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Gene Cloning, Expression, and Characterization of the Geobacillus Thermoleovorans CCR11 Thermoalkaliphilic Lipase

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Abstract

The gene for a Geobacillus thermoleovorans CCR11 thermostable lipase was recovered by PCR and cloned. Four genetic constructions were designed and successfully expressed in E. coli: (i) the lipase structural gene (lipCCR11) in the PinPoint Xa vector; (ii) the lipase structural gene (lipACCR11) in the pET-28a(+) vector; (iii) the lipase structural gene minus the signal peptide (lipMatCCR11) in the pET-3b vector; and (iv) the lipase structural gene plus its own promoter (lipProCCR11) in the pGEM-T cloning vector. The lipase gene sequence analysis showed an open reading frame of 1,212 nucleotides coding for a mature lipase of 382 residues (40 kDa) plus a 22 residues signal peptide. Expression under T7 and T7lac promoter resulted in a 40- and 36-fold increase in lipolytic activity with respect to the original strain lipase. All recombinant lipases showed an optimal activity at pH 9.0, but variations were found in the temperature for maximum activity and the substrate specificity among them and when compared with the parental strain lipase, especially in the recombinant lipases that contained fusion tags. Therefore, it is important to find the appropriate expression system able to attain a high concentration of the recombinant lipase without compromising the proper folding of the protein.

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Acknowledgements

Rodolfo Quintana-Castro (PhD) acknowledges his scholarship from the National Council on Technological Education (Cosnet) and the National Direction of Technological Education (DGEST-SEP). This work was supported by Grant 362.06-P from the National Direction of Technological Education (DGEST-SEP).

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Authors and Affiliations

  1. Instituto Tecnológico de Veracruz, Unidad de Investigación y Desarrollo en Alimentos, Av. Miguel A. de Quevedo 2779, Veracruz, 91897, Mexico

    Rodolfo Quintana-Castro, Gerardo Valerio-Alfaro, Hugo S. García & Rosamaría Oliart-Ros

  2. Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, Av. Diagonal 645, Barcelona, 08028, Spain

    Pilar Díaz

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  1. Rodolfo Quintana-Castro
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  2. Pilar Díaz
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  3. Gerardo Valerio-Alfaro
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  4. Hugo S. García
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  5. Rosamaría Oliart-Ros
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Correspondence to Rosamaría Oliart-Ros.

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Quintana-Castro, R., Díaz, P., Valerio-Alfaro, G. et al. Gene Cloning, Expression, and Characterization of the Geobacillus Thermoleovorans CCR11 Thermoalkaliphilic Lipase. Mol Biotechnol 42, 75–83 (2009). https://doi.org/10.1007/s12033-008-9136-6

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