Research work was objectively targeted to synthesize highly pure diacylglycerol (DAG) with glycerolysis of soybean oil in a solvent medium of t-butanol. Three commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM and Novozym 435) were screened, and Novozym 435 was the best out of three candidates. Batch reaction conditions of the enzymatic glycerolysis, the substrate mass ratio, the reaction temperature and the substrate concentration, were studied. The optimal reaction conditions were achieved as 6.23:1 mass ratio of soybean oil to glycerol, 40% (w/v) of substrate concentration in t-butanol and reaction temperature of 50 degrees C. A two-stage molecular distillation was employed for purification of DAG from reaction products. Scale-up was attempted based on the optimized reaction conditions, 98.7% (24h) for the conversion rate of soybean oil, 48.5% of DAG in the glycerolysis products and 96.1% for the content of DAG in the final products were taken in account as the results. (C) 2011 Elsevier Inc. All rights reserved.