本土光桿菌0805-P5G蛋白質分解酵素的純化、特性分析和殺蟲活性測試

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

張佑慈(chang you tz'u)

指導教授 ： 高穗生 ； 張竣維

亞洲大學/健康學院/生物科技學系碩士班/碩士(2007年)

摘要

本研究利用光桿菌菌株對脫脂奶與明膠平板之蛋白質水解能力，自13 株供試之本土菌株中，篩選4 株活性較佳之菌株，在進一步培養於NB、LB 和PP3T 等不同的液態培養基，觀察蛋白質分解酵素之活性。初步實驗結果顯示，光桿菌菌株0805-P5G 的蛋白質分解酵素於NB 液態培養液，在培養八天後其活性較佳。光桿菌的蛋白質分解酵素經由FPLC 與High Trap DEAE sepharose FF column 及HighTrap Q sepharose XL column 等連續步驟純化，可使蛋白質分解酵素純度提高。接著利用膠體酵素活性電泳分析（zymogram）得到0805-P5G菌株的蛋白質分解酵素，其蛋白質活性位置大約為50 kDa，以聚丙烯醯胺凝膠電泳（SDS-PAGE）確認其分子量，並進行N 端與LC MS/MS定序確認其為光桿菌蛋白質分解酵素。利用FPLC 進行蛋白質純化後測得蛋白質分解酵素最適溫度為60℃、最適酸鹼值為pH 8。此蛋白質分解酵素會受金屬螯合劑EDTA 及1, 10-phenanthtoline 之抑制，證實其為金屬蛋白質分解酵素。光桿菌蛋白質分解酵素經注射大蠟蛾處理後，發現具有殺蟲效果；光桿菌蛋白質分解酵素經餵食處理後，其對本土小菜蛾有口服殺蟲效果，但對美國品系的小菜蛾口服殺蟲效果較差。

關鍵字

光桿菌、短尾異小桿線蟲、蛋白質分解酵素

並列摘要

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.

並列關鍵字

Photorhabdus luminescens, Heterorhabditis spp., pro

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