03 Fakultät Chemie

Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/4

Browse

Publication Type: search.filters.UBSTyp.ZeitschriftenartikelAuthor: search.filters.author.Nestl, Bettina M.

Search Results

Now showing 1 - 2 of 2
  • Thumbnail Image
    ItemOpen Access
    Purification and characterization of recombinant expressed apple allergen Mal d 1
    (2020) Kaeswurm, Julia A. H.; Nestl, Bettina M.; Richter, Sven M.; Emperle, Max; Buchweitz, Maria
    Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fastprotein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated.
  • Thumbnail Image
    ItemOpen Access
    Inverting the stereoselectivity of an NADH‐dependent imine‐reductase variant
    (2021) Stockinger, Peter; Borlinghaus, Niels; Sharma, Mahima; Aberle, Benjamin; Grogan, Gideon; Pleiss, Jürgen; Nestl, Bettina M.
    Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the (R)‐selective IRED from Myxococcus stipitatus (NADH‐IRED‐Ms) yielding a NADH‐dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and (S)‐selectivity in asymmetric reductions has yet been reported. Herein, we applied semi‐rational enzyme engineering to switch the selectivity of NADH‐IRED‐Ms. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2‐methylpyrroline compared to the wild‐type and afforded the (S)‐amine product with >99 % conversion and 91 % enantiomeric excess. We also report the crystal‐structures of the NADPH‐dependent (R)‐IRED‐Ms wild‐type enzyme and the NADH‐dependent NADH‐IRED‐Ms variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.