BIOCHEMICAL-CHARACTERIZATION OF PURIFIED ZETA-CAROTENE DESATURASE FROM ANABAENA PCC-7120 AFTER EXPRESSION IN ESCHERICHIA-COLI

A novel enzyme, zeta-carotene desaturase from the cyanobacterium Anaba ena, which catalyzes the last two steps in a series of desaturations, was overexpressed in Escherichia coli. For the first time, this allowe d the purification of this enzyme and subsequent enzyme kinetic studie s. The enzyme was solubilized from the E. coli membranes by Chaps and purified to homogeneity by ammonium sulfate precipitation, ion-exchang e and hydrophobic interaction chromatography. The correct translationa l start was confirmed by N-terminal protein sequencing. Substrates for zeta-carotene desaturase apart from zeta-carotene are those carotenes which partially resemble the latter, like neurosporene and beta-zeaca rotene yielding lycopene and gamma-carotene, respectively as reaction products. Also cis isomers like pro-zeta-carotene were converted to th e correspondiong products. K-m values of 10 mu M were determined for b oth substrates zeta-carotene and neurosporene. The enzyme was inhibite d to some extent by the experimental herbicides J852 and LS80707 and a lso by diphenylamine which is a well-known inhibitor of the bacterial- type phytoene desaturase.