CaLB Catalyzed Conversion of ε-Caprolactone in Aqueous Medium. Part 1: Immobilization of CaLB to Microgels

The enzymatic ring-opening polymerization of lactones is a method of increasing interest for the synthesis of biodegradable and biocompatible polymers. In the past it was shown that immobilization of Candida antarctica lipase B (CaLB) and the reaction medium play an important role in the polymerization ability especially of medium ring size lactones like ε-caprolactone (ε-CL). We investigated a route for the preparation of compartmentalized microgels based on poly(glycidol) in which CaLB was immobilized to increase its esterification ability. To find the ideal environment for CaLB, we investigated the acceptable water concentration and the accessibility for the monomer in model polymerizations in toluene and analyzed the obtained oligomers/polymers by NMR and SEC. We observed a sufficient accessibility for ε-CL to a toluene like hydrophobic phase imitating a hydrophobic microgel. Comparing free CaLB and Novozym® 435 we found that not the monomer concentration but rather the solubility of the enzyme, as well as the water concentration, strongly influences the equilibrium of esterification and hydrolysis. On the basis of these investigations, microgels of different polarity were prepared and successfully loaded with CaLB by physical entrapment. By comparison of immobilized and free CaLB, we demonstrated an effect of the hydrophobicity of the microenvironment of CaLB on its enzymatic activity

Comparison of Candida antarctica Lipase B Variants for Conversion of ε-Caprolactone in Aqueous Medium-Part 2

Enzyme-catalyzed ring-opening polymerization of lactones is a method of increasing interest for the synthesis of polyesters. In the present work, we investigated which changes in the structure of Candida antarctica lipase B (CaLB) shift the catalytic equilibrium between esterification and hydrolysis towards polymerization. Therefore, we present two concepts: (i) removing the glycosylation of CaLB to increase the surface hydrophobicity; and (ii) introducing a hydrophobic lid adapted from Pseudomonas cepacia lipase (PsCL) to enhance the interaction of a growing polymer chain to the elongated lid helix. The deglycosylated CaLB (CaLB-degl) was successfully generated by site-saturation mutagenesis of asparagine 74. Furthermore, computational modeling showed that the introduction of a lid helix at position Ala148 was structurally feasible and the geometry of the active site remained intact. Via overlap extension PCR the lid was successfully inserted, and the variant was produced in large scale in Pichia pastoris with glycosylation (CaLB-lid) and without (CaLB-degl-lid). While the lid variants show a minor positive effect on the polymerization activity, CaLB-degl showed a clearly reduced hydrolytic and enhanced polymerization activity. Immobilization in a hydrophobic polyglycidol-based microgel intensified this effect such that a higher polymerization activity was achieved, compared to the “gold standard” Novozym® 435

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

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

Comparison of Candida antarctica Lipase B Variants for Conversion of ε-Caprolactone in Aqueous Medium—Part 2

Enzyme-catalyzed ring-opening polymerization of lactones is a method of increasing interest for the synthesis of polyesters. In the present work, we investigated which changes in the structure of Candida antarctica lipase B (CaLB) shift the catalytic equilibrium between esterification and hydrolysis towards polymerization. Therefore, we present two concepts: (i) removing the glycosylation of CaLB to increase the surface hydrophobicity; and (ii) introducing a hydrophobic lid adapted from Pseudomonas cepacia lipase (PsCL) to enhance the interaction of a growing polymer chain to the elongated lid helix. The deglycosylated CaLB (CaLB-degl) was successfully generated by site-saturation mutagenesis of asparagine 74. Furthermore, computational modeling showed that the introduction of a lid helix at position Ala148 was structurally feasible and the geometry of the active site remained intact. Via overlap extension PCR the lid was successfully inserted, and the variant was produced in large scale in Pichia pastoris with glycosylation (CaLB-lid) and without (CaLB-degl-lid). While the lid variants show a minor positive effect on the polymerization activity, CaLB-degl showed a clearly reduced hydrolytic and enhanced polymerization activity. Immobilization in a hydrophobic polyglycidol-based microgel intensified this effect such that a higher polymerization activity was achieved, compared to the “gold standard” Novozym® 435

CaLB Catalyzed Conversion of ε-Caprolactone in Aqueous Medium : Part 1: Immobilization of CaLB to Microgels

The enzymatic ring-opening polymerization of lactones is a method of increasing interest for the synthesis of biodegradable and biocompatible polymers. In the past it was shown that immobilization of Candida antarctica lipase B (CaLB) and the reaction medium play an important role in the polymerization ability especially of medium ring size lactones like ε-caprolactone (ε-CL). We investigated a route for the preparation of compartmentalized microgels based on poly(glycidol) in which CaLB was immobilized to increase its esterification ability. To find the ideal environment for CaLB, we investigated the acceptable water concentration and the accessibility for the monomer in model polymerizations in toluene and analyzed the obtained oligomers/polymers by NMR and SEC. We observed a sufficient accessibility for ε-CL to a toluene like hydrophobic phase imitating a hydrophobic microgel. Comparing free CaLB and Novozym® 435 we found that not the monomer concentration but rather the solubility of the enzyme, as well as the water concentration, strongly influences the equilibrium of esterification and hydrolysis. On the basis of these investigations, microgels of different polarity were prepared and successfully loaded with CaLB by physical entrapment. By comparison of immobilized and free CaLB, we demonstrated an effect of the hydrophobicity of the microenvironment of CaLB on its enzymatic activity

Comparison of Candida antarctica Lipase B Variants for Conversion of ε-Caprolactone in Aqueous Medium - Part 2

Enzyme-catalyzed ring-opening polymerization of lactones is a method of increasing interest for the synthesis of polyesters. In the present work, we investigated which changes in the structure of Candida antarctica lipase B (CaLB) shift the catalytic equilibrium between esterification and hydrolysis towards polymerization. Therefore, we present two concepts: (i) removing the glycosylation of CaLB to increase the surface hydrophobicity; and (ii) introducing a hydrophobic lid adapted from Pseudomonas cepacia lipase (PsCL) to enhance the interaction of a growing polymer chain to the elongated lid helix. The deglycosylated CaLB (CaLB-degl) was successfully generated by site-saturation mutagenesis of asparagine 74. Furthermore, computational modeling showed that the introduction of a lid helix at position Ala148 was structurally feasible and the geometry of the active site remained intact. Via overlap extension PCR the lid was successfully inserted, and the variant was produced in large scale in Pichia pastoris with glycosylation (CaLB-lid) and without (CaLB-degl-lid). While the lid variants show a minor positive effect on the polymerization activity, CaLB-degl showed a clearly reduced hydrolytic and enhanced polymerization activity. Immobilization in a hydrophobic polyglycidol-based microgel intensified this effect such that a higher polymerization activity was achieved, compared to the “gold standard” Novozym® 435