Abstract
Increasing the robustness of enzymes under operating conditions has been the goal of protein engineers for more than three decades, simply because this property is a prerequisite for applications in biotechnology. This chapter considers current approaches to protein engineering of enzyme robustness and the some of the properties of enzymes such as kinetic and thermodynamic thermostability, resistance to hostile organic solvents including ionic liquids, oxidative stability, and tolerance to different pH ranges. When turning away from "blind" directed evolution based on epPCR and/or DNA shuffling for enhancing thermostability, the B-FIT approach utilizing Saturation Mutagenesis (SM) and, optionally, Iterative SM (ISM) is one of several logical options. The basic idea is to utilize SM for rigidifying certain regions in a protein, which requires a criterion for choosing appropriate randomization sites. A novel and highly useful application of ISM was developed in which neither CASTing nor B-FIT was involved.
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