Commercial extracts from oro-pharyngeal tissues of goats and kids have been used as the source of pregastric lipase and have been processed to yield partially purified samples of the primary pregastric lipase. The activity of these lipases against tributyrylglycerol has been determined over a range of pH and temperatures. Optimum pH conditions for pregastric lipase ranged from pH 5.6 to 6.5 for goats and from pH 5.5 to 6.2 for kids, respectively; the optimum temperature ranged from 43 to 60 degrees C. Optima for kid lipase extended slightly below pH 5.5 and higher than 60 degrees C; which were the limits of the test conditions. The enzymes were also used as catalysts for the hydrolysis of monoacid triglycerides (C4:0 to C12:0) at 40 degrees C and pH 6.5; activity was maximum against tributyrylglycerol (C4:0). Values for the Michaelis-Menten constant, increased as carbon chain length of the carboxylic moiety on the triglycerides increased, but values were identical for pregastric lipases of both goats and kids. Anhydrous milk fat was hydrolyzed by the commercial extracts of pregastric lipases of goats and kids, and the resulting profiles for free fatty acids were very similar to one another and to the corresponding profile for a commercial sample of Parmesan cheese. There appear to be no significant differences in activity between the enzyme preparations from goats and kids.
Folate-modified cholesterol-bearing pullulan (FA-CHP) was synthesized by the reaction of folic acid [.beta]-2-aminoethylamide and 4-nitrophenyl chloroformate-activated cholesterol-bearing pullulan. The substitution ratio was estimated as 10% per glucoside units. Several combinations of FA-CHP, unmodified cholesterol-bearing pullulan (CHP) and doxorubicin (DOX) mixtures were tested for cancer-selective cytotoxicity in vitro. A combination of FA-CHP/CHP/DOX of 1:4:0.02 (weight ratio) had aggressive and selective effects on human epidermoid cancer KB cells, known to express a high level of folate receptor, while the same nanoparticle mixture had a weak effect on the growth of normal human fetal lung fibroblast TIG-1-20 cells.
Pregastric lipases from kid (KPGL) and goat (GPGL) were purified from the commercial extracts by different chromatographic procedures. The total recovery of activity for both purification methods was ca. 10%, and the specific activities of KPGL and GPGL were 533 and 546 U/mg, respectively, at pH 6.5, 35°C for tributyrylglycerol (TBG) as substrate in a casein/lecithin emulsion. The purification factors were 130-and 76-fold for the goat and kid lipases, respectively. The purified lipases from kid and goat showed the same 50 kDa protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an identical sequence for the first 11 amino acids. The optimal pH for the lipases was within the pH range 6-7, with maximal activity at pH 6.5. The stability of the purified lipases was decreased dramatically at pH > 6.5, but was enhanced by the addition of albumin. JAOCS 75, 411-416 (1998).
The goat pregastric lipase-catalyzed esterification of isopropylidene glycerol with caproic acid, to form isopropylidene glycerol caproate, followed a ping pong bi bi mechanism incorporating an acyl-enzyme intermediate. The maximum rate was estimated to be 96 µmol min -1 mg -1 in isooctane at 35°C, and the Michaelis-Menten constants for isopropylidene glycerol and caproic acid were 0.23 and 0.32 M, respectively. The catalyzed rate also correlated well with the partition coefficient of caproic acid between the organic and aqueous phases. The results suggest that the desolvation energy of the substrate from the bulk medium to the active site of the enzyme dominates the reaction rate for the enzyme-catalyzed reaction in organic media.Paper no. J9049 in JAOCS 76, 845-851 (July 1999).
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