Engineering a substrate-specific cold-adapted subtilisin.

One region predicted to be highly flexible for 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 300 K 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-directed 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 with 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, as investigated by molecular dynamic simulations, and global destabilization from differential scanning calorimetry 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.