Gene cloning, expression, and characterization of the Geobacillus Thermoleovorans CCR11 thermoalkaliphilic lipase.
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.
Quintana-Castro
Rodolfo
R
Instituto Tecnológico de Veracruz, Unidad de Investigación y Desarrollo en Alimentos, Veracruz, 91897, Mexico.
Díaz
Pilar
P
Valerio-Alfaro
Gerardo
G
García
Hugo S
HS
Oliart-Ros
Rosamaría
R
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
D011994:Recombinant Proteins; D008049:Lipase