Immobilization of pepsin on sand: preparation, characterization and application
Puvanakrishnan, R.; Bose, S.M.
Indian Journal of Biochemistry and Biophysics 21(5): 323-326
1984
ISSN/ISBN: 0301-1208 PMID: 6100590 Document Number: 238902
Pepsin was immobilized by binding to river sand sieved to a particle size of approx. 355 micro m (44 mesh), cleaned with conc. HNO3 and treated with 2% 3-aminopropyltriethoxysilane (APTS), 2% hexamethylenetetramine, 2% hexamethylenediamine or 2.5% melamine, and adding 3(3-dimethylaminopropyl)-carbodiimide hydrochloride, or by binding with titanium tetrachloride direct to sand. Enzyme activity was estimated by milk-clotting assay. APTS gave the greatest retained pepsin activity, 50.12%, and TiCl4 gave the lowest, 11.9; the other compounds gave 23-29% activity. The amount of bound pepsin was 2.5 mg/g for the APTS method vs. 0.81-1.84 mg/g for other methods. For native and immobilized enzymes resp., pH optimum was 1.8 and 1.5, optimum temp. 40 and 45 degrees C, Michaelis constant 1.3 and 1.8 mM. The immobilized enzyme had improved stability to pH 4-6 and 30-45 degrees C. The immobilized enzyme retained 100 and 90% of its activity when stored for 60 days dry or under 0.1 M citrate buffer, and 57 and 39% of its activity when kept for 60 days at room temp. under the same conditions. 75% of the initial activity was lost by washing 5 times in 6 M urea. Milk-clotting time was slightly increased by using the immobilized enzyme, from 40 to 40-55 s.