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Characterization of agarase activity from Burkholderia sp.

Grant number: 09/03772-9
Support Opportunities:Scholarships in Brazil - Master
Effective date (Start): August 01, 2009
Effective date (End): February 28, 2011
Field of knowledge:Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
Principal Investigator:Eliana Gertrudes de Macedo Lemos
Grantee:Thaís Carvalho Maester Casanova
Host Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , SP, Brazil

Abstract

Lipolytic enzymes have show enormous biotechnological potential, such as in enzyme mixtures for the production of detergents, the processing of leather, the production of cosmetics and other pharmaceuticals, perfumes and biodiesel. Most lipolytic enzymes are derived from microbes, present low toxicity, are easily biodegraded and are notably selective of chemicals. The present work was done as an attempt to find genes which codify lipolytic enzymes in a fosmid metagenomic library composed of petroleum hydrocarbons degradation microbe consortia with 4224 clones. Clones were selected according to lipolytic activity and were assessed by cultivation in Luria-Bertani (LB) medium supplemented with1% tributyrin (v/v), 1% gum arabic (w/v), 0.00125% chloramphenicol (v/v) and 0.001% arabinose (w/v). All cultures were maintained at 37ºC for 72 hours and then transferred at 4ºC. Assessment was done by observation of halos formed around the colonies, with 30 clones producing halo formations and identified as positives. Of these, two were selected and had their DNA sub cloned in pUC19 vectors. DNA from the sub libraries was sequenced, generating a complete contig for clone PL28.F10 that was compared to sequences from the National Center for Biotechnology Information (NCBI) bank by the ORF Finder program. An ORF coding for esterase/lipase of 303 amino acids with 61% of identity with uncunturable microorganism was found. Alignments were done using the Clustal W program and cladograms were constructed using MEGA 4, to compare the ORF found, ORF15, and the deposited sequences. Assessment of the cladograms showed that the clone presented the ORF15 similar to that of esterase/lipase family IV, because it was located in a unique branch, differing from the other representatives of this family. The alignments made possible the identification of active sites which represent the family, confirming the results obtained with the construction of the cladograms, although it was observed some changes in amino acid residues. When the same evaluation was done using patented sequences, the ORF15 showed similarities to patented BASF esterase/lipase (Patent WO0100842 ) related to biosynthesis of FK228 and its analogues used in cancer treatment and an unnamed protein of CAMBIA (Patent 6562958, accession number AAQ29806.1) used for diagnosis and therapy, which has conserved domain esterases/lipases.

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Academic Publications
(References retrieved automatically from State of São Paulo Research Institutions)
CASANOVA, Thaís Carvalho Maester. Screening for genomic sequences which codify lipolytic enzymes specialized in petroleum hydrocarbons degradation.. 2011. Master's Dissertation - Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI) São Paulo.

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