Procedure for the production of 6-Aminopenicillansäure by enzymatic splitting of Penicillinen

The invention concerns a procedure for the production of 6-Aminopenicillansäure (6-APS) by enzymatic splitting of Penicillinen. With present industrial production from 6-APS a benzyle penicillin (penicillin g) becomes - solution with a bacteria mud shifts, that the enzyme Penicilllnacylase (PER ANNUM), (E.C. 3.5.1.11) contains. By the effect of the enzyme the sideconstant Carl0onamidgruppierung of penicillin becomes abgespalten, without the 13-Lactamring is opened. On the effect of this enzyme the procedure of the German patent specification Nr.1.111778 for the production of 6-APS from penicillin G. is based. The use of the Bakterienschlarnmes, e.g. of E.coli ATCC 11105, has the following disadvantages: a) The bacteria mud contains except the intracellulären PER ANNUM further proteins and enzymes, as well as components from the growth medium or their conversion products, which developed with the fermentation. These impurities can be washed with the processing not completely from the crystallized 6-APS. b) The Bakterlenschlamm can be used only once. c) The bacteria mud contains impurities and other enzymes, penicillin G and/or 6-APS by opening of the flat steel bar-Lactemringes inactivates. d) The bacteria mud contains only small quantities to PER ANNUM the use of more EnzymmateriaI, e.g. for achievement of short Reakfionszeiten and thus better 6-APS-Ausbeuten with smaller content of foreign manufactured products is practically not possible. e) The operating yields at 6-APS depend on the varying PER ANNUM - education in the respective fermentation beginnings. f) The complete separation of the bacteria cells requires an additional job, which causes yield losses with the processing of the 6-APS-Ansätze. Zttr distance of protein-like impurities, which can cause all genes reactions, are necessary further cleaning steps (brlt. Patenrschriften Nr.1, 169,696; Nr.1, 078.847 and Nr.1, 114.311). All disadvantages mentioned are avoided, if one uses Penicillinacylase, which is insoluble by kovalente connection to a Mischpolymerlsat from acrylamide, N, N' until acrylamideacrylamide until acrylamide and maleic acid anhydride bound and in water in place of the bacteria mud. According to invention procedures for Herstelluug of 6-Aminopenicillansäure by enzymatic splitting of Penicillinen by means of kovalent to carriers bound Penicillinacylase is by it marked that one brings penicillin solutions at pH 6 to 8, preferably 7 to 7.8 and temperatures from 20 to 50, preferably 38°C, with suspensions of Penicillinacylasen, which are bound for until acrylamideacrylamide until acrylamide and maleic acid anhydride kovalent to a copolymer from acrylamide, N, N' 3 to 20 h in contact, which mixture neutralized and 6-Aminopenicillansäure in actually well-known isolates way. Attempts to manufacture 6-APS by enzymatic splitting with your Penicillinacylase are admit (German Offerdegungsschrlften 1917057, 1907365), could not however not into a technical yardstick be transferred. This has its reason above all therein that the specific activity of these Penicillinacylase is too small. It must be zngesetzt from there very much such carrier-bound Penicillinacylase, which leads however too in the technical yardstick only badly riättrbaren beginnings. The procedure according to invention lets itself transfer with as descriptive the above, by kovalente connection at a Mischpolymerlsat bound PeniciUinacylase easily also into the technical yardstick, since the specific activity is high. For enzymatic splitting by penicillin G only relatively little must be added to the aqueous solution the polymere-bound Penicillinacylase, in order to obtain a complete splitting of penicillin. The portion of polymere-bound enzyme is so small that the reaction mixture can be agitated also in the technical yardstick problem-free. The insoluble enzyme is suspended in a solution with 75 to 150000 E/tal penicillin, e.g. Penicillln G-potassium. Enzymatic splitting is accomplished at constant pH value within the range of 6 to 8. For the neutralization of the abgespaltenen Phenylessigsäure one uses aqueous alkali solutions, e.g. Kalloder caustic soda solution or organic amines, preferably tri ethyl amine. With tri ethyl amine e.g. the Aktivätät of the insoluble enzyme remains also after repeated employment. Favourable further attainable the according to invention yields are within the range of 86% and over it. Erfindtmgsgemäße enzymafische splitting is preferably accomplished at pH 7.8. At pH values above 8,0 PeniciUin or 6-APS under opening des/3-Lactamringes can be inactivated. At smaller pH values the equilibrium shifts in favor of the output product penicillin, so that splitting remains incomplete. Preferably at pH 7.8 one works. It is favourable to begin and increase toward end of the reaction slowly to 7,8 splitting at low pH value. The Reaktionsgeschwindlgkeit depends on the employment at carrier-bound enzyme. Like that a Spalttmgsansatz with a concentration of 100000 E/ml penicillin G-potassium is e.g. hydrolyzed after 12 h at pH 7.8 and 38°C completely to 6-APS and Phenylessigsäure, if per unit Penicilllnacylase 10s units penicillin G are used. (An enzyme unit (e) is defined as the activity, which hydrolyzes 1 #Mol penicillin G/min with 37°C to 6-APS and Phenylessigsäure). Per unit if Penicillinacylase 5 × 104 units penicillin G are used, complete splitting lasts 6 h. Also shorter response times are possible with still higher enzyme employment. Accordingly one must split the longer, the fewer enzymes is used. Spaltnngszeiten of more than 20 h lead however due to inactivating of penicillin and/or 6-APS to yield losses. The reaction temperature of enzymatic splitting amounts to preferably 38°C. At lower temperatures the activity of the enzyme decreases. If splitting is accomplished e.g. with 25°C, twice as much enzyme must be inserted as with 38°C, if same response times are to be obtained. The activity of the unsolvable carrier-bound Penicillinacylase decreases clearly in solutions with higher substrate concentration. Splitting a solution with 75000E/m1 PenicillinG lasts 12 h and with 150000 E [ml 15 h, if only the concentration is changed, not however the relationship enzyme to penicillin G. Preferably solutions with 75000 to 100000 E/ml penicillin G are used. The 6-APS lets itself isolate simply, if erf'mdungsgemäß used enzyme preparation is used. The portion to carrier-bound Penicillinacylase is with 0,05 Gew. - % so small that at the carrier hardly 6-APS can be adsorbed. The carrier-bound Penicillinacylase lets itself separate after splitting by Zentrifugation or filtration and begin several times for further splitting beginnings. The 6-APS formed with enzymatic splitting is kept crystallized from the reaction solution, which was separated from the unsolvable enzyme and concentrated then in the vacuum, by precipitation at the ISO-electrical point at pH 4.3. During erf'mdungsgemäßen splitting of PenieillinG with the unsolvable enzyme preparation substantially higher yields at 6-APS will receive than when using E.coli mud. Thus, as in the example is shown, 6-APS was isolated with repeated employment of the insoluble Penicillinacylase in an average yield from 87% d.Th. The purity of the preparations amounted to on the average 97%. In such a way manufactured 6-APS does not contain proteins as pollution. All genes side effects, which are attributed to proteins, are impossible with the 6-APS according to invention. The following examples describe the invention: B e i s p i e 1 1: Production of 6-Aminopenicillansäure (6-APS) 57 g damp, carrier-bound Penicillinacylase (spezitqsche activity 0.044 E/mg) and " 160 g penicillin G-potassium are given successively to 2500 ml water and agitated with 38°C. One keeps the mixture constant by addition of tri ethyl amine at pH 7,8. After 12 h no more tri ethyl amine is taken up. The carrier-bound enzyme is filtered off, washed afterwards with in each case 100tal water and 100ml 0,2m cook rolling solution and again used for further splitting beginnings. The filtrate including washing waters one restricts in the vacuum on 300 ml and fails the 6-APS at the ISO-electrical point at pH 4.3 in presence of 500 ml Methylisobutylketon. After 3h with 5°C one filters off and washes with 200 ml water and with 200 ml acetone after. One dries in the vacuum with 40°C. Schmp. 208°C, yield 88.3% d.Th. The carrier-bound Penicillinacylase was used successively like before descriptive ten times for enzymatic splitting of penicillin G. The obtained yields are below arranged: 2. Splitting 87.8 3rd splitting 86.3 4th splitting 88.2 splitting 87.5 6th splitting 86.9 7th splitting 88.1 8th splitting 87.5 9th splitting 85.0 splitting 86.5 87.2% production of the carrier-bound Penicillinacylase " 450g Acrylarnld, 22,5g N, N' until acrylamideacrylamide until acrylamide and! 50g maleic acid are solved in 3500mi 0.05 m-Phosphatpuffer, pH 7.6, and under N2 - inert gas with 150 ml 5% aqueous Propionsäurenitril solution and 150 ml 5% Ammoniuraperoxydlsulfatlösung shifts. The reaction mixture is warmed up to 80°C, held then 15 h at ambient temperature and pressed by a filter with a mesh size by 0.5 mm. After careful washing with water the polymer is freezingdried and heated up in the vacuum with 20 peat 2 h on 180°C. B e i s p i e 1 2: 110 g damp, carrier-bound Penicillinacylase (specific activity 0.044 E/mg) are agitated as in the example 1 descriptive with 160 g penicillin G-potassium in 2500 ml water 6 h at 38°C and pH 7.8. The yield at 6-APS amounts to 89.5% of the theory. B e i s p i e I 3: 30 g feucbte, trägergebnndene Penicillinacylase (specific activity 0.044 E/mg) are agitated as in the example 1 descriptive with 160 g penicillin G-potassium in 2500 ml water 20 h at 38°C and pH 7.8. The yield at 6-APS amounts to 86.1% d.Th.