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Expression and Characterization of a Novel Lipase from Aspergillus fumigatus with High Specific Activity

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Abstract

A novel lipase gene from Aspergillus fumigatus, afl1-1, was cloned and expressed with a molecular mass of 38 kDa in Escherichia coli for the first time. The recombinant lipase had a preference for short carbon chain p-nitrophenyl esters, especially toward C2 p-nitrophenyl ester and exhibited potent hydrolysis activity that had not been observed. The optimum pH and temperature of this new enzyme were 8.5 and 65 °C, respectively. The recombinant lipase (AFL1-1) is an alkaline enzyme which was stable in the pH range 6.0∼8.5 for 16 h (at 4 °C) and at 30∼50 °C for 1 h. It is an intracellular enzyme which was purified approximately 8.47-fold with an overall yield of 86.1% by single-step Ni-NTA affinity purification, with a very high specific activity of approximately 1.00 × 103 U mg−1 on a standard substrate of p-nitrophenyl acetate. The Michaelis–Menten kinetic parameters V max and K m of the lipase were 1.37 mM mg−1 min−1 and 14.0 mM, respectively. Ca2+ and other metal ions could not activate the lipase. According to the homology analysis and site-directed mutagenesis assay, the catalytic triad of the recombinant lipase was identified as Ser-165, Asp-260, and His-290 residues.

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Acknowledgments

This research was financially supported by China National Special Fund for State Key Laboratory of Bioreactor Engineering (No. 2060204) and Shanghai Leading Academic Discipline Project (No. B505).

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Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People’s Republic of China

    Jiao-Jiao Shangguan, Yu-Qiang Liu, Jian Zhao, Li-Qiang Fan, Su-Xia Li & Jian-He Xu

  2. Institute of Chinese Materia Medica, Shanghai University of Chinese Traditional Medicine, Shanghai, 201203, People’s Republic of China

    Fu-Jun Wang

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  1. Jiao-Jiao Shangguan
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  2. Yu-Qiang Liu
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Correspondence to Jian Zhao or Jian-He Xu.

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Shangguan, JJ., Liu, YQ., Wang, FJ. et al. Expression and Characterization of a Novel Lipase from Aspergillus fumigatus with High Specific Activity. Appl Biochem Biotechnol 165, 949–962 (2011). https://doi.org/10.1007/s12010-011-9311-2

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