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Chapter 1 briefly introduces lipids and enzymes, followed by a more specific discussion of lipases. Relevant literature details are reviewed on conformational notations, lipase selectivity, the properties of milkfat lipids, and enzyme roles in lipid metabolism. The confusions and wide-ranging nature of this field of study are outlined.
In Chapter 2, chemicals, apparatus and instrumentation, computer software, and standard or basic methodologies are described. In particular, procedures relating to the purification and assay of the enzyme have been included. Synthesis of acyl propanediol substrates and general extraction techniques are given.
Chapter 3 describes two degrees of purification for lamb pregastric lipase. The first employed techniques including anion exchange chromatography and freeze-drying for the preparation of a stable and soluble material, suitable for carrying out precision kinetic studies. The efficiency of this partial purification was analysed, and the properties of the lipase- and esterase- active fractions were contrasted. The second purification sequence for LPGL allowed identification of the enzyme by N-terminal amino acid sequencing, and allowed confirmation of a MW of 51 kDa. Other proteins which were also identified included two esterases (by kinetic properties) and two metabolic proteins, calreticulin and serum albumin (by N-terminal sequencing). Suggestions were made on likely MW values for the two esterases.
Chapter 4 discusses the analysis of enzyme kinetic data, and the means of establishing parameters for the function of best fit. The history of pregastric lipase assays using tributyrylglycerol is briefly reviewed, and the effect of emulsion preparation is outlined. A full Michaelis-Menten equation characterisation of the activity of LPGL was carried out over a range of values of pH and temperature, such that the conditions of maximal enzyme activity were delineated. The total data were smoothed and presented as a 3-D surface plot, showing the Vmax of LPGL against tributyrylglycerol as a function of pH and T. It became evident that the catalysis was kinetically selective for mono-hydrolysis only, and porcine pancreatic lipase behaved differently under the same conditions.
Chapter 5 further investigates the kinetic properties of LPGL and examines the lipid chain-length preference of LPGL against a range of triacylglycerol substrates with differing fatty acids. The butyryl diesters of 1,2- and 1,3- propanediol were also investigated, and despite similar affinities, definite reactive preferences were observed. The hydrolysis was again observed to be rate-selective for the release of one fatty acid. The potential role of metal ions in enzyme activity is discussed. The effects of EDTA, as well as the level of associated metals, were assessed for the partially purified LPGL. Some other factors affecting activity are briefly examined and discussed.
Chapter 6 introduces and develops the use of 13C NMR spectroscopy for quantitative speciation of mixtures of the acyl esters of glycerol and propanediol. This included verification of spectrometer linearity, as well as linearity of the signal representation of each species. Also discussed is the choice of CHCl3 as the solvent for rapid and gentle extraction of aqueous emulsions. Confirmation of the shift patterns and shift assignments for these compounds is presented and extensively discussed in Appendix I.
Chapters 7 and 8 draw upon the extraction and spectroscopic methods of Chapter 6 to delineate the positional selectivity of LPGl-catalysed hydrolysis of, respectively, emulsified tributyrylglycerol and dibutyryl-1,2-propanediol. Development of a system rate parameter, and the use of varying rates of reaction, has allowed distinction of the products of catalysis from those of acyl transfer. From the primary product speciation, the positional preferences of the LPGL could be outlined.
Chapter 9 also used the methods of Chapter 6 to examine uncatalysed acyl transfer reactions in hydrolysis products. The general indications of the literature are discussed. Typical composition mixtures, extracted from catalysed reactions have been examined under similar conditions but in the absence of enzyme, in order to establish parameters for acyl transfer in some short-chain partially acylated compounds.
Chapter 10 uses optical rotation methods to conclude the specification of the stereoselectivity of LPGL with triacylglycerol substrates. There is evidence that the literature values of specific optical activity for 1,2-dibutyryl-sn-glycerol are too low, but it is, nonetheless, likely that LPGL exhibits a high degree of stereoselectivity. |
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