Functional metagenomics and cultured-based approaches for the discovery of cold-active proteolytic enzymes from the Antarctic region

Abstract

The Antarctic region is a new frontier as a natural source for bio-prospecting purposes. Its extreme cold temperature may provide unique microbial enzyme characteristics that have valuable potential for industrial and biotechnological applications. However, the majority of microbial cannot be cultured due to their complex structure of life and unfavourable media composition and growth conditions. In this study, two different approaches were designed to ensure the total discovery of proteases that are active and able to work at relatively low temperatures. The first approach was the culture-based technique. Soil samples from the Antarctic region were screened for protease activity on skim milk agar at 4 °C. Bacteria that showed a clear halo zone around the colonies were isolated and identified through 16S rDNA sequencing. Bacteria that showed rapid halo zone formation within 24 hours were further screened for its growth rate condition using one-factor-at-a-time (OFAT). Then, crude protease of each strain was extracted during the late logarithmic phase for enzymatic assay. Strain with significant enzyme activity was optimized using Response Surface Method (RSM) for maximal growth rate. The second approach was the metagenomics approach. Amplicon metagenomics sequencing was performed to analyse the bacteria community and the discovery of cold-active protease was completed through functional metagenomics. A total of 46 bacteria strains with positive protease activity were isolated and phylogenetic analysis showed that 88% were from Pseudomonas sp., 9% from Arthrobacter sp. and 3% from Paenibacillus sp. OFAT result showed that 10 selected strains displayed the highest growth rate at 20 °C, pH 7 and 4% (w/v) of NaCl. The enzymatic assay showed that crude enzyme extracted from strain SC8 exhibited significantly higher activity than the positive control (protease from bovine pancreas) at -20 °C and 20 °C. Furthermore, RSM suggested that the optimized conditions for the growth of strain SC8 were at 20.5 °C, pH 6.83, and 2.05% (w/v) of NaCl. Bacterial community analysis using amplicon metagenomics sequencing showed that each sample (ROB, ROS, and SC) were dominated by the uncultured bacteria. A clone with positive protease activity was isolated and the fosmid was extracted and sequenced. Contigs of NODE_42 with 893 bp showed significant matched to Peptidase M23 and PG binding 1 protein families. In silico analysis showed that this predicted protease exhibited 27.07% similarity of the template enzyme with PDB ID 3SLU. The growth condition of isolated bacteria was influenced by the site surroundings and the coastal environment. These bacteria were categorized as psychrotolerants and the majority of them can tolerate acidic conditions. Optimum bacterial growth conditions were important to maximize enzyme production and activity. Thus, strain SC8 has the potential for bioprospecting for the development of large-scale production of cold-active protease in the future. Overall, for this project, we successfully discovered our targeted cold-active protease. This preliminary research is vital to unearth all potential protease for bioprospecting purposes. In the future, purification of this enzyme is essential to precisely inspect the enzyme activity.