Considerable amounts of data have demonstrated that protein stabilizations can be achieved by rational or combinatorial engineering or combination of both. Some of rational protein stabilization strategies include “entropic stabilization” through rigidification by mutations, introduction of disulfide bridges, introduction of salt bridges, introduction of clusters of aromatic-aromatic interactions, and engineering of subunit interfaces of multimeric enzymes. On the other hand, comparative studies on a large number of naturally found or engineered enzymes with high stability have revealed that there exist many different ways to enhance the protein stability through mutation and recombination. This underscores the difficulty in developing a general strategy to enhance the protein stability and the importance of individual structural context, which were further confirmed by directed evolution studies. Thermophilic protein has been used as a versatile scaffold for creation of thermophilic binding protein. Introduction of a binding site through a few mutations throughout the thermophilic protein successfully lead to the creation of thermophilic binding protein. However, little attention has been given to the attempt to create thermophilic enzyme by insertion of target enzyme into the thermophilic scaffold. In the research presented here, we explored possibility of creating thermostable enzyme by insertion of target enzyme into thermophilic maltose binding protein (ThMBP). Beta-lactamase (BLA) as a model target protein was inserted into several ThMBPs at locations suggested from previous research where insertion of BLA into mesophilic maltose binding protein (MBP) created maltose binding-dependent BLA activity. BLA-ThMBP complexes exhibited no BLA activity at temperatures below 60 oC but significant activities above 60 oC. Two-fold increase in BLA activity with increasing temperature by 10 oC was observed up to 90 oC. Effect of temperature on BLA activities was reversible even after several temperature cycles. Thermostabilities of BLA inserted into two different ThMBPs will be compared. Effects of the location of the insertion site on thermostability will be discussed.

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