Semi‐rational engineering of an amino acid racemase that is stabilized in aqueous/organic solvent mixtures

Abstract

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.

Immobilization of an amino acid racemase for application in crystallization‐based chiral resolutions of asparagine monohydrate

“…This method generates reaction substrates highly enriched in unwanted enantiomer and therefore low amounts of enzyme are need [7]. The main limitation regarding the biocatalyst was stability in organic solvent mixtures, which has been recently showed to significantly improve by enzyme engineering [33]. In dynamic kinetic resolution, the racemization must be at least as fast as the enantioselective reaction for the coupling to be effective [34].…”

Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments

“…This has been successfully applied for the directed evolution of β-glucoronidase, 112 aminoacyl-tRNA synthetase, 57 and the multidrug transporter EmrE. 56 Another innovative workflow in microfluidics-based ultrahigh-throughput screening for enzyme activity employs i m m o b i l i z a t i o n o n s o l i d p a r t i c l e s (beads) 28,32,61,70,71,[107][108][109][110]140,226,248,259,261 and is shown in Figure 9C. A variety of different systems have been used in a fashion compatible to enzyme engineering.…”

“…Hollow-core polyelectrolyte-coated chitosan alginate microcapsules (HC-PCAMs) have been similarly endowed with selective permeability and used to demonstrate enrichment of a sortase (employing a large particle sorter (COPAS, complex object parametric analyzer and sorter) instead of FACS) . Alternative materials provide routes to producing hydrogel beads as microspheres: alginate can be solidified with cations on-chip (Figure ) − or by laminar jetting into a bath, − and polyacrylamide can be cross-linked. , Beads based on hydrogels and other materials (e.g., polystyrene or paramagnetic composites) can also be used as a template to generate near-monodisperse droplets that tightly wrap around the bead via vortexing − or pipetting through filter tips , into an oil phase, avoiding microfluidic devices altogether.…”

“…Another innovative workflow in microfluidics-based ultrahigh-throughput screening for enzyme activity employs immobilization on solid particles (beads) ,,,,,− ,,,,, and is shown in Figure C. A variety of different systems have been used in a fashion compatible to enzyme engineering.…”

“…61 (ii) Another technique to couple genotype and phenotype is based on monodisperse nL-sized hydrogels that can be formed by laminar jet breakup. 68 Hydrogels couple genotype and phenotype, for example, by retaining a fluorescent bacterial host, 70,71 enabling sorting by FACS or by gas formation inducing a density shift. 226 (iii) Reaction partners can also be displayed on DNA-carrying microbeads enabling the coupling of genotype and phenotype (microbead display).…”