(188co) Enzyme Immobilization: Predictive Structure-Function Relationships for Effective Enzyme-Linker-Surface Complexes

Immobilized enzymes can be used as biosensors because their ability to detect highly specific substrates. Enzymes, alone, are susceptible to degradation and loss of function in solution; however, when immobilized to a surface, the enzyme’s lifetime and stability can increase, while the surface-chemistry can promote enzyme-substrate interactions. Our goal is to fundamentally understand the interactions between the enzyme, linker, and surface during covalent, site-specific immobilization that can be detrimental to its activity and apply our knowledge to successfully immobilize under studied enzymes. We begin our study with the well characterized, model enzyme, T4 Lysozyme, by exploring the effects on enzymatic activity of different site-directed cysteine mutations for linker attachment. Each point mutation changes the orientation of the enzyme when immobilized, either promoting or inhibiting substrate interactions. We also examine the effects of different linker-surface chemistries that complement desired enzyme activity. Based on the findings of a well characterized enzyme, such as T4 Lysozyme, we can begin to formulate heuristics for efficient enzyme immobilization techniques and apply our newfound knowledge to an uncharacterized enzyme, cytochrome P450 (CYP201A2), for biosensor development of nuclear fuel cycle activities.