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Molecular cloning and characterization of a thermostable lipase from deep-sea thermophile Geobacillus sp. EPT9

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Abstract

A gene (1,254 bp) encoding a lipase was identified from a deep-sea hydrothermal field thermophile Geobacillus sp. EPT9. The open reading frame of this gene encoded 417 amino acid residues. The gene was cloned, overexpressed in Escherichia coli, and the target protein was purified to homogeneity. The purified recombinant enzyme presented a molecular mass of 44.8 kDa. When p-nitrophenyl palmitate was used as a substrate, the recombinant lipase was optimally active at 55 °C and pH 8.5. The recombinant enzyme retained 44 % residual activity after incubation at 80 °C for 1 h, which indicated that Geobacillus sp. EPT9 lipase was thermostable. Homology modeling of strain EPT9 lipase was developed with the lipase from Bacillus sp. L2 as a template. The core structure exhibits an α/β-hydrolase fold and the typical catalytic triad might consist of Ser142, Asp346, and His387. The enzymatic activity of EPT9 lipase was inhibited by addition of phenylmethylsulfonyl fluoride, indicating that it contains serine residue, which plays an important role in the catalytic mechanism.

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Acknowledgments

This work was financially supported by the Foundation for Innovative Research Team of Jimei University, China (No. 2010A006), National Natural Science Foundation of China (No. 31371751), and Science and Technology Program of Xiamen, China (No. 201303120001).

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Correspondence to Huinong Cai.

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Zhu, Y., Li, H., Ni, H. et al. Molecular cloning and characterization of a thermostable lipase from deep-sea thermophile Geobacillus sp. EPT9. World J Microbiol Biotechnol 31, 295–306 (2015). https://doi.org/10.1007/s11274-014-1775-0

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  • DOI: https://doi.org/10.1007/s11274-014-1775-0

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