Title

Optimization of Keratinase production by Bacillus thuringeinsis strain Bt407 isolated from poultry soil .

Submitter and Co-author information

Victoria Sibyl Uttangi Ms, University of MumbaiFollow

Standing

Graduate (Masters)

Type of Proposal

Poster Presentation

Challenges Theme

Safeguarding Healthy Great Lakes

Your Location

Windsor

Faculty Sponsor

NA

Abstract/Description of Original Work

Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” which is recalcitrant to the commonly known proteolytic enzymes. Soil samples were collected from different poultry shops were enriched for keratinase producers on Whole feather agar containing whole feathers as a sole Carbon and Nitrogen source. Among 11 bacterial isolates, 6 isolates showed protease activity. The best keratinase producing bacterium K10 was selected and identified as Bacillus thuringiensis strain Bt407, based on morphological, cultural, biochemical characteristics and 16S rRNA sequence analysis. The isolate exhibited maximum keratinase production (94.52U/ml) in a optimized feather meal medium containing Feather meal (2%), Yeast extract (1%), Starch (1%), MgSO4 6H2O (0.003%), CaCl2 (0.5mM), KH2PO4 (0.5%), K2HPO4 (0.3%), NaCl (0.5%), pH 7, inoculated with 1% v/v pre-grown cell mass and incubated at 37°C on rotary shaker (120 rpm) for 48 hours. The optimum enzyme activity was observed at 55°C and pH 8. Metal ions like Ca+2 , Mg+2 , and Ba+2 were seen to enhance enzyme activity whereas Cd+2, Cu+2 , Fe+3, Hg+2 and Zn+2 were observed to inhibit enzyme activity. Inhibitors such as SDS helped to retain the activity of the enzyme while 2- mercaptoethanol, DMSO and EDTA were seen to inhibit the enzyme activity. The molecular weight of the keratinase was found to be 33kDa by SDS-PAGE method. Zymography was carried out to show protease activity of the keratinase. Depilatory action of keratinase on goat skin was also demonstrated. The applications of the enzyme as a detergent additive and enzyme hydrolyzed feather meal in bacteriological medium as nitrogen source were also studied. Of the tested keratinous materials used as substrates, the production of enzyme was seen to be greater in the presence of human nails than human hair.

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Optimization of Keratinase production by Bacillus thuringeinsis strain Bt407 isolated from poultry soil .

Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” which is recalcitrant to the commonly known proteolytic enzymes. Soil samples were collected from different poultry shops were enriched for keratinase producers on Whole feather agar containing whole feathers as a sole Carbon and Nitrogen source. Among 11 bacterial isolates, 6 isolates showed protease activity. The best keratinase producing bacterium K10 was selected and identified as Bacillus thuringiensis strain Bt407, based on morphological, cultural, biochemical characteristics and 16S rRNA sequence analysis. The isolate exhibited maximum keratinase production (94.52U/ml) in a optimized feather meal medium containing Feather meal (2%), Yeast extract (1%), Starch (1%), MgSO4 6H2O (0.003%), CaCl2 (0.5mM), KH2PO4 (0.5%), K2HPO4 (0.3%), NaCl (0.5%), pH 7, inoculated with 1% v/v pre-grown cell mass and incubated at 37°C on rotary shaker (120 rpm) for 48 hours. The optimum enzyme activity was observed at 55°C and pH 8. Metal ions like Ca+2 , Mg+2 , and Ba+2 were seen to enhance enzyme activity whereas Cd+2, Cu+2 , Fe+3, Hg+2 and Zn+2 were observed to inhibit enzyme activity. Inhibitors such as SDS helped to retain the activity of the enzyme while 2- mercaptoethanol, DMSO and EDTA were seen to inhibit the enzyme activity. The molecular weight of the keratinase was found to be 33kDa by SDS-PAGE method. Zymography was carried out to show protease activity of the keratinase. Depilatory action of keratinase on goat skin was also demonstrated. The applications of the enzyme as a detergent additive and enzyme hydrolyzed feather meal in bacteriological medium as nitrogen source were also studied. Of the tested keratinous materials used as substrates, the production of enzyme was seen to be greater in the presence of human nails than human hair.