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PEDS Advance Access published online on February 3, 2004
Protein Engineering Design and Selection, doi:10.1093/protein/gzh019
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Received September 25, 2003
Revised December 15, 2003
Accepted January 5, 2004
Oxford University Press

Article

Engineering a substrate specific cold adapted subtilisin

Nikolaj Tindbaek 1, Allan Svendsen 1*, Peter Rahbek Oestergaard 1, and Henriette Draborg 1

1 Molecular Biotechnology, Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark


  Abstract

One region predicted to be highly flexible of a psychrophilic enzyme, TA39 subtilisin (S39) was transferred in silico to the mesophilic subtilisin, Savinase (EC 3.4.21.62), from Bacillus lentus (clausii). The engineered hybrid and Savinase were initially investigated by molecular dynamic simulations at 300K to show binding region- and global flexibility. The predicted S39 region consists of 12 residues, which due to homology between the subtilisins, results in a total change of eight residues. By site-pdirected modifications the region was transferred to the binding region of Savinase thus a Savinase-S39 hybrid, named H5, was constructed.

The designed hybrid showed the same temperature optimum and pH profile as Savinase, but H5 had higher specific activity on the synthetic substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) at all temperatures measured and at the same time, H5 showed a decrease in thermostability. The H5 hybrid showed broader substrate specificity, measured at room temperature, due to an increase in catalytic efficiency on AAPF, AAPA and FAAF compared to Savinase (N-succinyl-XXXX-pNA; XXXX = AAPF, AAPA and FAAF). The H5 hybrid showed increased activity at low temperature, increased binding region and global flexibility investigated by molecular dynamic simulations and global destabilisation from DSC measurements. These psychrophilic characteristics indicated an increase in binding site flexibility, probably due to the modifications P129S, S130G, P131E and thus we show, that it is possible to increase low temperature activity and global flexibility by engineered flexibility in the binding region.

Keywords: cold adaptation/protein engineering/protein stability/psychrophilic enzymes/molecular dynamics


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