反应详情

温度

60 °C

5 kg of wet cell paste or the corresponding crude or purified extract obtained by fermenting the strain MG1655/pGM716 or the strain DH5α/pGM716 were resuspended in approximately 20 liters of 30 mM phosphate buffer at pH 7 and mixed with 980 liters of phosphate buffer in which had been dissolved at elevated temperature 10.1 kg of adenine (final concentration approximately 74.6 mM) and 18.3 kg of Ara-U (final concentration approximately 74.6 mM). The mixture was maintained at 60° C., with agitation, for 4 hours to obtain a bioconversion yield of approximately 70%. The cell paste was recovered in order to be used again in subsequent reactions by dilution at elevated temperature and diafiltration in accordance with the procedure described above. The ultrafiltrate was concentrated to a volume of approximately 1000 liters, cooled to collect the precipitate constituted by Ara-A which was subsequently crystallised from water to obtain approximately 14 kg of Ara-A with a degree of purity higher than 99.5%. According to an alternative procedure, in which the cells were not recovered and the diafiltration step was omitted, at the end of the reaction the mixture was heated to approximately 90° C. and filtered at elevated temperature to eliminate the cells, and the filtrate was cooled to precipitate Ara-A contaminated with non-reacted adenine (approximately 20 g of adenine per 100 g of Ara-A). 14 kg of Ara-A (total yield 65%) having a degree of purity higher than 99.5% were finally obtained after crystallisation from reaction of 1000 liters. Similar results were obtained starting from a mixture of the cell pastes or the corresponding crude or purified extracts obtained by fermenting the recombinant E. coli strains MG1655/p707 or MG1655/p708 and the strains DH5α/pGM707 or DH5α/pGM707 overexpressing UdP and PNP, respectively.

1. custom5 kg of wet cell paste or the corresponding crude or purified
2. extractionextract
3. customobtained
4. customto obtain a bioconversion yield of approximately 70%
5. customThe cell paste was recovered in order
6. concentrationThe ultrafiltrate was concentrated to a volume of approximately 1000 liters
7. temperaturecooled
8. customto collect the precipitate
9. customwas subsequently crystallised from water