African Journal of Biotechnology Vol. 5 (24), pp. 2433-2438, 16 December 2006   

ISSN 1684–5315 © 2006 Academic Journals        

Full Length Research Paper

Purification and characterization of a protease from Thermophilic bacillus strain HS08

HUANG Guangrong^1,2*, YING Tiejing¹, HUO Po² and JIANG Jiaxing³

¹College of Biosystems Engineering and Food science, Zhejiang University, Hangzhou, Zhejiang 310027, China.

²School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, Zhejiang 310012, China.

³School of Life Science, China Jiliang University, Hangzhou, Zhejiang 310018, China.

*Corresponding author E-mail: grhuang@126.com, Tel: 0086 571 8512 4599. Fax: 0086 571 8512 1955.

Accepted 19 October, 2006

The purification and characterization of a thermophilic neutral protease from Thermophilic bacillus strain HS08, originally isolated from a soil sample collected from the Tulufan Crater of China, is presented in this paper. The purification steps included ammonium sulfate precipitation, with columns of DEAE-Sepharose anion exchange chromatography and Sephacryl S-100HR on AKTA purifier 100 protein liquid chromatography. The method gave a 4.25 fold increase of the specific activity and had a yield of 5.1%. The molecular weight of the protease was found to be around 30.9 kDa by SDS-PAGE technique. The optimal pH and optimal temperature of the protease were at pH 7.5 and 65^oC, respectively. The protease was found stable during the 1 h incubation at 5°C. The protease activity showed wide range of variation in the presence of different reagents: it was inhibited remarkably by EDTA or PMSF and was almost activated by 2 mM Zn²⁺, even though it was only marginally inhibited by other inhibitors. We concluded that the protease was a Zn²⁺-acitived serine protease. Substrates specificity tests indicated that azocasein was the best substrate among the three substrates tested (azocasein, casein, and BSA).

Key words: Neutral protease, purification, characterization, Thermophilic bacillus, thermophilic protease, serine protease.