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KMID : 0545120150250111880
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Journal of Microbiology and Biotechnology
2015 Volume.25 No. 11 p.1880 ~ p.1893
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Characterizing LipR from Pseudomonas sp. R0-14 and Applying in Enrichment of Polyunsaturated Fatty Acids from Algal Oil
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Yang Wen Juan
Xu Li
Zhang Hou Jin
Yan Yun Jun
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Abstract
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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 60oC 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 50oC for 12 h. In addition, LipR was activated by Ca2+, Mg2+, Ba2+, and Sr2+, while strongly inhibited by Cu2+, Zn2+, Mn2+, 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.
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KEYWORD
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Genome walking, subfamily I.3 lipase, thermostability, hydrolysis, long-chain polyunsaturated fatty acids, diacylglycerols
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