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首页> 外文期刊>Enzyme and Microbial Technology >Engineering the D-amino acid oxidase from Trigonopsis variabilis tofacilitate its overproduction in Escherichia coli and its downstreamprocessing by tailor-made metal chelate supports
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Engineering the D-amino acid oxidase from Trigonopsis variabilis tofacilitate its overproduction in Escherichia coli and its downstreamprocessing by tailor-made metal chelate supports

机译：工程化Trigonopsis variabilis的D-氨基酸氧化酶，以促进其在大肠杆菌中的过量生产以及通过量身定制的金属螯合载体进行下游加工

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摘要

The DAO1 gene of the yeast Trigonopsis variabilis encoding a D-amino acid oxidase (DAAO) has been cloned, sequenced, and overexpressed in Escherichia coli once the intron, which interrupts the reading frame, was eliminated by polymerase chain reaction mutagenesis. Moreover, to facilitate the purification of DAAO, a fully active tagged enzyme was constructed by engineering a six histidine tail in the N-terminal region of the protein. Unexpectedly, the resulting His-DAAO could not be purified by metal-chelate chromatography by using supports containing copper or zinc since the adsorption process inactivates the enzyme that was so strongly bound to these supports that it could be eluted only after boiling the matrix with 4% sodium dodecyl sulfate. However, we were able to purify the enzyme from a crude extract in a single step by using a tailor-made metal chelate support containing a very low density of cobalt ligands. Interestingly, the enzyme bound to this support remained active, opening a new scenario to investigate the design of an industrial process based on its immobilization in this support. This is the first time that this important industrial enzyme has been successfully modified by protein engineering to facilitate its downstream processing. Therefore, our biotechnological approach not only provides the tools to develop more efficient industrial processes, such as the production of 7-amino cephalosporanic acid from cephalosporin C, by using a highly active DAAO preparation, free from undesirable contaminant enzymatic activities, but also illustrates the importance of a careful design of the metal chelate support for optimizing the purification of His-tagged proteins.
机译：一旦通过聚合酶链反应诱变消除了破坏阅读框的内含子，就已经在大肠杆菌中克隆了编码D-氨基酸氧化酶（DAAO）的变异三角酵母酵母DAO1基因，并对其进行了测序和过表达。此外，为了促进DAAO的纯化，通过在蛋白质的N末端区域改造6个组氨酸尾部，构建了具有完全活性的标记酶。出乎意料的是，所得的His-DAAO无法通过使用金属螯合物色谱法使用含铜或锌的载体进行纯化，因为吸附过程会灭活与这些载体牢固结合的酶，以至于只有在将基质与4一起煮沸后才能洗脱％十二烷基硫酸钠。但是，我们能够通过使用特制的包含非常低密度钴配体的金属螯合载体，一步一步从粗提物中纯化酶。有趣的是，与该支持物结合的酶仍然保持活性，从而开启了一个新的场景，以基于其在该支持物中的固定化来研究工业过程的设计。这是该重要的工业酶首次通过蛋白质工程成功地修饰以促进其下游加工。因此，我们的生物技术方法不仅提供了开发更有效的工业流程的工具，例如使用高活性的DAAO制剂从头孢菌素C生产7-氨基头孢菌酸，而且没有不良的酶活性，而且还说明了精心设计金属螯合物载体对于优化His标记蛋白的纯化的重要性。

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