抽象的

Keratinolytic activity from new recombinant fusant AYA2000, an endophytic Micromonospora spp.

Ahmed M.El-Bondkly, Mervat M.A.El-Gendy

Two different endophytic strains ESRAA1997 and ALAA2000 were isolated from the Egyptian herbal plant Anastatica hierochuntica. The two strains produced alkaline serine protease and they were identified based on their phenotypic and chemotypic characteristics as two different strains of Micromonospora spp. Both strains grew and produced keratinase using different keratinous waste substances as the sole source of carbon and nitrogen. In our study, the activity and properties of keratinase enzymes of the both wild strains ESRAA1997 and ALAA2000 have been altered by genetic recombination through protoplast fusion between them, leading to a potent keratinolytic fusant Micromonospora spp. AYA2000 with improved properties (activity, stability, specificity and tolerance to inhibitors). Whereas using a mixture of yeast extract, peptone and malt extract as a supplement to the bovine hair medium increased keratinase production by 48%, addition of 1% glucose suppressed enzyme production byMicromonospora spp.AYA2000. The enzyme was purified by ammoniumsulphate precipitation,DEAE-cellulose chromatography followed by gel filtration. The molecular mass, determined using SDS–PAGE, was 39kDa. AYA2000. The enzyme exhibited remarkable activity towards all keratinous wastes used and could also adapt to a broad range of pH and temperature with optima at pH 11 and 60C. The enzyme was not influenced by chelating reagents,metal ions or alcohols. These propertiesmake AYA2000 keratinase an ideal candidate for biotechnological application.