Isolation and purification of the protease from bacteria

• Main Authors: CHEN-CHU WEI, 陳俊維
• Other Authors: none
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/60176447855562509670

碩士 === 大仁科技大學 === 生物科技研究所 === 99 === This research begins by screening the environment for native bacterial strains in the soil by testing for protease activity. The bacterium is the Gram-positive and aerobic, which is classified to bacillus. Identification of the strain was by way of the 16S rDNA. Sequence alignment of the Bacillus strain was analyzed by gene bank, that showed the similarities up to 98%. This strain named Bacillus subtilis PTD25. The strain can grow between 15- 45℃ temperatures and pH 5- 9. The strain exhibited an optimum grow at pH 7.5 and 45℃. In the medium, casein, gelatin and milk can increase protease secretory volume. Adjunction of the casein can induce the most protease production at 30℃. After incubation at 40℃, the concentrated supernatant was crude enzyme. The first of all used phosphate buffer at pH 7.0 to dialysis. It used anion exchange chromatography column for purification. Salt gradients was analyzed to found activity of protease. Used SDS-polyacrylamide gel electrophoresis and zymography to confirm the molecular weight and purification of protein. The results shown its apparent molecular mass was approximately 28 kDa. Further to promote purity of enzyme for proceed the research of particularity.