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

• Main Author: Höck, Heidi
• Other Authors: Schwaneberg, Ulrich, Möller, Martin
• Format: Doctoral or Postdoctoral Thesis
• Language: English
• Published: 2020
• Subjects: info:eu-repo/classification/ddc/570; enzyme engineering; lipase; microgel; Antarc*; Antarctica
• Online Access: https://publications.rwth-aachen.de/record/785579; https://publications.rwth-aachen.de/search?p=id:%22RWTH-2020-03325%22

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, ...