[Agr. Biol. Chem., Vol. 36, No. 1, p. 112---119,1972]

Studies of the Peptide Antibiotic Suzukacillin

Part II

By Takaaki OOKA and Isao TAKEDA TechnicalResearch Laboratory, Asahi ChemicalIndustry Co., Ltd., Nakadai-cho3-27, Itabashi-kuTokyo ReceivedJuly 26, 1971

Suzukacillin produced by Trichoderma viride 6301 strain was purified and shown to be separated into two components A and B on thin-layer chromatography. The component A was isolated and crystallized from the mixture of components by alumina column chro matography. The component A is composed of six amino acids, Gly, Glu, Ala, Pro, Val, Leu and an unknown amino acid. This unknown amino acid was identified as a-amino isobutyric acid. It is supposed that a-amino isobutyrie acid is biosynthesized mainly from L-valine by the isotopic experiments. Suzukacillin formation by Trichoderma viride 6301 was stimulated by the addition of L-Asn, GABA, L-Ser, Gly and L-Arg into the medium.

Trichoderma viride 6301 strain producing Suzu ments were purchased as reagents from Tokyo Kasei kacillin was isolated by the author's laboratory. Co., Ltd. The following radioactive compounds were This organism accumulated noted amount of surplied by Dai-ichi Chemical Co., Ltd., with the in the peptide antibiotic. It was detected on dicated specific activities (ƒÊCi per mmole): Starch- TLC that the peptide antibiotic Suzukacillin (U)14C (30.8), pyruvic acid-(U)-14C (31.5), glycine- consists of two components A and B. A is (U)14C (45.2), L-alanine-(U)-14C (107), L-glutamic acid- (U)-14C (165), L-proline-(U)-14C (165), L-valine-(U)-14C a major and B is a minor component. These (165), L-leucine-(U)-14C (270), L-serine-(U)-14C (99), results were reported in previous paper." It ƒ¿-amino isobutyric acid-1-14C (56), L-threonine-(U)-14C is supported that component A is composed (132), L-aspartic acid-(U)-14C (132), y-amino butyric from six kinds of known amino acids and one acid-(U)14C (10.8), L-arginine-(U)-14C (210) and L-homo- unknown amino acid. It is considered that serine-(U)-t4C (32). the molar ratio of the amino acids of com Micro-organisms and culture conditions. Trichoderma ponent A is the constituent of Gly1, Glue, Alai, viride 63C1 strain which has been stocked by Iyophiliza Pro,, Val,, Leu1 and an unknown amino acid. tion was used in all experiments. Trichoderma viride This paper describes the purification of com was cultured with 50 ml of basal medium composed ponent A from the cultured broth and the of 200 soluble starch, l% polypeptone and 0.500 yeast identification of an unknown amino acid in extracts in 500 ml of shaking flasks at 30°C on re component A, and furthermore discusses the ciprocal shaker. In the experiments of the additions biosynthesis of an unknown amino acid by the of amino acids, 0.5% of polypeptone and 0.5% of incorporation of 14C-amino acids. Effects of the each amino acid were used in place of 100 of poly Suzukacillin formation by the addition of the peptone in the basal medium. various kinds of amino acids is also discussed. Assay methods of antimicrobial activities of Suzukacilln.

The total activities of both the supernatant of cultured MATERIALS AND METHODS broth and the extracts from cell treated with methanol were assayed with pulp disc method on the agar plates

Materials. All amino acids used in these experi using Sarcina lutea as test organism. Purified crystal Studies of the Peptide Antibiotic Suzukacillin. Part II 113 of component A was standardized in this assay. The Isolation and purification of Suzukacillin. The out- differences of antimicrobial substances produced by line of procedure of isolation and purification of Suzuka Trichoderma viride cultured in addition of various amino cillin is shown in Fig. 1. The mycelium was separated acids were examined by bioautography. from 10 liters of cultured broth by centrifugation.

FIG. 1. Isolation Procedure of Suzukacillin. 114 T. OOKA and 1. TAKEDA

This mycelium was extracted three times with 500 ml of methanol. Approximately 8 liters of supernatant were extracted with two-fold volume of 1-butanol. The organic solvent phases obtained from the myce lium and from the supernatant were collected and concentrated with rotary evaporator in vacuo. After the concentration, approximately 20 g of yellowish white powders were obtained. This powders were obtained. This powders were dissolved into 100 ml of methanol, after the removal of insoluble substances by centrifugation, methanol soluble fraction was con centrated to 40 ml in vacuo. This methanol solution FIG. 2. The Elution Pattern by Alumina Column was decolorized with active carbon powder. With the Chromatography. addition of distilled water adjusted to pH 3 with HCl The separation of component A, B and others to the decolorized methanol solution, white precipitates was detected by thin-layer chromatography which occured and were collected by centrifugation. These was developed with solvent system as follows; chloroform: methanol (10 : 4) and detected by the precipitates were dissolved again in methanol, pre cipitation was repeated by addition of the distilled reaction of C12-KI-starch.8) water adjusted to pH 3. After repeating this pro cedure, approximately 10 g of white precipitates were dimethyl-1, 4-bis-[2-(4-methyl-5-phenyloxazolyl)]-ben- obtained. Ten-volumes of ether was added to the zene). a-Amino isobutyric acid-l-14C was detected with methanol solution containing 10 g of the white pre autoradiography. cipitates, and ether insoluble precipitates there oc curred were collected by centrifugation. This pre RESULTS AND DISCUSSION cipitates were washed two times with ether. After removal of ether, 8.5 g of white powders were obtained. Crystallization of component A in Suzukacillin These powders of 8.5 g were dissolved into 80 ml of Approximately 6.3 g of crude substance con methanol, and were charged on the column packed taining component A was obtained from 8.5 g 120 g of alumina powder followed by the elution with 800 ml of methanol. Component A was separated of the white powder charged on alumina co from other components of Suzukacillin. The elution lumn. This crude substance was dissolved pattern of these components at thin-layer chromato into the hot chloroform and was crystallized graphy is shown in Fig. 2. by the addition of acetone. Four g of white crystals were obtained from the solution of Identification of radioactive amino acids incorporated into Suzukacillin. Ten microcuries of each radioac chloroform and acetone. This crystal showed tive compound were added to 50 ml of the basal me single spot on thin-layer chromatography with dium prior to the cultures with Trichoderma viride 63Cl. solvent system as follows: chloroform : metha After the end of cultures, the component A was iso nol=10:4 by volume. Melting point of this lated and purified from the cultured broth by the crystal was 259---261°C (decomposed), and op procedure mentioned in materials and methods. The tical rotation was [ƒ¿]18D-8.5 (c=1.0 in methanol). purified component A was hydrolysed with 6 N HCl Its infra-red spectrum is shown in Fig. 3. at 105°C for 20 hr. The hydrolysates which removed the excess HCl were developed on two-dimensional Identification of an unknown amino acid paper chromatography. After the location of each amino acid was detected with ninhydrin reagent, the In order to isolate an unknown amino acid, spots of amino acids were respectively cut into pieces, 2 g of the crystal of component A was hydro and the radioactivities of amino acids were counted lysed in 6 N HCl at 105°C for 18 hr. After by liquid scintillation spectrometer (Packard: model hydrolysation, excess HCl was almost eliminat No. 3002) in a toluene-popop solution (popop solution ed on boiling water bath from the hydrolysates, consists of 5 g of 2, 5-diphenyloxazole and 0.5g of and these hydrolysates were chromatographed Studies of the Peptide Antibiotic Suzukacillin . Part II 115

tified by IR and NMR spectra as a-amino isobutyric acid. IR and NMR spectrum are respectively shown in Figs. 5 and 6. From the

FIG. 3. IR Spectrum of Component A of Suzukacil lin.

on Dowex 50-X8 ion exchanger. The elution pattern of amino acids of hydrolysates is shown in Fig. 4. An unknown amino acid was eluted following to proline as shown in Fig. 4. The FIG. 5. IR Spectrum of the Unknown Amino Acid. (1) Authentic a-amino isobutyric acid. (2) Unknown amino acid.

FIG. 4. Chromatography of Amino Acids of Suzuka cillins Hydrolyzed.

Dowex-50-X8 ion exchanger was used for the separation of the constituent amino acids of Suzuka cillin hydrolyzed. FIG. 6. NMR Spectrum of the Unknown Amino Acid D2O. unknown amino acid fractions were collected and concentrated by evaporator in vactto. One analysis of elementary composition, this com- hundred and twenty milligrams of crude pow pound was identified with the theoretical ele ders of unknown amino acid obtained by con mentary composition of a-amino isobutyric centration was purified by repeating the same acid (Table I). column chromatography mentioned above. As the results, 60 mg of white powders which show TABLE I. ANALYSISOF ELEMENTARY single spot on paper chromatography with COMPOSITION solvent system as follows; 1-butanol: acetic acid: water=4:1:5 by volume, were obtained. These white powders were dissolved into water, and crystallized by the addition of ethanol. After recrystallization, 40 mg of crystal were collected and subjected to the following an alyses. This unknown amino acid was iden 116 T. OOKA and I. TAKEDA

Incorporation of radioactive amino acid into Suzu kacillin The purposes of the experiment was to in vestigate how various amino acids were in corporated into Suzukacillin and to discuss the biosynthesis of a-amino isobutyric acid. As mentioned in Materials and Methods, 10 ƒÊCi of each amino acid was added to the medium at an early phase of cultures. The procedures of detection of radioactive amino acids in com ponent A are shown in Materials and Methods. The results of distribution of radioactive amino acids incorporated into component A are shown in Table II, about the constituent amino acids and in Table III, about the non-constituent amino acids and other compounds. From these results, it was indicated that a part of the constituent amino acids was directly incorpo rated into component A. It was evident from PHOTOGRAPH1. Autoradiogram of a-Amino Iso radioautogram that a-amino isobutyric acid-l- butyric Acid. 14C was mainly incorporated into a-amino iso After Trichodermaviride 63C1 was cultured by the addition of 10t Ci of a-amino isobutyricacid butyric acid in component A (Photograph. 1). -1-14C,component A was isolated and hydrolyzed On the other hand, starch-(U)-14C, pyruvic (6N HCl, 105°C,20 hr). Hydrolysates was de acid-(U)-14C, Gly-(U)-14C and L-Asp-(U)-14C veloped in two-dimensionalpaper chromatography were also incorporated into a-amino isobutyric with the solvent systemof 1-butanol:acetic acid: acid. L-Val-(U)-14C, however, showed the high water (4: 1 : 5). specific incorporation into a-amino isobutyric acid. From this result, it is supported that Effects of the addition of amino acidsfor Suzukacil a-amino isobutyric acid is biosynthesized from tin formation valine through isobutyric acid as an inter- From the experiments of incorporation of mediate. radioactive amino acids to Suzukacillin, some

TABLE II. DISTRIBUTION OF RADIOACTIVITIES INCORPORATED INTO THE AMINO ACIDS OF COMPONENT A

ƒ¿-AiB: ƒ¿-amino isobutyric acid. Studies of the Peptide Antibiotic Suzukacillin. Part II 117

TABLE III. DISTRIBUTION OF RADIOACTIVITIES INCORPORATED INTO THE AMINO ACIDS OF COMPONENT A

GABA: Y-amino butyric acid. amino acids were incorporated to the consti stituent amino acids are shown in Table IV, tuent amino acids of Suzukacillin. According and of the additions of the non-constituent to these results, it was considered that Suzuka amino acids are shown in Table V Glycine cillin formation would be affected by addition and a-amino isobutyric acid among the con of amino acids to the medium. As mentioned stituent amino acids were effective for the in Materials and Methods, 0.5% of polypeptone and 0.5.0 of each amino acid were used in TABLE V. EFFECT OF AMINO ACID FOR place of 1.0% of polypeptone in the culture SUZUKACILLIN FORMATION of Trichoderma viride. The results of Suzuka cillin formation in the additions of the con-

TABLE IV. EFFECT OF AMINO ACIDS FOR SUZUKACILLINFORMATION Basal medium is composed of 2-0 of soluble starch, 1%of polypeptone and 0.2° of yeast extract. 0. Y-o of polypeptone in this basal medium was displaced with 0.5 of each amino acid. In this experiment, the constituent amino acid of component A was ad ded to the medium. The activity is shown with the amounts of total Suzukacillin formation in both supernatant and mycelium.

Basal medium is the same one shown in Table III. GABA: Y-amino butyric acid. DL-ƒ¿AB: DL-a-amino butyric acid. Activity is the same expression shown in Table III. 118 T. OOKA and I. TAKEDA

Suzukacillin formation. But the additions of affect the growth of mycelium. ƒÁ-Amino buty

L-valine, L-leucine and L-glutamic acid showed ric acid and a-amino isobutyric acid showed rather inhibitory effects. L-Asparagine, r- the stimulative effects for the Suzukacillin for

amino butyric acid, L-arginine and L-serine, mation, but DL-a-amino butyric acid showed as non-constituent amino acids, were more the negative effect for the Suzukacillin forma

effective for the Suzukacillin formation. From tion. As one reason, it is considered that D- these results of the incorporation of radioac form of a-amino butyric acid inhibited the

tive amino acids, the stimulative amino acids Suzukacillin formation activity. The addition for Suzukacillin formation, except for L-serine of L-methionine, L-cysteine, L-tyrosine and L- and L-asparagine, were not directly incorporat tryptophane suppressed perfectly the Suzuka

ed into the constituent amino acids of Suzuka cillin formation, but in these cases, the my cillin. L-Methionine, L-cysteine, L-tyrosine celium grew the same as the basal medium. and L-tryptophane completely inhibited the From these results, it was evident that there Suzukacillin formation, but the growth of my are no relations between the Suzukacillin for celium was not inhibited. mation and the growth of mycelium. As re-

ported by other investigators" about the rela tions between the biosyntheses of cell protein DISCUSSION and peptide antibiotics, it is supposed that the

It is well known that Trichoderma species pathway of biosynthesis of Suzukacillin is in- excrete a cellulase, and some strains of Tricho dependent of that of mycelium. a-Amino derma produce the antibiotics"" or inorganic isobutyric acid which is one of non-metabolic acid.' Trichoderma viride 63C1 isolated by the amino acid, has been isolated from the anti authors can not utilize cellulose as a carbon biotics before."" From the results of radio source. This strain produces considerable isotopic experiment, it has been shown that a-amino isobutyric acid is derived from starch amount of peptide antibiotic Suzukacillin. , When this strain was cultured by the additions pyruvic acid, L-valine, L-serine, L-aspartic acid of the amino acids, such as --amino butyric and glycine. Among the radioisotopic com- acid, L-asparagine, L-arginine, glycine and L- pounds tested, L-valine added into the culture serine, 3 to 5 g of Suzukacillin per liter were medium was incorported specifically into a- accumulated in the cultured broth. It is not amino isobutyric acid. It is considered that evident that the reasons why these amino acids a-amino isobutyric acid is derived from iso promote the Suzukacillin formation. These butyryl-CoA through the degradative pathway amino acids such as L-asparagine, r-amino of L-valine. butyric acid and L-arginine may be utilized as amino donors, or may accelerate the enzyme Acknowledgment. The authers wish to express their thanks to Director Dr. R. Wakasa of this laboratory system of Suzukacillin biosynthesis. Glycine , and to professor Dr. T. Uemura for the interest , en and L-serine are also effective in the Suzuka couragement and the suggestions during the course cillin formation. From the radioisotopic ex of this work. periments, it is supposed that glycine is directly incorporated into Suzukacillin, and have an important role in the biosynthesis of Suzuka REFERENCES cillin. The good effects of L-serine may be 1) T. Ooka, Y. Shimojima, T. Akimoto related to those of glycine. On the other hand, , I. Takeda, S. Senoh and J. Abe, Agr. Biol. Chem., 30, 700 the addition of L-valine was inhibitory for (1966). Suzukacillin formation, in spite of a consti 2) P. W. Brain, P. J. Curtis, H. G. Hemming, and tuent amino acids in Suzukacillin, but did not J. C. MacGowan Ann. Applied Biol., 33, 190 (1964). Studies of the Peptide Antibiotic Suzukacillin. Part II 119

3) J. R. Johnson, W. F. Bruce and J. D. Dutche, J. 6) F. Reusser, J. Biol. Chem., 242, 243 (1967). Am. Chem. Soc., 65, 2005 (1943). 7) G. W. Kenner and R. C. Sheppard, Nature, 181, 4) O. Terada, K. Ohishi and S. Kinoshita, Nippon 48 (1958). Nogeikagaku Kaishi, 34, 166 (1960). 8) H. N. Rydon and P. W. G. Smith, ibid., 169, 5) N. Cornell, J. E. Snoke, Biochim. Biophys. Acta, 922 (1952). 91, 533 (1964).