High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization

International Journal of Molecular Sciences

Jian-Rong Wang¹  Yang-Yuan Li¹  Shu-De Xu¹  Peng Li¹  Jing-Shan Liu¹ 

[1] Guangdong VTR Bio-Tech Co., Ltd., Zhuhai 519060, Guangdong, China; E-Mails:

关键词: Rhizopus oryzae;    lipase;    Pichia pastoris;    expression;   

DOI  :  10.3390/ijms15010203

来源: mdpi

【 摘 要 】

A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca²⁺ and inhibited by Hg²⁺ and Ag⁺. The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0).

【 授权许可】

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