Recombinant Expression And Application Of Fructose Valine Oxidase

In recent years, glycated hemoglobin in the blood (HbAlc)has been used as an important indicator of early diagnosis and prevention of diabetes, which can reflect blood glucose levels during a period in vivo.Fructosyl valine oxidase (FVO) can catalyze the oxidative decomposition of the glycated protein or glycated amino acid,and produce hydrogen peroxide, amino acid and other ingredients.Therefore, this enzyme can be used to achieve the enzymatic detection of glycated hemoglobin with colorimetric quantitative method,which Is widely used in the treatment of diabetes.In this paper, the fructose valine oxidase gene FVO was obtained in a synthetic way.Then expressed it in eukaryotic and prokaryotic.In eukaryotic expression,constructed the eukaryotic expressive vector Bacmid-pFastBacl-FVO,and Used Bac-to-Bac baculovirus expression system to carry out the homologous recombination, expressing the target protein in Sf9cells. After Western Blot test, got a treaty band near43kDa,which proved the successful expression.In prokaryotic expression,constructed the prokaryotic expression vector pET-15b-FVO,after sequenced correctly, induced the expression of recombinant bacteria and optimized expressive conditions,got the optimal indued temperature16℃,the best medium pH6.0,optimal induced concentration0.8mM, the optimal induced time24h, the optimal transfer volume1:20,the optimal bacteria OD1.0,the optimal induction speed180rpm.Purified the soluble target protein with the His-Binding-resin column,then got8mg of soluble fructosyl valine oxidase per liter of fermentation broth, the purity ware greater than85%.After purification, the enzyme activity of sample was measured, and its specific activity is2U/mg,the optimal temperature for the activity was30℃to37℃, the optimal pH was6.0to7.0.