J. VGen. Appl. Microbiol. ol. 10, No. 2, 1964. PRODUCTION OF NUCLEIC ACID-RELATED SUBSTANCES BY FERMENTATIVE PROCESSES. V. ACCUMULATION OF INOSINIC ACID BY AN ADENINE-AUXOTROPH OF MICROCOCC US GL UTAMIC US KIYOSHI NAKAYAMA, TAKEO SUZUKI, ZENROKU SATO and SHUKUO KINOSHITA Tokyo Research Laboratory, Kyowa Hakko Kogyo Co., Ltd. Machida, Tokyo ReceivedMarch 21, 1964 Utilization of an microbial auxotrophic mutant was successfully applied to the production of several amino acids by f ermentative methods (1). This led us to an attempt for the use of microbial auxotrophic mutants for the fermentative production of nucleic acid-related substances, some of which have recently received attention as seasoning materials (2-4). Xantho- sine accumulation by Aerobacter aerogenes P 14(5) was observed at a level of 6 mg per ml (6). The present paper is concerned with the accumulation of 5'-inosinic acid in culture media by an adenine-auxotrophic mutant of the glutamic acid-producing organism, Micrococcus glutamicus (7, 8). MATERIALS AND METHODS Microorganism. Micrococcus gltamicus No. 534-348, an adenine- requiring auxotrophic mutant of M. gltamicus was used in this study. It was previously isolated in this laboratory (9). Cultural conditions. Seed medium used for the investigation of inosinic acid accumulation had the following composition: 2% glucose, 0.5% yeast extract, 1 °o peptone, 0.25% NaCI at pH 7.2. Flasks containing the seed medium were inoculated with the bacterium and incubated at 28° for 24 hr. The flask contents were used as an inoculum (10% v/v) for the fermentation media. Unless otherwise stated, fermentation was conducted at 28° in a 250 ml Erlenmeyer flask containing 30 ml media. The flasks were agitated on a rotary shaker at a velocity of about 220 rpm. The cell suspension used as inoculum in testing the growth response of the bacterium to some compounds was prepared by scraping cells grown on yeast extract-nutrient agar slants into distilled water. Quantitative analysis of inosinic acid. The quantity of inosinic acid in culture broths was determined by optical density of extracts of inosinic acid spots on the paper chromatogram of the culture filtrate. Paper chromatography was performed on Toyo No. 51 A filter paper, by using the following solvent systems: (a) isobutyric acid--acetic acid -1 N NH4OH 133 134 NAKAYAMA, SuzuKI, SATO and KINOSHITA VOL. 10 (10:1 : 5) ; (b) isopropylalcohol - conc. NH40H - water (6:3:2); (c) isoamylal- cohol - tetrahydrof urf urylalcohol -0.08 M potassium citrate (1:1:1). In most cases solvent system (a) was used but systems (b) and (c) were also used in some cases where such substances as fluorescent compounds formed in the broth interf erred with the estimation of inosinic acid. Infrared analysis. Infrared spectra were obtained by the KBr pellet technique with an IRS type infrared spectrophotometer, Japan Spectroscopic Co., Tokyo. RESULTS Accumulation of UV absorbing substances In the course of the investigation of the accumulation of UV-absorbing cii} afaii a hn aiivn+rnnhir+ mnfan+c il/l nloutnnn'r'2ie Nm K2d_2LIS2xxvaa fmird Table 1. Comparison of UV-absorbing substance with inosinic acid and hypoxanthine by paper chromatography (RI-value). LOV LiSU 3UU Wave length (mp) Fig. 1. UV-absorption spectra of the extracts of paper chromatogram spot. Production of nucleic acid-related substances 135 to produce some UV-absorbing substances in culture media. The accumulation of two compounds was demonstrated by paper chromatography. The minor component was identified as hypoxanthine by its chromatographic behaviour in 4 solvent systems (Table 1) and its UV-absorption spectra. Wave length (ml') Fig. 2. UV-absorption spectra of the hydrolysate of the extract from inosinic acid spot. The main component was tentatively identified as inosinic acid by its chromatographic behaviour (Table 1) and UV-absorption spectra of the spot extracted from a paper chromatogram (Fig. 1). Furthermore, the extract was hydrolyzed with hydrochloric acid (final concentration 1 N) by heating in a boiling water bath for 90 min. The hydrolysate thus prepared showed characteristic curves of hypoxanthine as shown in Fig. 2. The presence of organic phosphorous compound was confirmed by methods similar to those described by BANDURSKI(10). Isolation and identificaton of inosinic acid accumulated in culture broth The main substance, supposed as sodium inosinate, was isolated in crystalline form from the culture broth by the process shown in Fig. 3. The material obtained from the broth was dissolved in water at a concentration of 100, tg per ml and hydrolyzed on a boiling water bath after addition of one part of 4 N sodium hydroxide. The amount of the phosphoric acid released by hydrolysis was assayed by the method of FISKE-SUBBAROW(11). As shown in Fig. 4, the material from culture broth gave the same decomposition curve as authentic sodium inosinate. The phosphoric acid content in the material was estimated to be 0.93 mole, assuming the molecular formula C1o111N403P • Na2.7.5H2O. On the other hand, the content of the phosphoric acid in the authentic sodium inosinate was assayed to be 0.96 mole. The content of ribose in the compound was 136 NAKAYAMA, SuzuKI, SATO and KINOSHITA VOL. [0 Fig. 3. Isolation procedure of inosinic acid from culture filtrates. found to be 0.82 mole and in authentic sodium inosinate, 0.80 mole as measured by the orcinol method. The UV-absorption spectrum of the material was identical with that of sodium inosinate. The behavior of the material on paper chromatography and on alkaline hydrolysis distinguished it from 3'(or 2')-inosinic acid as described above. Further confirmation of the site of attachment of phosphoric acid residue in ribose was accomplished by periodate oxidation (12,13) as follows. Fifty milligrams of the material was dissolved into 0.01 M sodium metaperiodate solution. After standing in dark at room temperature for 60 min, 90 min and 4 hr respectively, aliquots (each 5 ml) was taken and treated with 10 ml each of 20 per cent saturated sodium bicarbonate solution, potassium iodate solution and 0.01 M sodium arsenite solution. After mixing thoroughly by shaking and standing for 15 min, the residual sodium arsenite was titrated Production of nucleic acid-related substances 137 Fig. 4. Alkaline hydrolysis of sodium inosinate. Sample solutions were hydrolyzed with 2 N NaOH on b oiling water bath. with 0.01 M iodine solution and the quantity of the consumed metaperiodate caluculated. The material used up about 1 mole of metaperiodate per 1 mole of substrate as based on the molecular formula C10H11N4O8P • Na2.7.5H2O. Sodium salts of 5'-inosinic acid, 5'-adenylic acid and adenosine also used up about 1 mole of metaperiodate per 1 mole compound. Adenosine-3'-phosphate did not consume any periodate at all. The sample obtained from culture filtrate and the authentic sodium inosinate had identical infrared spectra as shown in Fig. 5. Fig. 5. Infrared absorption spectrum of sodium inosinate. - authentic sample -- sample from culture broth 138 NAKAYAMA, SuzuKI, SATO and KIN05HITA VoL. 10 Growth response to adenine derivatives M. glutamicus No. 534-348 was perviously proved to be adenine-less mutant by auxanographic methods (9). The strain was found to be aden- ine-specific auxotroph by the experiment shown in Table 2. The growth res- ponse to various adenine derivatives in media containing high concentrations of glucose and peptone is shown in Fig. 6. At equivalent molar concentrations, the culture responded to adenine most effectively. Adenosine, adenylic acid and adenosine triphosphate were also effective in this order in supporting the growth of the bacterium. The bacterium requires several amino acids to attain good growth (14). Therefore, peptone was added in Table 2. Growth response of M. glutamicus 534-348 to adenine and other basesa Fig. 6. Growth response of M. glutamicus 534348 to adenine-derivatives. Basal medium: glucose 10%, NH4C1 0.7%, KH2PO4 0.1%, K2HPO4 0.2%, MgSO4.7H2O 0.05%, peptone 4°0, biotin 30 pg/liter, trace element soln. 1 ml/liter, and CaCO3 0.9 g/30 ml. the media used in the experiment shown in Fig. 6. In media which contain little or no peptone, the growth was poor and inhibition of growth by high concentrations of adenine was noted. This phenomenon was also observed when adenosine was used, although higher concentrations were required than adenine. The competition between purine and pyrimidine bases has been known Production of nucleic acid-related substances 139 in the growth of the purine- or pyrimidine-auxotrophic microorganism (15, 18). Therefore, certain purine and pyrimidine bases were added at concentrations of 1.6 x 10_3 M and 8 x 10-4 M into media containing 1.25><10_5 M adenine, and the growth of the culture was measured. Guanine and 2, 6-diaminopurine promoted the growth, but no compound competed with adenine in this experiment. Histidine showed a slight adenine effect sparing, as in Esche- richia coli (19) and Lactobacillus casei (20). Influence of biotin and adenine on inosinic acid accumulation The marked effect of growth factor on the accumulation of metabolic product in auxotrophic mutant has been frequently noticed. Therefore, the effects of biotin and adenine on inosinic acid accumulation was studied using synthetic media. The results are shown in Fig. 7. In the medium containing higher concentration of biotin (30pg per liter) better growth and better inosinic acid accumulation were attained than in the medium containing lower one (2.5pg per liter). The range of the concentration of adenine being appropriate for inosinic acid accumulation was broader in high biotin medium than in low biotin medium. The accumulation of naenine jig/miJ Fig. 7. Effect of biotin and adenine concentrations on IMP-and glutamic acid-production and on growth of M.