Influence of the hydrostatic pressure and pH on the asymmetric 2‐hydroxyketone formation catalyzed by Pseudomonas putida benzoylformate decarboxylase and variants thereof

Abstract

Benzoylformate decarboxylase (BFD) from Pseudomonas putida is a thiamine diphosphate-dependent (ThDP) enzyme that catalyzes the asymmetric C--C bond formation to (S)-2-hydroxypropiophenone [(S)-HPP] starting from benzaldehyde and acetaldehyde. The enantioselectivity of BFD was shown to be a function of temperature and substrate concentration. It can additionally be changed by site-directed mutagenesis on hot spot positions in the active site. In this article, we present the effect of hydrostatic pressure up to 250 MPa on the enantioselectivity for the recombinant wtBFD as well as for the variants BFD F464I, BFD A460I, and BFD A460I-F464I. A general tendency toward lower amounts of (S)-HPP could be observed at increasing pressures. For two of these variants an increase in pressure even caused an inversion in the enantioselectivity and thus increasing enantiomeric excesses, respectively. A pressure-induced increase in enantioselectivity could therefore be observed for the first time in biocatalysis to the best of our knowledge. Furthermore, the pH is shown to be a parameter that also significantly influences the enantioselectivity of the reaction mentioned above.

Immobilization and characterization of benzoylformate decarboxylase from Pseudomonas putida on spherical silica carrier

“…Influence of pressure on the enantioselectivity of the immobilized enzyme A significant pressure effect on the enantioselectivity of the BFD A460I-F464I variant was found in previous investigations. With increasing pressure, a general tendency towards higher amounts of (R)-2-HPP could be observed [20]. While the previous investigations were done with free enzyme, in this work the influence of pressure on the enantioselectivity of the carrier bound enzyme was investigated.…”

“…Furthermore, a change of selectivity can be the result of steric effects through pressure-induced structural changes. In previous investigations, we found a significant pressure effect on the enantioselectivity of the BFD A460I-F464I variant [20]. Whereas the previous investigations were done with free enzyme, in this work the influence of pressure on the enantioselectivity of the carrier-bound enzyme was investigated.…”

“…Fermentation of transformed Escherichia coli SG13009 cells hosting the BFD plasmids was carried out as described in detail by Berheide et al [20]. The enzyme was purified as described by Siegert et al [21] but using a Q-Sepharose FF column (500 mL bed volume) at pH 7.5.…”

Influence of reaction conditions on the enantioselectivity of biocatalyzed C–C bond formations under high pressure conditions

“…In Berheide et al (2010) we have recently demonstrated that hydrostatic pressure and pH are important factors to influence the enantioselectivity of BFD-catalyzed C-C bond formation yielding 2-HPP. As a general trend we observed an increase in (R)-2-HPP synthesis, consequently a decrease in the ee of (S)-2-HPP under high pressure conditions up to 250 MPa.…”

“…To sum up, besides site-directed mutagenesis, temperature and substrate concentration were shown as parameters affecting the enantioselectivity of BFD. Recently, Berheide et al (2010) has showed that hydrostatic pressure is also able to influence the BFD-catalyzed asymmetric C-C bond formations.…”