Optimization Of Fermentation For Pentostatin Biosynthesis

Pentostatin is a kind of purine analog and is found to be a potent and tight-binding in-hibitor of adenosine deaminase (ADA). The accumulation of deoxyadenosine-triphosphate and deoxyadenosine ADA deficient lymphocytes which leads the inhibition of DNA or RNA synthesis might result in cytotoxicity in leukaemic cells. In1998, FDA approved pentostatin as the anti-tumor drug with its product name Nipent. Pentostatin was used to treat acute leukaemia such as Hairy cell leukemia, Chronic lymphocytic leukemia, Graft-versus-host disease. Waldenstrom’s macroglobulinemia.Currently, the methods of synthesis of pentostatin include chemical synthesis and bio-synthesis. The total synthetic route of chemical synthesis was cumbersome with higher raw material costs and lower yields. There are a few research articles about biosynthesis so far and its production was unsatisfactory. The methods of fermentation of pentostatin and de-tection were discussed in this study. The main results in this study were as follows:In the first part of this study, we established the detection of pentostatin in fermenta-tion broth with high performance liquid chromatography. Using the column of Agilent Eclipse XDB-C18(4.6×250mm,5μm). the mobile phase comprised methanol and10mmol/L ammonium acetate solution. The flow rate of mobile phase was0.8mL/min with the proportion of methanol changing with time; An UV-VIS detector equipped with a wavelength of283nm was employed. The injection volume was5μL, with the columns temperature being25℃. On these chromatographic conditions, the limit of detection and limit of quantitation were20.5μg/L, respectively. The linearity of the calibration process was y=0.0833x-0.2939with R2=1. RSD values of precision and stability were0.10%and0.61%. respectively. The average recovery was99.12%with RSD values of0.73%. The results of LC-MS techniques analysis indicated the method that using HPLC for detecting pentostatin was feasible.In the second part of this study, we determined the optimal medium and fermentation parameters in pentostatin submerged fermentation by Streptomyces sp. ATCC39365. Seed medium components described as follows (g/L):malt extract powder10, yeast extract4, glucose4.(NH4)3PO42. The liquid volume was280mL per1000mL flask, temperature30℃. rotate speed250r/min. seed culture time48h. Fermentation medium (g/L):malt ex-tract powder30, yeast extract12, glucose4. The liquid volume was5L per7L flask and inoculum size was10%. Temperature,0-24h33℃,24-312h32℃,312-403h35℃; DO35%; pH7.6adjusted with citric acid. Under these fermentation conditions, pentostatin concentration in fermentation broth reached100mg/L, which is2.96times of that before optimization and1.8times of the highest production reported in literature.