Preparation And Properties Of Adenylate Kinase-spidroin Biomimetic Complexes

The research of enzyme immobilization technology and its carrier materials has always been the focus in the field of enzymology.Recombinant spider silk has been studied as the immobilized carrier material for its high mechanical stability and biodegradability.In this paper,adenylate kinase（adk）as a model protein was linked to the spidroin sequence.The adk gene was infused with Rep and CT domain,from Aciniform and Minor ampullate spidroin in spider Araneus ventricosus,respectively,to construct a biomimetic spidroin,adk-RC.The conditions of inductive expression and purification of adk-RC and adk were investigated.The optimal expression conditions of adk-RC was 0.1 m M IPTG addition at16°C for 18 h,while that of adk was 0.1 m M IPTG addition at 25℃for 10 h.After purification by Immobilized Metal ion Affinity Chromatography（IMAC）,the purified adk-RC obtained in elution by Tris-HCl buffer with 75 m M imidazole,which was lower than adk.After further ammonium sulfate precipitation,a high-purity adk-RC obtained.Activity of adenylate kinase was detected by High Performance Liquid Chromatography（HPLC）of purified adk-RC.Enzymatic properties of adk and adk-RC were further detected by by microplate assay.The optimal temperature and p H of adk-RC was 37℃and 7.5,while that of adk was 50℃and7.5.Specific activity of adk-RC,8.06 mol·min^-1·mmol^-1,was 3.86 times more than that of adk,2.09 mol·min^-1·mmol^-1.Both of adk-RC and adk were stable at 50℃for 48 h,and maintained most of activity at p H of 9.0 and 10.0,respectively.The overall thermal and p H stability of adk-RC were lower than that of adk.Although more fragile treated by divalent metal ions and organic reagents,specific activity of adk-RC was better than that of adk.After freeze drying,the remained enzyme activity of adk-RC was 2 times more than that of adk.Despite more decrease for relative activity,after freeze drying,specific activity of adk-RC was still better than adk.The kinetic reaction studies was investigated that the V_maxvalue of adk-RC and adk was 12.346μmol·L^-1·min^-1and 37.453μmol·L^-1·min^-1,respectively,and the K_mvalue of both was 0.227 mmol·L^-1and 0.888 mmol·L^-1,respectively.Moreover,the catalytic efficiencies(k_cat/K_m)were determined for adk-RC and adk were 12427.22 L·mmol^-1·min^-1and34882.94 L·mmol^-1·min^-1,respectively,which indicated that both substrate affinity and catalytic efficiency of adk-RC were increased.A new strategy of immobilizing enzymes was proposed and a biomimetic spidroin,adk-RC,was successfully constructed,expressed and purified.The results of adk-RC of enzymatic properties suggested that the strategy of enzyme immobilization is a potential method,which can be used in the industrial field,as a new technology for immobilizing enzymes.