Specifically immobilised aldo/keto reductase AKR1A1 shows a dramatic increase in activity relative to the randomly immobilised enzyme.
The difference between site-specific and random immobilisation of the aldo/keto reductase AKR1A1 was explored. AKR1A1 was recombinantly expressed as a thioester by the intein strategy. The thioester was selectively modified with a biotin label by the expressed protein ligation method, and subsequent immobilisation on streptavidin templates was performed. Adsorption of wild-type AKR1A1 to streptavidin templates and of biotinylated AKR1A1 to uncoated templates was used to study randomly immobilised enzymes. Investigation of the kinetic parameters revealed remarkably improved activity for the site-specifically immobilised enzyme, which was comparable to that of the wild-type enzyme in solution and 60-300-fold greater than that of the randomly immobilized enzymes. Furthermore, the enzyme was surprisingly stable. No loss of activity was observed for over a week, and even after 50 days more than 35% of activity was maintained.
Holland-Nell
Kai
K
Institute of Biochemistry, University of Leipzig, Brüderstrasse 34, 04103 Leipzig.
Beck-Sickinger
Annette G
AG
D000429:Alcohol Oxidoreductases / Q000737:chemistry*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D011492:Protein Hydrolysates / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
D004800:Enzymes, Immobilized; D008024:Ligands; D011492:Protein Hydrolysates; D001710:Biotin; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; D014357:Trypsin