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Polymers 2018, 10(5), 524; https://doi.org/10.3390/polym10050524

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

1
DWI—Leibniz Institute for Interactive Materials and Institute of Biotechnology, RWTH Aachen University, Forckenbeckstraße 50, D-52056 Aachen, Germany
2
DWI—Leibniz Institute for Interactive Materials and Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Forckenbeckstraße 50, D-52056 Aachen, Germany
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Received: 10 April 2018 / Revised: 26 April 2018 / Accepted: 10 May 2018 / Published: 14 May 2018
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Abstract

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. View Full-Text
Keywords: Candida antarctica lipase B; enzymatic ring-opening polymerization; enzyme engineering; microgel; immobilization Candida antarctica lipase B; enzymatic ring-opening polymerization; enzyme engineering; microgel; immobilization
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Höck, H.; Engel, S.; Weingarten, S.; Keul, H.; Schwaneberg, U.; Möller, M.; Bocola, M. Comparison of Candida antarctica Lipase B Variants for Conversion of ε-Caprolactone in Aqueous Medium—Part 2. Polymers 2018, 10, 524.

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