title: Isolation of a novel alkaline-stable lipase from a metagenomic library and its specific application for milkfat flavor production.
abstract: BACKGROUND
  Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF.
  
  
  RESULTS
  A novel lipase Est_p6 was isolated from a metagenomic library and was expressed highly in E.coli. Bioinformatic analysis indicated that Est_p6 belongs to lipolytic enzyme family IV, the molecular weight of purified Est_p6 was estimated at 36 kDa by SDS-PAGE. The hydrolytic activity of the enzyme was stable under alkaline condition and the optimal temperature was 50°C. It had a high specific activity (2500 U/mg) toward pNP butyrate (pNP-C4), with K(m) and V(max) values of 1.148 mM and 3497 μmol∙min⁻¹∙mg⁻¹, respectively. The enzyme activity was enhanced by DTT and was not significantly inhibited by PMSF, EDTA or SDS. This enzyme also showed high hydrolysis specificity for myristate (C14) and palmitate (C16). It seems that Est_p6 has safety for commercial LMF flavor production and food manufacturing processes.
  
  
  CONCLUSIONS
  The ocean is a vast and largely unexplored resource for enzymes. According the outstanding alkaline-stability of Est_p6 and it produced myristic acid and palmitic acid more efficiently than other free fatty acids in lipolyzed milkfat. This novel lipase may be used to impart a distinctive and desirable flavor and odor in milkfat flavor production.
authors.forename: Qing
authors.initials: Q
authors.lastname: Peng
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.forename: Meng
authors.initials: M
authors.lastname: Shang
authors.forename: Jinjin
authors.initials: J
authors.lastname: Huang
authors.forename: Guohua
authors.initials: G
authors.lastname: Guan
authors.affiliation: State Key Laboratories for Agro-biotechnology and College of Biological Sciences, China Agricultural University, Beijing 100193, P, R, China. yingli528@vip.sina.com.
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.forename: Bo
authors.initials: B
authors.lastname: Shi
mesh_terms: D000595:Amino Acid Sequence; D019295:Computational Biology; D004926:Escherichia coli / Q000378:metabolism; D005421:Flavoring Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015723:Gene Library; D019015:Geologic Sediments / Q000382:microbiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D056186:Metagenomics; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005421:Flavoring Agents; D011994:Recombinant Proteins; D019814:Myristic Acid; D019308:Palmitic Acid; D008049:Lipase