Development of engineered lipases for enhanced surface binding and polymerization applications by directed evolution

Höck, Heidi; Schwaneberg, Ulrich (Thesis advisor); Möller, Martin (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


The field of enzymatic catalysis is of growing importance for the development of sustainable and environmentally friendly processes in chemical, pharmaceutical and food industry due to mild reaction conditions. One central question in our society is the recyclability and circular economy of plastics. Lipases have a great potential for polymerization towards bio-degradable polyesters. Since ester hydrolysis is the natural reaction of lipases, enzyme engineering tools were used to optimize the Candida antarctica Lipase B (CaLB) towards an improved catalytic activity for polymerization of ε caprolactone in "green solvents". Directed evolution and rational design were used for generating optimized catalysts.This work does not only focus on the enzyme as a catalyst itself, but also takes considerations about expression optimization in yeast, screening methods, as well as enzyme immobilization strategies. A good enzyme engineering strategy relies on a stable expression system and a good screening system. In the second chapter the secretion factor MFα was mutated by directed evolution in order to improve the secretion of CaLB in Saccharomyces cerevisiae. The directed evolution campaign of the secretion factor MFα yielded in 2.4 fold higher production than the natural secretion factor. The developed protocol allowed a very high mutation frequency due to high manganese concentrations in the error-prone PCR. One bottleneck in enzyme engineering is the need of a measurable substrate, which is comparable with the target substrate. In chapter 3 the model enzyme CaLB was immobilized on a gold chip to later on perform an electrical impedance spectroscopy during catalytic reaction and observe changes in the spectrum of the substrate solution. Three different approaches for immobilization of CaLB were tested and compared: (i) adsorption; (ii) covalent binding; (iii) anchor peptides. A 3 fold stronger lipase activity was measured due to directed immobilization of the enzyme on the gold surface. Anchoring peptides, e.g. LCI, showed in comparison to the other two immobilization methods the biggest potential for immobilization on a gold surface. The scientific question in chapter 4 was, if enzyme engineering can be used to suppress the natural reaction of lipases, hydrolysis of ester, to gain a catalyst which is able perform polymerization of ε-caprolactone towards polycaprolactone in an aqueous environment. Two engineering approaches were followed in combination with the immobilization of the enzyme in a microgel to create a microenvironment, with a lower local water concentration, supporting the reaction. It was shown that the increase of the surface hydrophobicity of the lipase surface by rational design had a positive impact on the polymerization capability. The increase of the hydrophobic lid structure resulted in 1.2 fold and the deglycosylated variant in 1.7 fold better performances in polymerization compared to non-immobilized lipase. Immobilization in a hydrophobic microgel boosted this effect and a higher polymerization activity was obtained.