title: Characterizing LipR from Pseudomonas sp. R0-14 and Applying in Enrichment of Polyunsaturated Fatty Acids from Algal Oil.
abstract: In this study, Pseudomonas R0-14, which was isolated from Arctic soil samples, showed a clear halo when grown on M9 medium agarose plates containing olive oil-rhodamine B as substrate, suggesting that it expressed putative lipase(s). A putative lipase gene, lipR, was cloned from R0-14 by genome walking and Touchdown PCR. lipR encodes a 562-amino-acid polypeptide showing a typical α/β hydrolase structure with a catalytic triad consisting of Ser153-Asp202-His260 and one α-helical lid (residues 103-113). A phylogenetic analysis revealed that LipR belongs to the lipase subfamily I.3. LipR was successfully expressed in Escherichia coli, purified, and biochemically characterized. Recombinant LipR exhibited its maximum activity towards p-nitrophenyl butyrate at pH 8.5 and 60°C with a Km of 0.37 mM and a kcat of 6.42 s(-1). It retained over 90% of its original activity after incubation at 50°C for 12 h. In addition, LipR was activated by Ca(2+), Mg(2+), Ba(2+), and Sr(2+), while strongly inhibited by Cu(2+), Zn(2+), Mn(2+), and ethylenediaminetetraacetic acid. Moreover, it showed a certain tolerance to organic solvents, including acetonitrile, isopropanol, acetone, methanol, and tert-butanol. When algal oil was hydrolyzed by LipR for 24 h, there was an enrichment of n-3 long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (1.22%, 1.65-fold), docosapentaenoic acid (21.24%, 2.04-fold), and docosahexaenoic acid (36.98%, 1.33-fold), and even a certain amount of diacylglycerols was also produced. As a result, LipR has great prospect in industrial applications, especially in food and/or cosmetics applications.
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P.R. China.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Yang
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P.R. China.
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P.R. China.
authors.forename: Houjin
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P.R. China.
authors.forename: Yunjun
authors.initials: Y
authors.lastname: Yan
mesh_terms: D001110:Arctic Regions; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D020536:Enzyme Activators / Q000032:analysis; D004791:Enzyme Inhibitors / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000235:genetics* / Q000378:metabolism*; D009821:Oils / Q000378:metabolism*; D010802:Phylogeny; D011549:Pseudomonas / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013696:Temperature
chemical_list: D003470:Culture Media; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005231:Fatty Acids, Unsaturated; D009821:Oils; D011994:Recombinant Proteins; D008049:Lipase
keywords: Genome walking; diacylglycerols; hydrolysis; long-chain polyunsaturated fatty acids; subfamily I.3 lipase; thermostability