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

Author： Wang Jian-Rong   Li Yang-Yuan   Xu Shu-De   Li Peng   Liu Jing-Shan   Liu Dan-Ni  

Publisher： MDPI

E-ISSN： 1422-0067|15|1|203-217

ISSN： 1422-0067

Source： International Journal of Molecular Sciences, Vol.15, Iss.1, 2013-12, pp. : 203-217

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Abstract

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).

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