| Methionine-lyase is a kind of multifunctional enzyme,which can catalyze various substances such like L-methionine, L-cysteine, and homocysteine. It can be widely used in adjuvant treatment of cancer and cardiovascular disease diagnosis.The Pichia pastoris expression system has been constructed and the pure methionine lyase has been obtained successfully, meanwhile exploring the enzymatic properties and the application of the test kit.The methionine lyase gene has been cloned, then constructed the Pichia pastoris expression vector pPIC9K-mg11, after sequencing and transforming into GS115, high-copy recombinant strains were screened by using the mRNA of trichomonas vaginalis as a template.Through Inducibling the expression of recombinant proteins, optimizing the expression conditions, the best pH value is6.0, optimum temperature for induction is28℃, the best dosage is0.5%methanol,the purified protein purity greater than85%recovery rate of greater than52.8%purification, per liter of fermentation broth can obtain60.42mg of recombinant protein.For L-methionine, L-cysteine and homocysteine as substrate,the specific activity of enzyme were0.77μmol/min/mg,0.83476μmol/min/mg,1.19863μmol/min/mg; the Km values were0.432mM,1.98mM,2.36mM. Optimal conditions for activity of enzyme is from35℃to37℃, the optimal pH value is from6.0to7.0.There are no different in trial test results with the imported kit qualitatively.It lay a solid foundation for localization of homocysteine detection kit and the adjuvant treatment of cancer clinical and the early diagnosis and prevention of cardiovascular disease. |
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