Cultivation of recombinant Escherichia coli to achieve high cell density with a high level of penicillin G acylase activity.

Y C Liu, L C Liao, W T Wu
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Abstract

A mutant strain of E. coli EP1 harbouring pGL-5 was employed to develop a process for producing penicillin G acylase (PGA). In comparison with different carbon sources in the medium, it was found that the specific levels of PGA activity obtained in the glucose medium were the lowest. which was likely due to catabolic repression. Phenylacetic acid (PAA) was previously reported to be an regulatory inducer for PGA production, whereas in this study, the addition of PAA repressed both cell growth and enzyme expression. In a fed-batch culture, the increase of specific PGA activity followed the pattern of the cell concentration during the early to middle cell growth phase. With application of pure oxygen aeration and an appropriate medium design, the cell concentration reached 162 (g wet weight/l), which was 2.4 times higher compared to that of the original operation, and a specific PGA activity of 37 (IU/g wet weight) was achieved after 12 h of cultivation.

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培养重组大肠杆菌,获得高细胞密度和高水平的青霉素G酰化酶活性。
利用携带pGL-5的大肠杆菌EP1突变株,建立了生产青霉素G酰化酶(PGA)的工艺。通过对培养基中不同碳源的比较,发现葡萄糖培养基中获得的PGA活性比水平最低。这可能是由于分解代谢抑制。苯基乙酸(PAA)是PGA生成的调节诱导剂,而在本研究中,PAA的添加抑制了细胞生长和酶的表达。在补料批培养中,PGA特异性活性的增加遵循细胞生长早期到中期细胞浓度的规律。采用纯氧曝气和适当的培养基设计,细胞浓度达到162 (g湿重/l),比原操作提高了2.4倍,培养12 h后PGA比活性达到37 (IU/g湿重)。
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