No abstract

In this study, we investigated the influence of different modes of magnetic mixing on effective enzyme activity of aspartate ammonia-lyase from Pseudomonas fluorescens immobilized onto epoxy-functionalized magnetic nanoparticles by covalent binding (AAL-MNP). The effective specific enzyme activity of AAL-MNPs in traditional shake vial method was compared to the specific activity of the MNP-based biocatalyst in two devices designed for magnetic agitation. The first device agitated the AAL-MNPs by moving two permanent magnets at two opposite sides of a vial in x-axis direction (being perpendicular to the y-axis of the vial); the second device unsettled the MNP biocatalyst by rotating the two permanent magnets around the y-axis of the vial. In a traditional shake vial, the substrate and biocatalyst move in the same direction with the same pattern. In magnetic agitation modes, the MNPs responded differently to the external magnetic field of two permanent magnets. In the axial agitation mode, MNPs formed a moving cloud inside the vial, whereas in the rotating agitation mode, they formed a ring. Especially, the rotating agitation of the MNPs generated small fluid flow inside the vial enabling the mixing of the reaction mixture, leading to enhanced effective activity of AAL-MNPs compared to shake vial agitation.

In this study, yeast strains were screened and immobilized in a form preserving the multifaceted biocatalytic activity. Immobilization of the silica-supported whole cells of various yeasts, such as Lodderomyces elongisporus, Pichia carsonii, Candida norvegica, and Debaryomyces fabryi, by sol−gel entrapment resulted in easy-to-handle biocatalysts that mediated efficiently different types of synthetic reactions. In the present study, the enantiotope selective reduction of prochiral ketones 1a−d and the acyloin condensation of benzaldehyde 3 were studied, representing two remarkably diverse types of biotransformation. The yeast cell biocatalystsin the presence of fresh or recovered NADH cofactorcould be applied for continuous-flow bioreduction of ketones 1a−d with moderate to good yields (20 to 92%) and excellent enantiomeric purity (>99%). Immobilized L. elongisporus and P. carsonii cells could also mediate acyloin condensation of benzaldehyde 3 in batch as well as in continuousflow mode. The switchable biocatalytic activity of the immobilized yeast cells was demonstrated by consecutive biotransformations under continuous-flow conditions involving reduction of phenylacetone 1a to (S)-phenylpropane-2-ol [(S)-2a] first, followed by conversion of benzaldehyde 3 to (R)-1-hydroxy-1-phenylpropan-2-one [(R)-4] and reduction of 1a to (S)-2a again by using the same packed-bed bioreactor.

A number of class I lyase-like enzymes, including aromatic ammonia-lyases and aromatic 2,3-aminomutases, contain the electrophilic 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) catalytic moiety. This study reveals that Pseudomonas fluorescens R124 strain isolated from a nutrient-limited cave encodes a histidine ammonia-lyase, a tyrosine/phenylalanine/histidine ammonia-lyase (XAL), and a phenylalanine 2,3-aminomutase (PAM), and demonstrates that an organism under nitrogen-limited conditions can develop novel nitrogen fixation and transformation pathways to enrich the possibility of nitrogen metabolism by gaining a PAM through horizontal gene transfer. The novel MIO enzymes are potential biocatalysts in the synthesis of enantiopure unnatural amino acids. The broad substrate acceptance and high thermal stability of PfXAL indicate that this enzyme is highly suitable for biocatalysis.

Aspartate ammonia-lyases (AALs) catalyze the non-oxidative elimination of ammonia from l-aspartate to give fumarate and ammonia. In this work the AAL coding gene from Pseudomonas fluorescens R124 was identified, isolated, and cloned into the pET-15b expression vector and expressed in E. coli. The purified enzyme (PfAAL) showed optimal activity at pH 8.8, Michaelis-Menten kinetics in the ammonia elimination from l-aspartate, and no strong dependence on divalent metal ions for its activity. The purified PfAAL was covalently immobilized on epoxy-functionalized magnetic nanoparticles (MNP), and effective kinetics of the immobilized PfAAL-MNP was compared to the native solution form. Glycerol addition significantly enhanced the storability of PfAAL-MNP. Inhibiting effect of the growing viscosity (modulated by addition of glycerol or glucose) on the enzymatic activity was observed for the native and immobilized form of PfAAL, as previously described for other free enzymes. The storage stability and recyclability of PfAAL-MNP is promising for further biocatalytic applications.