Characterization of the diacylglycerol acyltransferase activity in the lipid body fraction from an oleaginous fungus

Abstract

Diacylglycerol acyltransferase (DGAT) was examined as a key enzyme for triacylglycerol (TG) accumulation of an oleaginous fungus, Mortierella ramanniana var. angulispora. Subcellular fractionation of the fungus showed that DGAT activity was highest in the lipid body fraction, which occupied 77% of the recovered DGAT activity. DGAT activity in the lipid body fraction was much higher than that in the membrane fraction in terms of both total activity and specific activity. Similar results were obtained with another homogenization method. After repeated washing of the lipid body fraction, DGAT activity in the lipid body fraction was still larger than those in other fractions. The lipid body fraction contained larger amounts of lipids, especially TG and diacylglycerol. Moreover, the lipid body fraction had a specific set of polypeptides at 24, 29, and 59 kDa. These analyses of lipid and polypeptide composition suggested that the lipid body fraction represented a specific intracellular structure, presumably the lipid body. DGAT activity in the lipid body fraction had a similar characteristics to that in the membrane fraction, although some differences in sensitivity to SH-reagents were observed. Increase in DGAT activity in the lipid body fraction was observed when lipids were accumulated in the fungus. On the other hand, DGAT activity in the lipid body fraction decreased when lipids were accumulated with an increase in carbon to nitrogen ratio in media.