title: A novel eurythermic and thermostale lipase LipM from Pseudomonas moraviensis M9 and its application in the partial hydrolysis of algal oil.
abstract: BACKGROUND
  Lipases are regularly used in biotechnology to catalyse the hydrolysis of triglycerides and the synthesis of esters. Microbial lipases in particular have been widely used in a variety of industrial applications. However, the current commercial microbial lipases cannot meet industrial demand due to rapid inactivation under harsh conditions. Therefore, in order to identify more stable enzymes, we isolated novel eurythermic and thermostable lipase(s) from Pseudomonas moraviensis M9.
  
  
  METHODS
  Cloning of lipM was based on Touchdown PCR and genome walking, and then recombinant LipM was purified by guanidine hydrochloride and the nickel-nitrilotriacetic acid resins affinity chromatography. Finally, the hydrolysis of algal oil by LipM was analyzed by gas chromatograph-mass spectrometer, thin layer chromatography and gas chromatograph.
  
  
  RESULTS
  The lipM gene was first cloned from Pseudomonas moraviensis M9 via Touchdown PCR and genome walking. Sequence analysis reveals that LipM is a member of subfamily I.3 of lipases, and the predicted amino acid sequences of LipM has 82 % identity to lipase LipT from Pseudomonas mandelii JR-1, and 54 % identity to lipase PML from Pseudomonas sp. MIS38 and lipase Lip I.3 from Pseudomonas sp. CR-611. LipM was expressed in Escherichia coli, purified from inclusion bodies, and further biochemically characterized. Purified LipM differed significantly from previously reported subfamily I.3 lipases, and was eurythermic between 10 °C-95 °C. LipM activity was enhanced by Ca(2+), Sr(2+), Mn(2+), and Ba(2+), but sharply inhibited by Cu(2+), Zn(2+), Co(2+), Ni(2+), and EDTA. Compared with other lipases, LipM exhibited medium tolerance to methanol, ethanol, and isopropanol. When applied for hydrolysis of algal oil, LipM could enrich 65.88 % polyunsaturated fatty acids, which include 1.25 % eicosapentaenoic acid, 17.61 % docosapentaenoic acid, and 47.02 % docosahexaenoic acid with derivative glycerides containing 32.46 % diacylglycerols.
  
  
  CONCLUSIONS
  A novel eurythermic I.3 subfamily lipase with high tolerance and stability was identified from Pseudomonas moraviensis and biochemically characterized. It will not only improve our understanding of subfamily I.3 lipases, but also provides an ideal biocatalyst for the enrichment of polyunsaturated fatty acids. Pseudomonas moraviensis have been investigated as a potential resource of lipases.
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. kuailechangzai9988@126.com.
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. caohai230@163.com.
authors.forename: Hai
authors.initials: H
authors.lastname: Cao
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. xuli1881@yahoo.cn.
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. hjzhang@hust.edu.cn.
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. yanyunjun@hust.edu.cn.
authors.forename: Yunjun
authors.initials: Y
authors.lastname: Yan
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015525:Fatty Acids, Omega-3 / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019216:Metals, Heavy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology*; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D015525:Fatty Acids, Omega-3; D019216:Metals, Heavy; D008049:Lipase