Characterization of protease from Aquimarina Salinaria

• Main Authors: Lin,Chi-Ni, 林奇霓
• Other Authors: Kuo,Jen-Min
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/60876619142280172648

碩士 === 國立高雄海洋科技大學 === 水產食品科學研究所 === 100 === The objective of the present study is to purify and characterize the protease from Aquimarina salinaria which was newly identified and isolated from a salt pan in southern Taiwan. Aquimarina salinaria is a Gram (-)bacteria, and is able to grow between 15-37℃, pH 7.0-9.0, salinity 2-5%. The optimal growth conditions were at 25 ℃, pH 8.0 or salinity 3%. Two liters of a 4-day culture of Aquimarina salinaria were centrifuged at 20,000×g and the supernatant was used as crude protease. The crude enzyme was then purified with TFF (Tangential Flow Filtration, MWCO of 10k) system and Q-HP ion exchange chromatography. The overall recovery of enzyme activity was 22.9% with a 11-fold after purification, and Km and Vmax was 0.05 mM and 123.5 U/mg-min, respectively, using azo-casein as substrate. The optimal pH and temperature of the enzyme was 8.0 and 45℃, respectively, and stable at temperature below 50℃ or pH ranged 7.0 to 9.0. Metal ion of Cu2+&Hg2+,SDS (sodiumdodecylsulphate) and β-mercaptoethanol could inhibit enzyme activity while Ca2+, Mg2+, Triton X-100 slight activate the enzyme activity. Data suggested disulfide bond may play an important role in the enzyme structure. The enzyme activity of the partially purified proteinase was completely inhibited by EDTA (ethylene diamine teraacetates), indicating this enzyme was a metalloproteinase. Moreover, the partially purified proteinase showed 62.8% of enzyme activity at 8% NaCl. After storage at 4, 25, or -20℃ for 4 weeks, the partially purified enzyme all retained 90.9% of enzyme activity. keyword：Aquimarina salinaria , purification , protease