Lipase (Candida sp. 99-125) was immobilized by a two-step process, adsorption and subsequent entrapment. The formation mechanism of immobilized lipase was investigated by SEM and EDS. With the support of diatomite, the internal network of immobilized lipase showed more porosity and was fully developed. The dimension of the network was more uniform in the present of gelatine. The maximum biodiesel yield was 92% at 40 degrees C in a 24 h reaction. The biodiesel yield was maintained at about 70% after 11 repeated batch reactions. This enzyme immobilization method has potential applications in biodiesel and other lipase catalyzed synthesis process. (C) 2016 Elsevier Ltd. All rights reserved.