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PublicationFuel
Year2016
Volume177
Pages123-129
International

Rapid determination of the synthetic activity of lipases/esterases via transesterification and esterification zymography

Authors:Roberto Fernandez-Lafuente,
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos
Jaqueline G. Duartea+,  Kassia Leone-Ignacioa+, Jose Andre C. da Silvab, Roberto Fernandez-Lafuentec and Denise M.G. Freirea,*
 
a Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Cidade Universitária, Centro de Tecnologia, Bl. A, Sl. 549, Ilha do Fundão, 21949-900, Rio de Janeiro, Brasil.
b Cenpes, Centro de Pesquisas Leopoldo Miguez, Petrobras, Rio de Janeiro Brasileiro
cDepartamento de Biocatálisis. ICP-CSIC. C/ Marie Curie 2. Campus UAM-CSIC. Cantoblanco. 28049 Madrid (Spain).


Abstract
 
 
A new simple and extremely versatile zymography method has been developed to
determine the specific enzymes that are responsible of the transesterificationor esterification activities of different enzymatic crude preparations.   The method consists in building a transesterification or esterification reaction
medium according to the goal of the research and later identifying the protein
bands that are the responsible of these catalytic activities just by the 
precipitation of the product over the protein band. In this work, the
substrates used were a) for transesterification reaction: methyl esters from 
castor oil (methyl ricinoleate) and trimethylolpropane (TMP); b) for 
esterification reaction: oleic acid and TMP or ethanol. After submitting the 
crude enzyme preparations to  SDS-PAGE experiments, the SDS was removed from 
the gels by incubation in 5 mM sodium phosphate containing 1% (w/v) Triton X 100 at pH 7,0 and re-equilibrated in 5 mM sodium phosphate pH 7 before being immersed in the respective reaction media for 24h (TMP reactions) or 2h (ethanol reaction) at 40°C. This method has permitted to determine that in many instances the protein responsible of the reaction did not correspond to the main band of the enzyme preparation, in some cases it is even a very minority band (e.g., the case of Pseudomonas sp. Type XIII in the transesterification zymogram between methyl ricinoleate and trimethylolpropane two protein bands that can 
be hardly visualized when stained for protein were responsible of a high percentage of the crude activity). The protocol may be potentially used as a 
high-throughput screening of lipases or esterases that catalyzes the 
synthesis reaction for biodiesel and biolubricants production. 
Keywords:biodiesel, Esterification reactions, transesterification reactions, zymography, SDS-PAGE, lipase and biolubricants
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