스트레스에 따른 Vibrio anguillarum의 β-ketoacyl-ACP reductase (Fab G)의 발현과 생화학적 특성

Abstract

Fatty acid biosynthasis is the creation of fatty acids from acetyl-CoA and malonyl-CoA precursors through the action of enzymes named fatty acid biosynthases. It is an important part of the lipogenesis process, which - together with glycolysis - stands behind creation fats from blood sugar in living organism. The genes encoding several key fatty acid biosythetic enzymes (called the fab cluster) are clustered in the order plsX - fabH - fabD - fabG - acpP - fabF. There are two kinds of fabG in vibrio anguillarum and they are present in chromosomal DNA Ⅰ and Ⅱ respectively. The fabG1 in chromosomal DNA Ⅰ is known as fab cluster and fabG2 gene in chromosomal DNA II is known as OSDR which is down regulated protein under stress conditions. In this study, two kind of fabG gene from Vibrio anguillarum were cloned in pET-22b(+) vector and the fabG protein was overexpressed in Escherichia coli BL21 (DE3). SDS-PAGE demonstrated that a protein correspond to the fabG was overexpressed and migrated to a molecular mass of 27 KDa. Additionally, we measured fabG activity to determine whether our sequence analysis correctly predicted the function of the fabG protein. The activity of fabG1 showed 10 times higher than fabG2 when Acetoacetyl-CoA (AAC) and Ethylacetoacetate(EAA ) used as substrates.Expression under various stress condition revealed that the activity of fabG2 was reduced. And expression of fabG1 was stable at 1M NaCl, pH5, pH10 whereas expression at 37 and 25℃ was reduced which was confirmed by RT-PCR.