关键词: 海藻酸钠, 咖啡酸-3-O-甲基转移酶, 固定化

Abstract: The recombinant Chuanxiong caffeic acid-3-O-methyltransferase (LCCOMT) was induced by IPTG from E.coli BL21 (DE3) containing pET28a-LCCOMT,and purified by Ni²⁺ affinity column chromatography and identified by mass spectrometry.The recombinant LCCOMT was immobilized by calcium alginate gel.The optimal immobilization conditions,including optimum temperature,pH value,Km and Vmax,and the effect of reaction batch on the activity of immobilized enzyme were investigated by single factor experiment.The determined conditions were as follows∶CaCl₂ concentration at 2.5 g/L,sodium alginate concentration at 1.5%,immobilization time at 2 h,mass ratio of carrier to enzyme at 40 000∶1.According to the orthogonal experiment,The optimal conditions were as follows∶CaCl₂ concentration at 2.5 g/L,sodium alginate concentration at 2%,immobilization time at 1 h,mass ratio of carrier to enzyme at 35 000∶1.In this condition,the recovery rate of immobilized enzyme was 75.43%.Compared with the free enzyme,the optimum catalytic temperature and pH of immobilized enzyme increased by 0 ℃ and 0.5,and reached at 37.5 ℃ and 7.5,respectively.Compared with free enzyme,Km and Vmax increased by 0.4 and 0.74,respectively.The immobilized enzyme maintained 50% of original enzyme activity after 6 times for continuous using.

Key words: calcium alginate gel, caffeic acid-3-O-methyltransferase, immobilization