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  • Fermentation process for growth of E coli BL DE

    2019-11-07

    Fermentation process for growth of E. coli BL2(DE3)/pBMS–P3Hcys was carried out in 25- and 380-L fermentors containing 15- and 250-L medium, respectively. Cell yield of about 100g/L (wet cells) was achieved by feeding glycerol and Hy-yest during fermentation. Typical fermentation parameters are shown in Fig. 2. The optical density at 600nm reached about 60 corresponding to 110g/L cell concentration. Glycerol concentration reached 3g/L. Cells were harvested before glycerol was completely depleted, as we found that complete depletion of glycerol led to lower activity of cells. Periodically during the fermentation process Chymostatin mg were harvested from 200mL of broth and biotransformation was carried out for 1.5h. The activity of conversion of l-proline to cis-3-hydroxy-L praline coincided with cell growth. Direct use of the fermentation broth at the end of fermentation process in the subsequent biotransformation process could be achieved by supplementing 1L of broth with 0.881g ascorbic acid, 0.278g FeSO4·7H2O, 17.1g α-ketoglutarate, 20mL of 1M potassium phosphate buffer and 10g of l-proline. The biotransformation process was carried out at 300rpm, 1vvm aeration at 30°C. Maximum conversion obtained under these conditions was only about 20–25%. Cells harvested from fermentation runs were stored at −70°C freezer prior to being used for hydroxylation of l-proline in a 1L reaction as described in Section 2. Using 10% cell suspensions in distilled water, biotransformation was carried out at 10g/L substrate input without any feed and without maintaining pH during the biotransformation. After 50h biotransformation process only 2.88g of cis-3-hydroxy-l-proline was obtained with overall yield of 28.8%. The pH was increased to 9.2 from the initial pH of 5.8 at the start of the biotransformation process. When the cells were suspended in 50mM phosphate buffer, pH 7.5 and the pH was maintained at 7.5 during biotransformation process, a reaction yield of 63% (6.3g of cis-3-hydroxy-l-proline) was obtained after 48h (Table 1) indicating that maintenance of pH is required during biotransformation process. In order to further improve the biotransformation process, a feeding of α-ketoglutarate and ascorbate was attempted. The biotransformation process was carried out in a 20-L fermentor. The reaction mixture contained 20% (w/v, wet cells) cells of rec-E. coli in 50mM phosphate buffer (pH 7.0), 10mM ascorbic acid, 2mM FeSO4·7H2O, 90mM α-ketoglutarate, and 10g/L proline. The feed of α-ketoglutarate (8g/h) and ascorbic acid (3g/h) was started upon initiation of the biotransformation at 30°C, 2vvm aeration and 250rpm agitation. The feeding was discontinued at 32h. Under the above conditions, 9.1g/L cis-3-hydroxy-l-proline was obtained (Table 2) after 60h. From 1-L of the bioreaction mixture 8.01 cis-3-hydroxy-l-proline 2 was obtained with an e.e. >99.7% and d.e. of 99.9%. Product 2 was isolated as cis-3-hydroxy-boc-l-proline (Scheme 2) in 64% yield (9.016g). Finally cis-3-hydroxy-boc-l-proline was converted to cis-3-hydroxy-l-proline methyl ester in overall 58% yield as described in Section 2. In this report we have described the preparation of synthetic gene encoding proline 3-hydroxylase and cloning and overexpression of proline 3-hydroxylase protein in recombinant E. coli. A process for the conversion of l-proline to cis-3-hydroxy-l-proline by cell suspensions of recombinant E. coli has been demonstrated and scaled-up. cis-3-Hydroxy-l-proline was chemically converted to cis-3-hydroxy-l-proline methyl ester, a key intermediate required for synthesis of a potential Selective Androgen Receptor Modulators under development as a treatment for age-related functional decline.
    Acknowledgement
    Introduction Fibrosis is a major cause of mortality and morbidity and has been estimated to contribute to at least one third of all deaths worldwide and even 45% of deaths in the developed world [1,2]. Idiopathic pulmonary fibrosis (IPF) is one of the most progressive types of organ fibrosis, with a five-year survival rate of 30–50%, poorly understood etiology, and few treatment options [3].