fulltext.study @t Gmail

Simultaneous purification and immobilization of mushroom tyrosinase on an immunoaffinity support

Paper ID Volume ID Publish Year Pages File Format Full-Text
36540 45136 2005 8 PDF Available
Title
Simultaneous purification and immobilization of mushroom tyrosinase on an immunoaffinity support
Abstract

The use of immobilized and stable enzymes has immense potential in the enzymic analysis of clinical, industrial and environmental samples. But their widespread use is limited due to the high cost of their production. In the present study, an effort has been made to immobilize tyrosinase directly from ammonium sulphate precipitated proteins of the mushroom (Agaricus bisporus) on a polyclonal antibody bound to Seralose 4B support. Polyclonal antibodies were raised in male albino rabbits by injecting commercially available mushroom tyrosinase in the presence of Freund's adjuvants. Antibodies were purified from antisera by ammonium sulphate fractionation followed by DEAE-cellulose chromatography. Two distinct bands of light and heavy chains of purified IgG, appeared on SDS-PAGE. The homogeneity of the purified IgG was further confirmed by Ouchterlony double immunodiffusion. One milliliter of cyanogen bromide-activated Seralose 4B bound 9.0 mg purified IgG and retained nearly 573 tyrosinase units. Immunoaffinity bound tyrosinase was more stable against heat and pH inactivation compared to the soluble enzyme. Immobilized enzyme exhibited no change in temperature optima between 30–35 °C whereas soluble tyrosinase has temperature optima of 35 °C. Immunoaffinity bound tyrosinase retained greater fraction of activity on both sides of temperature-optima compared to soluble enzyme. However, the broadening in pH-optima was observed from pH 5.5 to 6.0 for immobilized enzyme. Moreover, immobilized tyrosinase preparation exhibited remarkably high resistance against denaturation induced by urea and water-miscible organic solvents.

Keywords
DMF, dimethyl formamide; DMSO, dimethyl sulphoxide; DEAE, diethyl aminoethyl; SDS, sodium dodecyl sulphateTyrosinase; Immobilization; Stabilization; Organic solvents; Polyclonal antibodies; Immunoaffinity support; Seralose 4B
First Page Preview
Simultaneous purification and immobilization of mushroom tyrosinase on an immunoaffinity support
Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 40, Issue 7, June 2005, Pages 2379–2386
Authors
, , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering