the Mn 2+ ion inhibition of citric acid production by the KCU 520 strain and the high citric acid yield by the mutant strain GS-III of A. niger even in the presence of Mn 2+. 相似文献
11.
The effects of amino acids on IMP production were examined with a mutant strain, KY10895, derived from Corynebacterium ammoniagenes KY13374. l-Proline improved the productivity of IMP more than any other amino acid. The optimum concentration of l-proline for IMP production was 1–2% and the IMP productivity was about 70% more than that in the control medium. The effects of l-proline analogs on IMP production were also examined with the mutant KY10895. DL-3,4-Dehydroproline inhibited IMP production. Mutants resistant to growth inhibition by dl-3,4-dehydroproline were derived from strain KY10895. Among mutants thus obtained, strain H-7335 had the highest productivity. The intracellular concentrations of l-proline in strain H-7335 were higher than those of the parental strain, KY10895. These findings indicated that an increase in intracellular l-proline was linked with an increase of IMP productivity and strengthening the l-proline synthesis of a strain was an effective method for obtaining a hyper-producer of IMP. 相似文献
12.
1. Suitable agar plate media were selected for isolation of nucleotide producing strains, by salvage synthesis, from natural sources. Since this agar medium contains a high concentration of phosphates, manganese and glucose, it is specific for these bacteria.2. With this plate medium, 113 bacterial strains accumulating 5′inosinic acid (IMP) or IMP-like substances were isolated effectively from feces of a variety of birds and mammals and from soils.Some of the strains isolated were recognized to accumulate other nucleotides, purine bases and sugars, such as guanine nucleotides, XMP, xanthine, ribulose or xylnlose, with or without hypoxanthine in the media.3. Five strains of IMP accumulating bacteria were identified; two were classified as Brevibacteriurm, two as Corynebacterium and one as Arthrobacterium species by taxonomical studies. But their characteristics did not completely coincide with those of bacteria described in Bergey’s manual.4. One of the IMP producing bacteria isolated, culture No. 21–26, actually consisted of two separate strains, namely No. 21–26–101 and No. 21–26–102. The highest production of IMP or guanine nucleotides was obtained, when each strain was inoculated together to the fermentation medium from each seed culture in the same inoculum size.5. The nucleotide productions by No. 21–26–101 or No. 21–26–102 with authentic strains were examined by the mixed culture technique. It was found that production of IMP or guanine nucleotides by Brevibacterium ammoniagenes ATCC 6871 was stimulated remarkably in the presence of No. 21–26–102. 相似文献
13.
A seed medium and a fermentation medium for nucleotide fermentations such as 5′ IMP, 5′GMP (plus GDP and GTP) and 5′AMP (plus ADP and ATP) with Brevibacterirm ammoniagenes ATCC 6872 were entirely chemically defined, with the use of a mixture of five amino acids.As a result, the presence of Zn 2+, Fe 2+ and Ca 2+ in addition to Mn 2+ was found to be essential for the nucleotide fermentations. In particular, Zn 2+ levels as well as Mn 2+ affected nucleotide productions remarkably. Various fermentations proceeded favorably only when suboptimum levels of manganese (20~30 μg/liter) and zinc (100~200 μg/liter) were simultaneously present. This effect of trace metals was attributed to the fact that the excretion of R5P, a precursor of nucleotides, and those enzymes catalyzing reactions synthesizing nucleotides from R5P, ATP and purine bases were greatly stimulated by trace metals in cooperation with two vitamins, Ca-pantothenate and thiamine, and presumably high concentrations of phosphate and magnesium.Furthermore, it was revealed that some metals were able to control the amounts of nucleotides accumulated when they were added to the broth during fermentation. For example, Hg 2+ and Ag + could increase the amounts of 5′GMP or 5′AMP, and decrease those of GTP and ATP.Growth responses of Brevibacterium ammoniagenes ATCC 6872, capable of accumulating purine nucleotides, were investigated by the use of completely defined media. Casamino acids required for its growth could be replaced by a mixture of l-histidine, l-homoserine, glycine, d, l-alanine and l-lysine. A completely defined medium for nucleotide productions was thus established by the use of this mixture. High levels of phosphate inhibited growth markedly, and this inhibition was overcome by the simultaneous addition 1) of hign levels of Mg2+ and 2) of Mn2+, 3) pantothenate and 4) thiamine. Ca2+ had also a stimulatory effect on the growth. Therefore, a clear growth response to Mn2+ levels and the requirement of the two vitamins for growth emerged only under the conditions of high phosphate and magnesium salts. These 4 factors were found entirely the same as factors essential for nucleotide accumulations by Br. ammoniagenes. 相似文献
14.
The presence of psicofuranine in the fermentation medium caused the accumulation of a copious amount of 5′–XMP by Brevibacterium ammoniagenes. The accumulation of 5′–XMP in the medium was considered to be due to the inhibition of converting 5′–XMP to 5′–GMP by psicofuranine, which is known as a specific inhibitor of XMP aminase.It was previously reported that in 5′–IMP fermentation with Br. ammoniagenes pantothenate and thiamine, in addition to biotin which was required for the growth of the microorganism, were exclusively required. This requirement for both vitamins was also observed in 5′–XMP production induced by the antibiotic.The addition of manganese in excess to the fermentation medium promoted the bacterial growth greatly and inhibited IMP production, whereas XMP production induced by piscofuranine was not affected by the addition of excess manganese.The accumulation of XMP induced by the antibiotic was completely suppressed by the presence of purine derivatives such as guanine, and xanthine derivatives, and partially by hypoxanthine.5′–XMP was identified by chemical and enzymatic analyses and by UV absorption spectrum. 相似文献
15.
A major problem involved in the direct fermentation of nucleotides is their breakdown by phosphohydrolases. Thus, adenine auxotrophs of most microorganisms produce hypoxanthine and/or inosine rather than inosine 5′-monophosphate (IMP) while guanine auxotrophs excrete xanthosine rather than xanthosine 5′-monophosphate (XMP). Examination of a Bacillus subtilis mutant producing hypoxanthine plus inosine revealed at least four phosphohydrolases, three of which could attack nucleotides. Even when the extracellular nucleotide phosphohydrolase was inhibited by Cu +2 and its surface-bound alkaline phosphohydrolase was repressed and inhibited by inorganic phosphate, or removed by mutation, the breakdown products were still the only products of fermentation. Under these conditions, the third enzyme, a surface-bound non-repressible nucleotide phosphohydrolase was still active. It appears, at least in B. subtilis, that excretion is dependent upon breakdown by this enzyme and if hydrolysis does not occur, excretion of purine nucleotides is feedback inhibited by the resultant high intracellular IMP concentration. Corynebacterium glutamicum mutants, on the other hand, can excrete intact nucleotides, and direct fermentations for IMP, XMP, and GMP have been described. An examination of phosphohydrolases in a GMP-producing culture revealed no extracellular or surface enzymes. Disruption of the cells resulted in liberation of cellular phosphohydrolase activity with a substrate specificity remarkably similar to the flavorenhancing properties of the 5′-nucleotides. The order of decreasing susceptibility was GMP, IMP, XMP; AMP was not attacked. 相似文献
16.
A novel process for producing inosine 5′-monophosphate (5′-IMP) has been demonstrated. The process consists of two sequential
bioreactions; the first is a fermentation of inosine by a mutant of Corynebacterium ammoniagenes, and the second is a unique phosphorylating reaction of inosine by guanosine/inosine kinase (GIKase). GIKase was produced
by an Escherichia coli recombinant strain, MC1000(pIK75), which overexpressed the enzyme up to 50% of the total cellular protein. The overproducing
plasmid, pIK75, which was randomly screened out from deletion plasmids with various lengths of intermediate sequence between
the E. coli trpL Shine-Dalgarno sequence, derived from the vector plasmid, and the start codon of the GIKase structural gene. In pIK75, the
start ATG was placed 16 bp downstream of the trpL Shine-Dalgarno sequence under the control of the E. coli trp promoter. Fermentation of inosine and its phosphorylation were sequentially performed in a 5-l jar fermenter. At the end
of inosine fermentation by C. ammoniagenes KY13761, culture broth of MC1000(pIK75) was mixed with that of KY13761 to start the phosphorylating reaction. Inosine in
the reaction mixture was stoichiometrically phosphorylated, and 91 mM 5′-IMP accumulated in a 12-h reaction. This new biological
process has advantages over traditional methods for producing 5′-IMP.
Received: 7 April 1997 / Received last revision: 18 July 1997 / Accepted: 27 July 1997 相似文献
17.
In order to establish industrial production of 5′-inosinic acid (5′-IMP), a permeability mutant, KY13171, of Brevibacterium ammoniagenes, which accumulated 7 to 8 grams of 5′-IMP per liter and 4 to 6 grams of hypoxanthine (Hx) per liter (calculated as 5′-IMP), was improved by a genetical procedure. Further improved mutants were selected stepwise through repeating mutational work. The finally selected mutant. KY13369, accumulated 20 to 27 grams of 5′-IMP per liter, but not Hx.Increased productivity of 5′-IMP and decreased productivity of Hx were not caused by the changes in 5′-IMP degrading activity, because these activities were not significantly different among the mutants. These results appear to indicate that the increased accumulation of 5′-IMP may be caused by the improvement in membrane permeability for 5′-IMP. However, the changes in phospholipid and fatty acid compositions were not enough to explain the increased permeability. 相似文献
18.
5′Xanthylic acid was efficiently converted to 5′guanine nucleotides (5′GMP, 5′GDP, and 5′GTP) without being degraded to guanine via 5′GMP by decoyinine resistant mutants of strain KY 13315 which had been isolated from Brevibacterium ammoniagenes and was practically devoid of 5′nucleotide degrading activity. The concentration of phosphate in the medium showed a profound effect on the ratio of the accumulated 5′guanine nucleotides, making it possible to direct the fermentation towards 5′GMP or 5′GTP. A direct accumulation of 5′guanine nucleotides from carbohydrate was possible by mixed cultivation of a 5′XMP accumulating strain and a 5′XMP converting mutant. A maximum concentration of 9.67 mg of 5′guanine nucleotides per ml was obtained directly from glucose in such a mixed culture. 相似文献
19.
Cell division of the wild type strain Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 which requires 1 M Mn 2+ for balanced growth was inhibited by addition of 20 mM hydroxyurea (HU) or 10 mM p-methoxyphenol (MP) to a Mn 2+-supplemented fermentation medium at an appropriate time. Scanning electron microscopy (SEM) showed a restricted elongation characteristic of arrest of the cell cycle in coryneform bacteria. The cultures treated with HU or MP had, respectively, a fourfold or sixfold enhanced accumulation of NAD + by a salvage biosynthetic pathway. An assay of nucleotide-permeable cells for ribonucleotide reductase activity using [ 3H-CDP] as substrate revealed a pre-early and complete decline of DNA precursor biosynthesis not found in the untreated control. Overproduction of NAD + is an alternative to the conventional fermentation process using Mn 2+ deficiency. A simple model is presented to discuss the metabolic regulation of the new process based on the presence of a manganese ribonucleotide reductase (Mn-RNR) in the producing strain. 相似文献
20.
Improvements were found in the inosine productivity of Brevibacterium ammoniagenes KY 13714, which is an adenine leaky and 6-mercaptoguanine resistant mutant. A highly productive mutant, KY 13761, was selected after the addition of 6-methyIthiopurine resistance and guanine requiring character to KY 13714 and after repeating single colony isolation.Culture conditions for the practical production of inosine were investigated using KY 13761. It was found that the concentrations of phosphate, magnesium, and adenine were important. Carbon sources and natural nutrients also showed profound effects on inosine accumulation. Especially, effective was the feeding of inverted molasses and urea for production of inosine. Under optimal conditions, 31 mg of inosine per ml was accumulated after 42 hr cultivation in a 5 liter jar fermenter at 32°C. A growth-associated type of accumulation was confirmed in inosine production with KY 13761. 相似文献
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