Robust ω-Transaminases by Computational Stabilization of the Subunit Interface

Qinglong Meng, Nikolas Capra, Cyntia M. Palacio, Elisa Lanfranchi, Marleen Otzen, Luc Z. Van Schie, Henriëtte J. Rozeboom, Andy Mark W.H. Thunnissen, Hein J. Wijma, Dick B. Janssen*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

46 Citaten (Scopus)
228 Downloads (Pure)


Transaminases are attractive catalysts for the production of enantiopure amines. However, the poor stability of these enzymes often limits their application in biocatalysis. Here, we used a framework for enzyme stability engineering by computational library design (FRESCO) to stabilize the homodimeric PLP fold type I ω-transaminase from Pseudomonas jessenii. A large number of surface-located point mutations and mutations predicted to stabilize the subunit interface were examined. Experimental screening revealed that 10 surface mutations out of 172 tested were indeed stabilizing (6% success), whereas testing 34 interface mutations gave 19 hits (56% success). Both the extent of stabilization and the spatial distribution of stabilizing mutations showed that the subunit interface was critical for stability. After mutations were combined, 2 very stable variants with 4 and 6 mutations were obtained, which in comparison to wild type (Tm app = 62 °C) displayed Tm app values of 80 and 85 °C, respectively. These two variants were also 5-fold more active at their optimum temperatures and tolerated high concentrations of isopropylamine and cosolvents. This allowed conversion of 100 mM acetophenone to (S)-1-phenylethylamine (>99% enantiomeric excess) with high yield (92%, in comparison to 24% with the wild-type transaminase). Crystal structures mostly confirmed the expected structural changes and revealed that the most stabilizing mutation, I154V, featured a rarely described stabilization mechanism: namely, removal of steric strain. The results show that computational interface redesign can be a rapid and powerful strategy for transaminase stabilization.

Originele taal-2English
Pagina's (van-tot)2915-2928
Aantal pagina's14
TijdschriftACS Catalysis
Nummer van het tijdschrift5
StatusPublished - 6-mrt.-2020

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