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Semi‐rational engineering of an amino acid racemase that is stabilized in aqueous/organic solvent mixtures
Author(s) -
Femmer Christian,
Bechtold Matthias,
Panke Sven
Publication year - 2020
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.27449
Subject(s) - chemistry , aqueous solution , pseudomonas putida , solvent , protein engineering , thermophile , biocatalysis , acetonitrile , organic chemistry , amino acid , enzyme , methanol , combinatorial chemistry , catalysis , reaction mechanism , biochemistry
Enzymes are industrially applied under increasingly diverse environmental conditions that are dictated by the efforts to optimize overall process efficiency. Engineering the operational stability of biocatalysts to enhance their half‐lives under the desired process conditions is a widely applied strategy to reduce costs. Here, we present a simple method to enhance enzyme stability in the presence of monophasic aqueous/organic solvent mixtures based on the concept of strengthening the enzyme's surface hydrogen‐bond network by exchanging surface‐located amino acid residues for arginine. Suitable residues are identified from sequence comparisons with homologous enzymes from thermophilic organisms and combined using a shuffling approach to obtain an enzyme variant with increased stability in monophasic aqueous/organic solvent mixtures. With this approach, we increase the stability of the broad‐spectrum amino acid racemase of Pseudomonas putida DSM 3263 eightfold in mixtures with 40% methanol and sixfold in mixtures with 30% acetonitrile.