title: High-level expression of pro-form lipase from Rhizopus oryzae in Pichia pastoris and its purification and characterization.
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 Ca(2+) and inhibited by Hg(2+) and Ag(+). The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0).
authors.forename: Jian-Rong
authors.initials: JR
authors.lastname: Wang
authors.forename: Yang-Yuan
authors.initials: YY
authors.lastname: Li
authors.forename: Shu-De
authors.initials: SD
authors.lastname: Xu
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.forename: Jing-Shan
authors.initials: JS
authors.lastname: Liu
authors.affiliation: Guangdong VTR Bio-Tech Co., Ltd., Zhuhai 519060, Guangdong, China. danni_vtr@163.com.
authors.forename: Dan-Ni
authors.initials: DN
authors.lastname: Liu
mesh_terms: D061249:Batch Cell Culture Techniques; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012233:Rhizopus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D014462:Ultrafiltration
chemical_list: D005656:Fungal Proteins; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D008049:Lipase