title: Enhanced enzyme stability through site-directed covalent immobilization.
abstract: Breakthroughs in enzyme immobilization have enabled increased enzyme recovery and reusability, leading to significant decreases in the cost of enzyme use and fueling biocatalysis growth. However, current enzyme immobilization techniques suffer from leaching, enzyme stability, and recoverability and reusability issues. Moreover, these techniques lack the ability to control the orientation of the immobilized enzymes. To determine the impact of orientation on covalently immobilized enzyme activity and stability, we apply our PRECISE (Protein Residue-Explicit Covalent Immobilization for Stability Enhancement) system to a model enzyme, T4 lysozyme. The PRECISE system uses non-canonical amino acid incorporation and the Huisgen 1,3-dipolar cycloaddition "click" reaction to enable directed enzyme immobilization at rationally chosen residues throughout an enzyme. Unlike previous site-specific systems, the PRECISE system is a truly covalent immobilization method. Utilizing this system, enzymes immobilized at proximate and distant locations from the active site were tested for activity and stability under denaturing conditions. Our results demonstrate that orientation control of covalently immobilized enzymes can provide activity and stability benefits exceeding that of traditional random covalent immobilization techniques. PRECISE immobilized enzymes were 50 and 73% more active than randomly immobilized enzymes after harsh freeze-thaw and chemical denaturant treatments.
authors.affiliation: Department of Chemical Engineering, Brigham Young University, Provo, UT, USA.
authors.forename: Jeffrey Chun Yu
authors.initials: JC
authors.lastname: Wu
authors.affiliation: Department of Chemical Engineering, Brigham Young University, Provo, UT, USA.
authors.forename: Christopher Hayden
authors.initials: CH
authors.lastname: Hutchings
authors.affiliation: Department of Chemical Engineering, Brigham Young University, Provo, UT, USA.
authors.forename: Mark Jeffrey
authors.initials: MJ
authors.lastname: Lindsay
authors.affiliation: Department of Chemical Engineering, Brigham Young University, Provo, UT, USA.
authors.forename: Christopher James
authors.initials: CJ
authors.lastname: Werner
authors.affiliation: Department of Chemical Engineering, Brigham Young University, Provo, UT, USA. Electronic address: bundy@byu.edu.
authors.forename: Bradley Charles
authors.initials: BC
authors.lastname: Bundy
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D019149:Bioreactors; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli; D055772:Green Chemistry Technology; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D014508:Urea / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D004800:Enzymes, Immobilized; D014508:Urea; D009113:Muramidase
keywords: Biocatalysis; Cell-free protein synthesis.; Enzyme immobilization; Enzyme stability; Green manufacturing; Non-canonical amino acids