| 摘要: | 本研究以蝦頭殼粉(SHP)為主要碳/氮源，經TKU008發酵後生產蛋白酶，純化並進行生化性質分析及其應用。TKU008篩選自台灣南部土壤，具蛋白酶及幾丁質酶生產能力，經鑑定為Pseudomonas sp.。在培養條件為1% SHP、0.1% K2HPO4、0.05%MgSO4．7H2O於pH7、100mL之液態培養基在30℃下震盪培養5天後，蛋白酶活性最佳。其發酵上清液經硫酸銨沉澱、DEAE-Sepharose及Phenyl Sepharose 6 Fast Flow等層析步驟，可分離出分子量約23kDa的蛋白酶，其最適反應pH為7、最適反應溫度為60℃、pH安定性為pH6-9及熱安定性為<50℃。此蛋白酶活性受EDTA完全抑制，為金屬型蛋白酶。對酪蛋白、彈性蛋白、人類白蛋白、血紅蛋白具不同程度的水解能力；在測試的界面活性劑中均相當穩定。應用方面則探討TKU008蛋白酶對蝦頭粉(SHP)、烏賊殼粉(SPP)的去蛋白能力，培養六天後其蛋白質去除率可達75%和85%。
This study reports the purification, characterization and application of a protease from Pseudomonas sp. TKU008, which takes shrimp head shells powder (SHP) as main carbon nitrogen source. TKU008 was isolated from Taiwan south soil, which can produce chitinase and protease. The optimized condition for protease production was found when the culture was shaken at 30℃ for 5 days in 100mL of medium (pH7) containing 1% SHP, 0.1% K2HPO4, 0.05%MgSO4．7H2O. One protease was purified by chromatography procedures of DEAE-Sepharose, and Phenyl Sepharose 6 Fast Flow. The molecular mass of the protease determined by SDS-PAGE was approximately 23 kDa. The optimum pH, optimum temperature, pH stability, and thermal stability of protease were pH7, 60℃, pH6-9 and <50℃, respectively. The protease was characterized as a metalloprotease, due to it was inactivated by EDTA. And it had different specific proteolytic activity with casein, elastin, human albumin, hemoglobin as the substrates. When the protease was treated of various surfactants, its activity was stable. We also discuss the deproteinization ability of this enzyme when grown in different mediums such as SHP, squid shell powder (SPP). The percent of protein removal for SHP and SPP after 6-day incubation were 75% and 85%, respectively. |
