Purification of recombinant hydantoinase and L-N-carbamoylase from Arthrobacter aurescens expressed in Escherichia coli: comparison of wild-type and genetically modified proteins

Two enzymes, hydantoinase (HyuH) and L-N-carbamoylase (HyuC), are required for the biocatalytic production of natural and unnatural, optically pure L- amino acids starting from D,L-5-monosubstituted hydantoins using the so cal led 'hydantoinase-method'. For the preparation of immobilized enzymes, whic h omit several drawbacks of whole cell biocatalysts, purified or at least e nriched HyuH and HyuC have to be provided. In order to simplify existing pu rification protocols several genetically modified derivatives of HyuH and H yuC from Arthrobacter aurescens DSM 3747 have been cloned and expressed in E. coli. A fusion protein consisting of maltose-binding protein (MalE) and HyuH resulted in an enhanced solubility of the hydantoinase, which easily f orms inclusion bodies. On the other hand the fusion protein could easily be purified with high yield (76%) by just one chromatographic step (amylose r esin) and the complex purification protocol of the wild-type enzyme could t herefore be simplified and shortened significantly. Interestingly, the spec ific activity of the MalE-HyuH fusion protein was as high as the wild-type enzyme despite that the molecular mass was doubled. A second modification o f HyuH carrying a histidine-tag was efficiently bound to a metal affinity m atrix but inactivated completely during elution from the column at either l ow pH or in the presence of imidazole. In the case of HyuC, an aspartate-ta g has been added to the biocatalyst to allow an integrated purification-imm obilization procedure since this enzyme is immobilized efficiently only via its carboxylic groups. The diminished isoelectric point of the Asp-tagged HyuC resulted in a simplified purification procedure. Compared to the wild- type enzyme expressed in E. coli HyuC-Asp(6) was shifted off the elution ra nge of the contaminating proteins and higher purification factors were obta ined even in the capturing step. In contrast to HyuH, it was possible to pu rify a L-N-carbamoylase carrying a histidine-tag to apparent homogeneity us ing immobilized metal affinity chromatography. Therefore, the existing thre e step purification protocol was reduced to one chromatographic step and th e yield of this relatively unstable protein enhanced remarkably. (C) 2000 E lsevier Science BN. All rights reserved.