This is a study from our lab, which is both published and patented (both as a process and as a product, in multiple countries including the USA, Australia and four countries in Europe), since it relates to a new process by which different characteristics of proteins performing the same function in different domains of life can be ‘mixed-and-matched’ or ‘recombined’ in ways that are quite predictable and robust. Briefly, we have evolved a technique with which to ‘transplant’ entire active surfaces between proteins if the surfaces in question are made up of beta strands forming beta sheets. Basically, the approach is to reengineer the surface of one protein with residues (and groups of residues) derived from a different protein, when the positions of the alpha carbon atoms of the said residues in the structures of the two proteins can be superimposed with a very low root mean square deviation of position. The approach is somewhat similar to the technique of veneering which has been used for antibodies; it is a systematic approach that allows transplantation of any beta sheet surface between proteins sharing some structural homology. In this paper, and using this technique, we have re-surfaced the entire substrate-binding and catalytically-active surface (the active surface) of a thermophile endoglucanase with an active surface pre-evolved on a mesophile endoglucanase. The resultant protein folds and functions with the temperature optimum of the mesophile homolog (i.e., it is meso-active) although it shows activity at temperatures much higher than any at which its mesophile ancestor ever had the scope to function. It also has the structural stability of the thermophile homolog (i.e., it is thermo-stable), with the transplantation of a ~60 residue comprising non-contiguous surface into a protein containing ~225 residues. So, the protein’s stability is like that of its thermophile ancestor. The determined (and deposited) crystal structure of the transplant-carrying protein clearly shows the success in the re-sculpting of the active surface that leads to the creation of a meso-active, thermo-stable protein. The study demonstrates that it is not only the denaturation of a protein with temperature that causes the rise of activity with temperature to reverse after a certain point, during heating, but rather the fact that every enzyme’s active surface has a pre-evolved optimum temperature of function which cannot be changed by providing it with a more stable structural scaffold, because the increased vibrations of side-chains with increase in temperature tend to increase catalytic rates but decrease binding of substrate.

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