title: Specifically and reversibly immobilizing proteins/enzymes to nitriolotriacetic-acid-modified mesoporous silicas through histidine tags for purification or catalysis.
abstract: Six nitriolotriacetic-acid-modified ordered mesoporous silicas (NTA-OMPSs) with different pore sizes and surface features for specific and reversible protein immobilization were fabricated and characterized. Specific immobilization of a genetically engineered undecaprenyl pyrophosphate synthase (UPPs) from cell lysate and a chemically modified His-tagged horseradish peroxidase (HRP) in these Ni-NTA-OMPSs through histidine coordination to the nickelated NTA was demonstrated and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Negligible leakage of these enzymes over a wide range of acidic conditions was observed. Moreover, histidine tags with different lengths (His6, His4, His3, and His2) applied to HRP were evaluated to find the minimum length for effective complexation. Enzymatic assessment studies indicated that the pore size of the OMPSs has minimal influence on the enzymatic activity, whereas chemical entities such as unreacted mercapto groups tailored on the interior surfaces of the OMPSs played certain roles in inhibiting the enzymatic activity and stability. On MCF-S-NTA, SBA-S-NTA, and film-S-NTA, which contained unreacted mercaptopropyl groups on the interior surface, immobilized His-tagged HRP showed lower catalytic activity and stability than on MCF-NTA, film-NTA, and SBA-NTA. Selective hydroxylation of optically pure L-tyrosine to (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid (L-DOPA) by the immobilized HRP was also demonstrated.
authors.affiliation: Department of Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617 Taiwan ROC.
authors.forename: Yu-Chung
authors.initials: YC
authors.lastname: Lin
authors.forename: Ming-Ren
authors.initials: MR
authors.lastname: Liang
authors.forename: Yu-Chen
authors.initials: YC
authors.lastname: Lin
authors.forename: Chao-Tsen
authors.initials: CT
authors.lastname: Chen
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry; D002384:Catalysis; D006639:Histidine / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000737:chemistry*; D016062:Porosity; D012822:Silicon Dioxide / Q000737:chemistry*
chemical_list: D055767:Immobilized Proteins; D006639:Histidine; D012822:Silicon Dioxide; D006735:Horseradish Peroxidase; D019883:Alkyl and Aryl Transferases; C036826:trans-pentaprenyltranstransferase; D009571:Nitrilotriacetic Acid