Thesis (M.Sc.) - Cairo University - Faculty of Agriculture - Department of Genetics

The main objective of this work is to select and genetically improve keratinase producing bacteria in order to reduce environmental feather pollution and convert it to useful products. Fourty two bacteria were isolated, all were screened for keratinolytic activity. Among those, one showed high keratinase specific activity of 28.88 U/ml, and was identified as Bacillus spp. Morphological, biochemical characteristic, growth and molecular level 16s rRNA indicated 99% similarity with Bacillus subtilis sub spiziziniie. Therefore, it was named Bacillus subtilis sub spiziziniie N23. this isolate was mutagenized using N-methyl-N{u2032}-nitro-N- nitrosoguanidine (NTG) mutagenesis and resulted in a high mutant no.12 as it produced 34.92 U/ml. In addition, five collected Bacillus strains were mutagenized using NTG mutagenesis: Bacillus licheniformis N5 had 40.76 U/ml, resulted in a high mutant no.58 (49.16 U/ml), Bacillus pumilis I1 had 27.76 U/ml, resulted in a high mutant no.17 (39.56 U/ml), Bacillus megaterium 7A37 had 23.80 U/ml, resulted in a high mutant no.7 (52.04 U/ml), Bacillus cereus 6A15 had 20.88 U/ml, resulted in a high mutant no.16 (37.12 U/ml). and Bacillus licheniformis 5A3 had 36.64 U/ml, resulted in a high mutant no.42 that had 44.20 U/ml. The highest mutants in keratinase specific activities were nos.58 and 7 and were used for protoplast fusion trail. Two fusants were grown on selective media. Fusant no.1 had 41.84 U/ml, fusant no.2 had 41.44 U/ml in keratinase specific activities. Keratinase gene from the chromosomal DNA of isolate Bacillus subtilis N23 Mutant no.12 was successfully amplified by PCR using keratinase specific primer, cloned into pGEM- Teasy cloning vector and transferred to Escherichia coli DH5Ü. Sequence analysis showed four transformants, two transformants designated as E.coli DH5Ü pGEM ker Bacillus subtilisN23-1,2 and two transformants designated as E.coli DH5Ü pGEM ker Bacillus subtilisN23-M12-1,2. The ker gene is succesfuly expressed in E. coli DH5Ü. Escherichia coli DH5Ü pGEM ker Bacillus subtilisN23 and Escherichia coli DH5Ü pGEM ker Bacillus subtilisN23-M12 produced extracellular keratinase specific activities of 52.16 and 53.78 U /ml, respectively

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