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Int. J. Mol. Sci. 2014, 15(1), 203-217; doi:10.3390/ijms15010203
Article

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

1,2,†
, 1,2,†
, 1,2
, 1,2
, 1,2
 and 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 These authors contributed equally to this work.
* Author to whom correspondence should be addressed.
Received: 27 September 2013 / Revised: 11 December 2013 / Accepted: 13 December 2013 / Published: 24 December 2013
(This article belongs to the Section Bioinorganic Chemistry)
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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).
Keywords: Rhizopus oryzae; lipase; Pichia pastoris; expression Rhizopus oryzae; lipase; Pichia pastoris; expression
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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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