Protein Engineering of Enzyme Robustness Relevant to Organic and Pharmaceutical Chemistry and Applications in Biotechnology

Abstract

Enzymes used as catalysts in chemistry and biotechnology generally should have sufficient thermostability, resistance to hostile organic solvents, oxidative stability, and tolerance to different pH ranges under operating conditions. This chapter first describes modern enzyme immobilization techniques and other bioprocess methods that ensure protein robustness. Then, different protein engineering methods are analyzed. These include directed evolution techniques such as epPCR, DNA shuffling, saturation mutagenesis at residues having large B-factors (B-FIT), and iterative saturation mutagenesis (ISM). Particularly effective for many enzymes is rational protein engineering, in which appropriate site-specific mutagenesis is performed, leading to new hydrogen bonds or H-bond networks on the enzyme surface, and introduction of cysteines at the correct sites, which enables S–S bridges. All such mutagenic manipulations lead to protein rigidification and enhanced robustness.