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首页> 外文期刊>Journal of Biotechnology >Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification.
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Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification.

机译：综合开发一种有效的生物工艺，用于在大肠杆菌中进行细胞外生产青霉素G酰基转移酶及其后续的一步纯化。

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摘要

An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange media, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level at 871U/g DCW, of which more than 90% was localized in the extracellular medium. In addition, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. The formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. Clarified culture medium was applied to a strong anion-exchange (Q) column and PAC was purified by non-retentive separation, where most contaminant proteins bound to the chromatographic media with PAC being collected as the major component in the flow-through fraction. After removing the contaminant oligopeptides using ultrafiltration, purified PAC with a specific activity of 16.3U/mg was obtained and the overall purification factor for this one-step downstream purification process was up to 3 fold
机译：开发了有效生产和纯化青霉素G酰基转移酶（PAC）的集成生物工艺。 PAC在基因工程大肠杆菌菌株中过表达，分泌到培养基中，收获并通过阴离子交换色谱一步一步纯化。在这项研究中开发的培养基具有足够低的电导率，可以将细胞外部分直接应用于阴离子交换色谱柱，同时提供维持细胞生长和PAC过表达所需的所有营养。其目的在于（i）提供足够的渗透压和缓冲能力，（ii）最小化离子种类以促进细胞外蛋白与阴离子交换介质的结合，以及（iii）提高PAC的表达水平和分泌效率。采用这种培养基配方，PAC的比活度达到了871U / g DCW的高水平，其中90％以上位于细胞外培养基中。此外，发现渗透压和诱导条件对于最佳培养性能至关重要。与PAC过表达相关的包涵体的形成倾向于阻止细胞生长，从而导致潜在的细胞溶解。将澄清的培养基应用于强阴离子交换（Q）柱，并通过非保留性分离纯化PAC，在该色谱中，与色谱介质结合的大多数杂质蛋白均以PAC为流通组分的主要成分收集。使用超滤去除污染物寡肽后，获得比活性为16.3U / mg的纯化PAC，这一一步下游纯化过程的总纯化因子高达3倍

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