화학공학소재연구정보센터
Biochemical and Biophysical Research Communications, Vol.458, No.4, 883-889, 2015
SUMO-fusion, purification, and characterization of a (+)-zizaene synthase from Chrysopogon zizanioides
An uncharacterized plant cDNA coding for a polypeptide presumably having sesquiterpene synthase activity, was expressed in soluble and active form. Two expression strategies were evaluated in Escherichia coli. The enzyme was fused to a highly soluble SUMO domain, in addition to being produced in an unfused form by a cold-shock expression system. Yields up to similar to 325 mg/L-1 were achieved in batch cultivations. The 6x-His-tagged enzyme was purified employing an Ni2+-IMAC-based procedure. Identity of the protein was established by Western Blot analysis as well as peptide mass fingerprinting. A molecular mass of 64 kDa and an isoelectric point of pI 4.95 were determined by 2D gel electrophoresis. Cleavage of the fusion domain was possible by digestion with specific SUMO protease. The synthase was active in Mg2+ containing buffer and catalyzed the production of (+)-zizaene (syn. khusimene), a precursor of khusimol, from farnesyl diphosphate. Product identity was confirmed by GC MS and comparison of retention indices. Enzyme kinetics were determined by measuring initial reaction rates for the product, using varying substrate concentrations. By assuming a Michaelis-Menten model, kinetic parameters of K-M = 1.111 mu M (+/- 0.113), v(max) = 0.3245 mu M min(-1) (+/- 0.0035), k(cat) = 2.95 min(-1), as well as a catalytic efficiency k(cat)/K-M = 4.43 x 10(4) M-1 s(-1) were calculated. Fusion to a SUMO moiety can substantially increase soluble expression levels of certain hard to express terpene synthases in E. coli. The kinetic data determined for the recombinant synthase are comparable to other described plant sesquiterpene synthases and in the typical range of enzymes belonging to the secondary metabolism. This leaves potential for optimizing catalytic parameters through methods like directed evolution. (C) 2015 Elsevier Inc. All rights reserved.