A new alkaline lipase obtained from the metagenome of marine sponge Ircinia sp.
Microorganisms associated with marine sponges are potential resources for marine enzymes. In this study, culture-independent metagenomic approach was used to isolate lipases from the complex microbiome of the sponge Ircinia sp. obtained from the South China Sea. A metagenomic library was constructed, containing 6568 clones, and functional screening on 1 % tributyrin agar resulted in the identification of a positive lipase clone (35F4). Following sequence analysis 35F4 clone was found to contain a putative lipase gene lipA. Sequence analysis of the predicted amino acid sequence of LipA revealed that it is a member of subfamily I.1 of lipases, with 63 % amino acid similarity to the lactonizing lipase from Aeromonas veronii (WP_021231793). Based on the predicted secondary structure, LipA was predicted to be an alkaline enzyme by sequence/structure analysis. Heterologous expression of lipA in E. coli BL21 (DE3) was performed and the characterization of the recombinant enzyme LipA showed that it is an alkaline enzyme with high tolerance to organic solvents. The isolated lipase LipA was active in the broad alkaline range, with the highest activity at pH 9.0, and had a high level of stability over a pH range of 7.0-12.0. The activity of LipA was increased in the presence of 5 mM Ca(2+) and some organic solvents, e.g. methanol, acetone and isopropanol. The optimum temperature for the activity of LipA is 40 °C and the molecular weight of LipA was determined to be ~30 kDa by SDS-PAGE. LipA is an alkaline lipase and shows good tolerance to some organic solvents, which make it of potential utility in the detergent industry and enzyme mediated organic synthesis. The result of this study has broadened the diversity of known lipolytic genes and demonstrated that marine sponges are an important source for new enzymes.
Marine Biotechnology Laboratory, State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, People's Republic China.
Jing
J
Su
Fengli
F
Zhang
Wei
W
Sun
Valliappan
V
Karuppiah
Guangya
G
Zhang
Zhiyong
Z
Li
Qun
Q
Jiang
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis; D004795:Enzyme Stability; D015698:Genomic Library; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D054892:Metagenome; D008969:Molecular Sequence Data; D011161:Porifera / Q000382:microbiology*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D004269:DNA, Bacterial; D008049:Lipase