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 )...

Full description

Saved in:
Bibliographic Details
Published in:Molecular biotechnology 2009-05, Vol.42 (1), p.75-83
Main Authors: Quintana-Castro, Rodolfo, Díaz, Pilar, Valerio-Alfaro, Gerardo, García, Hugo S., Oliart-Ros, Rosamaría
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacillaceae - enzymology
Bacteria
Biochemistry
Biological Techniques
Biotechnology
Cell Biology
Chemistry
Chemistry and Materials Science
Cloning
Genes
Genetics
Human Genetics
Hydrogen-Ion Concentration
Lipase - chemistry
Lipase - genetics
Lipase - metabolism
Protein Conformation
Protein Science
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Sequence Analysis, DNA
Studies
Temperature
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 T7 lac 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.
ISSN:1073-6085
1559-0305
DOI:10.1007/s12033-008-9136-6