title: Combinatorial screening for enzyme-mediated coupling. Tyrosinase-catalyzed coupling to create protein--chitosan conjugates.
abstract: In nature, tyrosinase-generated o-quinones are commonly involved in processes that lead to functional biomaterials. These biomaterials are chemically complex and have been difficult to analyze. Furthermore, the cascade of reactions involving o-quinones is poorly understood, and it has been difficult to mimic ex vivo for materials processing. We report the use of a combinatorial approach to learn how tyrosinase and low molecular weight phenolic precursors can be used to generate biologically active protein-polysaccharide conjugates. Specifically, we screened various phenolic coupling precursors and various reaction conditions for the coupling of proteins onto the polysaccharide chitosan. Several natural phenols were identified as appropriate precursors for the coupling of polyhistidine tagged organophosphorus hydrolase (His-OPH) onto chitosan films. OPH activity was retained upon coupling and subsequent studies indicated that the histidine tag was not necessary for coupling. Using conditions identified for His-OPH coupling, we observed that various biologically active proteins (cytochrome c, OPH, and His-CAT) could be coupled onto chitosan films. The glycosylated protein horseradish peroxidase was not effectively coupled onto chitosan under the conditions studied. In all cases studied, we observed that coupling required a phenolic precursor, suggesting that tyrosinase is unable to couple by reaction with surface tyrosyl residues of the target protein. In conclusion, this study illustrates a combinatorial approach for the "discovery" of conditions to couple biologically active proteins onto chitosan through natural, quinone-based processes.
authors.affiliation: Center for Agricultural Biotechnology, 5115 Plant Sciences Building, University of Maryland Biotechnology Institute, College Park, Maryland 20742, USA.
authors.forename: T
authors.initials: T
authors.lastname: Chen
authors.forename: R
authors.initials: R
authors.lastname: Vazquez-Duhalt
authors.forename: C F
authors.initials: CF
authors.lastname: Wu
authors.forename: W E
authors.initials: WE
authors.lastname: Bentley
authors.forename: G F
authors.initials: GF
authors.lastname: Payne
mesh_terms: D001672:Biocompatible Materials / Q000737:chemistry*; D002686:Chitin / Q000031:analogs & derivatives* / Q000737:chemistry*; D048271:Chitosan; D006639:Histidine / Q000737:chemistry; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D010084:Oxidation-Reduction; D010636:Phenols / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011809:Quinones / Q000737:chemistry
chemical_list: D001672:Biocompatible Materials; D010636:Phenols; D011506:Proteins; D011809:Quinones; D002686:Chitin; C033223:polyhistidine; D006639:Histidine; D048271:Chitosan; D014442:Monophenol Monooxygenase; D010744:Phosphoric Monoester Hydrolases