Additional stabilization of stem bromelain coupled to a thermosensitive polymer by uniform orientation and using polyclonal antibodies.
Stem bromelain was covalently coupled to a thermosensitive polymer of N-isopropylacrylamide (p(NIPAm)) either through the amino groups of the enzyme (randomly coupled) or via the lone oligosaccharide chain (uniformly coupled). The enzyme coupled via the oligosaccharide chain exhibited better access to the substrate casein as compared to the preparation in which the amino groups formed the point of contact between the enzyme and the polymer. Native bromelain exhibited a pH optimum of 8.0 and a broad pH-activity profile. The polymer-coupled preparations exhibited broader pH-activity profiles and shifting of pH optimum to 10.0 at 35 degrees C. At 25 degrees C, the shifting of pH optimum was observed for the randomly coupled enzyme only. The temperature-activity profiles of bromelain coupled to p(NIPAm) also showed appreciable broadening and the preparations retained greater fraction of maximum activity above the temperature optimum. The optimum temperature of the uniformly oriented preparation also rose to 70 degrees C. Inactivation rates of the polymer-coupled bromelain were remarkably low at 60 degrees C as compared to the native protease, and binding of antibromelain antibodies improved the resistance to inactivation of the polymer-coupled preparations. The cleavage patterns of hemoglobin and IgG by the native bromelain and the polymer-coupled preparations were comparable.
Mahmood
Rubab
R
Interdisciplinary Biotechnology Unit and Department of Biochemistry, Aligarh Muslim University, Aligarh, India. rubab1@rediffmail.com
Saleemuddin
M
M
D000178:Acrylamides / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001963:Bromelains / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D004795:Enzyme Stability; D006454:Hemoglobins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D011817:Rabbits; D013379:Substrate Specificity; D013696:Temperature
D000178:Acrylamides; D000911:Antibodies, Monoclonal; D002364:Caseins; D006454:Hemoglobins; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011108:Polymers; D001963:Bromelains; C067295:N-isopropylacrylamide; C091760:stem bromelain