Producton of Natural Pencllns by Strans of Pencllum chrysogenutn a J. FUSK and ЬЕ. WELWRDOVÁ ^Department of Mcrobology and Bochemstry, Slovak Techncal Unversty, Bratslava b Botka, Slovenská Ľupča Receved November 2, 968 n revsed form prl 3, 969 New strans wth hgher penclln G and V producton were solated usng mutagenc factors n ther mutual combnaton. The penclln producton by these strans was dependent upon the carbon source n medum. ndvdual strans reacted dfferently on the change of the carbon source. n the absence of precursors the strans produced a dfferent number of natural pencllns. drect relatonshp between ncreasng producton of pencllns and the concentraton of 6-amnopencllanc acd n the fltrate was not establshed. Penclln producton by strans of Pencllum chrysogenum depends on the sort and concentraton of added carbon source. good antbotc producton occurs n a lactose medum (one-shot addton of lactose). Smlar producton s obtaned n the meda contanng saccharose or glucose when these sugars are added gradually nto medum [ 3]. The formaton of ndvdual types of pencllns s nfluenced by the added precursor. n the meda wthout precursor, the types of syntheszed pencllns as well as the amounts of produced 6-amnopencllanc acd (6-P) and other compounds reactng wth phenyl acetyl chlorde, are dependent upon the used stran [4 6]. The purpose of the present work was to compare the results obtaned wth our new strans wth those acheved n other laboratores. Producton strans Materals Expermental and methods Strans of Pencllum chrysogenum wth hgher antbotc producton were solated after exposng the spores of selected strans to UV-lght and smultaneously to the acton of V-methyl(dchlorodethyl)amne hydrochlorde. The latter mutagenc factor was added to agar medum on whch the affected spores were further cultvated. The agar medum was supplemented wth 0 25% (v/v) of the fltrate obtaned after the Pencllum chrysogenum fermentaton. Compounds present n the fltrate promoted the smultaneous adapton of strans to new growng condtons n manufacture [7]. Thus attaned strans were mantaned accordng to Backus and Stauffer [8]. 704 Chem. zvest 23, 704 708 (969)
PRODUCTON OF NTURL PENCLLNS Fermentaton The producton medum had followng composton: lactose 5.8 g, glucose 0.5 g, corn steep (50% dry weght) g, peanut meal 2 g, calcum carbonate 0.65 g, sodum thosulfate 0.24 g, phenylacetc acd 0.35 g all dssolved n tap water and made up to 00 ml. ph of the medum before sterlzaton was 6.4. When saccharose was used as carbon source, glucose and lactose were omtted. Saccharose was added nto medum by portons each 24 hours (0.6 g per 60 ml). n experments n whch the formaton of 6-P was followed a synthetc medum wthout precursor and natve ntrogen sources was used [3]. Condtons of cultvaton The mcroorgansm was grown n 500 ml flasks contanng 60 ml of the medum on a rotary shaker (220 rev./mn., r = 5.5 cm). Bo-assay of pencllns Penclln G and V were estmated by dffuson plate method usng Bacllus subtls SDPC ( : 220) as a testng mcroorgansm [9]. 6-mnopencllanc acd and compounds reactng wth phenyl acetyl chlorde were determned by the same method after forgong transformaton nto benzylpenclln [0]. Chromatography of pencllns Paper chromatograms were developed n water saturated ether. Chromatography chamber was saturated wth the system for 4 hours at 24 C before development. boautographc method on agar plates seeded wth Bacllus subtls was used for localzaton of antbotcs on chromatograms. Results and Dscusson The producng abltes of the mproved strans of Pencllum chrysogenum No. 2/70 and No. 9/38 [7] were verfed by cultvaton n lqud meda. The results obtaned wth these new strans were compared wth those attaned usng the orgnal stran. The strans grown under the same condtons showed a dfferent overall penclln G and V producng abltes. n the lactose medum, the penclln G and V producton by the strans No. 2/70 and No. 9/38 was found to be 0%, resp. 35% hgher than the producton by the stran No. 65 (Fg., curves 2, 2 and 3). n the presence of saccharose, the mutual relatonshp of penclln G and V producton of tested strans was altered. The penclln V producton of the stran No. 2/70 represented 36% and that of the stran No. 9/38 7% aganst the 00% producton of the stran No. 65. The tme, at whch maxmum concentraton of penclln V appeared n meda contanng saccharose or lactose, vared wth all three strans. The replacement of lactose wth saccharose dd not nfluence the penclln V producton by the stran No. 65. Ths was expressvely altered, however, n the case of the stran No. 2/70 and No. 9/38 (Fg. ). Based upon above fndngs, we can suppose that the producng ablty n the course of stran mprovement should be evaluated usng meda whch contan the same carbon source as wll be used n producton medum n manufacture. The producton of above strans was also examned n a synthetc medum wthout precursor [3]. Concentraton of natural pencllns was determned by plate dffuson method [9] and the overall antbotc actvty was expressed n benzylpenclln Chem. zvest 23, 704 708 (969) 705
J. FUŠK, E. WELWRDOVÄ ^ д^^ ^- д / "ď ^ У /4 ^~*r~ ~ff< ^ * r'- d -~ >< C^ (f * - ^ ^""^ \ б \ V 0- \ 4 _ Fg.. Producton of penclln V by strans of Pencllum chrysogenum n lactose medum:. stran No. 65; 2. stran No. 2/70; 3. stran No. 9/38 and n saccharose medum: 4. stran No. 65; 5. stran No. 2/70; 6'. stran No. 9/38. unts. 6-P was reacted wth phenyl acetyl chlorde to gve benzylpenclln whch was estmated together wth pencllns n the second test by the same method. The dfference between those two determnatons represented the content of 6-P n the fltrate. drect relatonshp between penclln and 6-P has not been observed. The penclln V producton of both strans No. 2/70 and No. 9/38 ncreased. The producton of 6-P of the former stran ncreased too, but that of the latter one was dmnshed. These fndngs mght ndcate that 6-P s not a precursor of penclln [, 2]. Contrary to our results (Tabelle ), Ostrouchov and Kuznecov [3] reported a drect relatonshp between the rato of pencllns n the meda wth or wthout precursor and the overall producng ablty of a stran. Table Producton of penclln G and 6-amnopencllanc acd by strans of Pencllum chrysogenum n lactose medum Stran Penclln G г p* Dducton.u. /ml medum wth precursor 0 medum wthout precursor 0 Rato bja - 00 P oducton of 6-P n medum wthout precursor.u./ml c 65 2/70 9/38 4200 ± 40 4600 ± 60 5600 ± 80 470 ± 0 420 ± 60 50 ± 70.2 9.5 9.2 380 ± 40 260 ± 00 480 ± 60 a) Producton of penclln G. b) Producton of natural pencllns expressed as penclln G. c) Producton of 6-P expressed as penclln G. 706 Chem. zvest 23, 704 708 (969)
PRODUCTON OF NTUEL PENCLLNS fter reachng the maxmum antbotc actvty n the synthetc medum wthout precursor, the natural pencllns present n the fltrate were examned by paper chromatography. The number of pencllns appeared to be dfferent for each tested stran and to be smaller for strans wth enhanced producton of penclln n the meda wth precursor (Fg. 2). Fg. 2. Paper chromatography of natural pencllns by strans of Pencllum chrysogenum:. stran No. 65; 2. stran No. 2/70; 3. stran No. 9/38. The obtaned results ndcate that there s no drect relatonshp between overall producton of pencllns G and V by ndvdual strans and the used carbon source. Producng ablty of new strans n course of ther further mprovement should be evaluated therefore on the same carbon source whch wll be used n manufacture. The stran whch exhbted an enhanced antbotc actvty n the meda wth precursor, formed a less number of natural pencllns n the meda wthout precursor. The fact, that the ncreasng producton of pencllns was found to be unrelated to the producton of 6-P, supports the opnon that 6-P s not a drect ntermedate n the bosynthess of the penclln molecule. References. Soltero F. V., Johnson M. J., ppl. Mcrobol. 2, 4 (954). 2. Fuska J., Kuhr., Benda., vanov L., Chem. Zvest 7, 533 (963). 3. Lure L. M., Levtov M. M., ntbotk 8, 677 (963). 4. Ballo., Chan E. В., Dentce d ccada F., Nature 83, 80 (959). 5. Lure L. M., Levtov M. M., Mkrobologja 33, 308 (963). 6. Wolf E. C, rnsten H. R. V., Bochem. J. 76, 375 (960). 7. Fuska J., Welwardová E., Czechoslov. Patent 25 8 (965). 8. Backus M. P., Stauffer J. E., Mycologa 47, 429 (959). 9. Balan J., Betna V., Bológa 4, 53 (959). 0. Betna V., Balan J., Haľama D., Martonová K., J. ntbot. (Tokyo), Ser., 4, 67 (96). Chem. zvest 23, 704 70S (969) 707
J. FUŠK, E. WELWRDOVÁ. Bu'lock J. D., Bogeness of ntbotc Substances. (Z. Vaněk and Z. Hošťálek, Edtors.) P. 6. Publshng House of the Czechoslovak cademy of Scences, Prague, 965. 2. braham E. P., Newton G. G. F., ntbotcs. (D. Gottleb and P. D. Shaw, Edtors.) Vol., p. 8. Sprnger-Verlag, Berln, 967. 3. Ostrouchov.., Kuznecov V. D., ntbotk, 33 (963). Translated by P. Bely 708 Chem. zvest 23, 704 708 (969)