BACKGROUND: Ultrasonication has been proposed as a promising technique for enzymatic transesterification. In contrast, excess ultrasonication causes enzyme inactivation. This paper describes enzymatic transesterification to produce fatty acid methyl ester (FAME) from rapeseed oil using Callera Trans L-TM and an original two-compartment reactor. The reactor was composed of a mechanically stirred compartment (ST) and an ultrasound irradiation compartment (US). The reaction solution was recirculated between the ST and the US. The enzyme was exposed to ultrasonication only in the US. The reactor system has the option to control the direct irradiation period of ultrasonication to soluble enzyme, regulated by the mean residence time in the US. RESULTS: The production of FAME with ultrasound irradiation gave a final yield of 91wt% after 15 h. The reaction rate was enhanced up to 2-fold through the use of the two-compartment reactor compared with liquid lipase catalyzed transesterification without any ultrasound treatment. The V-max with ultrasound irradiation was 2.3-fold higher than that of the ultrasound-free system, while the Km remained at almost the same level. The reaction rate and the conversion increased with shorter mean residence time in the US. CONCLUSION: The advantages of the two-compartment reactor were shown to produce biodiesel (FAME) resulting in acceleration of the enzyme reaction by ultrasound irradiation. Reaction enhancement was maximized by using a separate compartment of the reactor. A shorter mean residence time of reaction solution in the US and higher ultrasound power successfully realized a higher production rate of FAME. (c) 2016 Society of Chemical Industry