Engineering the D-amino acid oxidase from Trigonopsis variabilis to facilitate its overproduction in Escherichia coli and its downstream processing by tailor-made metal chelate supports

The DAO1 gene of the yeast Trigonopsis variabilis encoding a D-amino acid o xidase (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 purific ation 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 chromatograp hy by using supports containing copper or zinc since the adsorption process inactivates the enzyme that was so strongly bound to these supports that i t could be eluted only after boiling the matrix with 4% sodium dodecyl sulf ate. However, we were able to purify the enzyme from a crude extract in a s ingle step by using a tailor-made metal chelate support containing a very l ow density of cobalt ligands. Interestingly, the enzyme bound to this suppo rt 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 mod ified by protein engineering to facilitate its downstream processing. There fore, our biotechnological approach not only provides the tools to develop more efficient industrial processes, such as the production of 7-amino ceph alosporanic acid from cephalosporin C, by using a highly active DAAO prepar ation, free from undesirable contaminant enzymatic activities, but also ill ustrates the importance of a careful design of the metal chelate support fo r optimizing the purification of His-tagged proteins. (C) 1999 Elsevier Sci ence Inc. All rights reserved.