284236 Enzyme Biocatalysis Orientation Control During Immobilization by Unnatural Amino Acid Incorporation

Monday, October 29, 2012: 12:48 PM
Westmoreland Central (Westin )
Bradley C. Bundy1, Jeffery C. Wu2, Mark T. Smith1 and Chad T. Varner1, (1)Chemical Engineering, Brigham Young University, Provo, UT, (2)Microbiology and Molecular Biology, Brigham Young University, Provo, UT

The $3+ Trillion global chemical industry is built predominantly upon catalytic and increasingly biocatalytic processing.  The enzyme biocatalysis market has increased by 15% annually to the current $5+ billion market and has many advantages including high chemo-, regio-, and stereospecificity and sustainability.  However, biocatalysis technology is often limited by factors such as enzyme instability and difficulties recovering and reusing the enzyme.  Enzyme immobilization has been shown in many cases to overcome these limitations.  While a number of immobilization methods have been developed (entrapment, cross-linking, absorption, covalent attachment, etc.), covalent attachment is particularly promising due to enzyme leaching and transport limitations of other methods.  Unfortunately, enzymes are composed of only 20 amino acids which complicate our ability to control the exact site where the enzyme is covalently attached to the surface without significant mutagenesis.  Here we present the controlled site-specific incorporation of the unnatural amino acid propargyloxyphenylalanine to directly attach proteins and enzymes to surfaces at a specified orientation for optimal performance.  The immobilization method requires only a single insertion or substitution of the unnatural amino acid which can be positioned at virtually any location on the enzyme.  The biocompatible copper-catalyzed azide alkyne cycloaddition is used to attach our unnatural amino acid containing protein to azide groups exposed on the solid support’s surface.  With this method we have demonstrated orientation controlled covalent immobilization at high attachment efficiency and retained activity.

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