Overexpression of Acid Protease of Saccharomycopsis fibuligera in Yarrowia lipolytica and Characterization of the Recombinant Acid Protease for Skimmed Milk Clotting
2010
Yu, Xin-Jun, Ocean University of China, Qingdao, China | Li, Hui-Juan, Shandong University of Science and Technology, Qingdao, China | Li, Jing, Ocean University of China, Qingdao, China | Chi, Zhen-Ming, Ocean University of China, Qingdao, China
The gene encoding an acid protease natively produced by Saccharomycopsis fibuligera was cloned and overexpressed in Yarrowia lipolytica and the resultant recombinant acid protease was purified and characterized. The molecular mass of the purified enzyme was estimated as 94.8 kDa by gel filtration chromatography. The optimal pH and temperature of the purified acid protease were 3.5 and 33℃, respectively, and the enzyme was very stable over a pH range of 1.0~3.0. The recombinant acid protease was activated by Zn²+, but was inhibited by Hg²+, Fe²+, Fe³+, and Mg²+, EDTA, EGTA, iodoacetic acid, and pepstatin. The purified recombinant acid protease from the positive transformant 71 had high milk clotting activity, suggesting that it may be used as a rennet substitute in the cheese industry.
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