Application of a Rational Crystal Contact Engineering Strategy on a Poly(ethylene terephthalate)-Degrading Cutinase

Walla B, Dietrich AM, Brames E, Bischoff D, Fritzsche S, Castiglione K, Janowski R, Niessing D, Weuster-Botz D (2025)

Publication Type: Journal article

Publication year: 2025

Journal

Bioengineering MDPI

Book Volume: 12

Article Number: 561

Journal Issue: 6

DOI: 10.3390/bioengineering12060561

Abstract

Industrial biotechnology offers a potential ecological solution for PET recycling under relatively mild reaction conditions via enzymatic degradation, particularly using the leaf branch compost cutinase (LCC) quadruple mutant ICCG. To improve the efficient downstream processing of this biocatalyst after heterologous gene expression with a suitable production host, protein crystallization can serve as an effective purification/capture step. Enhancing protein crystallization was achieved in recent studies by introducing electrostatic (and aromatic) interactions in two homologous alcohol dehydrogenases (Lb/LkADH) and an ene reductase (NspER1-L1,5) produced with Escherichia coli. In this study, ICCG, which is difficult to crystallize, was utilized for the application of crystal contact engineering strategies, resulting in ICCG mutant L50Y (ICCGY). Previously focused on the Lys-Glu interaction for the introduction of electrostatic interactions at crystal contacts, the applicability of the engineering strategy was extended here to an Arg-Glu interaction to increase crystallizability, as shown for ICCGY T110E. Furthermore, the rationale of the engineering approach is demonstrated by introducing Lys and Glu at non-crystal contacts or sites without potential interaction partners as negative controls. These resulting mutants crystallized comparably but not superior to the wild-type protein. As demonstrated by this study, crystal contact engineering emerges as a promising approach for rationally enhancing protein crystallization. This advancement could significantly streamline biotechnological downstream processing, offering a more efficient pathway for research and industry.

Authors with CRIS profile

Stefanie Fritzsche Lehrstuhl für Bioverfahrenstechnik Kathrin Castiglione Lehrstuhl für Bioverfahrenstechnik

Involved external institutions

Technische Universität München (TUM) DE Germany (DE) Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (HMGU) / Helmholtz Munich DE Germany (DE)

How to cite

APA:

Walla, B., Dietrich, A.M., Brames, E., Bischoff, D., Fritzsche, S., Castiglione, K.,... Weuster-Botz, D. (2025). Application of a Rational Crystal Contact Engineering Strategy on a Poly(ethylene terephthalate)-Degrading Cutinase. Bioengineering, 12(6). https://doi.org/10.3390/bioengineering12060561

MLA:

Walla, Brigitte, et al. "Application of a Rational Crystal Contact Engineering Strategy on a Poly(ethylene terephthalate)-Degrading Cutinase." Bioengineering 12.6 (2025).

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