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 Pm4CL1 of Pinus massoniana Lamb were performed for the first time. The Pm4CL1 cDNA was overexpressed in Escherichia coli Rosetta-gami™ cells. The recombinant Pm4CL1 protein expression was most effective when it was induced with 1 mM isopropyl α-d-thiogalactopyranoside for 2 h at 30°C. The 6× His-tagged recombinant Pm4CL1 protein about 60 kDa was purified by agarose coupled with Ni2+-NTA affinity chromatography. The optimal pH and temperature of the reaction were 7.6 and 35°C, respectively. The K m and V Max values of the purified Pm4CL1 enzyme for 4-coumaric acid were calculated to be 64.35 μM and 30.92 μM min−1 μg−1, respectively.