title: Immobilization of alkaline phosphatase on solid surface through self-assembled monolayer and by active-site protection.
abstract: Retaining biological activity of a protein after immobilization is an important issue and many studies reported to enhance the activity of proteins after immobilization. We recently developed a new immobilization method of enzyme using active-site protection and minimization of the cross-links between enzyme and surface with a DNA polymerase as a model system. In this study, we extended the new method to an enzyme with a small mono-substrate using alkaline phosphatase (AP) as another model system. A condition to apply the new method is that masking agents, in this case its own substrate needs to stay at the active-site of the enzyme to be immobilized in order to protect the active-site during the harsh immobilization process. This could be achieved by removal of essential divalent ion, Zn2+ that is required for full enzyme activity of AP from the masking solution while active-site of AP was protected with p-nitrophenyl phosphate (pNPP). Approximately 40% of the solution-phase activity was acquired with active-site protected immobilized AP. In addition to protection active-site of AP, the number of immobilization links was kinetically controlled. When the mole fraction of the activated carboxyl group of the linker molecule in self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 6-mercapto-1-ethanol was varied, 10% of 12-mercaptododecanoic acid gave the maximum enzyme activity. Approximately 51% increase in enzyme activity of the active-site protected AP was observed compared to that of the unprotected group. It was shown that the concept of active-site protection and kinetic control of the number of covalent immobilization bonds can be extended to enzymes with small mono-substrates. It opens the possibility of further extension of the new methods of active-site protection and kinetic control of immobilization bond to important enzymes used in research and industrial fields.
authors.forename: En-Feng
authors.initials: EF
authors.lastname: Gao
authors.forename: Kyung Lhi
authors.initials: KL
authors.lastname: Kang
authors.forename: Jeong Hee
authors.initials: JH
authors.lastname: Kim
mesh_terms: D000327:Adsorption; D000469:Alkaline Phosphatase / Q000737:chemistry*; D003460:Crystallization / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000138:chemical synthesis*; D008422:Materials Testing; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure*; D010316:Particle Size; D011485:Protein Binding; D013499:Surface Properties
chemical_list: D004800:Enzymes, Immobilized; D000469:Alkaline Phosphatase