SE190881C1

SE190881C1 -

Info

Publication number: SE190881C1
Authority: SE
Publication date: 1964-01-01

Description

Uppfinnare: AV Kaufmann och K Bauer Prioritet begiird !ran den 24 september 1959 (Forbundsrepubliken Tysk.land) Det har foreslagits, att framstalla 6-aminopenicillansyra darigenom, att man later sus-pensioner eller extrakt av sadana penicillinspaltande bakterier, vilka foretradesvis angripa amidbindningen I 6-stallning i penicillinmolekylen, inverka pa penicilliner. Inventors: BY Kaufmann and K. Bauer Priority given to September 24, 1959 (Federal Republic of Germany). 6-position in the penicillin molecule, affect penicillins.

Med hansyn till lampligheten av den for penicillinspaltningen erforderliga bakteriemassan, är det ells inte likgiltigt, under vilka betingelser organismerrta kultiverm Om man inympar en naringslosning med ett pHvarde av 7,0, som forutom de for odlingen av mikroorganismerna erforderliga jonerna som N-kalla innehaller ammoniumjoner, kaseinhydrolysat eller klittpepton och som energioch C-kalla innehaller glykos, laktos eller sackaros, med en. lamplig bakteriestam, sa erhailer man efter kultivering under luftning vid c :a ° C visserligen ett relativt Mgt utbyte av bakteriecellmaterial, men dessa cellers penicillinspaltande formaga dr dock liten. En viss okninig av den onskade enzymatiska aktiviteten erhaller man vid anvandning av glycerin eller salter av organiska syror, sasom mjolksyra eller barnstenssyra som energioch C-kalla. In view of the suitability of the bacterial mass required for the penicillin cleavage, it is otherwise not indifferent, under what conditions organically cultivated. , casein hydrolyzate or dune peptone and containing energy and C-cold containing glucose, lactose or sucrose, with a. suitable bacterial strain, so after cultivation during aeration at about ° C, a relatively high yield of bacterial cell material is obtained, but the penicillin cleavage of these cells is small. A certain increase in the desired enzymatic activity is obtained by the use of glycerin or salts of organic acids, such as lactic acid or succinic acid as energy and C-cold.

Det har visat sig, all man kan hop: den penicillinspaltande aktiviteten hos bakterier, vilka, innan an i bakteriecellvaggarna fast bundna enzymerna bringas inverka pa penicillin, ladles under luftning i narlosningar, vilka endast innehalla obetydliga mangder forjasbara kolhydrater, dvs. icke hogre halt av sadarta kolhydrater an den som forefinnes i 2-procentig majsstopvatska, om odlingen genomfores i narvaro av fenylattiksyra eller derivat darav och far fortga till .dess att en vasentlig mangd av enzymerna alstrats. It has been found that all that can be said is: the penicillin-cleaving activity of bacteria, which, before the enzymes bound in the bacterial cell walls are brought to act on penicillin, is charged under aeration in solutions which contain only insignificant amounts of digestible carbohydrates, ie. no higher content of such carbohydrates than that present in 2% maize stopping liquid, if the cultivation is carried out in the presence of phenylacetic acid or derivatives thereof and may proceed to the point that a substantial amount of the enzymes has been generated.

Dupl. kl. 6 a: 22/04; 6 b: 16/03 Fenylattiksyran eller dess derivat anvandes harvid i mangder av 0,002-2 Vo. Dupl. at 6 a: 22/04; 6 b: 16/03 The phenylacetic acid or its derivatives are used in amounts of 0.002-2 Vo.

I enlighet med fOreliggande uppfinning 1-ter sig bakteriecellmassa med bra penicillinspaltningsformaga utvinnas darigenom att man som huvudsaklig energi- och kvavekalla anvander aminosyrablandningar i form av aggvitehydrolysat eller peptidblandningar i form av peptoner eller annu hOgmolekylarare proteiner for framstallning av narlosningen. Det ãr andamalsenligt, att forsatta sadana narlosningar, forutom med de vanliga oorga- niska jonerna, sasomPO4---, Mr, K+, Na, Cl, aven med en vitamin- och tillvaztamnesblandning, t. ex. jastvatten, i mindre mangd. In accordance with the present invention, bacterial cell mass with good penicillin cleavage ability is recovered by using amino acid mixtures in the form of aggregate hydrolyzate or peptide mixtures in the form of peptones or other higher molecular weight proteins for the production of narlose as the main energy and nitrogen source. It is appropriate to continue such solutions, in addition to the usual inorganic ions, such as PO4 ---, Mr, K +, Na, Cl, even with a mixture of vitamins and additives, e.g. fermented water, in small quantities.

Genom tillsats av fenylattiksyra, dess salter och derivat till nArlosningen, kan man hoja den Onskade enzyrnatiska aktiviteten hos bakterierna. Dessutom kan man ytterligare hoja bakteriemassans penicillinspaltningsformaga genom att man i sadana kulturer under bakterietillvaxten forutom luft aven inledcr kol- Enligt uppfinningen anvandes foretradesvis majsstopvatska som huvudsaklig bestandsdel i narbadden. Majsstopvatskan innehaller val lampade energi- och kvavekallor, vitaminer och tillvaxtamnen jamte de ovannamnda oorganiska jonerna. Denna grundnaringslosning forsattes i enlighet med uppfinningen med fenylattiksyra, fenylacetamid, fenacetursyra, fenacetylglutaminsyra eller andra derivat av fenylattiksyra. Koldioxiden kan antingen blandas med den for kulturernas syreforsiirjning erforderliga luften eller ocksa separat inledas i de vaxande kulturerna. Temperaturen I kulturerna kan uppga till 20-45° C. Lamp- 2— — ligen later man kulturerna VaXa under 12-20 timmar vid 30-35° C. By adding phenyl acetic acid, its salts and derivatives to the solution, one can increase the desired enzymatic activity of the bacteria. In addition, the penicillin cleavage form of the bacterial mass can be further increased by introducing carbon dioxide liquid as the main constituent of the narbadden in such cultures during bacterial growth in addition to air. The corn stopper contains selective lamp energy and nitrogen sources, vitamins and growth substances as well as the above-mentioned inorganic ions. This primer solution was continued in accordance with the invention with phenyl acetic acid, phenylacetamide, phenaceturic acid, phenacetylglutamic acid or other derivatives of phenyl acetic acid. The carbon dioxide can either be mixed with the air required for the oxygen supply of the cultures or also be introduced separately into the growing cultures. The temperature in the cultures can be up to 20-45 ° C.

Efter denna tid avskiljas lampligen bakteriecellerna och tvattas. Darefter kan man lata cellerna inverka pa penicillin. Eftersom det penicillinspaltande enzymet ãr fast bundet till bakteriernas cellvaggar och inte som cyto- plasmatisk bestandsdel i cellerna, kan man efter avslutad inverkan atervinna ensymmaterialet ur penicillinspaltningsblandningarna och Her anvanda detsamma for ytterligare satser. After this time, the bacterial cells are conveniently separated and washed. The cells can then be allowed to act on penicillin. Since the penicillin-cleaving enzyme is firmly bound to the cell walls of the bacteria and not as a cytoplasmic component in the cells, after completion of the action, the enzyme material can be recovered from the penicillin-cleavage mixtures and used here for further batches.

Lampligheten for utvalda bakterier att genomfora spaltningen kan undersokas pa Rdjande enkla satt under anvandning av en till teknikens standpunkt horande metod (P. R. Bachelor, E. P. Doyle, J. H. C. Mayler & G. N. Robinson, Nature, London, 183 (4656), 257 av den 24 juni 1959). ml av en fosfatbuffertlosning med pH = 7,0, innehallande 10000 I. E. penicillin G/ml, forsattes med samma volymmangd av en baktieriesuspension i en fosfatbufferNsning med pH = 7,0, och forvaras, efter tillsats av 0,2 % toluen, under 5 timmar vid en temperatur av 37° C. The suitability of selected bacteria to carry out the cleavage can be examined in a very simple manner using a prior art method (PR Bachelor, EP Doyle, JHC Mayler & GN Robinson, Nature, London, 183 (4656), 257 of June 24, 1959). ). ml of a phosphate buffer solution with pH = 7.0, containing 10,000 IU penicillin G / ml, was added with the same volume of a bacterial suspension in a phosphate buffer solution of pH = 7.0, and stored, after the addition of 0.2% toluene, for 5 ml. hours at a temperature of 37 ° C.

Bakteriesuspensionen framstalles pa foljande satt. 100 ml narbuljong inympas i en renkultur av den bakterie, som skall provas, och odlas skakmaskin under 18 timmar vid en temperatur av 28-32° C. Darefter a.veentrifugeras bakterierna, tvattas i 40 ml fosfatbuffertlosning med pH = 7,0 och omsuspenderas i 10 ml fosfatbuffertlosning med pH = 7,0. The bacterial suspension is prepared in the following manner. 100 ml of broth is inoculated into a pure culture of the bacterium to be tested, and shaken for 18 hours at a temperature of 28-32 ° C. in 10 ml of phosphate buffer solution with pH = 7.0.

Efter det att bakteriecellerna under 5 timmar fatt inverka pa penicillin G bestammes i den till cellfrihet centrifugerade losningen den kvarvarande penicillinhalten pa mikrobiologisk vag. After the bacterial cells have been exposed to penicillin G for 5 hours, the residual penicillin content is determined in the microbiological solution in the solution centrifuged to cell freedom.

For olika bakteriestammar visar det sig harvid, att losningen innehaller hela den ursprungliga aktiviteten. I detta fall ha bakterie- cellerna icke utvecklat nagon enzymatisk verkan med avseende pa penicillin G. Bakterierna aro av derma anledning icke lampade att anvanda fOr framstallning av penicillinspaltande enzym, att losningen är inaktiv eller uppvisar en starkt reducerad aktivitet. I detta fall later man under iskylning och under samtidig tillsats av natriumbikarbonat 500 mg fenylacetylklorid inverka pa lOsningen. Losningen under-sakes darefter pa nytt. Om man vid denna undersOkning icke finner flagon okning i aktivitet jamforelse med den icke acylerade losningen, sa kan man harav draga den slutsatsen, att penicillinet under inverkan av penicillinas eller liknande enzyrn nedbrutits irreversibelt, finner en fullstandig reaktivering av penicillinverkan till 10000 I. E./m1 losning, sd framgar det av forsoket, att penicillin G un der enzyminverkan spaltats pa sadant sat, att 6-aminopenicillansyra och fenylattiksyra bildats. De i ,detta fall provade bakterierna lam-pa sig pa ett framtradande salt for enzymatisk utvinning av 6-aminopenicillansyra fran firmer endast delvis reaktivering av den ursprungliga penicillinaktiviteten av 10 000, I.E./m1 losning, sa framgar det av detta forsok, att foratom det fenylattiksyran avspaltande enzymet aven penicillinas var fOr handen i bakterierna. Sadana bakterier aro lampade for framstallning av 6-aminopenicillansyra_ endast i sadana fall, da det fOreliggande penicillinaset endast inaktiverar en liten del av penicillinet eller da det lyckas att i vittgaende grad selektivt eliminera penicillinasaktiviteten. For different bacterial strains, it turns out that the solution contains the entire original activity. In this case, the bacterial cells have not developed any enzymatic action with respect to penicillin G. For this reason, the bacteria are not suitable for use in the production of penicillin-cleaving enzyme, that the solution is inactive or has a greatly reduced activity. In this case, 500 mg of phenylacetyl chloride are allowed to act on the solution under ice-cooling and with the simultaneous addition of sodium bicarbonate. The solution is then re-examined. If in this study no flake increase in activity is found in comparison with the non-acylated solution, then it can be concluded that the penicillin under the influence of penicillinase or similar enzyme is degraded irreversibly, a complete reactivation of the penicillin effect to 10000 IU / ml solution is found. Thus, it appears from the experiment that penicillin G under the enzyme action is cleaved in such a way that 6-aminopenicillanic acid and phenylacetic acid are formed. The bacteria tested in this case laminate on a prominent salt for enzymatic recovery of 6-aminopenicillanic acid from firms only partially reactivating the original penicillin activity of 10,000 IU / ml solution, so it appears from this experiment that The phenylacetic acid cleaving enzyme of penicillinase was present in the bacteria. Such bacteria are suitable for the production of 6-aminopenicillanic acid only in such cases, when the present penicillinase inactivates only a small part of the penicillin or when it succeeds in extensively selectively eliminating the penicillinase activity.

Det ovan beskrivna provningsforfarandet kan ocksa komma till anvandning i modifierad form genom att man undersoker den enzymatiska aktiviteten i forhallande till penicillin G i cellfria kulturfiltrat. The test procedure described above can also be used in modified form by examining the enzymatic activity in relation to penicillin G in cell-free culture filtrates.

Exempel 1. 160 liter 2-volymprocentig majsstopvatska injusteras med KOH till pH 7,0 och upphettas under 30 minuter 120° C. Efter avkylning klaras losningen genom centrifuge-ring och steriliseras, efter tillsats av 0,% glykos, under 40 minuter vid 110° C i jaskarlet. Denna narlosning inympas efter avkylning med 400 ml av en 18-timmars skakningskultur av E.coli ATCC 11105. Den vatskeformiga blandningen: luftas darefter med 150 liter luft per minut vid en omroringshastighet av 150 varv/minut och kultiveras under 17 timmar vid 31° C. Example 1. 160 liters of 2% by volume maize stopper liquid are adjusted with KOH to pH 7.0 and heated for 30 minutes at 120 ° C. ° C in the jask. This release is inoculated after cooling with 400 ml of an 18-hour shake culture of E.coli ATCC 11105. The liquid mixture: then aerated with 150 liters of air per minute at a stirring speed of 150 rpm and cultured for 17 hours at 31 ° C. .

Bakteriecellerna avcentrifugeras Iran kulturlosningen, tvattas i 16 liter 1/15-m fosfatbufferilosning med pH 6,0 °eh omsuspenderas efter aveentrifugeringen i 16 liter 1/5-m fosfatbuffertlasning med pH 7,0. I denna suspension upploses torr penicillin G till en lioncentration av 5000 IE per ml varjamte 0,4 % toluen tillsattes. Ansattningen forvaras under 12 timmar vid 37° C och innehaller efter denna tid fortfarande 4200 IE penicillin G per ml suspension. Filtratet fran ett prov av denna suspension omsattes enligt i och for sig kanda metoder med fenylacetylklorid och later sig pa sd satt ateraktiveras upp till 4850 IE penicillin G/ml. The bacterial cells are centrifuged in the Iran culture solution, washed in 16 liters of 1/15-m phosphate buffer solution at pH 6.0 °, and resuspended after aveentrifugation in 16 liters of 1/5-m phosphate buffer solution at pH 7.0. In this suspension, dry penicillin G is dissolved to a lion concentration of 5000 IU per ml and 0.4% toluene is added. The batch is stored for 12 hours at 37 ° C and after this time still contains 4200 IU penicillin G per ml suspension. The filtrate from a sample of this suspension is reacted with phenylacetyl chloride according to methods known per se and can thus be reactivated up to 4850 IU penicillin G / ml.

Exempel 2. 160 liter steril majsstOpvatska, framstalld pa samma satt som i exempel 1, dock utan tillsats av glykos, inympas med 400 ml av en 18-timmars skakningskultur av E.coli ATCC 11105. Den vatskeformiga blandningen luftas med 150 liter luft per minut vid en omroringshastighet av 150 varv/minut och kultiveras under 17 timmar vid 31° C. Example 2. 160 liters of sterile corn steep liquor, prepared in the same manner as in Example 1, but without the addition of glucose, are inoculated with 400 ml of an 18-hour shaking culture of E.coli ATCC 11105. The liquid mixture is aerated with 150 liters of air per minute. at a stirring speed of 150 rpm and cultivated for 17 hours at 31 ° C.

Bakteriecellerna avcentrifugeras frail kulturlOsningen, tvattas i 16 liter 1/15-m fosfatbuffertlosning med pH 6,0 och omsuspenderas, efter avcentrifugering, i 16 liten 1/5-m fosfatbuffertlosning av pH 7,0. I denna suspension — —3 upploses torr penicillin G upp till en koncentration av 5000 IE per ml, varjamte 0,4 % toluen tillsattes. Den vatskeformiga bland- ningen Hiles under 5 timmar vid 37° C och innehaller efter denna tid fortfarande 600 IE penicillin G per ml suspension. Filtratet fran ett prov av denna suspension omsattes enligt i och for sig kanda forfaranden med fenylacetylklorid och later sig pa sa satt ateraktiveras upp till 4650 IE penicillin. G/ml. The bacterial cells are centrifuged from the culture solution, washed in 16 liters of 1/15-m phosphate buffer solution at pH 6.0 and resuspended, after centrifugation, in 16 small 1/5-m phosphate buffer solution of pH 7.0. In this suspension, dry penicillin G is dissolved up to a concentration of 5000 IU per ml, to which 0.4% of toluene is added. The liquid mixture hiles for 5 hours at 37 ° C and after this time still contains 600 IU penicillin G per ml suspension. The filtrate from a sample of this suspension was reacted with phenylacetyl chloride as known per se and allowed to reactivate up to 4650 IU penicillin. G / ml.

Av ovanstaende exempel framgar, att man med lag glykoshalt erhaller relativt h8g spalt- ning av penicillinet och aven relativt hogt utbyte av reaktiverat penicillin om man nojer sig med att spalta relativt sma mangder. The above example shows that with a low glucose content a relatively high cleavage of the penicillin is obtained and also a relatively high yield of reactivated penicillin if one is content to cleave relatively small amounts.

De ytterligare forbattrade resultat, som i enlighet med foreliggande uppfinning erhallas genom att &Bingen av bakterierna genom- fOres i narvaro av fenylattiksyra eller derivat darav, framga av nedanstaende exempel. The further improved results obtained in accordance with the present invention by carrying out the bacteria in the presence of phenyl acetic acid or derivatives thereof are shown in the following examples.

Exempel 3. 160 liter steril majsstopvatska, framstalld pit samma satt som i exempel 2, dock med tillsats av 0,2 % kaliumfenylacetat, inympas med 400 ml av en 18-timmars skakningskultur av E. cob. ATM 11105. Den vats- keformiga blandningen luftas med 150 liter luft per minut vid en omroringshastighet av 150 varv/minut och kultiveras under 17 timmar vid 31° C. Example 3. 160 liters of sterile corn stopper liquid, prepared in the same manner as in Example 2, but with the addition of 0.2% potassium phenyl acetate, inoculated with 400 ml of an 18-hour E. cob shaking culture. ATM 11105. The liquid mixture is aerated with 150 liters of air per minute at a stirring speed of 150 rpm and cultivated for 17 hours at 31 ° C.

Bakteriecellerna avcentrifugeras fran kulturlosningen, tvattas i 16 liter 1/15-m fosfat- buffertlosning av pH 6,0 och omsuspenderas, efter avcentrifugering, i 16 liter 1/15-m fosf atbuffertlOsning av pH 7,0. I denna suspension upploses torrt penicillin G till en koncentra- tion av 10 000 IE per ml, varjamte 0,4 % toluen tillsattes. Den vatskeformiga bland- ningen Mlles under 5 timmar vid 30° C och innehaller efter derma tid fortfarande 1500 IE penicillin G per ml suspension. Filtratet frail ett prov av denna suspension omsattes enligt I och for sig kanda forfaranden med fenylacetylklorid och later sig pa sit salt ateraktiveras upp till 9500 IE penicillin G/ml. The bacterial cells are centrifuged from the culture solution, washed in 16 liters of 1/15-m phosphate buffer solution of pH 6.0 and resuspended, after centrifugation, in 16 liters of 1/15-m phosphate buffer solution of pH 7.0. In this suspension, dry penicillin G is dissolved to a concentration of 10,000 IU per ml, to which 0.4% toluene is added. The liquid mixture is milled for 5 hours at 30 ° C and after this time still contains 1500 IU of penicillin G per ml of suspension. The filtrate from a sample of this suspension was reacted according to procedures known per se with phenylacetyl chloride and allowed to reactivate on its salt up to 9500 IU penicillin G / ml.

Exempel 4. 160 liter steril majsstopvatska, framstalld pa gamma salt som i exempel 2, dock med tillsats av 0,2 % kaliumfenylacetat, inympas med 400 ml av en 18-timmars skakningskultur av E.coli ATCC 11105. Den vatske- formiga blandningen luftas med 150 liter luft per minut vid en omroringshastighet av 150 varv/minut och kultiveras nuder 17 timmar vid 31° C titan overtryek. Under den sammanlagda tillvaxttiden inledas genom en fran luftledningen i jaskarlet skild ledning 5 liter koldioxid per minut i kulturen. Example 4. 160 liters of sterile corn stopper liquid, prepared on gamma salt as in Example 2, but with the addition of 0.2% potassium phenyl acetate, are inoculated with 400 ml of an 18-hour shaking culture of E.coli ATCC 11105. The liquid mixture is aerated with 150 liters of air per minute at a stirring speed of 150 rpm and cultured for 17 hours at 31 ° C titanium overpressure. During the total growth period, 5 liters of carbon dioxide per minute in the culture are initiated through a line separated from the overhead line in the jaskarlet.

Bakteriecellerna avcentrifugeras frail kulturlosningen, tvattas i 16 liter 1/15-m fosfatbuffertlosning av pH 6,0 och omsuspenderas, efter fornyad avskiljning i 16 liter 1/5-m fosfatbuffertlOsning av pH 7,5, som innehaller 100 000 IE penicillin G per ml och 0,4 % toluen. Den vatskeformiga blandningen Mlles under 7 timmar yid 30° C. Under denna tic! Mlles reaktionsblandningens pH-varde, vilket sitsom en foljd av den enzymatiskt fran peni- cillinmolekylen avspaltade fenylattiksyran forskjutas mot sura sidan, genom upprepade tillsatser av koncentrerad Na2CO3-16sning mel- la.n 7,0 och 7,5. Efter 7 timmars reaktion innehaller den vatskeformiga blandningen fort- farande 12000 IE penicillin G per ml suspen- sion. Filtratet fran ett prov av denna suspension omsattes enligt i och for sig kanda forfaranden med fenylacetylklorid och later sig pa sit salt atera.ktiveras upp till 91000 IE penicillin G/ml. The bacterial cells are centrifuged from the culture solution, washed in 16 liters of 1/15-m phosphate buffer solution of pH 6.0 and resuspended, after re-separation in 16 liters of 1/5-m phosphate buffer solution of pH 7.5, containing 100,000 IU penicillin G per ml. and 0.4% toluene. The liquid mixture is melted for 7 hours at 30 DEG C. During this tic! The pH value of the reaction mixture, which is shifted towards the acidic side, is shifted towards the acidic side by repeated additions of concentrated Na2CO3-16 solution between 7.0 and 7.5. After 7 hours of reaction, the liquid mixture still contains 12000 IU of penicillin G per ml of suspension. The filtrate from a sample of this suspension was reacted with phenylacetyl chloride according to methods known per se and allowed to reactivate on its salt up to 91000 IU penicillin G / ml.

Den vid detta tillvagagangssatt bildade 6- aminopenicillansyran kan med bra utbyte isoleras i form av ett farglOst, kristallint pulver. The 6-aminopenicillanic acid formed in this process can be isolated in good yield in the form of a colorless, crystalline powder.

Exempel 5. 160 liter steril majsstOpvatska, framstalld pa samma salt som i exempel 2, dock med tillsats av 0,2 % kaliumfenylacetat, inympas med 400 ml av en 18-timmars skakningskultur av E.coli ATCC 9637. Den vatske- formiga blandningen luftas med 150 liter luft per minut vid en omroringshastighet av 150) vary per minut och kultiveras under 17 tim- mar vid 310 C utan overtryck. Under den sammanlagda tillvaxttiden inledas genom en frail luftledningen i jaskarlet skild ledning 10 liter koldioxid per minut i kulturen. Example 5. 160 liters of sterile corn steep liquor, prepared on the same salt as in Example 2, but with the addition of 0.2% potassium phenyl acetate, are inoculated with 400 ml of an 18-hour shaking culture of E.coli ATCC 9637. The liquid mixture is aerated. with 150 liters of air per minute at a stirring speed of 150) vary per minute and cultivated for 17 hours at 310 C without overpressure. During the total growth period, 10 liters of carbon dioxide per minute are initiated through a frail overhead line in the jaskarlet separate line in the culture.

Bakteriecellerna avcentrifugeras fran kulturlosningen, tvattas 1 16 liter 1/15-m fosfat- buffertlosning av pH 6,0 oeh omsuspenderas, efter fornyad avskiljning, i 16 liter 1/5-m fosfatbuffertlosning av pH 7,5, som innehaller 50000 IE penicillin G per ml och 0,4 % toluen. The bacterial cells are centrifuged from the culture solution, washed in 16 liters of 1/15-m phosphate buffer solution of pH 6.0 and resuspended, after re-separation, in 16 liters of 1/5-m phosphate buffer solution of pH 7.5, containing 50,000 IU penicillin G per ml and 0.4% toluene.

Den vatslreformiga blandningen Hiles under 3 timmar vid 30° C. Under denna tid halles reaktionshlandningens pH-varde, vilket sasom en f8ljd av den enzymatiskt frail penicillinmolekylen avspaltande fenylattiksyran forskju- tes mot sura sidan, genom upprepade tillsatser av koncentrerad Na2CO3-losning mellan 7,0 och 7,5. Efter 3 tinunars reaktion innehaller den vatskeformiga blandningen fortfarande 4700 IE penicillin G per ml suspension. Filtratet fran ett prov av denna suspension omsat- tes enligt i och for sig kanda forfaranden med fenylacetylklorid och later sig darigenom geraktiveras upp till 46500 IE penicillin G/ml. The aqueous mixture is healed for 3 hours at 30 ° C. During this time the pH of the reaction mixture is maintained, which is shifted towards the acidic side as a result of the enzymatically free penicillin molecule cleaving the phenylacetic acid, by repeated additions of concentrated Na 2 CO 3 solution. 0 and 7.5. After 3 hours of reaction, the liquid mixture still contains 4700 IU of penicillin G per ml of suspension. The filtrate from a sample of this suspension was reacted according to per se known procedures with phenylacetyl chloride, thereby allowing itself to be reactivated up to 46500 IU penicillin G / ml.

Den vid denna ansattning bildade 6-aminopenicillansyran kan flu med bra utbyte isoIeras i form av ett farglOst, kristallint pulver. The 6-aminopenicillanic acid formed in this application can be isolated in good yield flu in the form of a colorless, crystalline powder.

Liknande resultat erhaller man ocksa vid anvandning av foljande stammar om man gar tillvaga pa liknande satt som beskrivits i ovanstaende exempel. Similar results are also obtained with the use of the following strains if one proceeds in a similar manner as described in the above example.

Proteus, Aerobacter aerogenes, salmonellaoch shigellaarter, Pseudomonas aureofaciens, Pseudomonas fluorescerts, Pseudomonas aeruginosa, Alcaligenes faecalis, Serratia marcescens, Micrococcus roseus, Microccoccus lysodeicticus, Sarcina lutea. — — Proteus, Aerobacter aerogenes, salmonella and shigella species, Pseudomonas aureofaciens, Pseudomonas fluorescerts, Pseudomonas aeruginosa, Alcaligenes faecalis, Serratia marcescens, Micrococcus roseus, Microccoccus lysodeicticus, Sarcina lutea. - -

Claims

Patent claim:

Set in the production of enzymes capable of cleaving 6-aminopenicillanic acid from penicillin G, wherein penicillin-cleaving bacteria are grown in aeration in aeration solutions, which contain only insignificant amounts of digestible carbohydrates, ie. no higher content of such carbohydrates than the soil is found in 2% maize substitute, may be due to the fact that the cultivation is carried out in the presence of phenyl acetic acid or derivatives thereof and that the cultivation is continued until a substantial amount of the enzymes has been generated.

2. Sat according to claim 1, may be characterized in that the phenyl acetic acid or its derivatives are used in amounts of 0.002-2 percent.

Salt according to patent claim 1 or 2, characterized in that during the cultivation carbon dioxide is introduced into the teddy bear solutions.

4. According to patent claims 1-3, it may be characterized in that maize starch water is used as the main component in the descriptions. Request publications: