Some new reactive polymers for the immobilization of enzymes.
From poly(vinyl alcohol) precursors, various reactive carriers for the immobilization of enzymes were synthesized. As insoluble starting polymers, the following products were used: poly(vinyl alcohol), gels crosslinked with terephthalaldehyde, hydrolyzed beads of crosslinked poly(vinyl acetate), poly(vinyl acetate-co- ethylene) tubes coated with poly(vinyl alcohol), and poly(vinyl alcohol)-containing synthetic pulp. Reactive groups introduced into these carriers or methods for their activation included the diazonium- and isothiocyanato group, and the glutardialdehyde-, BrCN, 2, 4, 6-trichloro-s-triazien, and p-benzoquinone methods. Furthermore, SH-specific reactive groups such as N-substituted maleimide groups or activated mixed disulfides with 2-thiopyridyl groups could be introduced into PVA-polymers. Enzymes like hydrolases (e.g. papain, trypsin, chymotrypsin, urease), oxidoreductases (e.g. glucose oxydase, catalase, glucose-6-phosphate dehydrogenase) as well as the example of transferase hexokinase coimmobilized with glucose-6-phosphate dehydrogenase, were immobilized by reactive poly(vinyl alcohol) carriers. The properties of the immobilized enzymes were investigated.
An experimental verification of the theory of diffusion limitation of immobilized enzymes.
Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) was covalently bound to Sepharose beads. Kinetic measurements with the substrate p-nitrophenyl phosphate gave the following results. Effectiveness factors were decreasing with increasing bound activity, decreasing substrate concentration, and increasing particle radius of the beads. With decreasing effectiveness factors, the apparent Michaelis constants were decreasing. Preparations with high bound activities did not obey Michaelis-Menten kinetics at substrate concentrations much higher than the apparent Michaelis constants. The experimental results accorded quantitatively with the theory of diffusion limitation (Engasser, J.M. (1978) Biochim. Biophys. Acta 526, 301-310).
Comparative study of the molecular species of chloropropanediol diesters and triacylglycerols in milk fat
In an effort to establish the origin of the fatty acid esters of 3-chloropropanediol, which recently have been isolated in small amounts from goat milk, we compared the molecular species composition of the chlorohydrin diesters and of goat milk triacylglycerols. The chloropropanediol diesters were found to be composed of molecular species containing C10−C18 fatty acids and corresponded closely in carbon number to those calculated for the long chain sn-1,2-diacyl-glycerol moieties of goat milk triacylglycerols. The molecular species of goat milk total triacylglycerols contained C4−C18 fatty acids. It is suggested that triacylglycerols and chloropropanediol diesters are derived from the same pool of long chain fatty acids. A molecular distillate of bovine milk fat did not contain chloropropanediol diesters, while the available samples of human milk fat were shown to contain alkyldiacylglycerols as the major components of a neutral lipid fraction corresponding in polarity to the chloropropanediol diesters.