Bioaffinity based oriented immobilization of stem bromelain.
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 degrees C 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.
Gupta
Pawan
P
Department of Biochemistry, Faculty of Life Sciences and Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India. pawan_g75@hotmail.com
Saleemuddin
M
M
D001963:Bromelains / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D003208:Concanavalin A / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D007700:Kinetics; D010936:Plant Extracts / Q000302:isolation & purification*; D018547:Plant Stems / Q000737:chemistry; D012685:Sepharose
D004800:Enzymes, Immobilized; D010936:Plant Extracts; D003208:Concanavalin A; D001963:Bromelains; D012685:Sepharose