Bile salt-stimulated human milk lipase characterisation and kinetic studies
Chapter One begins with an introduction to enzymology and leads into a discussion on BSSL beginning with the physical properties and the source of the enzyme. The literature concerning the kinetic properties of BSSL is reviewed, followed by a discussion on how the structure of enzymes influences their catalytic activity and specificity. The use of X-ray crystallographic techniques is addressed as a means of elucidating the three dimensional structure of enzymes. The chemicals, apparatus and standard methodologies used in this present investigation are described in Chapter Two. The means by which kinetic data measured for an enzyme-substrate system are analysed and compared are also discussed. Chapter Three describes the purification of bile salt-stimulated human milk lipase (BSSL) from whole human milk. A detailed study of the activity of the purified protein has been conducted against both lipid and ester substrates in order to monitor the progress of the purification. A further determination of the physical properties of the protein has also been conducted. Results from these studies have identified the protein as BSSL. In Chapter Four the methods used for determining the partial amino acid sequence of the enzyme are described. This study has revealed interesting homologies with enzymes from other species. The sequence of that part of the enzyme which includes the active site has been determined and has been found to be identical to the consensus sequence found in the active site of pancreatic lipase, serine proteases and cholinesterases. It may therefore be postulated that the similarity observed for some of the kinetic behaviour of enzymes arises from homology in their amino acid sequences and, in particular, those portions of the protein comprising the active site. The use of kinetic isotope effects, to gain insight as to the mechanism of BSSL catalysed hydrolysis of lipid substrates, is the subject of Chapter Five. A mechanism has been proposed which explains the observed effects and takes into account information from the literature. The mechanism also incorporates findings made from the amino acid sequence study and the literature reports on the residues involved in catalysis. Chapter Six begins within a literature survey of detergent less microemulsions and continues with an account of the kinetic properties of BSSL in this new and novel medium in which enzymic reactions involving substrates of low water solubility may be conducted. The advantage of this medium is that it allows one to monitor the reaction by spectrophotometric means. In more traditional methods of assay of lipid substrates this is not normally possible. This advantage has also been exploited in the study of the kinetics of BSSL against triolein in reversed micelles, which is the subject of Chapter Seven. A detailed description is given of an FT-IR technique which allows one to monitor the course of reaction of biologically important substrates.