Searching for new thermostable enzymes
Thermophilic microorganisms are a subject of intense research. Thermostable enzymes found in thermophilic bacteria are produced recombinantly and used both in industry and biotechnology. For example, the thermostable enzyme Taq-polymerase is used in polymerase chain reaction, a very sensitive and widespread tool for genetic analysis. The search for new thermostable enzymes continues. The project 'Exgenome Molecular Enzymes' (EXGENOMES) intended to develop new and improved enzymes for use in large-scale DNA synthesis. The target source for the new enzymes were self-replicating mobile genetic elements (phages, plasmids and transposons) from thermophilic bacteria. In addition to using existing genetic databases for thermophilic bacteria and phages, novel genetic material was obtained from hot springs in Iceland and sequenced. The candidate genes were selected, cloned and expressed. Produced enzymes were purified, characterised and tested. A total of 39 new enzymes were selected for cloning and expression, out of which 23 enzymes were produced and purified for activity evaluation. Eight types of thermostable enzymes were represented, such as DNA polymerases, protelomerases, lysozymes, helicase, RNAse H, Rec A, primase and single-stranded DNA binding protein (SSB). After activity evaluation, 14 enzymes were further purified and characterised, and finally 6 enzymes were developed into commercial products. These are: LysT lysozyme from Thermus phage Pro2631; Lys2119 lysozyme from Thermus phage Ph2119; RecA recombinase from Thermus phage T72; RadA recombinase from Pyrococcus woesei; Pol72 DNA polymerase from Thermus islandicus; and SSB M2 from metagenome. In addition, two new protelomerases, TelA and TelK, were cloned, expressed, purified and used for experiments in making new DNA vaccine constructs. New enzymes were described in six scientific publications, and two patents were filed. Development of new thermostable enzymes for commercial applications promises benefits for the smaller enterprises involved in the project. It also boosts EU competitiveness in the biotechnology sector for applications in health care and pharmaceutical industries.
Keywords
Thermostable enzymes, enzymes, biotechnology, thermophilic, molecular enzymes
