Specific effect of endonucleases from Brevibacterium ammoniagenes on DNA

The previously described deoxyribonucleases from Brevibacterium ammoniagenes have been characterized. It was shown that they are endonucleases with molecular weights of 60 000 (I), 10 000 (II) and 20 000 (III). The rate of endonuclease I effect on native DNA exceeded that on the denatured DNA 2-fold. The mechanism of its action is of a single hit type. The enzyme hydrolyzes two chains of DNA simultaneously in two symmetrical sites and splits the bond 5'-P to form fragments with terminal 5'-OH and 3'-P. Endonuclease I was characterized as deoxyribonucleate-3'-oligonucleotide hydrolase (EC 3.1.4.6).     

A Metaphosphate-dependent Nicotinamide Adenine Dinucleotide Kinase from Brevibacterium ammoniagenes

The enzyme utilizing metaphosphate for nicotinamide adenine dinucleotide phosphorylation was purified 500-fold from B. ammoniagenes and its properties were studied. The isolated enzyme appeared homogeneous on disc gel electrophoresis; its molecular weight was determined to be 9.0 × 10⁴ by gel filtration. This enzyme specifically phosphorylated nicotinamide adenine dinucleotide at the optimum pH at 6.0. Of phosphoryl donors tested, metaphosphate was most effective for the reaction, and adenosine-5′-triphosphate was less effective. The activity was inhibited by adenosine-5′-monophosphate, adenosine-5′-diphosphate or reduced pyridine nucleotides. The enzyme did not exhibit catalytic activity in the absence of a divalent cation. We concluded that the enzyme phosphorylating nicotinamide adenine dinucleotide in the presence of metaphosphate is distinct from adenosine-5′-triphosphate-dependent nicotinamide adenine dinucleotide kinase, and tentatively designated it metaphosphate-dependent nicotinamide adenine dinucleotide kinase.     

Purification and characterization of FAD synthetase from Brevibacterium ammoniagenes

The bifunctional enzyme FAD synthetase from Brevibacterium ammoniagenes was purified by a method involving ATP-affinity chromatography. The final preparation was more than 95% pure. The apparent molecular weight of the enzyme was determined as 38,000 and the isoelectric point as 4.6. Although previous attempts to separate the enzymatic activities had failed, ATP:riboflavin 5'-phosphotransferase and ATP:FMN-adenylyltransferase activities in B. ammoniagenes were believed to be located on two separate proteins with similar properties, possibly joined in a complex. The following evidence, however, suggests the presence of both activities on a single polypeptide chain. The two activities copurify in the same ratio through the purification scheme as presented. Only a single band could be detected when aliquots from the final purification step were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. Edman degradation of the protein yielded a single N-terminal sequence.     

Thermal regulation of fatty acid synthetase from Brevibacterium ammoniagenes.

The fatty acid composition of Brevibacterium ammoniagenes was affected by the temperature of growth. As the growth temperature was lowered, the proportion of unsaturated fatty acids increased. The fatty acid synthetase obtained from B. ammoniagense produced oleic acid as well as saturated fatty acids. The ratio of unsaturated to saturated fatty acids synthesized by this enzyme in vitro was dependent on the temperature of the enzyme reaction but not on the growth temperature of B. ammoniagenes from which the enzyme was prepared. These results suggest that the changes of composition in cellular fatty acids reflect the temperature dependence of the fatty acid synthetase.     

产氨短杆菌GMA-1112利用味精母液发酵生产肌苷

通过亚硝基胍或60 Coγ辐照等选育一株产氨短杆菌GMA 1 1 1 2 (Ade +Gua +VB1 +8 AGr+SGr+6 MPr) ,能以味精母液代替传统肌苷发酵添加酵母粉作为有机营养物 ,进行肌苷发酵 ,平均产肌苷率 2 5.4 0 g/L。具有降低发酵成本、提高产肌苷率等优点。     

Manganese transport in Brevibacterium ammoniagenes ATCC 6872.

Uptake of manganese by Brevibacterium ammoniagenes ATCC 6872 was energy dependent and obeyed saturation kinetics (Km = 0.65 microM; Vmax = 0.12 mumol/min per g [dry weight]). Uptake showed optima at 27 degrees C and pH 9.5. 54Mn2+ accumulated by the cells was released by treatment with toluene or by exchange for unlabeled manganese ions, via an energy-dependent process. Co2+, Fe2+, Cd2+, and Zn2+ inhibited manganese uptake. Inhibition by Cd2+ and Zn2+ was competitive (Ki = 0.15 microM Cd2+ and 1.2 microM Zn2+). Experiments with 65Zn2+ provided no evidence for Zn2+ uptake via the Mn2+ transport system.     

Purification and Properties of Pantothenate Kinase from Brevibacterium ammoniagenes IFO 12071

Pantothenate kinase (ATP: pantothenate 4′-phosphotransferase, EC 2.7.1.33) was purified about 200-fold from the cell extract of Brevibacterium ammoniagenes IFO 12071 by ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-150 gel filtration. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was calculated approximately 45,000. The enzyme catalyzed the formation of pantothenic acid 4′-phosphate and ADP from pantothenate and ATP in the presence of Mg²⁺ ATP could be substituted for, partly, by ITP, GTP, and UTP. The enzyme phosphorylated not only pantothenate, but also pantothenoylcysteine, pantetheine, and pantothenyl alcohol. Apparent Km values were 6.7×10^?5 m for pantothenate, 3.5×10^?5 m for ATP, and 10^?3 m for Mg²⁺. The reaction was inhibited by the intermediates of CoA biosynthesis, of which CoA itself was a most effective inhibitor. Other properties of the enzyme were also investigated.     

Nucleotide and thioredoxin specificity of the manganese ribonucleotide reductase from Brevibacterium ammoniagenes

The manganese-containing ribonucleotide reductase previously identified in gram-positive bacteria has been purified and its nucleotide specificity and other requirements were determined. The enzyme isolated from Brevibacterium ammoniagenes is a ribonucleoside-diphosphate reductase which, in the presence of allosteric effectors, reduces all four common substrates at comparable rates; very little activity is observed in the absence of effector nucleotides. Ribonucleoside triphosphates are reduced at 20% the rate of the diphosphates. Cytidine and uridine nucleotide reduction is specifically stimulated by ATP and dATP, adenylate reduction by dGTP, and guanosine nucleotide reduction by dTTP. Unlike the iron-containing ribonucleotide reductase systems, high concentrations of dATP do not inhibit substrate reduction. The new bacterial enzyme tolerates high salt concentrations (up to 250 mM ionic strength) and does not require divalent metal ions for activity in vitro. The presence of thioredoxin has been demonstrated in heat- and acid-treated protein extracts of B. ammoniagenes and the protein was purified to homogeneity. It is very similar to the thioredoxins isolated from other organisms in relative molecular mass (12,000), isoelectric point (4.3) and enzyme-activating properties. In the presence of 0.3 mM dithiothreitol, the bacterial thioredoxin can serve as hydrogen donor for B. ammoniagenes ribonucleotide reductase in vitro, indicating the presence of a functional ribonucleotide reductase-thioredoxin system in these bacteria. The properties described in this and in our preceding paper in this journal [Eur. J. Biochem. 170, 603-611 (1988)] suggest that the B. ammoniagenes ribonucleotide reductase is intermediate in structure and specificity between the deoxyadenosylcobalamin-dependent and the iron-containing enzyme classes and that it is adapted to the specific requirements of deoxyribonucleotide synthesis in this organism.     

Stereochemical studies of hydrogen incorporation from nucleotides with fatty acid synthetase from Brevibacterium ammoniagenes.

The biosynthesis of fatty acids from malonyl-CoA and acetyl-CoA was investigated with an enzyme preparation which was purified 100-fold from Brevibacterium ammoniagenes. Fatty acids synthesized in the presence of D2O and stereospecifically deuterated NADPH and NADH were isolated and analyzed by mass chromatography to examine the localization of deuterium in the molecule. The following results were obtained: 1) HB hydrogen of NADPH was used for beta-ketoacyl reductase. 2) HB hydrogen of NADH was used for enoyl reductase. 3) Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom) and some were also found on the odd-numbered methylene carbon atoms. 4) Hydrogen atoms from NADPH was found on the odd-numbered methylene carbon atoms (1 hydrogen per carbon). 5) Hydrogen atoms from NADH was also found on the odd-numbered methylene carbon atoms, but the number of incorporated hydrogen atoms was less than expected. The exchange of HB hydrogen of NADH with water catalyzed by enoyl reductase was suspected. 6) The exchange of methylene hydrogen atoms of malonyl-CoA with protons of water was suggested by 13C NMR analysis.     

固定化产氨短杆菌MA-2、黄色短杆菌MA-3反应动力学的研究

多年来虽然有不少学者对固定化细胞生产Ｌ 苹果酸的方法进行过探讨[1～ 6] ,但对过程动力学的研究报道并不多见[2 ,7] ,在富马酸铵转化体系中的表观动力学及本征动力学模型还未见报道 ,本文对富马酸铵转化体系中固定化产氨短杆菌ＭＡ 2、黄色短杆菌ＭＡ 3细胞的动力学进行了探讨 ,测定了两种固定化细胞的表观动力学常数 ,并进一步求解了相应的本征动力学常数 ,这一结果便于从理论上指导富马酸铵转化过程的工业化生产。1　材料和方法1 1　试剂富马酸 ,工业级 ,苏州合成化工厂 ,碳酸钙 ,工业级 ,泗联化工厂。1 2　菌株本文所用的菌株是由我院…     

The effect of manganese deficiency on lipid content and composition in Brevibacterium ammoniagenes

Summary Cells of Brevibacterium ammoniagenes grown under manganese deficient conditions contain less total lipids at the end of the logarithmic growth phase and the phospholipid content of these cells is lower over the whole fermentation period in comparison to those growing where the supply of manganese is sufficient.Phosphatidyl glycerol, cardiolipin, phosphatidyl inositol and phosphatidyl inositol mannoside were identified. There were quantitative, but no qualitative differences in the phospholipid composition. The phosphatidyl inositol mannoside content was greatly lowered under manganese deficiency, whereas the phosphatidyl glycerol and cardiolipin content were greatly increased.     

Temperature- and growth-phase-dependent changes in membrane fatty acid compositions of Brevibacterium ammoniagenes

Fatty acids newly synthesized by Brevibacterium ammoniagenes grown at different temperatures were analyzed. The assay temperature, not the growth temperature, was found to be the major factor affecting the unsaturated/saturated ratio of newly synthesized fatty acids in logarithmic-phase cells. However, in the stationary-phase cells the growth temperature also affected the product profile significantly; cells grown at 7 degrees C produced relatively more oleate and stearate and less palmitate and hexadecenoate when shifted up to 37 degrees C than did cells grown and assayed at 37 degrees C. The unsaturated/saturated ratio as well as average chain length of fatty acids also varied along with the progress of isothermal growth phase. These changes in fatty acid product profiles observed in vivo could be mimicked in vitro assays of the fatty acid synthetase by changing malonyl-CoA concentrations. Our results suggest that the malonyl-CoA concentration is a factor which, in addition to temperature, determines growth-phase-dependent and growth-temperature-dependent changes in the unsaturated/saturated ratios of fatty acids.     

Enzymatic bases for the fatty acid positioning in phospholipids of Brevibacterium ammoniagenes

Positional distribution of fatty acids in phospholipids from Brevibacterium ammoniagenes was analyzed to find that phosphatidylethanolamine consisted mainly of 1-saturated acyl 2-unsaturated acyl species while phosphatidylglycerol consisted mainly of 1-unsaturated acyl 2-saturated acyl species. Three acyltransferase systems were characterized in a membrane preparation--the acylations of glycerophosphate, 1-acyl-glycerophosphate, and 2-acyl-glycerophosphate--which appeared to be catalyzed by different enzymes. The distribution of fatty acids in the phosphatidylethanolamine molecule was not correlated simply with the specificities of these enzymes, but the relatively high specificity for palmitoyl-CoA of the glycerophosphate acyltransferase system to form 2-acyl-glycerophosphate, followed the relatively high specificity for oleoyl-CoA of the 2-acyl-glycerophosphate acyltransferase system, provided a basis for producing the major molecular species of phosphatidylglycerol.     

Effect of antibiotics on 5'-inosinic acid biosynthesis by a Brevibacterium ammoniagenes mutant

The effect of streptomycin, erythromycin, kanamycin and penicillin on the biosynthesis of 5'-inosinic acid (IMP) by the mutant strain Brevibacterium ammoniagenes was studied. It has been found that the efficiency of antibiotic action depends not only in its concentration but on the age of the culture. When the antibiotics were introduced into the culture broth at the beginning of fermentation, they inhibited the culture growth and accumulation of IMP in the cultural medium. Only after 36-72 hours of cultivation the addition of the antibiotics stimulated the biosynthesis. All the antibiotics tested when adding at the definite for each of them period of fermentation and at the definite concentration stimulated the accumulation of IMP. The stimulating effect appears to be connected with an increase in permeability. A considerable increase in the number of anormalous elongated and swollen cells and, as a rule, in the protein content of the cultural supernatant indicates the fact. Streptomycin and kanamycin were the most efficient antibiotics, as they increased the IMP yield from 10.4 to 17.5 g/l.     

Unbalanced Growth Death Due to Depletion of Mn2+ in Brevibacterium ammoniagenes

In the microbial conversion of added hypoxanthine to 5'-inosinic acid, Mn(2+) concentration in the growth medium is known to have a profound effect both on the yield of 5'-inosinic acid and the morphology of cells of Brevibacterium ammoniagenes. To elucidate the mechanism in which Mn(2+) was concerned with cell morphology and 5'-inosinic acid production, effects of Mn(2+) on the macromolecular synthesis were measured. It was found that Mn(2+) strongly governed deoxyribonucleic acid (DNA) synthesis and that, in the medium lacking Mn(2+), DNA synthesis was stopped at the level corresponding to one-fourth to one-third that in the medium supplemented with Mn(2+) (100 mug/liter). On the other hand, cellular ribonucleic acid and protein synthesis was quite indifferent to Mn(2+) concentration. Consequently, cells showed so-called "unbalanced growth death" after 10 hr of culture, losing the ability to form colonies while cell mass was increasing. The elongated cells turned into irregular forms (bulbous, club-shaped, etc.) which finally lysed. Two main reaction components in the conversion of hypoxanthine to 5'-inosinic acid, phosphoribosylpyrophosphate and hypoxanthine phosphoribosyltransferase, were liberated into the medium during lysis. The role of Mn(2+) in the synthesis of DNA and the role of the unbalanced growth death in the conversion of hypoxanthine to 5'-inosinic acid are discussed.     

Production of Ribotides of 4-Hydroxy- and 4-Aminopyrazolo[3, 4-d]pyrimidine by Brevibacterium ammoniagenes

Brevibacterium ammoniagenes ATCC 6872 was previously reported to accumulate large amounts of IMP, AMP, ADP, ATP, GMP, GDP and GTP from the corresponding purine bases. The organism was also reported to convert various derivatives of purine and 8-azapurine to the corresponding ribotides.

Using the similar process, ribotidation of pyrazolo[3, 4-d]pyrimidines was attempted, and it was found that the same organism was able to produce remarkable amounts of 4-hydroxy-1-β-d-ribofuranosylpyrazolo[3, 4-d]pyrimidine 5′-monophosphate (HPP-RP) from 4-hydroxypyrazolo[3, 4-d]pyrimidine (HPP, allopurinol) and 4-amino-1-β-d-ribofuranosylpyrazolo[3, 4-d]pyrimidine 5′-monophosphate and 5′-diphosphate from 4-amino-pyrazolo[3, 4-d]pyrimidine.

The crystals of HPP-RP (Na-salt) were isolated from the cultured broth of Br. ammoniagenes incubated with HPP, and characterized based on UV-spectra, IR-spectrum, NMR and others.

It was also found that HPP-RP was converted to the corresponding riboside by hydrolysis in aqueous solution (pH 4.0 ~ 9.0) for 6 hr at 140°C. The hydrolysis of HPP-RP was also accomplished with various organisms.