STRUCTURE-GUIDED COMPUTATIONAL ENGINEERING FOR BIOCATALYST STABILISATION
Omega-transaminases are attractive biocatalysts for the production of chiral amines but their industrial applicability is hampered by poor protein stability. A specialised protein engineering workflow, consisting of powerful protein folding energy calculations and guided by experimental 3D protein structural data, allowed the construction of two highly robust and active variants of the omega-transaminase PjTA from Pseudomonas jessenii.
The industrial application of enzymes as biocatalysts is often limited by their poor tolerance towards harsh bioprocess conditions such as high temperature, presence of organic co-solvents and high reactant concentrations. Enzyme variants with improved stability can be obtained in the laboratory by directed protein evolution, but this is a highly time-consuming and expensive approach, requiring experimental screening of large mutant libraries. Rational protein engineering, guided by crystal structures of the enzyme under study, can improve workflow efficiency by generating smaller, focused libraries of variants with potentially stabilising mutations. In recent years, the power of these structure- guided rational protein engineering approaches has grown significantly due to the development of computer algorithms that can quantitatively predict the stability effects of mutations and can
efficiently and effectively search for potentially stabilising mutations within the vast sequence space accessible by random mutagenesis . A successful computational workflow called FRESCO (framework for rapid enzyme stabilisation by computational libraries) has been developed, which combines prediction of potentially stabilising mutations by folding energy calculations with in-silico screening by molecular dynamics calculations to construct small mutant libraries. Experimentally verified mutations are combined, resulting in large increases in enzyme stability .
Omega-transaminases are able to synthesise enantiopure chiral amines by catalysing the transfer of an amino group from a primary amino donor to a carbonyl acceptor with pyridoxal 5 -phosphate (PLP) as cofactor .
Fig. 40: Overall FRESCO protein engineering workflow that resulted in the highly stable and robust variants PjTA-R4 and PjTA-R6.