Asian Journal of Biotechnology 2 (1): 1-13, 2010 ISSN 1996-0700 © 2010 Knowledgia Review, Malaysia Corresponding Author: Sanna Tork, National Research Center, Department of Microbial Genetics, Dokki, Cairo, Egypt 1 Biochemical and Molecular Characterization of a New Local Keratinase Producing Pseudomomanas sp., MS21 S. Tork, M.M. Aly and L. Nawar 1,3 2,3 3 National Research Center, Department of Microbial Genetics, Dokki, Cairo, Egypt 1 Department of Botany, Faculty of Science, Tanta University, Tanta, Egypt 2 Department of Biological Sciences, Faculty of Science, 3 King Abd El-Aziz University, Saudi Arabia Abstract: This study aimed to isolate and identify a new local bacterial strain, able to completely degrade keratin-rich wastes into soluble and useful materials which can be used in many proposes. Bacterial keratinases are of particular interest because of their action on insoluble keratin substrates and generally on a broad range of protein substrates. These enzymes have been studied for de-hairing processes in the leather industry and hydrolysis of feather and keratin. Samples from poultry industry wastes, soil, water, fodder and feather were collected from different places in Jeddah, Saudi Arabia. Each sample was plated on feather meal agar plates containing 5 g LG feather as the sole carbon and nitrogen source and 1 the obtained colonies were selected, purified and their growth were detected on skimmed milk agar and feather meal broth media. The well grown isolates on feather meal agar which producing the largest clearing zone on skimmed milk plate were selected for keratinase assays. Out of 23 bacterial isolates, 7 isolates were selected. The best keratinase producing bacterium kera MS21 was selected and identified based on morphological, physiological and some biochemical characteristics. It was recorded as a species belonging to the genus Pseudomonas and identified as Pseudomonas sp. The results of identification were confirmed by 16S rDNA studies. Precipitation and purification of the keratinase enzyme in addition to factors affecting enzyme activity were studied. The enzyme molecular weight was determined to be of 30 KDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The optimum temperature and pH were determined to be 37°C and pH 8.0, respectively. The effect of some proteases inhibitors and activators were also studied. Key words: Keratinase, feather, 16S rRNA, Pseudomonas sp., SDS poly acrylamide, keratin azure INTRODUCTION Keratins represent the major constituents of skin and its appendages such as nail, hair, feather and wool (Lin et al., 1996). Keratins are grouped into hard keratins (feather, hair, hoof and nail) and soft keratins (skin and callus) according to the sulphur content. Hard keratins like chicken feather which are recognized as a solid wastes generated from poultry processing industry are insoluble and resistant to degradation by common proteolytic