Journal of Pure and Applied MicrobiologyVol. 8 No. 6

Statistical Optimization, Immobilization and Partial Purification of Keratinase Production from Newly Isolated Bacillus subtilis

Dina I. Abdel Meguid, Amany S. Youssef, Rabab M. Attia and Samy A. El-Assar

Department of Botany and Microbiology, Faculty of Science, Alexandria University, Muharam Bek, Alexandria, Egypt.

Received on 12 October 2014 and accepted on 29 November 2014

 

ABSTRACT

In the present study, keratinase production by Bacillus subtilis DALEX isolated from chicken feathers was evaluated. The bacterium showed a proteolytic activity when grown on skimmed milk agar plates. Best enzyme activity (1.26 U/mg protein) was obtained on using chicken feathers compared to other substrates such as wool and rabbit fur. Best activity (1.35 U/mg protein) was achieved when B.subtilis DALEX was grown in shaken cultures of Feather basal medium supplemented with 10g feathers meal and incubated at 37?C for 3 days. Lower activities were observed with static cultures or in solid state fermentation. Placket-Burman design was employed to reach the near optimum condition for keratinase activity. Immobilization of bacterial cells on different supports was employed to study the effect of immobilization on enzyme activity. The highest keratinase production (1.83 U/mg protein) was reached by immobilized B. subtilis DALEX with 4 % alginate concentration which was higher than that obtained with free cells (1.33 U/mg protein).The fraction precipitated at 85% acetone concentration was considered as the partially purified enzyme. Optimum enzyme protein and substrate concentrations were 2.5 mg and 10 mg / reaction mixture, respectively. The optimum reaction temperature for the immobilized purified keratinase was 45?C. Partial purified enzyme showed a maximum activity at a reaction pH of 9.0 and a relatively high activity in a pH range of 7.0 to 9.0. The semi-purified enzyme was strongly stable at 50?C for 15 min and showed thermal stability even after 30 min exposure to 50 ?C.

Keywords : Keratinase, Solid state fermentation, Placket-Burman, Immobilization, Partial purification.