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

Automated summary

Glycolate oxidase (CreGO) from Chlamydomonas reinhardtii was selected and its expression was improved through the use of GST, pET22b vector, and optimization of induction conditions. The fusion enzyme GST-Gly-Ser-Gly-CreGO-Y27S/V111G/V212R showed a six-fold increase in catalytic activity compared to the wild type. The oxidation of methyl glycolate was significantly enhanced using crude enzyme GST-Gly-Ser-Gly-CreGO-Y27S/V111G/V212R with catalase.