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 called 'hydantoinase-method'. For the preparation of immobilized enzymes, which omit several drawbacks of whole cell biocatalysts, purified or at least enriched HyuH and HyuC have to be provided. In order to simplify existing purification protocols several genetically modified derivatives of HyuH and HyuC 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 forms inclusion bodies. On the other hand the fusion protein could easily be purified with high yield (76%) by just one chromatographic step (amylose resin) and the complex purification protocol of the wild-type enzyme could therefore be simplified and shortened significantly. Interestingly, the specific 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 of HyuH carrying a histidine-tag was efficiently bound to a metal affinity matrix but inactivated completely during elution from the column at either low pH or in the presence of imidazole. In the case of HyuC, an aspartate-tag has been added to the biocatalyst to allow an integrated purification-immobilization 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-Asp6 was shifted off the elution range of the contaminating proteins and higher purification factors were obtained even in the capturing step. In contrast to HyuH, it was possible to purify a L-N-carbamoylase carrying a histidine-tag to apparent homogeneity using immobilized metal affinity chromatography. Therefore, the existing three step purification protocol was reduced to one chromatographic step and the yield of this relatively unstable protein enhanced remarkably.
Pietzsch
M
M
Institute of Biochemical Engineering, University of Stuttgart, Germany. pietzsch@ibvt.uni-stuttgart.de
Wiese
A
A
Ragnitz
K
K
Wilms
B
B
Altenbuchner
J
J
Mattes
R
R
Syldatk
C
C
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001173:Arthrobacter / Q000201:enzymology*; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006639:Histidine; D000581:Amidohydrolases; C104561:N-carbamoyl-L-amino-acid hydrolase; C020047:dihydropyrimidinase