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Agr. Biol . Chem., 39 (6), 1219 1975

Utilization of a Cyclic Dieter and Linear Oligomers of-Aminocaproic Acid by Achromobacter guttatus KI 72

Shinichi KINOSHITA, Sadao KAGEYAMA, Kazuhiko IBA,Yasuhiro YAMADA and Hirosuke OKADA

Department of Fermentation Technology, Osaka University,Yamada-kami, Suita-shi, Osaka

Received November 18, 1974

A microorganism, strain KI 72 capable of utilizing -aminocaproic acid cyclic dimer assole carbon and nitrogen sources was isolated from sludge and identified as Achromobacterguttatus. This bacteria utilized 1 % of the cyclic dimer in a day and was not inhibited bythe higher concentration of the dimer. The growth rate was independent of the cyclic dimerconcentration in the medium, but the maximum cell concentration increased with the increaseof substrate concentration. The cell yield was 0.7 mg dry cell/mg -aminocaproic acid cyclicdimer. Bacterial growth with the cyclic dimer as substrate was significantly stimulated bythe addition of yeast extract. Ferric chloride was also stimulatory. Maximal growth wasobtained in cultures incubated at pH 6 and at 33C. Synthesized nylon oligomers, ranging fromaminocaproic acid up to its linear hexamer, were found to be catabolized by this organism.

Waste water from nylon factories is likelyto contain -caprolactam, -aminocaproic acid(ACA), and linear and cyclic nylon oligomersof various lengths. These substances are notproduced biologically and are decomposed byfew organisms. The enzymes capable ofhydrolyzing these oligomers and degrading themonomers are interesting from a standpointof microbial adaptation.

Fukumura1,2) reported isolation of organismswhich hydrolized linear and cyclic oligomers ofACA except ACA cyclic dimer (ACA-c-dimer). Maekawa et al.3' showed that Pseu-domonas sp. catabolized ACA-c-dimer. Recently we reported that Achromobacter guttatusKF71, an isolate from sludge, metabolizedcaprolactam but not ACA-c-dimer4) and thatsubcellular fractions prepared from the bacterial cells converted -caprolactam to ACA.51

In this paper, a newly isolated microorganism Achromobacter guttatus K172, capableof utilizing ACA-c-dimer as the sole carbon andnitrogen sources is described. The generaltaxonomic characteristics of the bacterium andcertain aspects of the metabolism of variousACA oligomers, including the cyclic dimer,are reported.

MATERIALS AND METHODSMicroorganism. The microorganism employed was

isolated from sludge samples used as inocula for 3-mllots of basal medium with 0.2% -aminocaproic acidcyclic dimer (ACA-c-dimer). The cultures were incubated at 30C for 2 days on a reciprocal shaker, andthose showing better growth were successively sub-cultured several times. From the most rapidly growingculture a bacterium designated KI 72 was obtained bysingle colony i solation, and used throughout this study.

Culture. The bacterium was grown in a 500-m1Erlenmeyer flask containing 100-ml basal medium at30C on a rotary shaker unless mentioned specially.The basal medium consisted of 1 % ACA-c-dimer, 0.3KH2PO4, 0.1 % K2HPO4, 0.02% MgSO4E7H2O, and0.05 % yeast extract. For slant culture the basal medium was supplemented with 1.5 % agar. The seed culturewas prepared by incubating the bacteria from the slantculture for 2 days, and the seed was used for 2 % inoculum in an experiment.

Assay methods. Cell concentration measured turbidimetrically at 600 nm using a Hitachi spectrophotometer 101 was correlated with dry cell weight to providea reference curve. ACA and linear and cyclic oligomerwere detected by paper or thin layer chromatog raphy(TLC) by using n-butanol: acetic acid: water (4: 1: 2),or n-propanol: ethylacetate: 7% aqueous ammonia(6:1:4) as a solvent system. The cyclic dimer wasdetected by spraying with modified Dragendorff reagent,6) or standing in iodine vapor. The amino acids

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1220 S. KINOSHITA, S. KAGEYAMA, K. IBA, Y. YAMADA and H. OKADAwere developed by spraying with ninhydrin (1% inn-butanol). ACA and its linear oligomers were deter-mined by the modified ninhydrin method7) with heatingin an autoclave at 0.8 atm. Infrared spectroscopy(IR) was carried out with a Hitachi 215, nuclearmagnetic-resonance spectroscopy (NMR) with a JEOLPS 100, and mass spectra determinations with a HitachiRMU-6E.

Aminocaproic acid cyclic dimer. ACA-c-dimerwas a gift from Dr. Kotani, the Institute of Toyo Boseki,Co., Ltd., Katada, Shiga.

Aminocaproic acid linear oligomers. ACA-oligomers were synthesized according to Zahn and Hilde-brand.8) N-Carbobenzoxy--aminocaproic acid (ZEACA) was synthesized from ACA and carbobenzoxychloride. N-Carbobenzoxy--aminocaproic acid lineardimer (ZEACA2) was prepared from ZEACA, ACA,triethylamine, and ethyichloroformate in toluene. N-Carbobenzoxy--aminocaproic acid linear trimer (ZACAS) or tetramer (ZEACA4) was prepared from ZEACA2, and ACA or ACA2 by a similar method in tetrahydrofuran. Also the N-carbobenzoxy--aminocaproicacid linear pentamer (ZEACA5) or hexamer (ZEACAB)was made from Z ACA3 and ACA2 or ACAS by usingdimethylformamide as solvent. ACA2, ACA3, ACA4,ACA5, and ACA6 were prepared by decarbobenzoxylation of the corresponding N-carbobenzoxy derivativesby using hydrogenolysis on paladium charcoal in aceticacid. The identity of these synthesized samples wasconfirmed by melting point, IR spectram, and TLC.

Dimethylimino ether of ACA-c-dimer. ACA-c-dimer (10g) was suspended in 30 ml of benzene, 8.5 mlof dimethyl sulfate were added dropwise, and the mixture was refluxed for 20 hr with stirring. To the reaction mixture, 100ml of 15% Na2CO3 was added, andthe mixture was kept at 50C for 1 hr with stirring.After removal of insoluble crystals formed by the addition of 100ml of ethylacetate, the organic layer wasseparated and dried over anhydrous sodium sulfate. Byevaporating off the solvent, crystals of the crude productwere obtained (yield 3 g). For further purification,the crystals were dissolved in ether. After removingthe insoluble materials and concentrating the ethersolution, pure white crystals of the dimethylimino etherwere obtained. mp 85C. IR Nujolmaxcm-1: 1680(C=N), 1265, 1235, 1135. Mass spectra (mle): 254(M*), 239 (M+ -CH3), 207, 126, 96, 55, 41. NMR (inCDCl3) (ppm): 3.60 (6H, OCH3, singlet), 3.30 (4H,-CH2-N= , triplet), 1.10` 1.75 (12H, multiplet).

Other chemicals were obtained commercially.

RESULTS

A strain K172 was isolated from sludge by

FIG. 1. Time Course of the Growth of Achromobacterguttatus KI 72 on -Aminocaproic Acid Cyclic Dimer.For details see the text.

an enrichment culture technique, and thetaxonomic study was, carried out accordingto Bergey's Manual of Determinative Bacteriology.9) The flagella were observed underan electron microscope (Hitachi MS70). Itscharacteristics and some other properties weresimilar to those of Achromobacter guttatuslisted in Bergey's Manual. The isolate, K172was identified as Achromobacter guttatus, butdifferent from Achromobacter guttatus KF71

FIG. 2. Time Course of the Bacterial Growth onDifferent Concentrations of Substrate.A, Higher concentration ACA-c-dimerB, Lower concentration of ACA-c-dimerThe numbers indicate the initial concentration of thesubstrate (%). 0.1 % of the seed culture was added at0 time.

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which had been reported in our previouspaper" in ability to utilize -caprolactam.lizuka et al.10) isolated A. cycloclastus as ancaprolactam utilizing bacteria, but the strainK172 was different from it in acid productionfrom glucose.

Effect of culture conditionFigure 1 shows the time course of the

culture in 600ml of the basal medium in a3-liter Erlenmeyer flask inoculated with 1 mlof the seed culture and incubated on areciprocal shaker. The cell mass increasedfor 27 hr and ACA was formed with the in-crease in the cell mass. Though several kindsof amino acids were detectable in trace amountsby paper chromatography, all ninhydrin positive materials were calculated to be ACAin this figure. The pH increased graduallyfrom 6.2 to 7.2 after remaining constant for16 hr. On examination by paper chromatography, the culture filtrate yielded two largespots and small spots representing naturalamino acids, identified as alanine aspartic acid,glutamic acid, glycine, isoleucine, methionine,phenylalanine, threonine, and valine whenanalyzed by amino acid analyzer (HitachiKLA-3B).

The increase in cell mass in the basal mediumcontaining various concentrations of ACA-c-dimer are shown in Fig. 2. Differences inthe growth rate were not observed in mediacontaining more than 0.4% substrate. Thetime course of growth in media with less than0.4% substrate was measured, but no remarkable difference was detected. These results were attributed in part to the poorsolubility of the substrate (0.2% in water),and to the high affinity of the substrate forthe cell. When the initial substrate concentration and the maximum cell dry weightobtained were plotted, a linear relation wasobtained up to 1.5 %. The cell yield constantfrom this results was calculated to be 0.7 mgdry cell/mg substrate provided.The bacteria grew on the basal mediumwithout added organic nitrogen source exceptACA-c-dimer though slowly. This meant that

TABLE I. EFFECT OF VARIOUS NITROGENSOURCES ON GROWTH

a) 0.05% of each nitrogen source was added to thebasal medium prepared without yeast extract and10-4M FeCl3 was added.

FIG. 3. Effect of Incubation Temperature on theSpecific Growth Rate.The initial cell concentration was adjusted to O.D. 0.4and incubated in a water bath with reciprocal shaking.

FIG. 4. Effect of pH of the Medium on the SpecificGrowth Rate.The initial cell concentration was adjusted to O.D. 0.4.The initial pH was adjusted by adding KOH in mediumcontaining 20 ml of 0.5 M KH2PO4.

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1222 S. KINOSHITA, S. KAGEYAMA, K. IBA, Y. YAMADA and H. OKADAthe strain utilized ACA-c-dimer as sole carbonand nitrogen sources. Addition of inorganicnitrogen such as ammonium or nitrate to theabove medium was not stimulatory, whichsuggested that the nitrogen source of thebacteria was sufficiently supplied from ACA

- c-dimer. Addition of organic nitrogen sourcesuch as yeast extract, casamino acids orpeptone improved the growth remarkably(Table I).

Effect of metal ions on the growth of thebacterium was examined. Ferric chloride(10-4M) stimulated the growth twice as muchas that in the control medium when testedin the basal medium without yeast extract,but in the basal medium containing yeastextract stimulation was not so much. Ca,Zn, and Mn had no effect on the growth.Cu, Ni, Co, and Hg ions were inhibitory.

The temperature providing maximal growthwas 33C (Fig. 3). The bacteria did notgrow when incubated at temperatures higherthan 45C. The bacteria were grown inmedia of various pH values adjusted withhigh concentration of phosphate, and maximalgrowth rate was obtained at pH 6.0. Thechange of the medium pH was less than 0.1throughout this experiment. This results areshown in Fig. 4.

Substrate specificity of nylon oligomers forgrowthWhen isolate K172 was incubated in medium

containing various lengths of nylon oligomers,ACA and its linear oligomers supported thebacterial growth to various degrees, whichdecreased with increase in the degree ofpolimerization (Table II). This may resultfrom the decreasing solubility of the oligomers:trimer, tetramer, and pentamer; 1.8, 0.8, and0.01 % in water,"' respectively. The hexameris almost insoluble. Dimethylimino ether ofACA-c-dimer, which was prepared to givehigh solubility, was metabolized but no morethan effective than ACA-c-dimer. The isolateK172 grew on -caprolactam as well as thecyclic dimer.

TABLE II. EFFECT OF NYLON OLIGOMERS ANDTHEIR DERIVATIVES ON THE GROWTH

a) Each substrate (2 mg/ml) was added to the basalmedium. Abbreviations were given in MATERIALSAND METHODS.b) The cell weight was measured at 16 hr culture .c) Dimethylformamide was added at measurementof turbidity.

DISCUSSIONAchromobacter guttatus K172, isolated from

sludge, was found to utilize -caprolactam,ACA, its cyclic dimer, linear dimer, trimer,tetramer, pentamer, and hexamer of ACA, anddimethylimino ether of the cyclic dimer atvarious metabolic rates. Fukumura showed1,2),that a strain of Corynebacterium aurantiacummetabolized ACA, ACA2, ACA3, and ACA4,ACA-c-trimer. Another strain, Achromobacterguttatus KF714) could grow on -caprolactambut not the cyclic dimer. The cyclic dimer seemsto have a stable configuration with strongintramolecular hydrogen bonds and this isa reason to be resistant to biological attack.The crude extract of C. aurantiacum12) hadtwo enzymes which hydrolyzed cyclic andlinear oligomers of ACA. Also our recentresults showed that one enzyme hydrolyzedACA-c-dimer to ACA2, and the other ACA-linear-oligomers after extensive purification."'In the experiment on carbobenzoxy derivativesof ACA oligomers, the monomer and thedimer were not utilized but longer oligomerswere metabolized slowly. Acrylated ACAmonomer and dimer were not utilized.

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Acknowledgement. We thank Dr. N. Takada forhis kind suggestion for the identification of the bacteriaand Miss A. Esumi for her helpful technical assistance.

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907 (1954).8) H. Zahn and D. Hildebrand, Chem. Ber., 90, 320

(1957).9) R. S. Breed, E. G. D. Murray and N. R. Smith,"BergeyLs Manual of Determinative Bacteriology ,"

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10) H. lizuka, I. Tanabe, T. Fukumura and K. Kato,J. Gen. Appl. Microbiol., 13, 125 (1967).

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