Degradation of Nucleic Acids and their Related Compounds by Microbial Enzymes

Aspergillus quercinus (IFO 4363) was selected as the most suitable strain to produce 5′-mononucleotides from RNA among several species of Aspergillus which produced enzymes capable of degrading RNA into 5′-mononucleotides.

Aspergillus quercinus produced two kinds of RNA-depolymerases (designated as RNA-deploymerase I and II), phosphodiesterase, phosphomonoesterase and adenylic deaminase in the culture medium. The optimum pH of each enzyme was found to be about 4.5, 7.0, 5.0, 6.0 and 5.5, respectively. Maximal production of these enzymes in the culture medium occurred at 96, 96, 216, 168 and 264 hour culture, respectively. The culture filtrate of Aspergillus quercinus degraded RNA into 3′-mononucleotides at the pH lower than 6.0, into 5′-mono-nucleotides at the pH higher than 8.5 and into both mononucleotides at the pH range between 6.0 and 8.5. 5′-Inosinic acid was prepared from RNA by using the extra- and intracellular enzymes of Aspergillus quercinus.     

The influence of pH on growth kinetics of yeasts in the presence of benzoate as a sole carbon source

The inhibitory effect and substrate properties of benzoic acid were estimated for 25 yeast strains belonging to generaCondida, Hansenula, Hypopichia, Rhodosporidium, Rhodotorula, Saitoella andTrichosporon. Benzoic acid can serve as a sole carbon source for growth of yeasts belong to generaRhodotorula, Rhodosporidium andSaitoella in synthetic mineral media. Specific growth rate is strongly dependent both on the concentration of benzoate and the pH value of the cultivation media. Maximum specific growth rate on benzoate is observed in alkaline cultivation media at pH 7.0–7.5 whereas those for growth on glucose in mildly acidic media at pH 5.0. Some of the strains showed weak growth on benzoate even at pH 8.5. Some carotenoid-containing yeasts of the generaRhodotorula andRhodosporidium lost their ability to synthesize carotenoid pigments during growth in alkaline benzoate media.     

Extracytoplasmic phosphatases of selected species of Saccharomyces, Kluyveromyces and Rhodotorula

Phosphatase activities of yeasts belonging to the genera Saccharomyces, Kluyveromyces and Rhodotorula were studied. Rhodotorula rubra exhibited activities at acid, neutral and alkaline pH; the other yeasts only had activity at acid pH. Growing yeasts in a constant pH (4.5) medium decreased phosphatase activities in Saccharomyces and Kluyveromyces, while neutral activity was enhanced in Rhodotorula rubra which excreted more enzyme under these conditions. Washing cells with sucrose solutions lowered phosphatase activities in all yeasts, due to enzyme liberation. Acid phosphatase activities in isolated and purified cell walls were very small. Phosphatases thus appear not to be strongly bound to yeast cell walls.     

Substrate Specificity of Nuclease P1

Nuclease P₁ cleaved substantially all phosphodiester bonds in rRNA, tRNA, poly(I), poly(U), poly(A), poly(C), poly(G), poly(I)·poly(C), native DNA and heat-denatured DNA to produce exclusively 5′-mononucleotides. Single-stranded polynucleotides were much more susceptible than double-stranded ones. Influence of pH and ionic strength on the hydrolysis rate significantly varied with the kind of polynucleotides. The enzyme also hydrolyzed 3′-phosphomonoester bonds in 3′-AMP, 3′-GMP, 3′-UMP, 3′-CMP, 3′-dAMP, 3′-dGMP, 3′-dCMP and 3′-dTMP. Ribonucleoside 3′-monophosphates were hydrolyzed 20 to 50 times faster than the corresponding 3′-deoxyribonucleotides. Base preference of the enzyme for 3′-ribonucleotides was in the order of G>A>C≧U, whereas that for 3′-deoxyribo-nucleotides was in the order of C≧T>A≧G. The 3′-phosphomonoester bonds in nucleoside 3′, 5′-diphosphates, coenzyme A and dinucleotides bearing 3′-phosphate were hydrolyzed at a rate similar to that for the corresponding 3′-mononucleotides. Adenosine 2′-monophosphate was highly resistant, being split at less than 1/3,000 the rate at which 3′-AMP was split.     

Purification and Some Properties of Nucleases from Shii-take,Lentinus edodes

Three kinds of nuclease preparations, each of which having both endonuclease activity that formed 5′-mononucleotides and 3′-nucleotidase activity, were separated and partially purified from Shii-take, Lentinus edodes. Both enzyme activities of each preparation showed a similar thermostability and electrophoretic mobility on Polyacrylamide gel, and a competitive relationship was observed between RNA and 3′-AMP in their enzyme reactions. From these results, it is concluded that both enzyme activities of these three preparations reside in a single protein, respectively. They resemble one another in substrate specificity, cleavage pattern of RNA and thermostability, but are distinguishable from one another by molecular weight, electrophoretic mobility and optimum pH for degradation of RNA.     

An Inhibition and Kinetic Study of Acid Phosphatase in Paramecium caudatum and Paramecium tetraurelia1

ABSTRACT. Inhibition, inactivation, pH, and kinetic studies using both homogenates and purified lysosomal fractions of Paramecium caudalum and of P. tetraurelia were carried out to examine the lysosomal acid phosphatase (AcPase) and its relationship to p-nitrophenylphosphatase (pNPPase), glucose-6-phosphatase (G6Pase), and 5′-nucleotidase (AMPase). The results generally support the idea that Paramecium cells contain a distinct lysosomal AcPase with a broad substrate specificity. The hydrolysis of glucose-6-phosphate (G6P) and adenosine 5′-monophosphate (AMP) was shown to be due to this enzyme, suggesting that true G6Pase and AMPase may be lacking in these two species; however, some hydrolysis of AMP at pH 7.5 catalyzed by an unknown soluble enzyme distinct from alkaline phosphatase and Na⁺-K⁺-ATPase was observed. Since the hydrolysis of p-nitrophenylphosphate (pNPP) at acid pH was also shown to be due to AcPase alone, pNPPase could be used as a rapid assay for Paramecium AcPase. At an alkaline pH, however, this activity was catalyzed by an alkaline phosphatase located in the cytosol fraction. P. caudatum AcPase was shown to have kinetic properties similar to those of purified rat liver and human prostatic AcPase and to have relative substrate affinities in the order of G6P < β-glycerophosphate < pNPP < AMP. These different substrate affinities might account for the observed differences in the inhibition of the four lysosomal activities by NaF, L(+)-tartrate, and molybdate, all of which inhibited the hydrolysis of G6P, β-glycerophosphate, and pNPP competitively, but which exhibited a noncompetitive inhibition of a mixed type with the hydrolysis of AMP.     

pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase and trypsin

The pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase (Lys-C) and trypsin has been studied. The reaction was quantitatively monitored by measuring the incorporation of 18O atom into the alpha-carboxyl group of N(alpha)-acetyl-L-lysine from H2(18)O solvent. The optimum pHs of the carboxyl oxygen exchange reaction catalyzed by Lys-C and trypsin were found to be pH 5.0 and 6.0, respectively, which were significantly shifted toward acidic pHs compared to the most favorable pHs of their amidase activities for N(alpha)-acetyl-L-lysine amide in the pHs examined. Steady-state kinetics parameters were also determined for both enzymes at two different pHs, one at the pH optimum for their carboxyl oxygen exchange activity (pH 5-6) and the other at the favorable pH for their amidase activity (pH 8-9). Significantly lower Km (2-fold lower for Lys-C, 3-fold lower for trypsin), and higher kcat values (1.5-fold higher for Lys-C, 5-fold higher for trypsin) were obtained at the acidic pHs compared to the alkaline pHs, suggesting that Lys-C and trypsin have higher substrate binding affinities and higher catalytic rates at the acidic pHs than at the alkaline pHs. The higher carboxyl oxygen exchange activities at the acidic pHs were also confirmed with peptide substrates derived from apomyoglobin. These findings are significant toward the goal of improving the efficiency of the Lys-C and trypsin catalyzed 18O labeling reactions and are thus pertinent to improving the accuracy and reliability of quantitative proteomic experiments utilizing 18O labeling.     

Separation and Identification of UDP-N-Acetylhexosamine Derivatives Excreted by Penicillin-treated Corynebacterium alkanolyticum

N-Acetylhexosamine derivatives, which are intermediates of cell wall synthesis, were detected in UV-absorbing substances excreted by penicillin-treated Corynebacterium alkanolyticum. Gel filtration, using Sephadex G-25, separated N-acetylhexosamine derivatives to three components, each of which was purified by Dowex 1 × 2 column and paper chromatographies.

From the analytical studies, N-acetylhexosamine derivatives were found to be composed of UDP-N-acetylmuramic acid-(diaminopimelic acid, glutamic acid, alanine), UDP-N-acetylhexosaminuronic acid and UDP-N-acetylglucosamine.     

Constituents of Plant Leaf Wax Contained in Rice Callus Tissues

Two enzyme preparations having both nuclease and 3′-nucleotidase activities were partially purified from an extract of tea leaves. They resemble each other in most enzymatic properties, but are separated by DEAE-cellulose column chromatography.

The enzyme activities for RNA, native DNA, heat-denatured DNA and 3′-AMP of each preparation showed a high degree of similarity with respect to the following properties: pH stability, thermal stability and response to EDTA. Both enzymes were shown to be endonucleases (EC 3.1.30.2) which liberated 5′-mononucleotides and oligonucleotides from both RNA and DNA with the following relative rate of hydrolysis: RNA > native DNA = heat-denatured DNA.     

Degradation of Nucleic Acids and their Related Compounds by Microbial Enzymes

The distribution in microorganisms of extracellular enzymes which degrade RNA into 5′-mononucleotides was studied. The degradation products of RNA were determined by using 5′-nucleotidase and adenosine deaminase.

It was found that the enzymes were produced by various microorganisms belonging to Streptomyces, Bacillus, Fungi imperfecta such as Fusarium, Helminthosporium, etc., and Ascomycetes such as Neurospora, Glomerella, Aspergillus, etc.     

Ethyl carbamate precursor citrulline formation from arginine degradation by malolactic wine lactic acid bacteria

Major commercially available strains for induction of malolactic fermentation in wine were examined for arginine metabolism in a resting cell system at wine pH with the aim of evaluating their ability to excrete and utilize citrulline, a precursor of carcinogenic ethyl carbamate (urethane). All strains tested excreted citrulline from arginine degradation. Citrulline was stored intracellularly during growth in arginine rich medium and was released upon lysis of the cells. All strains were found to degrade citrulline as a sole amino acid and some of them were able to reutilize previously excreted citrulline.     

A simple, specific radiometric assay for 5'-nucleotidase.

A radiometric assay for 5′-nucleotidese (EC 3.1.3.5) has been developed, which is applicable for all 5′-nucleotide substrates. Various column materials and eluants were evaluated for their suitability in the separation of purine and pyrimidine bases and nucleosides produced in the reaction. Neutral alumina columns were found to be the best. The unadsorbed nucleosides and their bases could be quantitatively eluted with 0.1 m Tris-HCl, pH 7.4; subsequent elution of the 5′-nucleotide was then accomplished with 0.2 m sodium phosphate, pH 7.4. Differential measurement of 5′-nucleotidase can be accomplished in the presence of acid or alkaline phosphatases by inclusion of concanavalin A into the reaction mixture. It completely inhibits 5′-nucleotidase without effecting the phosphatases. The applicability of this assay has been demonstrated by studying the properties of 5′-nucleotidase present in a purified plasma membrane preparation from a rat tumor which is enriched with both 5′-nucleotidase and alkaline phosphatase.     

Purine and Pyrimidine 5′-Nucleotide Catabolism in Tetrahymena pyriformis W1

SYNOPSIS Deamination at pH 7.5 of adenosine, deoxyadenosine, cytidine and deoxycytidine by cell-free preparations of Tetrahymena pyriformis W was observed both in the presence and absence of fluoride. Deamination of 5′-AMP, 5′-dAMP, 5′-CMP, and 5′-dCMP was found only in the absence of fluoride. Dephosphorylation of the above nucleotides by acid phosphatases occurred at pH 4.5; reduced activity was noted at pH 7.5. Fluoride effectively blocked acid phosphatase activity at both pH values. This correlation of phosphatase and deaminase activities suggests a catabolic pathway for 5′-AMP and 5′-CMP whereby dephosphorylation precedes deamination. Radiolabelled substrates were used to test this hypothesis. The experiments were designed so that conversion of as little at 1.0% of the radiolabelled substrate to the deaminated product could be detected. No 5′-IMP or 5′-UMP, the expected deamination products of 5′-AMP and 5′-CMP, respectively, was recovered after incubation of the radiolabelled substrates with cell-free enzyme preparations. Thus, it appears that Tetrahymena has no 5′-AMP or 5′-CMP deaminases and that these compounds are deaminated only after conversion to nucleosides. Acid phosphatase activity toward 5′-GMP, 5′-dGMP, 5′-TMP, 5′-UMP, and 5′-XMP was also found.     

A Raman spectroscopic study on 5′-rGMP gel and self-aggregate

In order to analyze the melting behavior of 5′-rGMP gel at acidic pH and self-aggregate near neutral pH we have obtained Raman spectra of aqueous solutions of 5′-rGMP at various temperatures. At low temperature the intensities of Raman peaks at 502, 585, 1083, 1179, 1322, 1366, 1487, and 1578 cm^?1 decrease due to the formation of ordered structure (Raman hypochromism). In contrast, the peaks at 671, 725, 813, and 1338 cm^?1 become stronger at low temperature (Raman hyperchromism). The Raman hyperchromism of the 671- and 813-cm^?1 peaks have been explained in terms of detailed structural models. Recently, the 668- and 682-cm^?1 peaks in the Raman spectrum of aqueous 5′-rGMP solution have been attributed to the guanine ring breathing vibrations in C3′- and C2′-endo conformers [Benevides, J. B., Lemur, D. & Thomas, G. J., Jr. (1984) Biopolymers 23 , 1011–1024]. On the basis of this information our Raman data can be interpreted to suggest that the continuous helix model of 5′-rGMP gel is right-handed. The 1487-cm^?1 peak intensity has been used to monitor the melting profies at several pHs. Near neutral pH the melting profile shows a single transition, whereas at acidic pH it shows two transitions. From these observations we propose possible pathways for the melting of 5′-rGMP gel formed at acidic pH and self-aggregate formed near neutral pH.     

Phosphohydrolases and the production of 5′-nucleotides by direct fermentation

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.     

New Series of Mixed Pronucleotides. Synthesis and Anti-HIV Activities of Mononucleoside Phenyl SATE Phosphotriesters

Abstract

The synthesis and anti-HIV activities of phenyl S-pivaloyl-2-thioethyl (tBuSATE) phosphotriesters of AZT and d4T are reported. These compounds show similar activity compared to bis(tBuSATE) phosphotriesters and appear to be able to deliver the corresponding 5′-mononucleotides inside the cells.     

Medium pH, carbon and nitrogen concentrations modulate the phosphate solubilization efficiency of Penicillium purpurogenum through organic acid production

Aims: To study phosphate solubilization in Penicillium purpurogenum as function of medium pH, and carbon and nitrogen concentrations. Methods and Results: Tricalcium phosphate (CP) solubilization efficiency of P. purpurogenum was evaluated at acid or alkaline pH using different C and N sources. Glucose‐ and (NH₄)₂SO₄‐based media showed the highest P solubilization values followed by fructose. P. purpurogenum solubilizing ability was higher in cultures grown at pH 6·5 than cultures at pH 8·5. Organic acids were detected in both alkaline and neutral media, but the relative percentages of each organic acid differed. Highest P release coincided with the highest organic acids production peak, especially gluconic acid. When P. purpurogenum grew in alkaline media, the nature and concentration of organic acids changed at different N and C concentrations. A factorial categorical experimental design showed that the highest P‐solubilizing activity, coinciding with the highest organic acid production, corresponded to the highest C concentration and lowest N concentration. Conclusions: The results described in the present study show that medium pH and carbon and nitrogen concentrations modulate the P solubilization efficiency of P. purpurogenum through the production of organic acids and particularly that of gluconic acid. In the P solubilization optimization studies, glucose and (NH₄)₂SO₄ as C and N sources allowed a higher solubilization efficiency at high pH. Significance and Impact of the Study: This organism is a potentially proficient soil inoculant, especially in P‐poor alkaline soils where other P solubilizers fail to release soluble P. Further work is necessary to elucidate whether these results can be extrapolated to natural soil ecosystems, where different pH values are present. Penicillium purpurogenum could be used to develop a bioprocess for the manufacture of phosphatic fertilizer with phosphate calcium minerals.     

Studies on the Autolysis of Aspergillus oryzae

The conditions of autolysis of washed mycelia of Aspergillus oryzae were systematically examined as for temperature, pH, aeration, energy supply, and chemicals which stimulate autolysis. Below 45°C, the higher the temperature the faster was the rate of autolysis. Optimum pH of autolysis with special reference to the excretion of nucleic acid components and amino acids was 5. With the optimum conditions of autolysis settled by us, 90 to 100% of nucleic acids, 75% of protein, and 20% of sugars in the mycelia were excreted into the medium within three days.

In the presence of lipophilic compounds such as toluene and sodium salts of fatty acids, autolysis occurred much faster than in distilled water. Autolysis was inhibited by the addition of glucose and aeration.

Mycelia of Aspergillus oryzae were autolyzed in distilled water, in toluene-saturated water, or in acetate buffer, pH 5.4, at 30°C. The cytoplasmic materials disappeared from cells during autolysis, but the cell wall retained its shape even after autolysis. The disappearance of the cytoplasmic materials started from the inner part under an aerobic condition and from the outer part under an anaerobic condition. During the autolysis, 15% of the cellular proteins was excreted as free amino acids (60%) and peptides (15%). Glucose, ribose, glucosamine, and three unidentified sugars were found in autolyzate. After eighteen hours of autolysis stimulated by toluene, 81% of the cellular nucleic acids was excreted as uridine (28%), xanthine (24%), hypoxanthine (17%), and two other nucleosides or bases.     

Base compositional analysis of nanogram quantities of unlabeled nucleic acids.

After conversion of unlabeled DNA and RNA to 3′-mononucleotides accurate base compositional analysis can be performed on as little as 10 ng of the hydrolysate. The 3′-mononucleotides are first quantitatively postlabeled with [γ-³²P]ATP by T4 polynucleotide kinase and are then separated as mononucleoside diphosphates on Whatman DE-81 ion-exchange paper at pH 3.5 after hydrolysis of surplus [γ-³²P]ATP to ³²P₁. The locations of the four labeled nucleoside diphosphates are determined by autoradiography and the ratio of radioactivity in the four spots gives the base ratio of the sample.     

Altering the substrate specificity site of <Emphasis Type="Italic">Aspergillus niger</Emphasis> PhyB shifts the pH optimum to pH 3.2

Phytases are of biotechnological importance as animal feed additives for their ability to catalyze the hydrolysis of phosphate from phytate for absorption by simple-stomached animals, and to reduce their fecal phosphorus excretion. Aspergillus niger PhyB has high catalytic activity at low pHs around 2.5, but has little activity at the commonly observed gastric pH of young animals (3.0–3.5). Our objective was to determine if the pH optima of PhyB could be broadened to a more characteristic pH range in the stomach of young animals through site-directed mutagenesis. We created two mutants, E272K and E272Q, each with a single amino acid substitution of the same residue in the substrate specificity site. Mutants were designed to replace an acidic amino acid, with either a neutral amino acid (E272Q) or basic amino acid (E272K), and were overexpressed in the yeast Pichia pastoris. While the wild-type (WT) pH optimum was 2.5, mutant E272K shifted to a new optimum of pH 3.2. E272K had a concomitant reduction in K _m of 36-fold at pH 2.5 and 6-fold at pH 3.2 compared to the WT. Our results indicate that the pH optimum of PhyB can be altered to match the stomach pH, along with an improved substrate affinity.