The Degradation of Isopropylbenzene and Isobutylbenze Ne by Pseudomonas Sp
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Agr. Biol. Chem., 39 (9), 1781-1788,1975 The Degradation of Isopropylbenzene and Isobutylbenze ne by Pseudomonas sp. Yoshifumi JIGAMI,Toshio OMORIand Yasuji MINODA Departmentof AgriculturalChemistry, Faculty of Agriculture, The Universityof Tokyo,Tokyo ReceivedMarch 17, 1975 To clarify biodegradation pathways of isoalkyl substituted aromatic hydrocarbons, oxidation products of isopropylbenzene and isobutylbenzene by Ps. desmolytica S449B1 and Ps. convexa S107BI were examined. Oxidation products from isopropyl benzene were determined to be 3-isopropylcatechol and (+)-2-hydroxy-7-methyl-6-oxooctanoic acid. Isobutylbenzene was also oxidized to 3- isobutylcatechol and (+)-2-hydroxy-8-methyl-6-oxononanoic acid by the same strains. From these results, the existence of an unknown reductive step in the degradation of these isoalkyl substituted aromatic hydrocarbons and the initial oxidation of these aromatic hydrocarbons by the strains were made clear. The degradation pathways of isopropyl benzene and isobutylbenzene by these strains were discussed. In the previous paper," the authors de convexa S107B1 described in the previous paper's were scribed the isolation of isopropylbenzene used for study. assimilation bacteria and the identification of Cultural methods. The composition of the medium the isolated strains, S107B1 and S182B1. and the culture conditions used for isolation of pro Furthermore, the substrate specificity differ ducts were the same as those reported for the microbial ence between bacteria assimilating various oxidation of ƒ¿-methylstyrene and ƒÀ-methylstyrene.2) aromatic hydrocarbons was reported. To Chemical. Isopropylbenzene and isobutylbenzene examine biodegradation pathways of these were obtained from Tokyo Chemical Industry Co., Ltd. aromatic hydrocarbons and the effect of 3-Isopropylcatechol was purchased from Aldrich Chemical Co., Inc. U.S.A. substituent groups of substrates on decom posing pathways by microorganisms, the Analytical methods. Melting points were deter authors tried to isolate the intermediates from mined on a microscope hot stage and were reported culture broths. The effort resulted in the uncorrected. Optical rotations were measured in a JASCO DIP-S polarimeter. IR spectra were obtained isolation of new ring fission products from with a JASCO IR-S spectrometer. NMR spectra isopropylbenzene and isobutylbenzene by were recorded on a JEOL-JNM-4H spectrometer at Pseudomonas species. This paper describes 100 MHz in CDCl3 with tetramethylsilane as an the physical and chemical properties of these internal standard. Mass spectra were measured products and their chemical structures. From with a Hitachi RMU-6L mass spectrometer operating with an ionization energy of 70 eV. UV spectra were these results, the existence of an unknown measured on a Hitachi Model 124 spectrophotometer. reductive step in the degradation of these TLC were carried out with silica gel plates of 0.25 mm aromatic hydrocarbons was revealed. Fur thickness (Tokyo Kasei chromatogram sheet, type thermore, both the initial attack on these S073), which were developed with the solvent system isoalkyl substituted aromatic hydrocarbons by of benzene-dioxane-acetic acid (90: 25: 4, v/v). Spots the strains and their metabolic pathways were were detected by spraying the following reagents: bromocresol green for acidic compounds, diazotized discussed. benzidine for phenolic compounds and 2,4-dinitro- phenylhydrazine-HCl for keto compounds. MATERIALS AND METHODS Microorganisms. Ps. desmolytica S449B1 and Ps. 1782 Y . JIGAMI, T. OMORI and Y. MINODA cultivated in 30 liter jar fermentors containing RESULT 20 liters of liquid medium and 100 ml of iso propylbenzene by the same way mentioned S1. Oxidation products from isopropylbenzene previously.' After cultivation, 80 liters of In order to examine oxidation products culture broth were collected, centrifuged to from isopropylbenzene, I liter of culture broth remove the cells and concentrated in vacuo to of strain S107B1 and S449B1 grown on is 8 liters. The concentrated culture broth was opropylbenzene for 2 days was fractionated and extracted with ether and fractionated accord extracted with ether to obtain an acidic frac ing to the procedures shown in Fig. 1. tion by usual methods . The results by TLC From the weakly acidic fraction, a crude showed that the main products differed be Product 1 was separated by silica gel column tween strain S107B1 and S449B1. On the chromatography using benzene as solvent. chromatogram, Ps. desmolytica S449B1 show Each eluate (5ml) was applied to TLC. The ed two dominant spots on Rf 0.71 and Rf zone at Rf 0.71 was cut, removed and ex 0.38, while Ps . convexa S107BI showed the tracted with ether. Evaporation of the solvent main spot on Rf 0.38. By spraying reagents, gave a brown oil (220 mg). Product I showed the spot on Rf 0.71 (Product 1) was known to positive coloration to diazotized benzidine be a phenolic compound and the spot on Rf and negative coloration to bromocresol green 0.38 (Product 2) was known to be an acidic and 2,4-dinitrophenylhydrazine. Judging from compound with a carbonyl group . For isola the coloration of dark brown to diazotized tion of these products, strain S449B1 was benzidine, Product 1 was assumed to be a FIG. 1. Isolation Procedure for Product from Isopropylbenzene and Isobutylbenzene . Degradation of Isopropylbenzene and Isobutylbenzene by Pseudomonas 1783 diphenolic compound. The IR spectrum TABLE 1. PHYSICAL AND CHEMICAL PROPERTIES OF PRODUCT 2 (film, CHCl3 sol.) showed absorption peaks at 3480cm-1 (hydroxyl), 1380cm-1 and 1360 cm-1 (isopropyl), 780cm-1 and 730cm-1 (aromatic). The mass spectrum revealed a molecular ion peak at m/e 152 and the fragmentation exhibited prominent ion peaks at m/e 137 (M+-CH,), 123, 91, 77, 65, 51 and 43. From these data, Product 1 was assumed to be a diphenolic compound with an isopropyl group. Accordingly, in order to examine the presence of ortho dihydroxyl groups on benzene nucleus, the formation of insoluble lead compound was checked by the method of Evans.3) In this test, Product 1 in neutral solution with lead acetate gave a dark green insoluble lead salt. Thus, the presence of ortho dihydroxyl group was known. Furthermore, the NMR spectrum confirmed the structure of Product 1, including a sub stitution of aromatic protons. Signals were at 82•`83•Ž, was positive to bromocresol observed at ƒÂCDCl3Me4Si 1.18 (6H, doublet, J= green and 2,4-dinitrophenylhydrazine and nega tive to diazotized benzidine. The IR spec 6.5Hz, isopropyl methyl), 3.16 (1H, multiplet, isopropyl methine), 5.42 (2H, singlet, hydroxyl, trum (in Nujol) showed the presence of hydro xyl group (3400 cm 1), carbonyl group (1700 disappeared on shaking with D,O) and 6.50- 6.80 (3H, multiplet, aromatic). In this case, cm-1) and isopropyl group (1378cm-1 and the entire spectrum, especially a chemical 1170cm-1). In the mass spectrum, a mole cular ion peak was not observed and the shift and a spectral pattern of aromatic fragmentation exhibited prominent ion peaks protons, was identical with authentic 3-iso at m/e 170 (M+-H,O), 145 (M+-C,H,), 99 propylcatechol. Thus, the structure of Pro duct 1 was determined to be 3-isopropylcate (C3H7COC2H5+), 85 (C3H7COCH2+), 71 chol. (C3H7CO+) and 57. On treatment with acetic anhydride-pyridine, Product 2 formed a mono On the other hand, for isolation of Product acetylated compound whose mass spectrum 2, a strongly acidic fraction was applied to showed a molecular ion peak at m/e 230. silica gel column chromatography using chloro Accordingly, the molecular weight of Product form as solvent. Each eluate (10g) which was 2 was determined to be 188, which was in passed through a silica gel column (3 x 40cm) was applied to TLC and fractions which gave good agreement with the result of elementary analysis (C9H16O4). The transparence in the a single spot of Rf 0.38 were collected. UV region suggested the absence of a con Evaporation of the solvent gave a crude Product 2, which was dissolved in chloroform jugated double bond. Further, the NMR spectrum exhibited signals at ƒÂCDCl3Me4Si1.05 (6H, and passed through a silica gel column (I x doublet, J=7.0 Hz, isopropyl methyl), 1.82 20cm). Removal of the solvent after column chromatography resulted in the isolation of a (6H, multiplet, three methylenes), 1.90-.2.20 (2H, overlap of singlet hydroxyl and multiplet pale yellow crystal. Recrystallization from chloroform gave 1.83 g of colorless Product 2. isopropyl methine), 4.50 (1H, multiplet, hydro xy methine) and 4.90 5.20 (1H, broad singlet, Physical and chemical properties of Product 2 carboxylic). In this spectrum, however, the are summarized in Table I. Product 2 melted 1784 Y. JIGAMI, T. OMORI and Y. MINODA FIG. 2. NMR Spectrum of Acetylated Product 2 (in CDCl3). spectral patterns of signals at b 1.90 - 2.20 and triplet of methylene protons at b 2.52 (J= b 4.50 were not clear and the assignment was 7.0Hz). Considering the chemical shift and difficult. spectral pattern, the triplet at b 2.52 was To further confirm the structure, the NMR recognized as a methylene group adjacent to spectrum of acetylated Product 2 shown in both methylene and oxo groups and the Fig. 2 was analysed. Signals were observed multiplet at b 2.60 was recognized as a at brasi 1.09 (6H, doublet, J=7.0 Hz, iso methine group adjacent to both isopropyl propyl methyl), 1.50-2.00 (4H, multiplet, methyl and oxo groups. Accordingly, the two methylenes),2.12 (3H, singlet, acetate partial structures of (CH3)2CHCOCH2CH2- methyl), 2.40-2.80 (3H, overlap of triplet and -CH2CH(OH)- were clarified, which were methyleneand multipletisopropyl methine), also supported by the fragmentation of mass 5.00 (1H, triplet, J=6.5 Hz, acetoxymethine) spectrum. Thus, the structure of Product 2 and 9.45 (1H, singlet,carboxylic, not shown was determined to be 2-hydroxy-7-methyl-6- in Fig.