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

by Jian-Rong Wang 1,2,†, Yang-Yuan Li 1,2,†, Shu-De Xu 1,2, Peng Li 1,2, Jing-Shan Liu 1,2 and Dan-Ni Liu 1,2,*
1
Guangdong VTR Bio-Tech Co., Ltd., Zhuhai 519060, Guangdong, China
2
Guangdong Feed Additive Research and Development Center, Zhuhai 519060, Guangdong, China
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2014, 15(1), 203-217; https://doi.org/10.3390/ijms15010203
Received: 27 September 2013 / Revised: 11 December 2013 / Accepted: 13 December 2013 / Published: 24 December 2013
(This article belongs to the Section Biochemistry)
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). View Full-Text
Keywords: Rhizopus oryzae; lipase; Pichia pastoris; expression Rhizopus oryzae; lipase; Pichia pastoris; expression
MDPI and ACS Style

Wang, J.-R.; Li, Y.-Y.; Xu, S.-D.; Li, P.; Liu, J.-S.; Liu, D.-N. High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization. Int. J. Mol. Sci. 2014, 15, 203-217.

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