Enhancing the Catalytic Activity of Glycolate Oxidase from Chlamydomonas reinhardtii through Semi-Rational Design

Glycolate oxidase is a peroxisomal flavoprotein catalyzing the oxidation of glycolate to glyoxylate and plays crucial metabolic roles in green algae, plants, animals. It could serve as biocatalyst for enzymatic production glyoxylate, fine chemical with wide variety applications perfumery, flavor, pharmaceutical agrochemical industries. However, low catalytic activity native levels active enzyme heterologous expression limit its practical use industrial biocatalysis. Herein, from Chlamydomonas reinhardtii (CreGO) was selected through phylogenetic tree analysis, level soluble E. coli BL21(DE3) improved glutathione thioltransferase (GST), choice vector pET22b optimization induction conditions. The semi-rational design fusion GST-Gly-Ser-Gly-CreGO led superior variant GST-Gly-Ser-Gly-CreGO-Y27S/V111G/V212R kcat/Km value 29.2 s−1·mM−1, which six times higher than that wild type. In contrast GST-Gly-Ser-Gly-CreGO, 5 mg/mL crude together 25 μg/mL catalase catalyzed 300 mM methyl 8 h, increasing yield 50.4 93.5%.