A comprehensive study of critical parameters in the pig pancreatic lipase (PPL) catalysed transesterification of sunflower oil to novel biofuels integrating glycerol into their composition is reported. The influence of oil/alcohol ratio, temperature, quantity of enzyme and water added and pH have been investigated. The enzymatic activity of PPL was found to be greatly influenced by the pH, reaching notable activities at high pH values (10-12), in contrast to other lipases. The addition of small quantities of NaOH (up to 0.1 mL) as coadjuvant in the transesterification reaction enhances the activity of the enzymes. This remarkable behaviour, reported for the first time, may pave the way for the utilisation of these relatively cheap enzymes in large scale commercial biodiesel production. Besides, a novel biofuels containing glycerol into their composition as mono- and diglycerides using PPL as biocatalyst has been developed. (C) 2010 Elsevier Ltd. All rights reserved.