Expression, Characterization And Mutagenesis Study Of A Psychrophilic Lipase From Deep-sea Bacterium Shewanella Piezotolerans WP3

Lipases are versatile tools for biotechnological applications not only their potential to catalyze the hydrolysis but also the synthesis of many useful ester compounds. Numerous lipases have already been isolated and characterized from animals, plants, and microorganisms. Most of the lipases used in industry are microbial enzymes, which have been extensively characterized biochemically. Psychrophilic lipases derived from extremophiles of deep-sea have lately attracted attention as a result of their increasing use due to their high activity at low temperatures and thermoliability, which are favorable properties for the production of fatty acids and interesterification of fats in the food, pharmaceutical, and fine-chemical industries.A piezotolenrant and psychrotolenrant bacteria Shewanella piezotolerans WP3, showing lipolytic activity, was previously isolated from deep-sea sediments of 1914m depth in west Pacific Warm Pool. It could grow at a temperature range of 0-28°C and the best growth temperature was around 20°C. The open reading frame of Shewanella piezotolerans WP3 lipases consisted of 759 nucleotides, encoding a protein of 252 amino acids with a predicted molecular mass of approximate 30kDa. This lipase is a member of family V lipases, and had the highest homology with the esterases of Haemophilus influenzae Rd KW20. The catalytic triad of Shewanella piezotolerans WP3 lipases is composed of Ser82, Asp202 and His231. Additionally, the unknown conserved sequence of family V lipases could be found in N-terminal part of this lipase.Shewanella piezotolerans WP3 lipase is a psychrophilic enzyme with a low optimum temperature of 20°C, and retains 80% activity even at 4°C. It is a thermoliable and pH neutral lipase, which is able to hydrolyze short to medium chain PNP esters. Metal ions, various reagents and organic solvents could inhibit the hydrolytic activity of Shewanella piezotolerans WP3 lipase except for EDTA, which indicate the independence of metal ions. H-bonds, rather than disulfide bridges, are the most important interactions in the stability of protein structure.Structural modifications involved in cold-adaptation had been detected from structure analysis. A large amount of charged residues exposed at the protein surface, and a relative smaller hydrophobic core region. The lack of disulfide bridges, which are conserved in mesophilic and thermophilic lipases. A low proportion of arginine residues as compared to lysines, a low content in proline residues and a high content in glycine residues were also observed. All these properties should confer on the lipase a more flexible structure, in accordance with its low thermostability.For further study of the relationship between structure and activity or stability, a series of single amino acid mutants had been constructed by site-directed mutagenesis. For Shewanella piezotolerans WP3 lipase, Leu14 and Lys246 may be the key residues of cold activity, while Asp23 and Gly97 may be the key residues of thermostability. Conformation changes of lipase active center, may affect cold activity by changes of catalytic ability and substrates affinity, but also the thermostability, and even lead to accumulation of non-active products. It is also predicted that hydrophobic region of unknown conserved sequence and cold activity are closely relative, and protein surface region of that and thermostability are closely relative, while it play no role in substrates affinity.A high quality random mutagenesis of Shewanella piezotolerans WP3 lipase, containing around 10000 mutants, was constructed by error-prone PCR method. After screening, 2 of them were found to be favorable mutants with 1.8-fold and 1.6-fold catalytic efficiency(k_cat/K_m) of wild type lipase at low temperature.Shewanella piezotolerans WP3 lipase is the first lipase expressed and characterized from Shewanella strains. This study on the cold-adaption of lipase may facilitate the research of relationship between enzyme structure and activity(stability), and the protein engineering of biocatalysts.