000150788 001__ 150788
000150788 005__ 20251017144651.0
000150788 0247_ $$2doi$$a10.1002/pro.70040
000150788 0248_ $$2sideral$$a142860
000150788 037__ $$aART-2025-142860
000150788 041__ $$aeng
000150788 100__ $$aZeballos, Nicoll
000150788 245__ $$aLoop engineering of enzymes to control their immobilization and ultimately fabricate more efficient heterogeneous biocatalysts
000150788 260__ $$c2025
000150788 5060_ $$aAccess copy available to the general public$$fUnrestricted
000150788 5203_ $$aEnzyme immobilization is indispensable for enhancing enzyme performance in various industrial applications. Typically, enzymes require specific spatial arrangements for optimal functionality, underscoring the importance of correct orientation. Despite well‐known N‐ or C‐terminus tailoring techniques, alternatives for achieving orientation control are limited. Here, we propose a novel approach that tailors the enzyme surface with engineered His‐rich loops. To that aim, we first solve the X‐ray crystal structure of a hexameric alcohol dehydrogenase from Thermus thermophilus HB27 (TtHBDH) (PDB: 9FBD). Guided by this 3D structure, we engineer the enzyme surface with a new loop enriched with six His residues to control enzyme orientation. Molecular dynamics simulations reveal that the engineered loop's imidazole rings have greater solvent accessibility than those in native His residues, allowing for more efficient enzyme immobilization on certain metal chelate‐functionalized carriers. Using carriers functionalized with iron (III)‐catechol, the apparent Vmax of the immobilized loop variant doubles the immobilized His‐tagged one, and vice versa when both variants are immobilized on carriers functionalized with copper (II)‐imidodiacetic acid. His‐tagged and loop‐engineered TtHBDH show high operational stability reaching 100% bioconversion after 10 reaction cycles, yet the loop variant is faster than the His‐tagged one.
000150788 536__ $$9info:eu-repo/grantAgreement/ES/DGA-FEDER/B29-17R$$9info:eu-repo/grantAgreement/ES/DGA-FEDER/E34-R17$$9info:eu-repo/grantAgreement/ES/DGA/LMP139-21$$9info:eu-repo/grantAgreement/ES/DGA/LMP58-18$$9info:eu-repo/grantAgreement/EC/H2020/818089/EU/Artificial metabolic cells for biomanufacturing of bio-based chiral fine chemicals/METACELL$$9This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 818089-METACELL$$9info:eu-repo/grantAgreement/EC/H2020/829162/EU/Redesigning biocatalysis: Thermal-tuning of one-pot multienzymatic cascades by nanoactuation/HOTZYMES$$9This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No H2020 829162-HOTZYMES$$9info:eu-repo/grantAgreement/ES/MICINN AEI/PID2022-136362NB-I00$$9info:eu-repo/grantAgreement/ES/MICINN/PID2022-137973NB-I00
000150788 540__ $$9info:eu-repo/semantics/embargoedAccess$$aAll rights reserved$$uhttp://www.europeana.eu/rights/rr-f/
000150788 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/acceptedVersion
000150788 700__ $$aGinés-Alcober, Irene
000150788 700__ $$0(orcid)0000-0001-6815-6720$$aMacías-León, Javier
000150788 700__ $$aAndrés-Sanz, Daniel
000150788 700__ $$0(orcid)0000-0002-5838-0857$$aGonzález-Ramírez, Andrés Manuel
000150788 700__ $$aSánchez-Costa, Mercedes
000150788 700__ $$0(orcid)0000-0002-2202-3460$$aMerino, Pedro$$uUniversidad de Zaragoza
000150788 700__ $$0(orcid)0000-0002-3122-9401$$aHurtado-Guerrero, Ramón
000150788 700__ $$0(orcid)0000-0003-0031-1880$$aLópez-Gallego, Fernando
000150788 7102_ $$12013$$2765$$aUniversidad de Zaragoza$$bDpto. Química Orgánica$$cÁrea Química Orgánica
000150788 773__ $$g34, 2 (2025), e70040 [12 pp.]$$pProtein sci.$$tProtein science$$x0961-8368
000150788 8564_ $$s2605464$$uhttps://zaguan.unizar.es/record/150788/files/texto_completo.pdf$$yPostprint$$zinfo:eu-repo/date/embargoEnd/2026-01-22
000150788 8564_ $$s410148$$uhttps://zaguan.unizar.es/record/150788/files/texto_completo.jpg?subformat=icon$$xicon$$yPostprint$$zinfo:eu-repo/date/embargoEnd/2026-01-22
000150788 909CO $$ooai:zaguan.unizar.es:150788$$particulos$$pdriver
000150788 951__ $$a2025-10-17-14:36:35
000150788 980__ $$aARTICLE