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Functional Genomic and Culture Independent Approaches to Search for Protease Activity in Forest soils in Puerto Rico Zulei...
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  1. 1. Functional Genomic and Culture Independent Approaches to Search for Protease Activity in Forest soils in Puerto Rico Zuleika M. Corales-Tirado 1 , Rocio K. Rivera-Valentin 1 , and Dr. Carlos Rios-Velazquez 2 1 Industrial Biotechnology Program, University of Puerto Rico, Mayagüez, PR 2 Department of Biology, University of Puerto Rico, Mayagüez, PR Category: Biotechnology (1)Schloss, P.D., Handelsman, J. Biotechnological prospects from metagenomics. Current Opinion in Biotechnology 14:303–310, (2003). (2)Maurer, K.H. Detergent proteases. Current Opinion in Biotechnology 15:330–334, (2004). (3)Patrick, L., Eck, J. Metagenomics and industrial applications. Nature Reviews Microbiology 3:510-516, (2005). Rondon, M.R. et.al. Cloning the Soil Metagenome: a Strategy for Accesing the Genetic and Functional Diversity of Uncultured Microorganisms. Applied and Environmental Microbiology. June 2002, 2541-2547. References Methodology Results and Discussion Future Work Conclusions Acknowledgements <ul><li>BioMinds </li></ul><ul><li>Dr. Carlos Rios-Velazquez </li></ul><ul><li>Microbial Biotechnology and Bioprospecting </li></ul><ul><li>Laboratory- B266 </li></ul>Combining molecular biology, genetics, and the recognition that most microorganisms in the environment cannot be cultured by standard methods, emerges the field of metagenomics 1 . Metagenomics is the study of the genetics material that is directly isolated from enviroments samples, in this case soils from Puertorrican forests 1 . Different industries have diverse initiatives to probe the immense resource that is uncultivated microbial diversity. It is well known that biotechnology has a continuous requirement for novel genes, industrial enzymes, and compounds. Metagenomics or soil microorganisms have been the best supplier for these novel molecules with diverse functions 3 . Actually, the main enzyme with the greater demand in the industry is protease 2 . Casein is the predominant phosphoprotein (αS1, αS2, β, κ) that accounts for nearly 80% of proteins in milk, and its soluble portion is catalyzed by proteases. Our objective is the screening of two metagenomic libraries, from Puerto Rican Forests, in order to identify clones producing protease activity. <ul><li>Assay to detect enzyme production in E.coli EPI 300 </li></ul><ul><li>  </li></ul><ul><li>Figure 1). Protease assay in E.coli EPI300 - To detect the production of protease E.coli EPI 300 was streaked on plates containing LB agar plus 1% commercial nonfat dry milk. Plates were scored after 3 days for the presence of a clear halo. </li></ul><ul><li>II. Serial Dilutions </li></ul><ul><li>Figure 2). Serial Dilutions - Serial dilutions were performed in order to determine the ideal dilution factor to use with each metage4nomic library. </li></ul><ul><li>III. Screening of protease </li></ul><ul><li>Figure 3). Srceening of protease - The library was grown in LB-Broth+chloramphenicol (12.5 µg/ml) during 2 hours. Serial dilutions were performed. For detecting protease activity the clones were grown on plates containing LB agar+chloramphenicol plus 1% commercial nonfat dry milk, and scored after 3 days for the presence of a clear halo. </li></ul>The screening of enzymes from metagenomic libraries is a new strategy that is in process of development, which allows accessing the genetic diversity of those non-cultivable microorganisms, so increasing the availability of enzymes and biomolecules for industrial processes. In our laboratory, using function-based screening, we found about 20 potential clones producing protease enzyme, which will be analyzed through various techniques to check whether they’re producing the enzyme. The recognition that most microorganisms in the environment cannot be culture by standard methods stimulated the development of metagenomics, which is the genomic analysis of uncultured microorganisms. Modern biotechnology has a steadily increasing demand for novel bioactive compounds, biomolecules, and enzymes. Natural and genetic diversity of the soil metagenome has so far been the best supplier for these novel molecules. Enzymes production is one of the most important drivers of the industrial processing industry because it uses modern biology in the production process, and produces the biotechnological products needed for applications in other industrial areas. Some of these industrial enzymes are the proteases, which have important biotechnological applications and execute a large variety of functions. The proteases represent one of the three largest groups of industrial enzymes and have application in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes. Probably the largest application of proteases is in laundry detergents, where they help removing proteins based strains from clothing. Two approaches, the function-driven analysis and the sequence-driven analysis, have emerged to extract biological information from metagenomic libraries. Here, we used the function-driven analysis, which is initiated by identification of clones that express a desired trait, followed by characterization of the active clones by sequence and biochemical analysis. We screened three metagenomic libraries generated from forests in Puerto Rico. Serial dilutions of the libraries were spread on Petri plates supplemented with non-fat dry milk. A clear zone around the tested clones indicated clones with potential for protease activity. A total 20 potential clones were found which have being analyzed through biochemical and molecular techniques to confirm whether the activity is due to the cloned fragment. The screening of detergent proteases for industrial production has a great importance because detergent enzymes account for about 30% of the total worldwide enzyme production, and represent one of the largest and most successful applications of modern industrial biotechnology. Figure 4) . The test for the production of protease activity were carried out in E.coli EPI 300, in order to determine whether the strain was positive or negative to these tests. This strain showed growth in the culture media used to detect protease activity, however doesn’t produce any of this enzyme, thus is negative protease. I. Assay to detect enzyme production in E.coli EPI 300 II. Screening of protease Figurea 5) and 6) . Casein is the predominant phosphoprotein (αS1, αS2, β, κ) that accounts for nearly 80% of proteins in milk. To screening the protein degradation we used culture media with low fat dry milk. The screening was carried out in the metagenomic libraries of 14,000 and 600,000 clones. The library dilution used was 10 -4 , allowed us to test only ten thousands clones of each library. There are still many more clones that need to be tested. We found 20 possible clones of protease enzyme with apparently clear halo around colonies, but the process to confirm this is still in progress. 900  l NaCl 0.85% 100  l LB-broth+clones 10 -1 10 -2 10 -3 10 -4 10 -5 10 0 10 -1 10 -2 10 -3 10 -4 100  l 100  l 100  l 100  l 100  l Metagenomic Library 1mL 100  l 100  l 100  l 100  l Clones, LB-Broth+Chloramphenicol (12.5  l /ml), 2 hr, 37  C <ul><li>To obtain better results in future projects the protocols of the function-based screening should be improved and optimized. </li></ul><ul><li>Furthermore, will be used sequence-based screening, which is based on monitoring clone’s sequences. </li></ul>Abstract Introduction

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