Pressurized CO2 as a carboxylating agent for the biocatalytic ortho-carboxylation of resorcinol

Abstract

The utilization of gaseous carbon dioxide instead of bicarbonate would greatly facilitate process development for enzyme catalyzed carboxylations on a large scale. As a proof-of-concept, 1,3-dihydroxybenzene (resorcinol) was carboxylated in the ortho-position using pressurized CO₂ (∼30-40 bar) catalyzed by ortho-benzoic acid decarboxylases with up to 68% conversion. Optimization studies revealed tight pH-control and enzyme stability as the most important determinants.

Fig. 1. (a) Enzyme-catalyzed de/carboxylation of resorcinol (1). Carboxylated product 2 is a mixture of regio-isomeric 2,6- (2,6-dhba, 2a) and 2,4-dihydroxybenzoic acid (2,4-dhba, 2b) with a ratio of 3 : 4; (b) CO₂ pressure dependence of the carboxylation of 1 using 2,3-DHBD_Ao; (c) carboxylation activity of decarboxylases with CO₂ (30 bar) using 1 as a substrate; (d) stopped-flow measurements of the decarboxylation of 2,6-dhba (2a) with pressure-pretreated (≤1.5 kbar) 2,3-DHBD_Ao and 2,6-DHBD_Rs.

Fig. 2. (a) Decarboxylation activity of CO₂ pressure pretreated (10, 40 and 50 bar) 2,6-DHBD_Rs using 2a as a substrate; (b) DSF comparison of the pH dependent melting temperature of 2,3-DHBD_Ao and 2,6-DHBD_Rs in multicomponent buffer (pH 4 to 9) and citrate buffer (pH 4 to 5); (c) Influence of TRIS-HCl buffer concentration on the carboxylation of 1 using 2,6-DHBD_Rs and 2,3-DHBD_Ao under 50 bar CO₂ pressure.