Purification of NADP Dependent, 1,3,6,8 -Tetrahydroxynaphthalene Reductase from Verticillium dahliae brm - 1(ATCC 44571)

Abstract

NADPH dependent 1,3,6,8- tetrahydroxynaphthalene Reductase, one of the key enzymes of 1,8-dihydroxy melanin (DHN melanin) biosynthesis was isolated from verticillium dahliae brm-I (ATCC 44571) and purified to near homogeneity (as shown by SDS-PAGE), using ammonium sulphate fractionation, ion exchange chromatography on DEAE Cellulose, FPLC Mono Q anion exchange and Superose - 12 gel filtration chromatography, Fold purification and yield of the enzyme were found to be 1423 and 0.47% respectively, Specific activity ofthe final enzyme fraction was 79,7 (nmole/ mg.min) Approximate molecular weight of the purified enzyme was determined as-24 kDa by Superose-12 gel filtration chromatography, A satisfactory HPLC based enzyme assay was also developed for thepurification of enzyme