Characterization of Type IV Carboxylate Reductases (CARs) for Whole Cell-Mediated Preparation of 3-Hydroxytyrosol

Fragrance and flavor industries could not imagine business without aldehydes. Processes for their commercial production raise environmental and ecological concerns. The chemical reduction of organic acids to aldehydes is challenging. To fulfill the demand of a mild and selective reduction of carboxylic acids to aldehydes, carboxylic acid reductases (CARs) are gaining importance. We identified two new subtype IV fungal CARs from CAR (CAR) and CAR (CAR) in addition to literature known CAR CAR. Expression levels were improved by the co-expression of GroEL-GroES with either the trigger factor or the DnaJ-DnaK-GrpE system. Investigation of the substrate scope of the three enzymes revealed overlapping substrate-specificities. CAR and CAR showed a preferred pH range of 7.0 to 8.0 in bicine buffer. CAR showed highest activity at pH 6.5 to 7.5 in MES buffer and slightly reduced activity at pH 6.0 or 8.0. CAR appeared to tolerate a wider pH range without significant loss of activity. Type IV fungal CARs optimal temperature was in the range of 25-35 °C. CAR showed a melting temperature (T) of 55 °C indicating higher stability compared to type III and the other type IV fungal CARs (T 51-52 °C). Finally, CAR was used as the key enzyme for the bioreduction of 3,4-dihydroxyphenylacetic acid to the antioxidant 3-hydroxytyrosol (3-HT) and gave 58 mM of 3-HT after 24 h, which correlates to a productivity of 0.37 g L h.