P57: Tools for biocatalysis and bioremediation – high throughput methodology of searching for new enzymes

Bacteria during evolution created enormous biodiversity enabling them to spread around the whole planet and adopt their metabolism even to extreme environments. Recently next generation sequencing (NGS) methods gave technological breakthrough for exploration of bacterial biodiversity and enzymatic activity. Here we present procedure that enables efficient searching of new enzymes in high throughput manner. NGS of three metagenomic DNA samples isolated from soil samples highly contaminated with persistent pesticides, followed by bioinformatic analysis gave almost 2 mln contigs longer than 150bp, with largest contig 332 kb. Contig analysis after annotation of potential protein-coding sequences showed that more than 10% of potential ORF's might be potentially involved in xenobiotic degradation, what is 40% more when compared to metagenom coming from non contaminated samples. To explore plethora of enzymes revealed in bioinformatic approach, we developed methodology comprising high throughput gene cloning and protein purification. Biochemical features of newly cloned enzymes are determined by mass spectrometry. Here we show dozen dehalogenases that were cloned, purified and tested against persistent pesticides as DDT and HCH. All above confirm that developed strategy searching of new enzymes in soil metagenoms might be helpful in fast identification of catalysts having big practical importance for bioremediation and biotransformation.