Molecular Modification Of Glycolate Oxidase And Its Application In The Synthesis Of Methyl Glyoxylate

Methyl glyoxylate serves as an important intermediate in the synthesis of redipavir,a novel therapeutic drug of gene type I hepatitis C infection.It can also be used for the synthesis of D-pantolactone,one of the precursors in the synthesis of D-pantothenic acid.The chemical production of methyl glyoxylate requires high temperature and high pressure and often cause over-oxidation.In contrast to the chemical methods,the biological methods are milder and greener,and avoid the side reactions,demonstrating great potentials in the synthesis of methyl glyoxylate.Glycolate oxidase?GO,EC 1.1.3.15?can catalyze the oxidation of methyl glycolate to methyl glyoxylate.Usually,the native enzymes cannot meet the needs of production,e.g.,poor catalytic activity for specific substrate,whereas the glycolic acid oxidase with high activity is critical for the industrial production of methyl glyoxylate.In this study,the spinach-derived glycolate oxidase gene was heterologously expressed in E.coli,which was engineered to improve catalytic activity.Furthermore,the fusion expression was fulfilled to enhance the catalytic activity and promote the soluble expression.The catalase,hemoglobin and glycolate oxidase were brought together,in which the orientations of the enzymes and the linker peptides were optimized.Finally,a multifunctional fusion enzyme with high catalytic activity was obtained and used for the enzymatic synthesis of methyl glyoxylate.?1?The glycolate oxidase gene was amplified by error-prone PCR method,the PCR products was inserted into the expression vector p ET-28a,and then the resulting recombinant plasmids were introduced into E.coli BL21?DE3?competent cells.The recombinant cells expressing GO mutants were screened using Fe²⁺color reagent,and verified by liquid chromatograp,causing the mutant M267S/S362G with significantly improved catalytic activity.The two mutation sites were subjected to site-directed saturation mutation,and the superior combined mutant M267T/S362G was obtained,which catalytic activity was 1.78 times higher than that of the original enzyme.?2?In the fusion expression of the genes encoding glycolate oxidase GO,catalase CAT and hemoglobin Vs HGB,the orientations of hemoglobin-glycolate oxidase-catalase and catalase-glycolate oxidase-hemoglobin and the linker peptides of GSG and GGGGS were investigated,resulting in eight fusion genes constructed by overlapping PCR extension splicing method.And all eight fusion genes were successfully expressed in E.coli cells,which showed glycolate oxidase activity.In SDS-PAGE analyses,the bands corresponding to the fusion enzymes were consistent with the theoretical size of 112 k Da.The fusion enzymes was tested to catalyze the oxidation of methyl glycolate to methyl glyoxylate.In comparison with the single enzyme of glycolate oxidase,all eight fusion enzymes resulted in higher yields,of which the fusion enzyme Vs HGB-GSG-GO-GGGGS-CAT showed the highest catalytic activity.After 4 h reaction,the fusion enzyme Vs HGB-GSG-GO-GGGGS-CAT catalyzed the synthesis of methyl glyoxylate with a yield of 67.8%,which was 5.29 times higher than that of the single glycolate oxidase.?3?A fusion enzyme with mutant M267T/S362G(GO^Mut),catalase and hemoglobin were constructed,The induction conditions of the mutant fusion enzyme Vs HGB-GSG-GO^Mut-GGGGS-CAT were optimized.When the IPTG concentration was 0.6 m M and the induction temperature was 20?,the activity of induced expression of glycolate oxidase was up to 0.1035 U/m L.The crude extracts from the E.coli cells expressing the fusion enzyme Vs HGB-GSG-GO^Mut-GGGGS-CAT were used as biocatalyst.Using methyl glycolate as a substrate,various catalytic conditions including reaction time,oxygenation rate,reaction temperature,buffer p H were explored on the effect of methyl glyoxylate yield.The optimal reaction conditions were determined as follows:the oxygen rate was 1 L/h,the reaction temperature was15?,the buffer p H was 8.0,and the reaction time was 6 h.Under optimized reaction conditions,the oxidaiton of 200 m M methyl glycolate led to the yield of 95.37%.