|dc.description.abstract||Microorganisms have evolved to exist in diverse environments on earth, even under the harsh conditions of oil reservoirs several kilometres below the seafloor. The environmental conditions there are extreme in several aspects, such as high temperature, pressure, salinity, and presence heavy metals or other potentially toxic compounds. In order to live under these conditions, the microbes enzymes have evolved to withstand the extremes. Enzymes with such traits might be utilized for several biochemical industries.
In a previous project, microbial DNA from two oil reservoirs has been isolated and high throughput pyrosequencing used to create an extensive metagenomic DNA sequence database. Using bioinformatics tools, three putative gene sequences for thermostable peptidases were identified in this oil reservoir metagenomic sequence database. They showed a high degree of sequence homology to a known peptidase of the enzyme sub-classes, subtilisin-like protease, carboxypeptidase or aminopeptidases, and were named SLP01, CPT01 and ATP01.
Protein production of the recombinant proteins was performed in Escherichia coli strains BL21 (DE3) CodonPlus-RIPL and ER2566, using the plasmid vector pET21, at temperatures 16 and 37 °C. Desired protein production was verified using SDS-PAGE analysis. Production of target protein was observed for the APT01 and CPT01 candidate, although in an insoluble form. Some soluble APT01 was observed when the culture had been incubated at 16 °C.
Heat denaturation series was performed on the sample containing soluble APT01, at temperatures of 65 and 80 °C for a duration of 5, 15, 30, 60 and 120 minutes. The results showed APT01 to be thermostable at 65 °C, but none at 80 °C. An activity assay, using skim milk agar (MA) plates, was performed on all samples from the protein production. No activity was observed for any of the three candidates.||