期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 155, 期 -, 页码 1-7出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2018.10.012
关键词
Heterologous expression; Aspergillus oryzae lipase; Pichia pastoris; Characterization
类别
资金
- National Natural Science Foundation of China [31600639, 31660247]
- Education Department of Jiangxi Province [GJJ151211]
Pichia pastoris expression is a mature and efficient eukaryotic expression system. In this work, Aspergillus oryzae lipase (AOL, with the molecular mass of 28 kDa), which can perform highly stereoselective hydrolysis of (R, S)methyl 2-(4-hydroxyphenoxy) propanoate, was expressed in P. pastoris X-33. The specific activity of AOL was 432 U/mg, which was obtained by fed-batch cultivation in a 5 L bioreactor using a methanol feeding strategy. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut-off membrane and purified with DEAE-Sepharose (TM) FF ion-exchange chromatography and phenyl Seflnose (TM) 6 FF hydrophobic interaction chromatography. The purified lipase activity reached 5509 U/mg. AOL showed high activity toward short-chain triacylglyceride (C-4), and the optimum temperature and pH were 40 degrees C and 8.0, respectively. The purified enzyme activity was inhibited by Zn2+ and Cu2+. Moreover, the K-m and V-max values were 1 mM and 32.89 mmol/min, respectively.
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