Study On Improving The Catalytic Activity Of Heparin Smlgate 3-O-Sulfotransferase By Tational Design

Heparin drugs are commonly used in clinical anticoagulant drugs at present,how to efficiently synthesize heparin anticoagulant drugs through biosynthesis has always been a concern of people.3-oxygen-sulfotransferase I(3-OST-1)is a necessary tool enzyme for the synthesis of heparin sulfate with anticoagulant activity.Therefore,increasing the expression of 3-OST-1 enzyme,improving the activity of 3-OST-1 enzyme and expanding its substrate spectrum have far-reaching significance for the production of heparin drugs and the research and development of related drugs.In this study,rational design strategy was adopted to analyze the binding mode between 3-OST-1 and substrate through molecular docking and mutation energy simulation calculation,and 11 mutation strategies were designed to express the wild type and mutant strains of 3-OST-1 recombinant protein through Escherichia coli expression system.The soluble and active 3-OST-1 recombinant protein wild type and mutant strain were successfully expressed.The purification and enzymatic activity of the expressed product were studied.It was found that the wild-type and mutant strains of 3-OST-1 recombinant protein were expressed under the conditions of0.5 mM IPTG and 20 h induction at 16 ℃.After that,the purified recombinant protein was quantified and the enzyme activity was determined.It was found that the enzyme amount and enzyme activity of the mutant strains of 3-OST-1recombinant protein were increased 2-4 times compared with that of the wild type,and the enzyme activity was also increased tens of times.In conclusion,the prokaryotic system suitable for 3-OST-1 expression was successfully constructed in this study,and the enzyme catalytic activity of 3-OST-1 was improved through rational design,which is of great significance for improving the production efficiency of heparin drugs and exploring the way of heparin biosynthesis.