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.
