NO147452B - HYDROLASE PREPARATION FOR THE PREPARATION OF D-PHENYLGYLINE BY ENZYMATIC CONVERSION OF D, L-PHENYL HYDANTOIN - Google Patents
HYDROLASE PREPARATION FOR THE PREPARATION OF D-PHENYLGYLINE BY ENZYMATIC CONVERSION OF D, L-PHENYL HYDANTOIN Download PDFInfo
- Publication number
- NO147452B NO147452B NO77771998A NO771998A NO147452B NO 147452 B NO147452 B NO 147452B NO 77771998 A NO77771998 A NO 77771998A NO 771998 A NO771998 A NO 771998A NO 147452 B NO147452 B NO 147452B
- Authority
- NO
- Norway
- Prior art keywords
- preparation
- hydantoin
- hydrolase
- carbamyl
- phenylgyline
- Prior art date
Links
- 238000002360 preparation method Methods 0.000 title claims description 13
- 238000006243 chemical reaction Methods 0.000 title claims description 6
- 230000002255 enzymatic effect Effects 0.000 title claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 244000005700 microbiome Species 0.000 claims description 9
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- ZGUNAGUHMKGQNY-SSDOTTSWSA-N D-alpha-phenylglycine Chemical compound OC(=O)[C@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-SSDOTTSWSA-N 0.000 claims description 4
- 241000589516 Pseudomonas Species 0.000 claims description 4
- 150000001469 hydantoins Chemical class 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000003776 cleavage reaction Methods 0.000 claims description 2
- 230000007017 scission Effects 0.000 claims description 2
- 229940091173 hydantoin Drugs 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 230000007062 hydrolysis Effects 0.000 description 8
- 238000006460 hydrolysis reaction Methods 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000604 Hydrolases Proteins 0.000 description 7
- 102000004157 Hydrolases Human genes 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 241000589774 Pseudomonas sp. Species 0.000 description 5
- 230000007071 enzymatic hydrolysis Effects 0.000 description 5
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- WJRBRSLFGCUECM-UHFFFAOYSA-N hydantoin Chemical compound O=C1CNC(=O)N1 WJRBRSLFGCUECM-UHFFFAOYSA-N 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 150000008574 D-amino acids Chemical class 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 3
- 230000006340 racemization Effects 0.000 description 3
- 230000000284 resting effect Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- GIOUOHDKHHZWIQ-UHFFFAOYSA-N 2-(carbamoylamino)-2-phenylacetic acid Chemical compound NC(=O)NC(C(O)=O)C1=CC=CC=C1 GIOUOHDKHHZWIQ-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 150000003862 amino acid derivatives Chemical class 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- RKYHFLJACVNDIB-UHFFFAOYSA-N 2-(n-carbamoylanilino)acetic acid Chemical compound OC(=O)CN(C(=O)N)C1=CC=CC=C1 RKYHFLJACVNDIB-UHFFFAOYSA-N 0.000 description 1
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical compound CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 0.000 description 1
- LJCWONGJFPCTTL-UHFFFAOYSA-N 4-hydroxyphenylglycine Chemical compound OC(=O)C(N)C1=CC=C(O)C=C1 LJCWONGJFPCTTL-UHFFFAOYSA-N 0.000 description 1
- NXQJDVBMMRCKQG-UHFFFAOYSA-N 5-phenylimidazolidine-2,4-dione Chemical compound O=C1NC(=O)NC1C1=CC=CC=C1 NXQJDVBMMRCKQG-UHFFFAOYSA-N 0.000 description 1
- 108700023418 Amidases Proteins 0.000 description 1
- NXQJDVBMMRCKQG-SSDOTTSWSA-N D-5-phenylhydantoin Chemical compound O=C1NC(=O)N[C@@H]1C1=CC=CC=C1 NXQJDVBMMRCKQG-SSDOTTSWSA-N 0.000 description 1
- 102100036238 Dihydropyrimidinase Human genes 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- ZGUNAGUHMKGQNY-ZETCQYMHSA-N L-alpha-phenylglycine zwitterion Chemical compound OC(=O)[C@@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-ZETCQYMHSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 102000005922 amidase Human genes 0.000 description 1
- 229940053195 antiepileptics hydantoin derivative Drugs 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- MIOPJNTWMNEORI-UHFFFAOYSA-N camphorsulfonic acid Chemical compound C1CC2(CS(O)(=O)=O)C(=O)CC1C2(C)C MIOPJNTWMNEORI-UHFFFAOYSA-N 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 108091022884 dihydropyrimidinase Proteins 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Description
Foreliggende oppfinnelse vedrører et hydrolasepreparat for fremstilling av D-fenylglycin ved enzymatisk omdannelse av D,L-fenylhydantoin og etterfølgende avspaltning av karbamylresten fra det dannede D-karbamyl-fenylglycin, og det sær-egne ved hydrolasepreparatet i henhold til oppfinnelsen er at preparatet er erholdt fra mikroorganismer av slekten Pseudomonas som kan anvende hydantoiner som eneste nitrogenkilde, fortrinnsvis fra stammen CBS 145.75 eller CBS 146.75. The present invention relates to a hydrolase preparation for the production of D-phenylglycine by enzymatic conversion of D,L-phenylhydantoin and subsequent cleavage of the carbamyl residue from the formed D-carbamyl-phenylglycine, and the special feature of the hydrolase preparation according to the invention is that the preparation is obtained from microorganisms of the genus Pseudomonas which can use hydantoins as the sole source of nitrogen, preferably from the strain CBS 145.75 or CBS 146.75.
Foreliggende oppfinnelse vedrører således enzymkomplekser The present invention thus relates to enzyme complexes
som er i stand til å omdanne et racemisk hydantoin til et optisk aktivt aminosyrederivat, idet det ved hydrolysen anvendes spesielle mikroorganismer som bruker hydantoin som nitrogenkilde. Aminosyrederivatet (D-karbamyl-fenylglycinet) kan deretter på i og for seg kjent måte omsettes til det øns-kede D-fenylglycin. which is capable of converting a racemic hydantoin into an optically active amino acid derivative, as special microorganisms are used during the hydrolysis that use hydantoin as a nitrogen source. The amino acid derivative (D-carbamyl-phenylglycine) can then be converted to the desired D-phenylglycine in a manner known per se.
Et fåtall aminosyrer, spesielt fenylglycin og p-hydroksyfen-ylglycin er viktige utgangsforbindelser for fremstilling av forbindelser med vid anvendelse innen den farmasøytiske in-dustri. A small number of amino acids, especially phenylglycine and p-hydroxyphenylglycine, are important starting compounds for the production of compounds with wide application in the pharmaceutical industry.
Et antall forsøk er tidligere gjort på fremstilling av D-aminosyrer, men ingen av disse metoder kunne gjennomføres i indus-triell målestokk. A number of attempts have previously been made to produce D-amino acids, but none of these methods could be carried out on an industrial scale.
De kjemiske metoder som hittil er anvendt for separering av optiske isomerer er meget dyre og gir lavere utbytter. De er basert på bruken av kamfer-sulfonsyre. The chemical methods that have been used to date for the separation of optical isomers are very expensive and give lower yields. They are based on the use of camphor sulphonic acid.
En annen metode går ut på selektiv hydrolyse av D-acylamino-syre med acylaseenzym. D-acylasene er imidlertid forholds-vis sjeldne og alltid urene med hensyn til L-acylase og dette gjør metoden vanskelig og fører til oppnåelse av et produkt med dårlig optisk renhet. Another method involves the selective hydrolysis of D-acylamino acid with an acylase enzyme. However, the D-acylases are relatively rare and always impure with regard to L-acylase, and this makes the method difficult and leads to obtaining a product with poor optical purity.
Enzympreparatet i henhold til den foreliggende oppfinnelse The enzyme preparation according to the present invention
•tillater derimot fremstilling av en enkel stereo-isomer form av en aminosyre fra en racemisk forbindelse. En metode for enzymatisk separering av visse D,L-aminosyrer er allerede tidligere foreslått i det norske patentskrift 141. 689. Denne metode omfatter hovedsakelig trinnet med at man underkaster den racemiske form av hydantoin-forbindelser med den generelle formel •on the other hand, allows the preparation of a simple stereoisomeric form of an amino acid from a racemic compound. A method for the enzymatic separation of certain D,L-amino acids has already been previously proposed in the Norwegian patent document 141.689. This method mainly comprises the step of subjecting the racemic form of hydantoin compounds to the general formula
hvori in which
R er metyl eller isopropyl for en enzymatisk hydrolyse ved hjelp av dihydropyrimidinase ekstrahert fra kalvelever. R is methyl or isopropyl for an enzymatic hydrolysis by means of dihydropyrimidinase extracted from calf liver.
Hydrolysen foregår i henhold til skjemaet. The hydrolysis takes place according to the scheme.
hvoretter da karbamylresten kan avspaltes fra karbamylderivatet for erholdelse av et ønsket D-fenylglycin. after which the carbamyl residue can be cleaved from the carbamyl derivative to obtain a desired D-phenylglycine.
Den enzymatiske separering av racemiske forbindelser kan således gjennomføres med hydrolaser som tilveiebringes av mikroorganismer av slekten Pseudomonas som kan anvende hydantoin som eneste nitrogenkilde. The enzymatic separation of racemic compounds can thus be carried out with hydrolases provided by microorganisms of the genus Pseudomonas which can use hydantoin as the only nitrogen source.
Man tar således sikte på først å frembringe det spesifikke enzym som dannes under dyrkning av mikroorganismer av Pseudomonas-slekten med dette hydrolyseres hydantoinet til deri-vater av D-aminosyren, idet enzymet kan anvendes direkte som en bakterisuspensjon eller dyrkingsmedium, eller kan ekstra-heres fra cellene og dyrkingsmediet. The aim is therefore to first produce the specific enzyme that is formed during the cultivation of microorganisms of the genus Pseudomonas, with which the hydantoin is hydrolysed into derivatives of the D-amino acid, as the enzyme can be used directly as a bacterial suspension or culture medium, or can be extracted from the cells and culture medium.
Testen for den selektive hydrolyse av hydantoinderivater til D-aminosyrederivatet ved hjelp av enzympreparatet ble gjen-nomført på følgende måte: The test for the selective hydrolysis of hydantoin derivatives to the D-amino acid derivative using the enzyme preparation was carried out in the following way:
Bak teriestammene, isolert fra jord, planter, avfall av forskjellige typer, etc, såvel som stammer fra bakteriekollek-sjoner ble inokulert fra skrå-agar i 250 ml's kolber inneholdende 50 ml av følgende dyrkingsmedium: Behind the terial strains, isolated from soil, plants, waste of various types, etc., as well as strains from bacterial collections, were inoculated from slanted agar in 250 ml flasks containing 50 ml of the following culture medium:
Etter 20 til 24 timers inkubering (orbitalomrøring) ved 30°C ble 30 ml kolber inneholdende 100 ml av det samme medium inkubert med 5 ml av den ovennevnte forkultur. After 20 to 24 hours of incubation (orbital stirring) at 30°C, 30 ml flasks containing 100 ml of the same medium were incubated with 5 ml of the above pre-culture.
Etter 18 til 22 timers ytterligere inkubering ble den enzymatiske reaksjon med de hvilende celler gjennomført i prøve-rør med 10 ml's fosfatbuffer, 0,07 M, pH = 8,5 med 20 mikro-mol/ml 5-(D,L)-fenylhydantoin, det ble tilsatt 1 ml av bak-teriesuspensjonen (tørr vekt omtrent 50 mg pr. ml). Etter 30 min. inkubering ved 30°C ble reaksjonen med p-dimetylamino-benzaldehyd gjennomført for den kvantitative bestemmelse av det derved dannede karbamylderivat (J. Biol. Chem., 238, 3325 After 18 to 22 hours of further incubation, the enzymatic reaction with the resting cells was carried out in test tubes with 10 ml of phosphate buffer, 0.07 M, pH = 8.5 with 20 micromol/ml 5-(D,L)- phenylhydantoin, 1 ml of the bacterial suspension was added (dry weight approximately 50 mg per ml). After 30 min. incubation at 30°C, the reaction with p-dimethylaminobenzaldehyde was carried out for the quantitative determination of the carbamyl derivative thus formed (J. Biol. Chem., 238, 3325
(1963). De stammer som ble anvendt er gjengitt i tabell 1. (1963). The strains that were used are reproduced in table 1.
De av dem som er betegnet med nummere 942 og 945 er deponert i Centraalbureau voor Schimmelcultures hvor de er gitt beteg-nelsene CBS 145.75 henhv. 146.75. Those of them designated with the numbers 942 and 945 have been deposited in the Centraalbureau voor Schimmelcultures where they have been given the designations CBS 145.75 respectively. 146.75.
De bakteriestammer som er gjengitt i tabell 1 vokser godt i alle vanlige laboratoriemedia. De er alle i stand til å anvende fenyl-hydantoin som en eneste nitrogenkilde. Veks-ten finner sted ved en temperatur på fra 5 til 40°C med op-timal temperatur mellom 25 og 30°C. The bacterial strains listed in Table 1 grow well in all common laboratory media. They are all able to use phenyl-hydantoin as a sole nitrogen source. Growth takes place at a temperature of from 5 to 40°C with an optimum temperature between 25 and 30°C.
Med hensyn til klassifikasjonen ble skjemaet i Bergey's Manual, 7. utgave fulgt sammen med forslag av Lysenko, J. With regard to the classification, the scheme in Bergey's Manual, 7th edition was followed along with suggestions by Lysenko, J.
Gen. Microbiol. 25, 379 (1961) og Stanier, Palleroni, Dou-doroff, J. Gen. Microbiol. 43, 159-271 (1966). Gen. Microbiol. 25, 379 (1961) and Stanier, Palleroni, Doroff, J. Gen. Microbiol. 43, 159-271 (1966).
Det er funnet at under den enzymatiske hydrolyse av D-hydantoin ved alkalisk pH (7-10) foregår det en ikke-enzymatisk racemisering av det resterende L-hydantoin. Av denne grunn, og som en følge av den kontinuerlige fjernelse av D-hydantoinet ved den enzymatiske hydrolyse, vil alt karbamylderivatet oppnås i D-formen ved avsluttet reaksjon. It has been found that during the enzymatic hydrolysis of D-hydantoin at alkaline pH (7-10) a non-enzymatic racemization of the remaining L-hydantoin takes place. For this reason, and as a consequence of the continuous removal of the D-hydantoin by the enzymatic hydrolysis, all the carbamyl derivative will be obtained in the D form at the end of the reaction.
Identiteten av D(-)-N-karbamylfenylglycin ble vist etter krys-tallisering av reaksjonsproduktet, på basis av IR, NMR, masse-spektra og elementæranalyse. The identity of D(-)-N-carbamylphenylglycine was shown after crystallization of the reaction product, on the basis of IR, NMR, mass spectra and elemental analysis.
Den spesifikke rotasjon er JdJ^ = -137° (c = 1% i IN NH.OH) The specific rotation is JdJ^ = -137° (c = 1% in IN NH.OH)
D 4 D 4
tilsvarende den som er gjengitt i litteraturen. corresponding to that reproduced in the literature.
Racemiseringshastigheten av L-hydantoin er en funksjon av temperaturen og pH og er desto større ved høyere temperatur og mer basisk pH. Ved imidlertid å arbeide ved en pH i nær-heten av 7,5 er racemiseringshastigheten tilstrekkelig høy til ikke å begrense reaksjonshastigheten. The rate of racemization of L-hydantoin is a function of temperature and pH and is all the greater at higher temperature and more basic pH. However, by working at a pH in the vicinity of 7.5, the racemization rate is sufficiently high not to limit the reaction rate.
Temperaturen av den enzymatiske reaksjon kan holdes mellom 10 og 60°C men av praktiske grunner foretrekkes temperaturen mellom 25 og 40°C. The temperature of the enzymatic reaction can be kept between 10 and 60°C, but for practical reasons the temperature between 25 and 40°C is preferred.
Hydrolysen av D-fenyl-hydantoinet foregår ikke bare i nærvær av mikroorganismer som er dyrket eller i nærvær av intakte celler av disse, men også i ekstrakter av de ovenfor angitte mikroorganismer. Mikroorganismene kan dyrkes, f.eks. i et flytende næringsmedium for å oppnå en akkumulering av hydro-lase i cellene og DL-hydantoinene kan deretter tilsettes til dyrkingskulturen. Den enzymatiske reaksjon kan også gjennom-føres med såkalte hvilende celler. I dette tilfelle utvin-nes bakteriecellene fra dyrkningsmediet, vaskes og suspend-eres i et passende bufret medium hvortil det racemiske hydantoin tilsettes. The hydrolysis of the D-phenyl-hydantoin takes place not only in the presence of microorganisms that have been cultivated or in the presence of intact cells thereof, but also in extracts of the above-mentioned microorganisms. The microorganisms can be cultivated, e.g. in a liquid nutrient medium to achieve an accumulation of hydrolase in the cells and the DL-hydantoins can then be added to the culture. The enzymatic reaction can also be carried out with so-called resting cells. In this case, the bacterial cells are recovered from the culture medium, washed and suspended in a suitable buffered medium to which the racemic hydantoin is added.
I tillegg er det mulig å anvende preparater som inneholder hydrolasene, som f.eks. ekstrakter eller konsentrater av dem, rå eller rensede hydrolasepreparater som er oppnådd fra celler av de ovenfor angitte mikroorganismer. Endelig kan rå eller rensede hydrolaser anvendes, som er immobili-sert ved kombinasjoner med makromolekylære forbindelser. In addition, it is possible to use preparations containing the hydrolases, such as e.g. extracts or concentrates thereof, crude or purified hydrolase preparations obtained from cells of the above-mentioned microorganisms. Finally, crude or purified hydrolases can be used, which are immobilized by combinations with macromolecular compounds.
Følgende eksempler illustrerer oppfinnelsen. The following examples illustrate the invention.
EKSEMPEL 1 EXAMPLE 1
Til 100 ml av en flytende kultur i det ovenfor angitte medium av stammen Pseudomonas sp. 942 i en 500 ml kolbe tilsettes etter 24 timers inkubasjon (orbital omrøring) ved 30°C, 100 ml av en fosfatbuffer (0,14 M) pH = 8,5, som inneholdt 30 mikromol/ml 5-(D,1)-fenylhydantoin. To 100 ml of a liquid culture in the above medium of the strain Pseudomonas sp. 942 in a 500 ml flask is added after 24 hours of incubation (orbital stirring) at 30°C, 100 ml of a phosphate buffer (0.14 M) pH = 8.5, which contained 30 micromol/ml 5-(D,1) -phenylhydantoin.
Etter ytterligere 5 timers inkubasjon, under de samme betingelser, ble mengden av derved dannet N-karbamylfenylglycin bestemt . After a further 5 hours of incubation, under the same conditions, the amount of N-carbamylphenylglycine thus formed was determined.
Fra 530 mg 5-(D,L)-fenylhydantoin ble det dannet 525 mg N-karbamylfenylglycin, tilsvarende et utbytte på omtrent 90%. From 530 mg of 5-(D,L)-phenylhydantoin, 525 mg of N-carbamylphenylglycine were formed, corresponding to a yield of approximately 90%.
EKSEMPEL 2 EXAMPLE 2
En dyrkingsvæske ble fremstilt, med den ovennevnte sammenset-ning og inneholdende 1 g pr. liter 5-(D,L)-metylhydantoin. A culture liquid was prepared, with the above composition and containing 1 g per liter of 5-(D,L)-methylhydantoin.
pH ble innstilt til 7,2 med soda og mediet ble fordelt i 50 ml porsjoner i 250 ml kolber. Etter sterilisering ved 110°C i 30 min. ble kolbene inokulert med en kultur av Pseudomonas sp. 942 fra skrå-agar som inneholdt det samme medium The pH was adjusted to 7.2 with soda and the medium was distributed in 50 ml portions in 250 ml flasks. After sterilization at 110°C for 30 min. the flasks were inoculated with a culture of Pseudomonas sp. 942 from agar slants containing the same medium
med 2% agar (DIFCO) og inkubert ved 30°C i 20 timer med or-bitalomrøring (220 omdreininger pr. min.). with 2% agar (DIFCO) and incubated at 30°C for 20 hours with orbital agitation (220 rpm).
Med denne forkultur (optisk densitet ved 550 nm: 0,400: for-tynning 1:10) ble det inokulert 5 ml i 500 ml kolber som inneholdt 100 ml av det samme medium og kulturen ble inkubert ved 30°C med orbitalomrøring (220 omdreininger pr. minutt) i 18 timer (siste fase av eksponentiell vekst). Cellene ble så samlet ved sentrifugering (500 g, 20 mon.) og vaskes 3 ganger i bufret fysiologisk saltløsning og til slutt oppslemmet i en fosfatsaltbuffer pH = 8,5, 0,07 M med hvilende celler. With this pre-culture (optical density at 550 nm: 0.400: dilution 1:10) 5 ml were inoculated into 500 ml flasks containing 100 ml of the same medium and the culture was incubated at 30°C with orbital agitation (220 rpm . minute) for 18 hours (last phase of exponential growth). The cells were then collected by centrifugation (500 g, 20 months) and washed 3 times in buffered physiological salt solution and finally resuspended in a phosphate salt buffer pH = 8.5, 0.07 M with resting cells.
For den enzymatiske hydrolyse ble det inkubert ved 30°C med orbitalomrøring (220 omdreininger pr. minutt) i 250 ml kolber, 64 ml av reaksjonsblandingen sammensatt av: 260 mg bakterier (tørr vekt) og 20 mikromol/ml D,L-fenylhydantoin (= 3,52 mg/ml). Ved forskjellige tidsintervaller ble produk-tet fra hydrolysen, dvs. D-karbamylfenylglycin, bestemt med den kolorimetriske metode angitt i J. Biol. Chem. 238, 3325 (1963 ved 438 nm). Figuren viser resultatene, som prosent av teoretisk utbytte av det derved dannede karbamylderivat. For the enzymatic hydrolysis, 64 ml of the reaction mixture composed of: 260 mg bacteria (dry weight) and 20 micromol/ml D,L-phenylhydantoin ( = 3.52 mg/ml). At different time intervals, the product from the hydrolysis, i.e. D-carbamylphenylglycine, was determined with the colorimetric method indicated in J. Biol. Chem. 238, 3325 (1963 at 438 nm). The figure shows the results, as a percentage of theoretical yield of the resulting carbamyl derivative.
På abscissen er angitt tid i timer og på ordinaten prosent dannet D (-) -N-karbamylf enylglycin.. On the abscissa is the time in hours and on the ordinate is the percentage formed of D (-)-N-carbamylf enylglycine..
E KSEMPEL 3 EXAMPLE 3
Det ble fremstilt et halvsyntetisk medium med følgende sam-mensetning . A semi-synthetic medium with the following composition was produced.
Mediet ble fordelt i 50 ml porsjoner i 250 ml kolber og i 100 ml porsjoner i 500 ml kolber og sterilisert i 30 min. ved 110°C. The medium was distributed in 50 ml portions in 250 ml flasks and in 100 ml portions in 500 ml flasks and sterilized for 30 min. at 110°C.
For forkulturen ble 250 ml kolber inokulert fra skrå-agar med en kultur av Pseudomonas sp. 942-stammen og inkubert som i eksempel 2 i 24 timer ved 30°C. For the pre-culture, 250 ml flasks were inoculated from agar slants with a culture of Pseudomonas sp. 942 strain and incubated as in Example 2 for 24 hours at 30°C.
Fra denne kultur (optisk densitet ved 550 nm: 0.245: fortyn-ning 1:10) ble det inokulert 5 ml i 500 ml kolber. Etter en 19 timers inkubering ved 30°C, som ovenfor, ble de hvilende celler fremstilt som i eksempel 2. From this culture (optical density at 550 nm: 0.245: dilution 1:10) 5 ml were inoculated into 500 ml flasks. After a 19 hour incubation at 30°C, as above, the quiescent cells were prepared as in Example 2.
Enzymatisk hydrolyse ble gjennomført ved 30°C i en reaksjons-blanding som inneholdt i 24 ml buffer 280 mg bakterier (på basis av tørr vekt) og 20 mikromol/ml 5-(D,L)-fenylhydantoin. Etter 5, 10 og 15 min. ble mengden av det derved dannede karbamylderivat bestemt. Enzymatic hydrolysis was carried out at 30°C in a reaction mixture containing in 24 ml buffer 280 mg bacteria (on a dry weight basis) and 20 micromol/ml 5-(D,L)-phenylhydantoin. After 5, 10 and 15 min. the amount of the carbamyl derivative thus formed was determined.
EKSEMPEL 4 EXAMPLE 4
Bakterieceller ble fremstilt av stammen Pseudomonas sp. 942 som beskrevet i eksempel 2. En suspensjon av dem (42 mg pr. ml av tørre celler) i en fosfatsaltbuffer, 0,1 M, pH = 8 ble utsatt for mekanisk knusing ved anvendelse av en "Manto-Gaulin" homogenisator som arbeidet ved et trykk på 850 kg/cm 2 ved en temperatur under 24°C. Bacterial cells were prepared from the strain Pseudomonas sp. 942 as described in Example 2. A suspension of them (42 mg per ml of dry cells) in a phosphate salt buffer, 0.1 M, pH = 8 was subjected to mechanical crushing using a "Manto-Gaulin" homogenizer working at a pressure of 850 kg/cm 2 at a temperature below 24°C.
Cellemassen ble skilt fra ekstrakten ved sentrifugering ( 25, 000 g i 30 min.) . The cell mass was separated from the extract by centrifugation (25,000 g for 30 min.).
Til 950 ml fosfatsaltbuffer, pH = 7.7, inneholdende 2,12 g 5-(D,L)-fenylhydantoin ble det det tilsatt 50 ml av ekstrakten som inneholdt 8,500 U av enzymet. (1U er den mengden enzym som i en fosfatbuffer, pH =7,7 ved 30°C, inneholdende 12 mikromol/ml 5-(D,L)-fenylhydantoin, omdanner 1 mikromol/ml av substratet i løpet av 60 min.). To 950 ml of phosphate salt buffer, pH = 7.7, containing 2.12 g of 5-(D,L)-phenylhydantoin was added 50 ml of the extract containing 8,500 U of the enzyme. (1U is the amount of enzyme which, in a phosphate buffer, pH =7.7 at 30°C, containing 12 micromol/ml 5-(D,L)-phenylhydantoin, converts 1 micromol/ml of the substrate within 60 min.) .
Etter 1 time ved 30°C hadde det dannet seg 1,7 g D-karbamyl-derivat tilsvarende omtrent 80% total hydrolyse. After 1 hour at 30°C, 1.7 g of D-carbamyl derivative corresponding to approximately 80% total hydrolysis had formed.
EKSEMPEL 5 EXAMPLE 5
Som i eksempel 4 ble det tilsatt til 993 ml substrat, 7 ml av et preparat av den ekstrakt som var renset omtrent 7 ganger. Etter 1 time ved 30°C var det dannet 1,7 g D-karbamyl-derivat tilsvarende omtrent 80% av total hydrolyse. As in example 4, 7 ml of a preparation of the extract that had been purified approximately 7 times was added to 993 ml of substrate. After 1 hour at 30°C, 1.7 g of D-carbamyl derivative corresponding to approximately 80% of total hydrolysis had been formed.
EKSEMPEL 6 EXAMPLE 6
Under de samme betingelser som i eksempel 2, ble det etter 30 min. ved 3 0°C, med stammen Pseudomonas sp. 945, Under the same conditions as in example 2, after 30 min. at 30°C, with the strain Pseudomonas sp. 945,
fra 352 mg 5-(D,L)-fenylhydantoin, oppnådd 220 mg N-karbamylfenyl-glycin, tilsvarende omtrent 57% av det teoretiske utbytte. from 352 mg of 5-(D,L)-phenylhydantoin, obtained 220 mg of N-carbamylphenyl-glycine, corresponding to approximately 57% of the theoretical yield.
Claims (1)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NO77771998A NO147452C (en) | 1975-07-10 | 1977-06-08 | HYDROLASE PREPARATION FOR THE PREPARATION OF D-PHENYLGYLINE BY ENZYMATIC CONVERSION OF D, L-PHENYL HYDANTOIN |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IT25252/75A IT1039757B (en) | 1975-07-10 | 1975-07-10 | ENZYMATIC COMPLEXES FOR TRANSFORMING IDANTOIN RACEME INTO OPTICALLY ACTIVE AMINO ACIDS AND THEIR APPLICATION |
NO762324A NO147570C (en) | 1975-07-10 | 1976-07-02 | PROCEDURE FOR THE PREPARATION OF D-PHENYLGYLINE BY ENZYMATIC CONVERSION OF D, L-PHENYL HYDANTOIN |
NO77771998A NO147452C (en) | 1975-07-10 | 1977-06-08 | HYDROLASE PREPARATION FOR THE PREPARATION OF D-PHENYLGYLINE BY ENZYMATIC CONVERSION OF D, L-PHENYL HYDANTOIN |
Publications (3)
Publication Number | Publication Date |
---|---|
NO771998L NO771998L (en) | 1977-01-11 |
NO147452B true NO147452B (en) | 1983-01-03 |
NO147452C NO147452C (en) | 1983-04-13 |
Family
ID=27273413
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
NO77771998A NO147452C (en) | 1975-07-10 | 1977-06-08 | HYDROLASE PREPARATION FOR THE PREPARATION OF D-PHENYLGYLINE BY ENZYMATIC CONVERSION OF D, L-PHENYL HYDANTOIN |
Country Status (1)
Country | Link |
---|---|
NO (1) | NO147452C (en) |
-
1977
- 1977-06-08 NO NO77771998A patent/NO147452C/en unknown
Also Published As
Publication number | Publication date |
---|---|
NO147452C (en) | 1983-04-13 |
NO771998L (en) | 1977-01-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR940005654B1 (en) | Method of producing l-2-amino-4-(hydroxymethyphospinyl)-butylic acid | |
US4080259A (en) | Process of preparing L and D α-amino acids by enzyme treatment of DL-α-amino acid amide | |
NO147570B (en) | PROCEDURE FOR THE PREPARATION OF D-PHENYLGYLINE BY ENZYMATIC CONVERSION OF D, L-PHENYL HYDANTOIN | |
AU616933B2 (en) | Process for the preparation of optically active 2-arylpropionic acids | |
JP3717535B2 (en) | Novel microorganism and process for producing L-α-amino acid | |
US4774179A (en) | Process for preparing a 7-aminocephalosporanic acid compound | |
IL34956A (en) | Production of lipase | |
US4226941A (en) | Process for the optical resolution of d,l-2-amino-4-methylphosphinobutyric acid | |
DK165191B (en) | L-CARNITIN-PRODUCING MICROORGANISMS AND PROCEDURES FOR THE MANUFACTURE OF L-CARNITIN USING THE MICROORGANISMS | |
JPS585189A (en) | Novel amidohydrolase | |
GB2031896A (en) | A process for the optical resolution of D,L-2-amino-4- methylphosphinobutyric acid | |
JPH0411194B2 (en) | ||
AU674945B2 (en) | Enantioselective hydrolysis of ketoprofen esters by (beauveria bassiana) and enzymes derived therefrom | |
US5108914A (en) | Process for the synthesis of l-alpha-amino acids | |
CA2116003C (en) | Arylalkanoic acid resolution | |
GB1566088A (en) | Hydrlases for the hydrolysis of racemic hydantoins | |
NO147452B (en) | HYDROLASE PREPARATION FOR THE PREPARATION OF D-PHENYLGYLINE BY ENZYMATIC CONVERSION OF D, L-PHENYL HYDANTOIN | |
US20020037559A1 (en) | Process for producing n-protected d-proline derivatives | |
JPH01320991A (en) | Production of d-homophenylalanines | |
FR2620731A1 (en) | NOVEL ENZYMATIC SYSTEM, PROCESS FOR PREPARING SAME AND APPLICATION IN PARTICULAR FOR THE PREPARATION OF D-PARAHYDROXYPHENYLGLYCIN | |
US5496715A (en) | Process for preparing indigo | |
Sambale et al. | Microbial hydrolysis of amino acid carbamates | |
US5079167A (en) | Method of racemate resolution | |
NO155698B (en) | PROCEDURE FOR THE PREPARATION OF STEREOSPECIFIC CARBUM MOLD DERIVATIVES OF AAA HYDROXY ACIDS. | |
CZ76393A3 (en) | Micro-biological process for preparing derivatives of malonyl-7-aminocephalosporanic acid |