Overexpression and the enzymatic properties of the recombinant 4-coumarate: coenzyme A ligase, a key enzyme in lignin biosynthesis pathway

摘要

The investigation for the overexpression and the enzymatic properties of the Pm4CLl of Pinus mas-soniana Lamb were performed for the first time. The Pm4CLl cDNA was overexpressed in Escherichia coli Rosetta-garni cells. The recombinant Pm4CLl proteinexpression was most effective when it was induced with 1 mM isopropyl a-D-thiogalactopyranoside for 2 h at 30 deg C. The 6x His-tagged recombinant PmACLl protein about 60 kDa was purified by agarose coupled with Ni~(2+)-NTA affinity chromatography. The optimal pH and temperature of the reaction were 7.6 and 35 deg C, respectively. The K_m and Viviax values of the purified Pm4CLl enzyme for 4-coumaric acid were calculated to be 64.35 muM and 30.92 muM min~(-1) mu g~(-1) respectively.