Purification, characterization and insecticidal activity of protease from Photorhabdus luminescens 0805-P5G isolated from Taiwanmsword

• Main Authors: chang you tz'u, 張佑慈
• Other Authors: kao sui sheng
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/04113658148132888889

碩士 === 亞洲大學 === 生物科技學系碩士班 === 95 === Among 13 local Photorhabdus luminescens tested, 4 candidates with higher proteolytic activity assayed with skim milk and gelatin plates methods were selected for further testing. Three liquid cultures namely, NB, LB and PP3T were chosen to culture the candidates and their proteolytic activities were again measured. Results showed that after 8-day liquid cultured in NB medium, the proteolytic activity of isolated P. luminescens 0805-P5G reached to the peak. The purify of protease from P. luminescens 0805-P5G was greatly increased after a serial purification process by FPLC using High Trap DEAE sepharose FF column and High Trap Q sepharose XL column. Zymograpphic analysis was applied to confirm the molecule weight of protease, approximately 50 kDa, which was furtherly reconfirmed by SDS-PAGE. Further more, the peptide sequence of protease of P. luminescens 0805-P5G was identified by N-terminal sequencing analysis and LC MS/MS. The activity of purified protease was optimum at 60℃ and pH 8. Bioassay of P. luminescens protease against Galleria mellonella by injection showed that P. luminescens the high insecticidal activity. High oral toxicity for Plutella xyllostella （Taiwan strain in Taiwan）was found, however, the oral toxicity for Plutella xyllostella（American strain）was low. The protease was inhibited by EDTA and 1, 10-phenanthtoline and categorized as metalloprotease.