Keratinase production by proteolytic micro-organisms in activated sludge and farmyard waste.

Okoroma, Emeka, Purchase, Diane ORCID: https://orcid.org/0000-0001-8071-4385, Garelick, Hemda ORCID: https://orcid.org/0000-0003-4568-2300 and Abiola, Oduola (2008) Keratinase production by proteolytic micro-organisms in activated sludge and farmyard waste. In: Society for General Microbiology, Autumn Meeting, 8-11 September 2008, Trinity College, Dublin, Ireland. . [Conference or Workshop Item]

Abstract

Thirty-two microbial strains from activated sludge and farmyard waste
were isolated on feather-meal agar. Five of these isolates demonstrated
significant proteolytic activity raising prospects of biotechnological
application. The keratinolytic activity was assessed by casein-agar plate
assay and further confirmed by keratinase assay using keratin azure as
substrate. One of the five isolates (a Gram-positive cocci bacterium)
produced the highest keratinase activity within 24h at a cell density of
5.2 x 108 CFU/mL under the optimized conditions of pH 10, 37°C and
substrate concentration of 0.4 g/L. The crude extract gave an activity
of 14 U/mL. Addition of carbon (starch) and nitrogen (yeast extract
and peptone) sources caused a decline in enzymatic activity. The
culture supernatants was purified by ultra filtration, concentrated by
Amicon Ultra-15 centrifugal filter device and further purified by
HiTrap affinity chromatography column. The resulting fraction has a
purification factor of 108.7 and specific activity of 2000 U/mg. The
molecular weight of the enzyme was determined by Matrix-assisted
laser desorption/ionization (MALDI) to be 27.9 KDa.

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