Walla Brigitte, Dietrich Anna-Maria, Brames Edwin, Bischoff Daniel, Fritzsche Stefanie, Castiglione Kathrin, Janowski Robert, Niessing Dierk, Weuster-Botz Dirk
Biochemical Engineering, Department of Energy and Process Engineering, TUM School of Engineering and Design, Technical University of Munich, Boltzmannstraße 15, 85748 Garching, Germany.
Institute of Bioprocess Engineering, Department of Chemical and Biological Engineering, Friedrich-Alexander-Universität Erlangen-Nürnberg, Paul-Gordan-Straße 3, 91052 Erlangen, Germany.
Bioengineering (Basel). 2025 May 23;12(6):561. doi: 10.3390/bioengineering12060561.
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 (/ADH) and an ene reductase (ER1-L1,5) produced with . 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.
工业生物技术通过酶促降解，特别是使用叶枝堆肥角质酶（LCC）四重突变体ICCG，在相对温和的反应条件下为PET回收提供了一种潜在的生态解决方案。为了在使用合适的生产宿主进行异源基因表达后改进这种生物催化剂的高效下游加工，蛋白质结晶可作为有效的纯化/捕获步骤。最近的研究通过在两种同源醇脱氢酶（/ADH）和一种用……生产的烯还原酶（ER1-L1,5）中引入静电（和芳香）相互作用来提高蛋白质结晶。在本研究中，难以结晶的ICCG被用于晶体接触工程策略的应用，产生了ICCG突变体L50Y（ICCGY）。以前专注于在晶体接触处引入静电相互作用的赖氨酸-谷氨酸相互作用，这里将工程策略的适用性扩展到精氨酸-谷氨酸相互作用以提高结晶性，如ICCGY T110E所示。此外，通过在非晶体接触处或没有潜在相互作用伙伴的位点引入赖氨酸和谷氨酸作为阴性对照，证明了工程方法的原理。这些产生的突变体结晶情况与野生型蛋白相当，但并不优于野生型蛋白。如本研究所示，晶体接触工程成为一种合理增强蛋白质结晶的有前途的方法。这一进展可以显著简化生物技术下游加工，为研究和工业提供更有效的途径。
