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 immobilisotion of the oldo/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 immobilisotion on streptavidin templates was performed. Adsorption of wildtype 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 wildtype 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.