원문정보
초록
영어
The gene encoding β-carotene claving enzyme Blh from uncultured marine bacte rium 66A03, which cleaves β-carotene into two molecules of retinal, was codon optimization and then cloned and expressed in Escherichia coli. The expressed e nzyme was purified by His-tag affinity chromatography columns. The molecular mass of native enzyme was estimated as 68 kDa, to be of dimmer of 34 kDasub units. The optimum pH and temperature were determined to be 8.0 and 40 °C, r espectively. In thermostability experiments, the enzyme followed first-order kinet
ics of thermal inactivation, and the half-lives of the enzyme at 35, 40, 45, 50 an d 55°C were 17.61, 14.97, 12.47, 6.06 and 1.51 h, respectively. The Km and Vmax of the enzyme with β-carotene as substrate were 37 μM and 45 nmol retinal∙mg-1 min-1, respectively, values that correspond to a turnover number (kcat) of 3.6 min-1 and a catalytic efficiency (kcat / Km) of 97,000 M-1min-1.