Efficient Oxidation of Methyl Glycolate to Methyl Glyoxylate Using a Fusion Enzyme of Glycolate Oxidase, Catalase and Hemoglobin

Abstract

Possessing aldehyde and carboxyl groups, glyoxylic acid and its ester derivatives serve as platform chemicals for the synthesis of vanillin, (R)-pantolactone, antibiotics or agrochemicals. Methyl glycolate is one of the by-products in the coal-to-glycol industry, and we attempted its value-added use through enzymatic oxidation of methyl glycolate to methyl glyoxylate. The cascade catalysis of glycolate oxidase from Spinacia oleracea (SoGOX), catalase from Helicobacter pylori (HpCAT) and hemoglobin from Vitreoscilla stercoraria (VsHGB) was firstly constructed, despite poor catalytic performance. To enable efficient oxidation of methyl glycolate, eight fusion enzymes of SoGOX, HpCAT and VsHGB were constructed by varying the orientation and the linker length. The fusion enzyme VsHGB-GSG-SoGOX-GGGGS-HpCAT was proved to be best, which reaction yield was 2.9 times higher than that of separated enzymes. The enzyme SoGOX was further subjected to directed evolution and site-saturation mutagenesis. The reaction yield of the resulting variant M267T/S362G was 1.9 times higher than that of the wild type. Then, the double substitution M267T/S362G was integrated with fusion expression to give the fusion enzyme VsHGB-GSG-SoGOXmut-GGGGS-HpCAT, which crude enzyme was used as biocatalyst. The use of crude enzyme virtually eliminated side reactions and simplified the preparation of biocatalysts. Under the optimized conditions, the crude enzyme VsHGB-GSG-SoGOXmut-GGGGS-HpCAT catalyzed the oxidation of 200 mM methyl glycolate for 6 h, giving a yield of 95.3%. The development of efficient fusion enzyme and the use of its crude enzyme paved the way for preparative scale application on enzymatic oxidation of methyl glycolate to methyl glyoxylate.