Engineering enzymes for clinical diagnosis.
The enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3.) is a major limiting factor in the enzymatic creatinine determination because of its comparatively poor catalytic activity and stability in the native form. The gene from Pseudomonas putida coding for creatinase was cloned and used for overexpression of the protein in E coli and Pseudomonas. In addition, it was possible by means of 'random' mutagenesis in vivo and subsequent screening using an activity plate assay to isolate creatinase derivatives that are more stable towards detergents and elevated temperature under test kit conditions. This example shows that enzymes can be optimized for use in given assay conditions by mutagenesis of cloned DNA and suitable screening methods.
Schumacher
G
G
Boehringer Mannheim GmbH, Penzberg, Germany.
Hilscher
W
W
Möllering
H
H
Siedel
J
J
Buckel
P
P
D003001:Cloning, Molecular; D003404:Creatinine / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D010957:Plasmids / Q000235:genetics; D016958:Pseudomonas putida / Q000235:genetics*; D014512:Ureohydrolases / Q000096:biosynthesis / Q000235:genetics*
D003404:Creatinine; D014512:Ureohydrolases; C019944:creatinase