Bioaffinity Based Oriented Immobilization of Stem Bromelain |
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Authors: | Pawan Gupta M Saleemuddin |
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Institution: | (1) Department of Biochemistry, Faculty of Life Sciences and Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, 202002, India;(2) Department of Pharmacology, Basic Science Biomedical Engineering, University of Minnesota, 321 Church Street SE, 6-120 Jackson hall, Minneapolis, MN55455, USA |
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Abstract: | Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas comosus) and is unique in containing a single oligosaccharide chain attached to the polypeptide. This property allowed its affinity binding and favorable orientation on a Sepharose support pre-coupled with the lectin, concanavalin A (Con A). For comparison, bromelain was also immobilized by covalently coupling to the CNBr-activated Sepharose. The preparation obtained was more resistant to thermal inactivation as evident from the retention of over 50% activity after incubation at 60 for 100 min (as compared to 20% retained by the native enzyme and 30% retained by the covalently immobilized enzyme), exhibited a broader pH-activity profile with the enzyme retaining over 60% activity at pH 11 (as compared to over 25% retained by native and the enzyme immobilized covalently). The native, covalently-coupled and affinity-bound bromelains had apparent K
m values of 1.1, 2 and 0.54 mg/ml, respectively using casein as the substrate. The V
max values remained unaffected on immobilization. |
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Keywords: | Bromelain Concanavalin A Oligosaccharide Stability |
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