Purification, mofecular cloning and expression of the cDNA of bovine pregastric esterase

Abstract

In this work the preduodenal lipase of the calf, also called pregastric esterase (PGE) is studied. From the literature very little information on the catalytic mechanism of preduodenal lipases is available. Comparison of the biochemical properties and the primary structure of pregastric esterase, with those of other preduodenal and nonpreduodenal lipases, might contribute to an understanding of the catalytic mechanism of preduodenal Ii pases. In the first place a purification method for pregastric esterase was established. The enzyme was characterised and the influence of inhibitors binding to specific amino acids was studied. These data allowed determination of the functional groups essential for the lipolytic activity of pregastric esterase (Chapter 2). Secondly, a cDNA library of calf tongue tissue from the region containing the glands of von Ebner was prepared. The cDNA coding for pregastric esterase was amplified from this library by PCR, using primers based on conserved sequences in two other preduodenal lipases. After sequencing this cDNA, the nucleotide and deduced amino acid sequence was compared with other lipases (Chapter 3). Due to the ability of preduodenal lipases to act in an acidic environment, they are of great interest to facilitate fat digestion in patients suffering from pancreatic insufficiency. Therefore, the production of recombinant active pregastric esterase on an industrial scale would be very helpful. Furthermore because of increasing demands for crude pregastric esterase extracts, frequently used in the food industry, the recombinant enzyme could serve as substitute. To express functional pregastric esterase, its cDNA was cloned in expression vectors for the bacterium E. coli and the yeast Pichia Pastoris. The eukaryotic system has the advantage of carrying out post-translational modifications. To detect the recombinant enzyme, antibodies prepared against purified enzyme were used and the functionality of pregastric esterase was determined (Chapters 4 and 5).