PEDS Advance Access originally published online on July 21, 2009
Protein Engineering Design and Selection 2009 22(10):615-623; doi:10.1093/protein/gzp044
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Enzyme stabilization by domain insertion into a thermophilic protein
1Othmer-Jacobs Department of Chemical and Biological Engineering, Polytechnic Institute of New York University, 6 MetroTech Center, Brooklyn, NY 11201, USA 2Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA 3 Howard Hughes Medical Institute, Janelia Farm Campus, 19700 Helix Drive, Ashburn, VA 20147, USA
4 To whom correspondence should be addressed. E-mail: jkim{at}poly.edu
Insufficient kinetic stability of exoinulinase (EI) restricts its application in many areas including enzymatic transformation of inulin for production of ultra-high fructose syrup and oligofructan, as well as fermentation of inulin into bioethanol. The conventional method for enzyme stabilization involves mutagenesis and therefore risks alteration of an enzyme's desired properties, such as activity. Here, we report a novel method for stabilization of EI without any modification of its primary sequence. Our method employs domain insertion of an entire EI domain into a thermophilic scaffold protein. Insertion of EI into a loop of a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) resulted in improvement of kinetic stability (the duration over which an enzyme remains active) at 37°C without any compromise in EI activity. Our analysis suggests that the improved kinetic stability at 37°C might originate from a raised kinetic barrier for irreversible conversion of unfolded intermediates to completely inactivated species, rather than an increased energy difference between the folded and unfolded forms.
Keywords: exoinulinase/insertion/maltodextrin-binding protein/protein/stabilization
Received April 4, 2009; revised June 9, 2009; accepted June 16, 2009.