Agr. Biol. Chem., 39 (4), 879 `883, 1975 Degradation of N-Lauroyl-L-valine in Soil and the Effect of Sunlight and Ultraviolet Rays on N-Lauroyl-L-valinet Toshiro SHIDA, Yasuo HOMMA, Akira KAMIMURA and Tomomasa MISATO Central Research Laboratories, Ajinomoto Co., Inc., 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki 210, Japan and The Institute of Physical and Chemical Research, Wako-Shi Saitama 351, Japan Received December 3, 1974 The degradation of N-lauroyl-L-valine in soil and the effects of sunlight and ultraviolet radiation on this compound were studied. When N-Lauroyl-L-valine was added to the soil extract, a precipitate was observed. The precipitate was thought to be a salt formed from one calcium atom and two molecules of N-lauroyl-L-valine. These data indicated that N-lauroyl-L-valine was absorbed firmly in the soil. Qualitative analysis of compounds formed during incubation of soil with N-lauroyl-L-valine was investigated by gas chromato graphy. Laurie acid (C12) and capric acid (C10) were detected in the medium after incubation. The amino acid could be determined quantitatively by a microbial assay method. The experiment using N-lauroyl-L-valine (14C) indicated that 14CO2 was produced as a final product. These data suggested that N-lauroyl-L-valine was spilt to lauric acid and L-valine, and the lauric acid was degraded to CO2 through capric acid by (ƒà-oxidation. The parent compound, N-lauroyl-L-valine was stable to sunlight and ultraviolet rays. Compounds synthesized from amino acids and fatty acids may not bring about the pos sibility of environmental pollution because they are components of living organisms. We have studied the utilization of several amino acid derivatives as agricultural chemicals along this line. In the previous paper,1) we reported that various amino acid derivatives, as new pesticides, showed some preventive effect against rice blast disease. Among them N-lauroyl-L-valine (hereafter we refer to this compound as No. 5) was most effective to the disease. Further, it was reported that many strains of type cultured bacteria could utilize sodium N-lauroyl-L-valinate (No. 5-Na) as carbon and nitrogen sources for their growth. The metabolism of No. 5 was investigated in detail using Pseudomonas aeruginosa AJ 2116 (ATCC 10145), and lauric acid was identified by gas chromatography suggesting cleavage of N-acyl linkage in No. 5.2 ) õ Studies on the Control of Plant Diseases by Amino Acid Derivatives. Part X. It is important to investigate the decomposi tion of the chemicals in soil and the effect of light, since a large portion of chemicals sprayed falls to the soil and they are usually used out of doors. In this paper, the degradation of No. 5 in soil and the effect of sunlight and ultraviolet rays on No. 5 are investigated. MATERIALS AND METHODS Soil. Soil used throughout this work was mold obtained from a forest of Kanagawa prefecture, Japan. To the soil (15kg) 2g of (NH4)2SO4, 6g of K2S04, and 17g of a mixture of Ca(H2PO4)2 and CaSO4 2H2O (1:2) were added as fertilizers. Preparation of soil extract. A sample of soil (20g) containing fertilizers was shaken vigorously in 40ml of sterile water for 1hr at 25 Ž. The mixture was cen trifuged at 3000rpm and the supernatant was treated with a lump of absorbent. The filtrate was used as soil extract. It seemed that this soil extract was in fected by many microorganisms. Degradation of No. 5-Na in the soil extract. Sodium salt of No. 5 (No. 5-Na) (18mg) was dissolved in 300m1 of the soil extract and placed in an erlenmayer flask
880 T. SHIDA, Y. HOMMA, A. KAMIMURA and T. MISATO (500ml) with a cotton plug. The mixture was incubat ed for 15 days at 25 Ž without shaking. After incuba tion, unchanged No. 5 was determined colorimetrically by the pinacyanol chloride CHCI2 method.3) Assay of 14CO2 produced from No. 5(14C) in the soil extract. The soil extract (2.5ml) and 0.2ml of No. 5 (14C) solution (39946cpm) were mixed in a Warburg's vessel. The carbonyl carbon of lauroyl in No. 5 was labeled as shown in the previous paper.2) Incubation was performed for 19hr at 30 Ž. 14CO2 produced was trapped in 0.5ml of ethanolamine in a center well of the vessel. After incubation radioactive carbon dioxide dissolved in the soil extract was gasified by adding 0.5ml of 1 N H2SO4 in a side arm to the soil extract. Radioactivity of 14CO2 in ethanolamine was counted on a liquid scintillation counter. Quantitative determination of No. 5-Na was made by the method of Kamimura.3) Degradation of'no. 5 by light. No. 5-Na (40mg or 80mg) was dissolved in 2ml of ethanol on a Petri dish and dried up. A thin film of No. 5-Na was formed on the Petridish. This thin film on the Petridish was placed under a photochemical lamp (Toshiba H400-P, minimal wave length, 220nm) at 35cm in height or exposed to the sun. After the treatment this film was dissolved in water and filled up to 40ml. A concentration of No. 5 in the solution was 1000 or 2000ppm. The inhibitory activity against rice blast was determined by the method described in the previous paper.1) FIG. 1. Degradation of No. 5 in Soil Extract. Na remained at the end of this incubation period. In order to determine carbon dioxide as the end product of No. 5-Na degradation, soil extract was incubated with No. 5 (14C) in a vessel employed for Warburg's manometry. Radioactive carbon dioxide produced during the incubation was absorbed in 0.5ml of ethanolamine in a center well. The result is shown in Table T. Radioactivity of 14CO2 TABLE U. DEGRADATION OF No. 5 IN SOIL EXTRACT Identification of No. 5 by gas chromatography. Gas chromatographic identification of No. 5 and its de graded products was performed by the method described in the previous paper.2) Quantitative determination of L-valine. The amino acid was determined quantitatively by microbial assay method. RESULTS (1) Degradation of No. 5 in soil extract As reported previously,2) many bacterial strains can utilize No. 5-Na as carbon and nitrogen sources for their growth, and No. 5- Na utilizing bacteria were not limited widely in the natural world. As the first step of studies on the No. 5 degradation in soil, degra dation of No. 5 was examined in the soil extract containing various microorganisms. As shown in Fig. 1, No. 5-Na was degraded linearly for 7 days and only 10% of No. 5- Concentration of substrate 65ppm, 39946cpm/0.2ml. Reaction time 19hr. Reaction temperature 30 Ž. produced during the incubation was 5590cpm and radioactivity of No. 5-Na added at the begining of the incubation was 39946cpm. Therefore, 14CO2 produced was 14% of the ini tial activity.from these results, it seems that No. 5 was degraded rapidly to CO2 by micro organisms in the soil extract. (2) Formation of a precipitate by adding No 5-Na in soil extract When the concentration of No. 5-Na was from 100 to 600ppm in the soil extract, for mation of a precipitate was observed. No. 5- Na was added to the soil extract (soil: water=
Degradation of N-Lauroyl-L-valine in Soil 881 1:2) and shaked vigorously for 30 to 60 seconds and left at room temperature for 1.5 to 2.0hr. After filtration, No. 5-Na in the filtrate was determined quantitatively. Figure 2 shows and the mixture was shaken vigorously. After centrifugation, a precipitate formed was wash ed once with water. The precipitate was dried in an out gassed desicator at 50 to 60 Ž. The gray powder obtained was subjected to an emission spectroscopic examination. As shown in Table U, calcium was strongly emitted. Further, the precipitate was identi fied ultimate analysis (Table U). From these data, it seems that the precipitate was a salt formed from one calcium atom and two molecules of No. 5. (4) Degradation of No. 5 in the soil As mentioned above, No. 5 was degraded to CO2 in the soil extract containing various microorganisms. Further, it is clear that No. 5 was absorbed in soil, and that calcium salt FIG. 2. Change of No. 5-Na in Soil Extract, œ, Recovery No. 5;, Theoretical volume of No. 5. the relationship between the initial amount of No. 5-Na added to the soil extract and the concentration of dissolved compound in the extract. When 60ppm of No. 5-Na was added to the soil extract, the compound was quantita tively recovered in the filtrate. When 200 and 600ppm of No. 5-Na was added, amounts of the compound recovered in the filtrate were 146 and 175ppm respectively. The more amount of No. 5-Na was added, the less com pound was recovered in the filtrate. (3) Identification of a precipitate formed in the soil extract by adding No. 5-Na No. 5-Na was added to the soil extract at the final concentration of 2000ppm to 3000ppm, of No. 5 was formed. Then, it is interest to observe No. 5 degradation in soil. Experi mental conditions are identical to those using soil extract. Radioactivity of 0.2ml of No. 5 solution (65ppm) was 39946cpm., Soil (1g) in 2ml of sterile water was incubated with 0.2ml of No. 5 (14C) solution for 19hr at 30 Ž in a Warburg's manometer vessel. As shown in Table V, radioactivity of 14CO2 produced was 16539 cpm (41.3%). From this result, it is evident that No. 5 was degraded to CO2 rapidly in soil. (5) Identification of metabolites of No.5 Qualitative analysis of compounds formed during incubation of soil with No. 5 was investi gated by gas chromatography. As shown in Fig. 3, lauric acid (C12) was detected in the medium after the incubation (peak 2 in graph B), and another peak (peak 1 in graph B) was TABLE U. EMISSION SPECTROSCOPY OF PRECIPITATE AND ULTIMATE ANALYSIS
882 T. SHIDA, Y. HOMMA, A. KAMIMURA and T. MISATO FIG. 3. Gas Chromatograms of Incubation Mixture Containing Sodium N-Lauroyl-L-valinate and Soil. A, mixture before incubation; B, mixture after incubation for 120hr; C, Authentic caprylic acid (C8), capric acid (C10), and lauric acid (C12); Column, 10% SE-30 Gas Chrom Q 1/4 Inch. temp. 149 Ž. TABLE V. DEGRADATION OF No. 5 IN SOIL detected at the identical position with reten tion time of authentic capric acid (C10). A small shoulder on the left hand of peak I in the graph B could not be identified. Further 10.64mg/liter of L-valine was released from 65 mg/liter of No. 5-Na (Table W). From TABLE W. LIBERATION OF L-VALINE FROM SODIUM N-LAUROYL-L-VALINATE IN SOIL light on the chemicals. In the present in vestigation, we examined effects of ultraviolet rays and sunlight on No. 5-Na. As shown in Figs. 4 and 5, the irradiation of ultraviolet rays affected a little preventive value against rice blast. When an initial concentration of No. 5-Na was 2000ppm, residual No. 5-Na was 1350ppm after 15 days and preventive value was 81%. When an initial concentra tion was 1000ppm, residual No. 5-Na was 600ppm and residual preventive value was 83%. Figure 6 shows the effect of the ir radiation of sunlight on No. 5-Na. It is clear that the irradiation of sunlight did not degrade No. 5-Na at concentrations of 1000 and -: Not determined. these results, it seems that No. 5 was split to lauric acid and L-valine, and lauric acid might be degraded to CO2, through capric acid by ƒà-oxidation. (6) Effect of light on No. 5-Na Since fungicides are usually used in the open air, it is necessary to investigate effects of FIG. 4. Effect of the Irradiation of Ultraviolet Rays on No. 5-Na., 1000ppm;, 2000ppm.
B Degradation of N-Lauroyl-L-valine in Soil 883 result obtained in the present investigation indicates that No. 5 shows the same behavior. As shown in Table W, and Fig. 3, C10 and C12 fatty acids and L-valine were detected and the generation of CO2, was detected as the end product. It is recognized that N-acyl linkage in No. 5 was cleft and lauric acid and L-valine were produced. The fatty acid appears to be metabolized to CO2, through capric acid by ƒà-oxidation. This pathway is similar to that FIG. 5. Degradation of No. 5-Na by the Irradiation of Ultraviolet Rays., 1000ppm; œ, 2000ppm. FIG. 6. Effect of Sunlight on No. 5-Na., 1000ppm;, 2000ppm. found in P. aeruginosa AJ 2116. Many fungicides and organophosphorous insecticides such as blasticidin S and parathion are degraded rapidly by ultraviolet rays. 5,6,7,) However, No. 5 is stable against the irradia tions of sunlight as well as ultraviolet rays as shown in Figs. 4 and 6. This is expected enough because No. 5 has no photoenergy absorbing positions in the region of the wave length teted. This is one of the important characters of No. 5. The strong points of No. 5 are stable to sunlight and ultraviolet and biodegradable producing common natu rally-occurring compounds. The effects of lights on No. 5 in liquid phase will be reported elswhere. From these results, it is supposed that the use of this compound as a fungicide will not bring about the possibility of enviro nmental pollution. 2000ppm. Preventive values at both con centrations did not vary after the treatment. From these data, it is evident that No. 5-Na was stable to light. DISCUSSION It was recognized that No. 5-Na was absorbed firmly in soil and (No. 5). Ca was formed as shown in Fig. 2, and Table U. From this result, it seems that the release of this compound from soil is difficult. Various fungicides and herbicides such as blasticidin S and paraquat are absorbed in soil.4,5) The REFERENCES 1) Y. Homma, T. Shida and T. Misato, Ann. Phyto pathol. Soc. Japan, 39,(2) 90 (1973). 2) T. Shida, Y. Homma and T. Misato, Agr. Biol. Chem., 37,1027 (1973). 3) A. Kamimura, ibid., 37, 457 (1973). 4) B. A. G. Knight and T. E. Tomlinson, J. Soil. Sci., 18, 233 (1967). 5) "Environmental Toxicology of Pesticides," ed. by T. Misato and F. Matsumura and G. M. Boush, Academic Press Inc., New York, N.Y., 1972, p. 257. 6) J. Payton, Nature, 171, 355 (1953). 7) J. O. Frawley, J. W. Cook, J. R. Blake and O. G. Fitzhugh, J. Agr. Food Chem., 6, 28 (1958).