Immobilized Enzymes: Activity, Orientation, and Stability.

Abstract

Enzyme immobilization is an important tool for many industrial and medical fields applications as well as biosensors. Much work has been done developing types of immobilization, yet the field lacks a comprehensive understanding of the relationship between immobilized enzyme orientation and activity/stability. This work was done to build a more complete picture of the interplay between activity, stability, orientation, and surface characteristics for immobilized enzymes. In Chapter 2, we started by making uniform, chemically defined self-assembling monolayer (SAM) surfaces functionalized with maleimide to bind NfsB through single cysteine residues. Two orientation NfsB constructs were used in these experiments, H360C and V424C. Orientation was not found to change the specific activity. In Chapter 3, SAM surfaces functionalized with EG3-MAL, EG3-OH and EG3-ME terminated linkers were made to explore the effects of surface characteristics on immobilized NfsB. We found that for mixed surfaces a mole ratio of 1:10 EG3-MAL:EG3-OH and EG3-MAL:EG3-ME resulted in a significantly higher specific activity compared to a 1:1 or 1:20 mole ratio. In these experiments, T ½ was used as a measure of thermal stability. T ½ was increase , but was unaffected by orientation. Mixed surfaces with both EG3-ME and EG3-OH with EG3-MAL held at a constant concentration were created. These mixed surfaces were used to explore the relationship between surface hydrophobicity and the activity/stability of the immobilized enzyme. T ½ was unaffected by surface hydrophobicity while specific activity nearly doubled from 100% EG3-ME surface to 100% EG3-OH surface. A variant of NfsB was created placing the cysteine on an α-helix. The α-helix anchor site didn’t change the specific activity or T ½ of immobilized NfsB. The experiments described in Chapter 4 were performed to investigate the effect of surface crowding on specific activity and stability of immobilized NfsB. Multiple surface coverage conditions from a sparse monolayer to a densely packed monolayer were used for activity and T ½ measurements. Two NfsB variants V424C and S63C showed no change in specific activity or T ½ arising from changes in surface density. NfsB-H360C showed a decrease in specific activity and T ½ at lower surface density.